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  1. Aptamers Binding to c-Met Inhibiting Tumor Cell Migration.

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    Birgit Piater

    Full Text Available The human receptor tyrosine kinase c-Met plays an important role in the control of critical cellular processes. Since c-Met is frequently over expressed or deregulated in human malignancies, blocking its activation is of special interest for therapy. In normal conditions, the c-Met receptor is activated by its bivalent ligand hepatocyte growth factor (HGF. Also bivalent antibodies can activate the receptor by cross linking, limiting therapeutic applications. We report the generation of the RNA aptamer CLN64 containing 2'-fluoro pyrimidine modifications by systematic evolution of ligands by exponential enrichment (SELEX. CLN64 and a previously described single-stranded DNA (ssDNA aptamer CLN3 exhibited high specificities and affinities to recombinant and cellular expressed c-Met. Both aptamers effectively inhibited HGF-dependent c-Met activation, signaling and cell migration. We showed that these aptamers did not induce c-Met activation, revealing an advantage over bivalent therapeutic molecules. Both aptamers were shown to bind overlapping epitopes but only CLN3 competed with HGF binding to cMet. In addition to their therapeutic and diagnostic potential, CLN3 and CLN64 aptamers exhibit valuable tools to further understand the structural and functional basis for c-Met activation or inhibition by synthetic ligands and their interplay with HGF binding.

  2. Hepatocyte growth factor/cMET pathway activation enhances cancer hallmarks in adrenocortical carcinoma

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    Phan, Liem M.; Fuentes-Mattei, Enrique; Wu, Weixin; Velazquez-Torres, Guermarie; Sircar, Kanishka; Wood, Christopher G.; Hai, Tao; Jimenez, Camilo; Cote, Gilbert J.; Ozsari, Levent; Hofmann, Marie-Claude; Zheng, Siyuan; Verhaak, Roeland; Pagliaro, Lance; Cortez, Maria Angelica; Lee, Mong-Hong; Yeung, Sai-Ching J.; Habra, Mouhammed Amir

    2015-01-01

    Adrenocortical carcinoma (ACC) is a rare malignancy with poor prognosis and limited response to chemotherapy. Hepatocyte growth factor (HGF) and its receptor cMET augment cancer growth and resistance to chemotherapy, but their role in ACC has not been examined. In this study, we investigated the association between HGF/cMET expression and cancer hallmarks of ACC. Transcriptomic and immunohistochemical analyses indicated that increased HGF/cMET expression in human ACC samples was positively associated with cancer-related biological processes including proliferation and angiogenesis, and negatively correlated with apoptosis. Accordingly, treatment of ACC cells with exogenous HCG resulted in increased cell proliferation in vitro and in vivo while short hairpin RNA-mediated knockdown or pharmacological inhibition of cMET suppressed cell proliferation and tumor growth. Moreover, exposure of cells to mitotane, cisplatin, or radiation rapidly induced pro-cMET expression and was associated with an enrichment of genes (e.g., CYP450 family) related to therapy resistance further implicating cMET in the anticancer drug response. Together, these data suggest an important role for HGF/cMET signaling in ACC growth and resistance to commonly used treatments. Targeting cMET, alone or in combination with other drugs, could provide a breakthrough in the management of this aggressive cancer. PMID:26282167

  3. The EGFR/ErbB3 Pathway Acts as a Compensatory Survival Mechanism upon c-Met Inhibition in Human c-Met+ Hepatocellular Carcinoma.

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    Steven N Steinway

    Full Text Available c-Met, a high-affinity receptor for Hepatocyte Growth Factor (HGF, plays a critical role in tumor growth, invasion, and metastasis. Hepatocellular carcinoma (HCC patients with activated HGF/c-Met signaling have a significantly worse prognosis. Targeted therapies using c-Met tyrosine kinase inhibitors are currently in clinical trials for HCC, although receptor tyrosine kinase inhibition in other cancers has demonstrated early success. Unfortunately, therapeutic effect is frequently not durable due to acquired resistance.We utilized the human MHCC97-H c-Met positive (c-Met+ HCC cell line to explore the compensatory survival mechanisms that are acquired after c-Met inhibition. MHCC97-H cells with stable c-Met knockdown (MHCC97-H c-Met KD cells were generated using a c-Met shRNA vector with puromycin selection and stably transfected scrambled shRNA as a control. Gene expression profiling was conducted, and protein expression was analyzed to characterize MHCC97-H cells after blockade of the c-Met oncogene. A high-throughput siRNA screen was performed to find putative compensatory survival proteins, which could drive HCC growth in the absence of c-Met. Findings from this screen were validated through subsequent analyses.We have previously demonstrated that treatment of MHCC97-H cells with a c-Met inhibitor, PHA665752, results in stasis of tumor growth in vivo. MHCC97-H c-Met KD cells demonstrate slower growth kinetics, similar to c-Met inhibitor treated tumors. Using gene expression profiling and siRNA screening against 873 kinases and phosphatases, we identified ErbB3 and TGF-α as compensatory survival factors that are upregulated after c-Met inhibition. Suppressing these factors in c-Met KD MHCC97-H cells suppresses tumor growth in vitro. In addition, we found that the PI3K/Akt signaling pathway serves as a negative feedback signal responsible for the ErbB3 upregulation after c-Met inhibition. Furthermore, in vitro studies demonstrate that

  4. Inhibition of c-Met as a Therapeutic Strategy for Esophageal Adenocarcinoma

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    Gregory A. Watson

    2006-11-01

    Full Text Available The hepatocyte growth factor (HGF receptor c-Met is a tyrosine kinase receptor with established oncogenic properties. We have previously shown that c-Met is usually overexpressed in esophageal adenocarcinoma (EA, yet the implications of c-Met inhibition in EA remain unknown. Three c-Met-overexpressiog EA cell lines (Seg-1, Bic-1, Flo-1 were used to examine the effects of a c-Met-specific small molecule inhibitor (PHA665752 on cell viability, apoptosis, motility, invasion, downstream signaling pathways. PHA665752 demonstrated dose-dependent inhibition of constitutive and/or HGF-induced phosphorylation of c-Met, which correlated with reduced cell viability and inhibition of extracellular regulated kinase 1/2 phosphorylation in all three EA cell lines. In contrast, PHA665752 induced apoptosis and reduced motility and invasion in only one EA cell line, Flo-1. Interestingly, Flo-1 was the only cell line in which phosphatidylinositol 3-kinase (PI3K/Akt was induced following HGF stimulation. The PI3K inhibitor LY294002 produced effects equivalent to those of PHA665752 in these cells. We conclude that inhibition of c-Met may be a useful therapeutic strategy for EA. Factors other than receptor overexpression, such as c-Met-dependent PI3K/Akt signaling, may be predictive of an individual tumor's response to c-Met inhibition.

  5. The c-Met Inhibitor MSC2156119J Effectively Inhibits Tumor Growth in Liver Cancer Models

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    Bladt, Friedhelm, E-mail: Friedhelm.Bladt@merckgroup.com; Friese-Hamim, Manja; Ihling, Christian; Wilm, Claudia; Blaukat, Andree [EMD Serono, and Merck Serono Research and Development, Merck KGaA, Darmstadt 64293 (Germany)

    2014-08-19

    The mesenchymal-epithelial transition factor (c-Met) is a receptor tyrosine kinase with hepatocyte growth factor (HGF) as its only high-affinity ligand. Aberrant activation of c-Met is associated with many human malignancies, including hepatocellular carcinoma (HCC). We investigated the in vivo antitumor and antimetastatic efficacy of the c-Met inhibitor MSC2156119J (EMD 1214063) in patient-derived tumor explants. BALB/c nude mice were inoculated with MHCC97H cells or with tumor fragments of 10 patient-derived primary liver cancer explants selected according to c-Met/HGF expression levels. MSC2156119J (10, 30, and 100 mg/kg) and sorafenib (50 mg/kg) were administered orally as single-agent treatment or in combination, with vehicle as control. Tumor response, metastases formation, and alpha fetoprotein (AFP) levels were measured. MSC2156119J inhibited tumor growth and induced complete regression in mice bearing subcutaneous and orthotopic MHCC97H tumors. AFP levels were undetectable after 5 weeks of MSC2156119J treatment, and the number of metastatic lung foci was reduced. Primary liver explant models with strong c-Met/HGF activation showed increased responsiveness to MSC2156119J, with MSC2156119J showing similar or superior activity to sorafenib. Tumors characterized by low c-Met expression were less sensitive to MSC2156119J. MSC2156119J was better tolerated than sorafenib, and combination therapy did not improve efficacy. These findings indicate that selective c-Met/HGF inhibition with MSC2156119J is associated with marked regression of c-Met high-expressing tumors, supporting its clinical development as an antitumor treatment for HCC patients with active c-Met signaling.

  6. Expression of hepatocyte growth factor and its receptor c-Met in lens-induced myopia in guinea pigs

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    LI Xiu-juan; YANG Xiao-peng; WAN Guang-ming; WANG Yu-ying; ZHANG Jin-song

    2013-01-01

    Background Myopia is a common disorder and the incidence has increased yearly,but its pathogenesis remains unclear.The aim of this study was to investigate the possible role of hepatocyte growth factor (HGF) and its receptor c-Met in the development of lens-induced myopia in guinea pigs.Methods Sixty one-week-old guinea pigs were chosen.The right eyes were treated with-10.0 diopters (D) lenses as the lens-induced myopia group; the left eyes remained untreated as the control group.Six weeks later,refractive status and axial length were determined by streak retinoscopy and A-scan ultrasonography,respectively.The guinea pigs were killed and both eyes collected.Morphological changes were observed by hematoxylin and eosin staining.The expression levels of HGF,c-Met,and matrix metalloproteinase 2 (MMP-2) mRNA and protein in the posterior sclera were analyzed by RT-PCR and Western blotting,respectively.Results The lens-induced myopia group became myopic with a significant increase in axial length and a significant decrease in refraction.Compared with the control group,the posterior retina and sclera were thinner in the lens-induced myopia group.The expression levels of HGF and MMP-2 mRNA and protein and of phosphorylated c-Met protein were significantly higher in the posterior sclera of the lens-induced myopia group than in the control group (all P <0.05).In the lens-induced myopia group,the expression level of MMP-2 in the posterior sclera positively correlated with the expression level of HGF (r=0.902,P <0.05) and phosphorylated c-Met (r=0.885,P <0.05).Conclusion HGF/c-Met might play a role in the development of lens-induced myopia in guinea pigs by upregulating the expression of MMP-2.

  7. Osthole suppresses hepatocyte growth factor (HGF)-induced epithelial-mesenchymal transition via repression of the c-Met/Akt/mTOR pathway in human breast cancer cells.

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    Hung, Chao-Ming; Kuo, Daih-Huang; Chou, Chun-Hung; Su, Yen-Chao; Ho, Chi-Tang; Way, Tzong-Der

    2011-09-14

    Substantial activation of the HGF/c-Met signaling pathway is involved in the progression of several types of cancers and associated with increased tumor invasion and metastatic potential. Underlying HGF-induced tumorigenesis, epithelial to mesenchymal transition (EMT) shows a positive correlation with progression in patients. We previously determined that osthole is a potent fatty acid synthase (FASN) inhibitor. FASN is implicated in cancer progression and may regulate lipid raft function. We therefore examined whether osthole could block HGF-induced tumorigenesis by disrupting lipid rafts. Here, we found that osthole could abrogate HGF-induced cell scattering, migration, and invasion in MCF-7 breast cancer cells. Osthole also effectively inhibited the HGF-induced decrease of E-cadherin and increase of vimentin via down-regulation of phosphorylated Akt and mTOR. Interestingly, osthole blocked HGF-induced c-Met phosphorylation and repressed the expression of total c-Met protein in MCF-7 cells. In addition, C75, a pharmacological inhibitor of FASN, repressed the expression of total c-Met protein in MCF-7 cells. Consistent with a role for FASN, loss of c-Met in cells treated with osthole was prevented by the exogenous addition of palmitate. Briefly, our result suggests a connection between FASN activity and c-Met protein expression and that osthole is a potential compound for breast cancer therapy by targeting the major pathway of HGF/c-Met-induced EMT. PMID:21806057

  8. Expression of the proto-oncogenes c-met and c-kit and their ligands, hepatocyte growth factor/scatter factor and stem cell factor, in SCLC cell lines and xenografts

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    Rygaard, K; Nakamura, T; Spang-Thomsen, M

    1993-01-01

    We examined a panel of 25 small cell lung cancer (SCLC) cell lines and nude mouse xenografts for expression of the proto-oncogenes c-met and c-kit, and for expression of the corresponding ligands, hepatocyte growth factor (HGF) (also known as scatter factor (SF)), and stem cell factor (SCF......), respectively. Expression of mRNA was detected by Northern blotting, and c-met and c-kit protein expression was detected by Western blotting and immunocytochemistry. c-met and c-kit mRNA was expressed in 22 of the examined cell lines or xenografts, and coexpression of the two proto-oncogenes was observed in 20...... tumours. Expression of c-met and c-kit protein paralleled in the mRNA expression. HGF/SF mRNA was expressed in two of the examined tumours, and only one of these also expressed the c-met proto-oncogene. SCF mRNA was expressed in 19 of the examined tumours, and in 18 of these coexpression of c-kit and SCF...

  9. Detection of hepatocyte growth factor/scatter factor receptor (c-Met in axillary clearance after mastectomy for breast cancer using reverse transcriptase-polymerase chain reaction

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    El-Refaey H.K.

    2007-06-01

    Full Text Available The diverse biological effects of hepatocyte growth factor/scatter factor (HGF/SF are mediated by c-Met which is preferentially expressed on epithelial cells. Met signaling has a role in normal cellular activities, and may be associated with development and progression of malignant processes. In this study presence of Met in the axillary drainage from patients who underwent conservative operations for breast cancer, and its prognostic significance was examined. Sixty-two consecutive patients with invasive ductal carcinoma of the breast which were suitable for breast-conserving treatment participated in the study. The output of the drain that had been placed in the axilla during the operation was collected, and the presence of Met and β-actin were assessed by reverse transcriptase-polymerase chain reaction (RT-PCR assays. The data were compared with the pathological features of the tumor and the axillary lymph nodes, and with the estrogen and progesterone receptors status.RT-PCR of the axillary lymphatic drainage was positive for Met in 46 (74.2% of the patients and positive assays were correlated with increase in tumor size and grade of capillary and lymphatic invasion, as well as with lymph node metastasis (P < 0.02, for all comparisons. All 24 patients with axillary lymph node metastases in comparison with those without lymph node (57.9% metastases had positive assays for Met. While all ten patients with tumor involvement in the margins of the resection had positive assays for Met in their lymphatic fluid, only 36 out of 52 patients (69.2% were positive for met assay. Finally, Met showed negative correlations with positive estrogen and progesterone receptor assays (P<0.02.From the results of this study it may concluded that Met can be detected in the axillary fluids of patients with breast cancer and its expression in the axillary drainage may be a potential prognostic factor. This finding might be useful in therapeutic considerations since a

  10. Norstictic Acid Inhibits Breast Cancer Cell Proliferation, Migration, Invasion, and In Vivo Invasive Growth Through Targeting C-Met.

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    Ebrahim, Hassan Y; Elsayed, Heba E; Mohyeldin, Mohamed M; Akl, Mohamed R; Bhattacharjee, Joydeep; Egbert, Susan; El Sayed, Khalid A

    2016-04-01

    Breast cancer is a major health problem affecting the female population worldwide. The triple-negative breast cancers (TNBCs) are characterized by malignant phenotypes, worse patient outcomes, poorest prognosis, and highest mortality rates. The proto-oncogenic receptor tyrosine kinase c-Met is usually dysregulated in TNBCs, contributing to their oncogenesis, tumor progression, and aggressive cellular invasiveness that is strongly linked to tumor metastasis. Therefore, c-Met is proposed as a promising candidate target for the control of TNBCs. Lichens-derived metabolites are characterized by their structural diversity, complexity, and novelty. The chemical space of lichen-derived metabolites has been extensively investigated, albeit their biological space is still not fully explored. The anticancer-guided fractionation of Usnea strigosa (Ach.) lichen extract led to the identification of the depsidone-derived norstictic acid as a novel bioactive hit against breast cancer cell lines. Norstictic acid significantly suppressed the TNBC MDA-MB-231 cell proliferation, migration, and invasion, with minimal toxicity to non-tumorigenic MCF-10A mammary epithelial cells. Molecular modeling, Z'-LYTE biochemical kinase assay and Western blot analysis identified c-Met as a potential macromolecular target. Norstictic acid treatment significantly suppressed MDA-MB-231/GFP tumor growth of a breast cancer xenograft model in athymic nude mice. Lichen-derived natural products are promising resources to discover novel c-Met inhibitors useful to control TNBCs. PMID:26744260

  11. c-MET receptor tyrosine kinase as a molecular target in advanced hepatocellular carcinoma

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    Granito A

    2015-04-01

    Full Text Available Alessandro Granito,1 Elena Guidetti,1 Laura Gramantieri2,3 1Dipartimento di Scienze Mediche e Chirurgiche Università di Bologna, Bologna, Italy; 2Dipartimento dell'Apparato Digerente, Azienda Ospedaliero-Universitaria di Bologna, Bologna, Italy; 3Centro di Ricerca Biomedica Applicata (CRBA, Azienda Ospedaliero-Universitaria Policlinico S Orsola-Malpighi e Università di Bologna, Bologna, Italy Abstract: c-MET is the membrane receptor for hepatocyte growth factor (HGF, also known as scatter factor or tumor cytotoxic factor, a mitogenic growth factor for hepatocytes. HGF is mainly produced by cells of mesenchymal origin and it mainly acts on neighboring epidermal and endothelial cells, regulating epithelial growth and morphogenesis. HGF/MET signaling has been identified among the drivers of tumorigenesis in human cancers. As such, c-MET is a recognized druggable target, and against it, targeted agents are currently under clinical investigation. c-MET overexpression is a common event in a wide range of human malignancies, including gastric, lung, breast, ovary, colon, kidney, thyroid, and liver carcinomas. Despite c-MET overexpression being reported by a large majority of studies, no evidence for a c-MET oncogenic addiction exists in hepatocellular carcinoma (HCC. In particular, c-MET amplification is a rare event, accounting for 4%–5% of cases while no mutation has been identified in c-MET oncogene in HCC. Thus, the selection of patient subgroups more likely to benefit from c-MET inhibition is challenging. Notwithstanding, c-MET overexpression was reported to be associated with increased metastatic potential and poor prognosis in patients with HCC, providing a rationale for its therapeutic inhibition. Here we summarize the role of activated HGF/MET signaling in HCC, its prognostic relevance, and the implications for therapeutic approaches in HCC. Keywords: hepatocellular carcinoma, c-MET, clinical trials

  12. Piperlongumine and its analogs down-regulate expression of c-Met in renal cell carcinoma.

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    Golovine, Konstantin; Makhov, Peter; Naito, Sei; Raiyani, Henish; Tomaszewski, Jeffrey; Mehrazin, Reza; Tulin, Alexei; Kutikov, Alexander; Uzzo, Robert G; Kolenko, Vladimir M

    2015-01-01

    The c-Met protein, a transmembrane receptor tyrosine kinase, is the product of a proto-oncogene. Its only known ligand, hepatocyte growth factor (HGF), regulates cell growth, motility, migration, invasion, proliferation, and angiogenesis. The aberrant expression of c-Met is often associated with poor prognosis in multiple cancers, including renal cell carcinoma (RCC). Silencing or inactivation of c-Met leads to decreased viability of cancer cells, thereby making ablation of c-Met signaling an attractive concept for developing novel strategies for the treatment of renal tumors. Naturally-occurring products or substances are the most consistent source of drug development. As such, we investigated the functional impact of piperlongumine (PL), a naturally occurring alkaloid present in the Long pepper (Piper longum) on c-Met expression in RCC cells and demonstrated that PL and its analogs rapidly reduce c-Met protein and RNA levels in RCC cells via ROS-dependent mechanism. PL-mediated c-Met depletion coincided with the inhibition of downstream c-Met signaling; namely Erk/MAPK, STAT3, NF-κB and Akt/mTOR. As such, PL and PL analogs hold promise as potential therapeutic agents for the treatment of metastatic RCC and the prevention of postoperative RCC recurrence. PMID:25801713

  13. The oleocanthal-based homovanillyl sinapate as a novel c-Met inhibitor.

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    Mohyeldin, Mohamed M; Akl, Mohamed R; Ebrahim, Hassan Y; Dragoi, Ana Maria; Dykes, Samantha; Cardelli, James A; El Sayed, Khalid A

    2016-05-31

    The hepatocyte growth factor (HGF)/mesenchymal-epithelial transition factor (c-Met) signaling axis has gained considerable attention as an attractive molecular target for therapeutic blockade of cancer. Inspired by the chemical structure of S (-)-oleocanthal, a natural secoiridoid from extra-virgin olive oil with documented anticancer activity against c-Met-dependent malignancies, the research presented herein reports on the discovery of the novel olive-derived homovanillyl sinapate (HVS) as a promising c-Met inhibitor. HVS was distinguished for its remarkable potency against wild-type c-Met and its oncogenic variant in cell-free assays and confirmed by in silico docking studies. Furthermore, HVS substantially impaired the c-Met-mediated growth across a broad spectrum of breast cancer cells, while similar treatment doses had no effect on the non-tumorigenic mammary epithelial cell growth. In addition, HVS caused a dose-dependent inhibition of HGF-induced, but not epidermal growth factor (EGF)-induced, cell scattering in addition to HGF-mediated migration, invasion, and 3-dimensional (3D) proliferation of tumor cell spheroids. HVS treatment effects were mediated via inhibition of ligand-mediated c-Met activation and its downstream mitogenic signaling and blocking molecular mediators involved in cellular motility across different cellular contexts. An interesting feature of HVS is its good selectivity for c-Met and Abelson murine leukemia viral oncogene homolog 1 (ABL1) when profiled against a panel of kinases. Docking studies revealed interactions likely to impart high dual affinity for both ABL1 and c-Met kinases. HVS markedly reduced tumor growth, showed excellent pharmacodynamics, and suppressed cell proliferation and microvessel density in an orthotopic model of triple negative breast cancer. Collectively, the present findings suggested that the oleocanthal-based HVS is a promising c-Met inhibitor lead entity with excellent therapeutic potential to control

  14. (-)-Oleocanthal as a c-Met inhibitor for the control of metastatic breast and prostate cancers.

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    Elnagar, Ahmed Y; Sylvester, Paul W; El Sayed, Khalid A

    2011-07-01

    The proto-oncogene receptor tyrosine kinase c-Met encodes the high-affinity receptor for hepatocyte growth factor (HGF). Dysregulation of the HGF-c-Met pathway plays a significant oncogenic role in many tumors. Overexpression of c-Met is a prognostic indicator for some transitional cell carcinomas. Extra-virgin olive oil (EVOO) provides a variety of minor phenolic compounds with beneficial properties. (-)-Oleocanthal (1) is a naturally occurring minor secoiridoid isolated from EVOO, which showed potent anti-inflammatory activity via its ability to inhibit COX-1 and COX-2. It altered the structure of neurotoxic proteins believed to contribute to the debilitating effects of Alzheimer's disease. Computer-Assisted Molecular Design (CAMD) identified 1 as a potential virtual c-Met inhibitor hit. Oleocanthal inhibited the proliferation, migration, and invasion of the epithelial human breast and prostate cancer cell lines MCF7, MDA-MB-231, and PC-3, respectively, with an IC (50) range of 10-20 µM, and demonstrated anti-angiogenic activity via downregulating the expression of the microvessel density marker CD31 in endothelial colony forming cells with an IC (50) of 4.4 µM. It inhibited the phosphorylation of c-Met kinase IN VITRO in the Z'-LYTE™ assay, with an IC (50) value of 4.8 µM. (-)-Oleocanthal and EVOO can have potential therapeutic use for the control of c-Met-dependent malignancies. PMID:21328179

  15. Expression of c-met Stimulated by High Glucose in Human Renal Tubular Epithelial Cells and Its Implication

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    LI Xing; ZHANG Muxun

    2007-01-01

    The expression of c-met stimulated by high glucose in human renal tubular epithelial cells and the role of HGF/c-met system in diabetic nephropathy were examined. The proximal tubular epithelial cells were cultured in vitro under different conditions. MTT was used for the detection of cellular proliferation and RT-PCR was employed for measurement of c-met mRNA level. Our results showed that under different conditions, there were no significant differences in the proliferation of proximal tubular epithelial cells 12 h and 24 h after the culuture (P>0.05). The proliferation of proximal tubular epithelial cells showed a significant change 96 h after the culture and the cellular proliferation induced by hepatocyte growth factor (HGF) was very active (P<0.05). Moreover, no significant difference in the expression of c-met mRNA was found 12 h after the culture under different conditions (P>0.05), while 24 and 96 h after the culture, a persistent and significantly higher expression of c-met mRNA was found in HGF-induced proliferation. It is concluded that addition of exogenous HGF could inhibit the apoptosis induced by high-level glucose, promote the proliferation of proximal tubular epithelial cells, and induce the expression of c-met. Our study suggests that local up-regulation of HGF/c-met system plays an important role in the repair of renal damage in diabetic nephropathy.

  16. Piperlongumine and its analogs down-regulate expression of c-Met in renal cell carcinoma

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    Golovine, Konstantin; Makhov, Peter; Naito, Sei; Raiyani, Henish; Tomaszewski, Jeffrey; Mehrazin, Reza; Tulin, Alexei; Kutikov, Alexander; Uzzo, Robert G.; Kolenko, Vladimir M.

    2015-01-01

    The c-Met protein, a transmembrane receptor tyrosine kinase, is the product of a proto-oncogene. Its only known ligand, hepatocyte growth factor (HGF), regulates cell growth, motility, migration, invasion, proliferation, and angiogenesis. The aberrant expression of c-Met is often associated with poor prognosis in multiple cancers, including renal cell carcinoma (RCC). Silencing or inactivation of c-Met leads to decreased viability of cancer cells, thereby making ablation of c-Met signaling an...

  17. MicroRNA-449a is downregulated in non-small cell lung cancer and inhibits migration and invasion by targeting c-Met.

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    Wenting Luo

    Full Text Available MicroRNA-449a is expressed at a low level in several tumors and cancer cell lines, and induces G1 arrest, apoptosis, and senescence. To identify the function of miR-449a in non-small cell lung cancer (NSCLC, we discussed the potential relevance of miR-449a to clinicopathological characteristics and prognosis in NSCLC. We also investigated the impact of miR-449a on migration and invasion in NSCLC cells. The expression of miR-449a in NSCLC tissues and cell lines was detected using RT-qPCR. In vitro, gain-of-function, loss-of-function experiments, and fluorescence assays were performed to identify the potential target of miR-449a and the function of miR-449a in NSCLC cells. MiR-449a was downregulated in both NSCLC tissues and cell lines. Moreover, a low expression level of miR-449a appeared to be correlated with lymph node metastasis and poor survival. In vitro, miR-449 regulated cell migration and invasion in NSCLC cells as a potential tumor suppressor, at least in part by targeting c-Met. Furthermore, reciprocal expression of miR-449a and c-Met was shown in NSCLC tissue samples. This study indicates that miR-449a might be associated with NSCLC progression, and suggests a crucial role for miR-449a in NSCLC.

  18. Efficacy of c-Met inhibitor for advanced prostate cancer

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    Christensen James G

    2010-10-01

    Full Text Available Abstract Background Aberrant expression of HGF/SF and its receptor, c-Met, often correlates with advanced prostate cancer. Our previous study showed that expression of c-Met in prostate cancer cells was increased after attenuation of androgen receptor (AR signalling. This suggested that current androgen ablation therapy for prostate cancer activates c-Met expression and may contribute to development of more aggressive, castration resistant prostate cancer (CRPC. Therefore, we directly assessed the efficacy of c-Met inhibition during androgen ablation on the growth and progression of prostate cancer. Methods We tested two c-Met small molecule inhibitors, PHA-665752 and PF-2341066, for anti-proliferative activity by MTS assay and cell proliferation assay on human prostate cancer cell lines with different levels of androgen sensitivity. We also used renal subcapsular and castrated orthotopic xenograft mouse models to assess the effect of the inhibitors on prostate tumor formation and progression. Results We demonstrated a dose-dependent inhibitory effect of PHA-665752 and PF-2341066 on the proliferation of human prostate cancer cells and the phosphorylation of c-Met. The effect on cell proliferation was stronger in androgen insensitive cells. The c-Met inhibitor, PF-2341066, significantly reduced growth of prostate tumor cells in the renal subcapsular mouse model and the castrated orthotopic mouse model. The effect on cell proliferation was greater following castration. Conclusions The c-Met inhibitors demonstrated anti-proliferative efficacy when combined with androgen ablation therapy for advanced prostate cancer.

  19. FAK kinase activity is required for the progression of c-Met/β-catenin-driven HCC

    Science.gov (United States)

    Shang, Na; Arteaga, Maribel; Zaidi, Ali; Cotler, Scott J.; Breslin, Peter; Ding, Xianzhong; Kuo, Paul; Nishimura, Michael; Zhang, Jiwang; Qiu, Wei

    2016-01-01

    Background & Aims There is an urgent need to develop new and more effective therapeutic strategies and agents to treat hepatocellular carcinoma (HCC). We have recently found that deletion of Fak in hepatocytes before tumors form inhibits tumor development and prolongs survival of animals in a c-Met (MET)/β-catenin (CAT)-driven HCC mouse model. However, it has yet to be determined whether FAK expression in hepatocytes promotes MET/CAT-induced HCC progression after tumor initiation. In addition, it remains unclear whether FAK promotes HCC development through its kinase activity. Methods We generated hepatocyte-specific inducible Fak-deficient mice (Alb-creERT2; Fakflox/flox) to examine the role of FAK in HCC progression. We re-expressed wild-type and mutant FAK in Fak-deficient mice to determine FAK’s kinase activity in HCC development. We also examined the efficacy of a FAK kinase inhibitor PF-562271 on HCC inhibition. Results We found that deletion of Fak after tumors form significantly repressed MET/CAT-induced tumor progression. Ectopic FAK expression restored HCC formation in hepatocyte-specific Fak-deficient mice. However, overexpression of a FAK kinase-dead mutant led to reduced tumor load compared to mice which express wild-type FAK. Furthermore, PF-562271 significantly suppressed progression of MET/CAT-induced HCC. Conclusion Fak kinase activity is important for MET/CAT-induced HCC progression. Inhibiting FAK kinase activity provides a potential therapeutic strategy to treat HCC. PMID:27142958

  20. c-Met in pancreatic cancer stem cells: Therapeutic implications

    Institute of Scientific and Technical Information of China (English)

    Marta Herreros-Villanueva; Aizpea Zubia-Olascoaga; Luis Bujanda

    2012-01-01

    Pancreatic cancer is the deadliest solid cancer and currently the fourth most frequent cause of cancer-related deaths.Emerging evidence suggests that cancer stem cells (CSCs) play a crucial role in the development and progression of this disease.The identification of CSC markers could lead to the development of new therapeutic targets.In this study,the authors explore the functional role of c-Met in pancreatic CSCs,by analyzing self-renewal with sphere assays and tumorigenicity capacity in NOD SCID mice.They concluded that c-Met is a novel marker for identifying pancreatic CSCs and c-Methigh in a higher tumorigenic cancer cell population.Inhibition of c-Met with XL184 blocks self-renewal capacity in pancreatic CSCs.In pancreatic tumors established in NOD SCID mice,c-Met inhibition slowed tumor growth and reduced the population of CSCs,along with preventing the development of metastases.

  1. Olive phenolics as c-Met inhibitors: (-)-Oleocanthal attenuates cell proliferation, invasiveness, and tumor growth in breast cancer models.

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    Akl, Mohamed R; Ayoub, Nehad M; Mohyeldin, Mohamed M; Busnena, Belnaser A; Foudah, Ahmed I; Liu, Yong-Yu; Sayed, Khalid A Ei

    2014-01-01

    Dysregulation of the Hepatocyte growth factor (HGF)/c-Met signaling axis upregulates diverse tumor cell functions, including cell proliferation, survival, scattering and motility, epithelial-to-mesenchymal transition (EMT), angiogenesis, invasion, and metastasis. (-)-Oleocanthal is a naturally occurring secoiridoid from extra-virgin olive oil, which showed antiproliferative and antimigratory activity against different cancer cell lines. The aim of this study was to characterize the intracellular mechanisms involved in mediating the anticancer effects of (-)-oleocanthal treatment and the potential involvement of c-Met receptor signaling components in breast cancer. Results showed that (-)-oleocanthal inhibits the growth of human breast cancer cell lines MDA-MB-231, MCF-7 and BT-474 while similar treatment doses were found to have no effect on normal human MCF10A cell growth. In addition, (-)-oleocanthal treatment caused a dose-dependent inhibition of HGF-induced cell migration, invasion and G1/S cell cycle progression in breast cancer cell lines. Moreover, (-)-oleocanthal treatment effects were found to be mediated via inhibition of HGF-induced c-Met activation and its downstream mitogenic signaling pathways. This growth inhibitory effect is associated with blockade of EMT and reduction in cellular motility. Further results from in vivo studies showed that (-)-oleocanthal treatment suppressed tumor cell growth in an orthotopic model of breast cancer in athymic nude mice. Collectively, the findings of this study suggest that (-)-oleocanthal is a promising dietary supplement lead with potential for therapeutic use to control malignancies with aberrant c-Met activity. PMID:24849787

  2. A novel rabbit anti-hepatocyte growth factor monoclonal neutralizing antibody inhibits tumor growth in prostate cancer cells and mouse xenografts

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Yanlan; Chen, Yicheng; Ding, Guoqing; Wang, Mingchao; Wu, Haiyang; Xu, Liwei; Rui, Xuefang; Zhang, Zhigen, E-mail: srrshurology@163.com

    2015-08-14

    The hepatocyte growth factor and its receptor c-Met are correlated with castration-resistance in prostate cancer. Although HGF has been considered as an attractive target for therapeutic antibodies, the lack of cross-reactivity of monoclonal antibodies with human/mouse HGFs is a major obstacle in preclinical developments. We generated a panel of anti-HGF RabMAbs either blocking HGF/c-Met interaction or inhibiting c-Met phosphorylation. We selected one RabMAb with mouse cross-reactivity and demonstrated that it blocked HGF-stimulated downstream activation in PC-3 and DU145 cells. Anti-HGF RabMAb inhibited not only the growth of PC-3 cells but also HGF-dependent proliferation in HUVECs. We further demonstrated the efficacy and potency of the anti-HGF RabMAb in tumor xenograft mice models. Through these in vitro and in vivo experiments, we explored a novel therapeutic antibody for advanced prostate cancer. - Highlights: • HGF is an attractive target for castration-refractory prostate cancer. • We generated and characterized a panel of anti-HGF rabbit monoclonal antibodies. • More than half of these anti-HGF RabMAbs was cross-reactive with mouse HGF. • Anti-HGF RabMAb blocks HGF-stimulated phosphorylation and cell growth in vitro. • Anti-HGF RabMAb inhibits tumor growth and angiogenesis in xenograft mice.

  3. LoVo colon cancer cells resistant to oxaliplatin overexpress c-MET and VEGFR-1 and respond to VEGF with dephosphorylation of c-MET.

    Science.gov (United States)

    Mezquita, Belén; Pineda, Estela; Mezquita, Jovita; Mezquita, Pau; Pau, Montserrat; Codony-Servat, Jordi; Martínez-Balibrea, Eva; Mora, Conchi; Maurel, Joan; Mezquita, Cristóbal

    2016-05-01

    Oxaliplatin-resistant LoVo colon cancer cells overexpressing c-MET and VEGFR-1 were selected to study several signaling pathways involved in chemoresistance, as well as the effect of increasing amounts of VEGF in the regulation of c-MET. In comparison with chemosensitive LoVo colon cancer cells, oxaliplatin-resistant cells (LoVoR) overexpress and phosphorylate c-MET, upregulate the expression of transmembrane and soluble VEGFR-1 and, unexpectedly, downregulate VEGF. In addition, LoVoR cells activate other transduction pathways involved in chemoresistance such as Akt, β-catenin-TCF4 and E-cadherin. While c-MET is phosphorylated in LoVoR cells expressing low levels of VEGF, c-MET phosphorylation decreases when recombinant VEGF is added into the culture medium. Inhibition of c-MET by VEGF is mediated by VEGFR-1, since phosphorylation of c-MET in the presence of VEGF is restored after silencing VEGFR-1. Dephosphorylation of c-MET by VEGF suggests that tumors coexpressing VEGFR-1 and c-MET may activate c-MET as a result of anti-VEGF therapy.

  4. Curcumin inhibits activation of TRPM2 channels in rat hepatocytes

    Directory of Open Access Journals (Sweden)

    E. Kheradpezhouh

    2016-04-01

    Full Text Available Oxidative stress is a hallmark of many liver diseases including viral and drug-induced hepatitis, ischemia-reperfusion injury, and non-alcoholic steatohepatitis. One of the consequences of oxidative stress in the liver is deregulation of Ca2+ homeostasis, resulting in a sustained elevation of the free cytosolic Ca2+ concentration ([Ca2+]c in hepatocytes, which leads to irreversible cellular damage. Recently it has been shown that liver damage induced by paracetamol and subsequent oxidative stress is, in large part, mediated by Ca2+ entry through Transient Receptor Potential Melastatin 2 (TRPM2 channels. Involvement of TRPM2 channels in hepatocellular damage induced by oxidative stress makes TRPM2 a potential therapeutic target for treatment of a range of oxidative stress-related liver diseases. We report here the identification of curcumin ((1E,6E-1,7-bis(4-hydroxy-3-methoxyphenyl-1,6-heptadiene-3,5-dione, a natural plant-derived polyphenol in turmeric spice, as a novel inhibitor of TRPM2 channel. Presence of 5 µM curcumin in the incubation medium prevented the H2O2- and paracetamol-induced [Ca2+]c rise in rat hepatocytes. Furthermore, in patch clamping experiments incubation of hepatocytes with curcumin inhibited activation of TRPM2 current by intracellular ADPR with IC50 of approximately 50 nM. These findings enhance understanding of the actions of curcumin and suggest that the known hepatoprotective properties of curcumin are, at least in part, mediated through inhibition of TRPM2 channels.

  5. A novel antagonist anti-cMet antibody with antitumor activities targeting both ligand-dependent and ligand-independent c-Met receptors.

    Science.gov (United States)

    Gonzalez, Alexandra; Broussas, Matthieu; Beau-Larvor, Charlotte; Haeuw, Jean-François; Boute, Nicolas; Robert, Alain; Champion, Thierry; Beck, Alain; Bailly, Christian; Corvaïa, Nathalie; Goetsch, Liliane

    2016-10-15

    c-Met is a prototypic member of a sub-family of RTKs. Inappropriate c-Met activation plays a crucial role in tumor formation, proliferation and metastasis. Using a key c-Met dimerization assay, a set of 12 murine whole IgG1 monoclonal antibodies was selected and a lead candidate, m224G11, was humanized by CDR-grafting and engineered to generate a divalent full antagonist humanized IgG1 antibody, hz224G11. Neither m224G11 nor hz224G11 bind to the murine c-Met receptor. Their antitumor activity was investigated in vitro in a set of experiments consistent with the reported pleiotropic effects mediated by c-Met and, in vivo, using several human tumor xenograft models. Both m224G11 and hz224G11 exhibited nanomolar affinities for the receptor and inhibited HGF binding, c-Met phosphorylation, and receptor dimerization in a similar fashion, resulting in a profound inhibition of all c-Met functions in vitro. These effects were presumably responsible for the inhibition of c-Met's major functions including cell proliferation, migration, invasion scattering, morphogenesis and angiogenesis. In addition to these in vitro properties, hz224G11 dramatically inhibits the growth of autocrine, partially autophosphorylated and c-Met amplified cell lines in vivo. Pharmacological studies performed on Hs746T gastric cancer xenografts demonstrate that hz224G11 strongly downregulates c-Met expression and phosphorylation. It also decreases the tumor mitotic index (Ki67) and induces apoptosis. Taken together, the in vitro and in vivo data suggest that hz224G11 is a promising candidate for the treatment of tumors. This antibody, now known as ABT-700 and currently in Phase I clinical trials, may provide a novel therapeutic approach to c-Met-expressing cancers.

  6. A novel antagonist anti-cMet antibody with antitumor activities targeting both ligand-dependent and ligand-independent c-Met receptors.

    Science.gov (United States)

    Gonzalez, Alexandra; Broussas, Matthieu; Beau-Larvor, Charlotte; Haeuw, Jean-François; Boute, Nicolas; Robert, Alain; Champion, Thierry; Beck, Alain; Bailly, Christian; Corvaïa, Nathalie; Goetsch, Liliane

    2016-10-15

    c-Met is a prototypic member of a sub-family of RTKs. Inappropriate c-Met activation plays a crucial role in tumor formation, proliferation and metastasis. Using a key c-Met dimerization assay, a set of 12 murine whole IgG1 monoclonal antibodies was selected and a lead candidate, m224G11, was humanized by CDR-grafting and engineered to generate a divalent full antagonist humanized IgG1 antibody, hz224G11. Neither m224G11 nor hz224G11 bind to the murine c-Met receptor. Their antitumor activity was investigated in vitro in a set of experiments consistent with the reported pleiotropic effects mediated by c-Met and, in vivo, using several human tumor xenograft models. Both m224G11 and hz224G11 exhibited nanomolar affinities for the receptor and inhibited HGF binding, c-Met phosphorylation, and receptor dimerization in a similar fashion, resulting in a profound inhibition of all c-Met functions in vitro. These effects were presumably responsible for the inhibition of c-Met's major functions including cell proliferation, migration, invasion scattering, morphogenesis and angiogenesis. In addition to these in vitro properties, hz224G11 dramatically inhibits the growth of autocrine, partially autophosphorylated and c-Met amplified cell lines in vivo. Pharmacological studies performed on Hs746T gastric cancer xenografts demonstrate that hz224G11 strongly downregulates c-Met expression and phosphorylation. It also decreases the tumor mitotic index (Ki67) and induces apoptosis. Taken together, the in vitro and in vivo data suggest that hz224G11 is a promising candidate for the treatment of tumors. This antibody, now known as ABT-700 and currently in Phase I clinical trials, may provide a novel therapeutic approach to c-Met-expressing cancers. PMID:27144973

  7. Fibulin-3 negatively regulates ALDH1 via c-MET suppression and increases γ-radiation-induced sensitivity in some pancreatic cancer cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Kim, In-Gyu, E-mail: igkim@kaeri.re.kr [Department of Radiation Biology, Environmental Radiation Research Group, Korea Atomic Energy Research Institute, 989-111 Daedeok-daero, Yuseong-gu, Daejeon 305-353 (Korea, Republic of); Department of Radiation Biotechnology and Applied Radioisotope, Korea University of Science and Technology (UST), 989-111 Daedeok-daero, Yusong-gu, Daejeon 305-353 (Korea, Republic of); Lee, Jae-Ha [Department of Radiation Biology, Environmental Radiation Research Group, Korea Atomic Energy Research Institute, 989-111 Daedeok-daero, Yuseong-gu, Daejeon 305-353 (Korea, Republic of); Department of Radiation Biotechnology and Applied Radioisotope, Korea University of Science and Technology (UST), 989-111 Daedeok-daero, Yusong-gu, Daejeon 305-353 (Korea, Republic of); Kim, Seo-Yoen; Kim, Jeong-Yul [Department of Radiation Biology, Environmental Radiation Research Group, Korea Atomic Energy Research Institute, 989-111 Daedeok-daero, Yuseong-gu, Daejeon 305-353 (Korea, Republic of); Cho, Eun-Wie [Epigenomics Research Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806 (Korea, Republic of)

    2014-11-21

    Highlights: • FBLN-3 gene was poorly expressed in some pancreatic cancer lines. • FBLN-3 promoter region was highly methylated in some pancreatic cancer cell lines. • FBLN-3 inhibited c-MET activation and expression and reduced cellular level of ALDH1. • FBLN-3/c-Met/ALDH1 axis modulates stemness and EMT in pancreatic cancer cells. - Abstract: Fibulin-3 (FBLN-3) has been postulated to be either a tumor suppressor or promoter depending on the cell type, and hypermethylation of the FBLN-3 promoter is often associated with human disease, especially cancer. We report that the promoter region of the FBLN-3 was significantly methylated (>95%) in some pancreatic cancer cell lines and thus FBLN-3 was poorly expressed in pancreatic cancer cell lines such as AsPC-1 and MiaPaCa-2. FBLN-3 overexpression significantly down-regulated the cellular level of c-MET and inhibited hepatocyte growth factor-induced c-MET activation, which were closely associated with γ-radiation resistance of cancer cells. Moreover, we also showed that c-MET suppression or inactivation decreased the cellular level of ALDH1 isozymes (ALDH1A1 or ALDH1A3), which serve as cancer stem cell markers, and subsequently induced inhibition of cell growth in pancreatic cancer cells. Therefore, forced overexpression of FBLN-3 sensitized cells to cytotoxic agents such as γ-radiation and strongly inhibited the stemness and epithelial to mesenchymal transition (EMT) property of pancreatic cancer cells. On the other hand, if FBLN3 was suppressed in FBLN-3-expressing BxPC3 cells, the results were opposite. This study provides the first demonstration that the FBLN-3/c-MET/ALDH1 axis in pancreatic cancer cells partially modulates stemness and EMT as well as sensitization of cells to the detrimental effects of γ-radiation.

  8. Fibulin-3 negatively regulates ALDH1 via c-MET suppression and increases γ-radiation-induced sensitivity in some pancreatic cancer cell lines

    International Nuclear Information System (INIS)

    Highlights: • FBLN-3 gene was poorly expressed in some pancreatic cancer lines. • FBLN-3 promoter region was highly methylated in some pancreatic cancer cell lines. • FBLN-3 inhibited c-MET activation and expression and reduced cellular level of ALDH1. • FBLN-3/c-Met/ALDH1 axis modulates stemness and EMT in pancreatic cancer cells. - Abstract: Fibulin-3 (FBLN-3) has been postulated to be either a tumor suppressor or promoter depending on the cell type, and hypermethylation of the FBLN-3 promoter is often associated with human disease, especially cancer. We report that the promoter region of the FBLN-3 was significantly methylated (>95%) in some pancreatic cancer cell lines and thus FBLN-3 was poorly expressed in pancreatic cancer cell lines such as AsPC-1 and MiaPaCa-2. FBLN-3 overexpression significantly down-regulated the cellular level of c-MET and inhibited hepatocyte growth factor-induced c-MET activation, which were closely associated with γ-radiation resistance of cancer cells. Moreover, we also showed that c-MET suppression or inactivation decreased the cellular level of ALDH1 isozymes (ALDH1A1 or ALDH1A3), which serve as cancer stem cell markers, and subsequently induced inhibition of cell growth in pancreatic cancer cells. Therefore, forced overexpression of FBLN-3 sensitized cells to cytotoxic agents such as γ-radiation and strongly inhibited the stemness and epithelial to mesenchymal transition (EMT) property of pancreatic cancer cells. On the other hand, if FBLN3 was suppressed in FBLN-3-expressing BxPC3 cells, the results were opposite. This study provides the first demonstration that the FBLN-3/c-MET/ALDH1 axis in pancreatic cancer cells partially modulates stemness and EMT as well as sensitization of cells to the detrimental effects of γ-radiation

  9. Effect of c-Met Inhibitor on HGF-induced Ovarian Carcinoma Cell Migration

    Science.gov (United States)

    Lo, Chun-Min; Lo, Jun-Chih; Yip, Kay-Pong

    2010-03-01

    The dysregulation of hepatocyte growth factor (HGF) and its receptor, c-Met, in cell migration contributes to tumor invasion and metastasis in numerous cancers including ovarian cancer. Specific inhibitors against HGF/c-Met signaling like SU11274, therefore, may have important therapeutic potential for the treatment of cancers. Here, we applied electric cell-substrate impedance sensing (ECIS) and traction force microscopy to evaluate the effect of SU11274 on HGF-treated SKOV-3 ovarian cancer cells. Our results showed that, compared with control cells, HGF-treated cell monolayer displayed lower junctional resistance between cells, larger cell-substrate separation, and higher cell micromotion. In addition, individual HGF-treated SKOV-3 cells demonstrated weaker traction forces on the collagen-coated polyacrylamide substrate than did control cells. These changes lead to faster directional movement of HGF-treated cells, as demonstrated with wound healing assay. Treatment of SKOV-3 cells with SU11274 indicated significant inhibition of HGF stimulation on all assays tested.

  10. Olive phenolics as c-Met inhibitors: (--Oleocanthal attenuates cell proliferation, invasiveness, and tumor growth in breast cancer models.

    Directory of Open Access Journals (Sweden)

    Mohamed R Akl

    Full Text Available Dysregulation of the Hepatocyte growth factor (HGF/c-Met signaling axis upregulates diverse tumor cell functions, including cell proliferation, survival, scattering and motility, epithelial-to-mesenchymal transition (EMT, angiogenesis, invasion, and metastasis. (--Oleocanthal is a naturally occurring secoiridoid from extra-virgin olive oil, which showed antiproliferative and antimigratory activity against different cancer cell lines. The aim of this study was to characterize the intracellular mechanisms involved in mediating the anticancer effects of (--oleocanthal treatment and the potential involvement of c-Met receptor signaling components in breast cancer. Results showed that (--oleocanthal inhibits the growth of human breast cancer cell lines MDA-MB-231, MCF-7 and BT-474 while similar treatment doses were found to have no effect on normal human MCF10A cell growth. In addition, (--oleocanthal treatment caused a dose-dependent inhibition of HGF-induced cell migration, invasion and G1/S cell cycle progression in breast cancer cell lines. Moreover, (--oleocanthal treatment effects were found to be mediated via inhibition of HGF-induced c-Met activation and its downstream mitogenic signaling pathways. This growth inhibitory effect is associated with blockade of EMT and reduction in cellular motility. Further results from in vivo studies showed that (--oleocanthal treatment suppressed tumor cell growth in an orthotopic model of breast cancer in athymic nude mice. Collectively, the findings of this study suggest that (--oleocanthal is a promising dietary supplement lead with potential for therapeutic use to control malignancies with aberrant c-Met activity.

  11. The clinical and functional significance of c-Met in breast cancer: a review.

    Science.gov (United States)

    Ho-Yen, Colan M; Jones, J Louise; Kermorgant, Stephanie

    2015-04-08

    c-Met is a receptor tyrosine kinase that upon binding of its ligand, hepatocyte growth factor (HGF), activates downstream pathways with diverse cellular functions that are important in organ development and cancer progression. Anomalous c-Met signalling has been described in a variety of cancer types, and the receptor is regarded as a novel therapeutic target. In breast cancer there is a need to develop new treatments, particularly for the aggressive subtypes such as triple-negative and basal-like cancer, which currently lack targeted therapy. Over the last two decades, much has been learnt about the functional role of c-Met signalling in different models of breast development and cancer. This work has been complemented by clinical studies, establishing the prognostic significance of c-Met in tissue samples of breast cancer. While the clinical trials of anti-c-Met therapy in advanced breast cancer progress, there is a need to review the existing evidence so that the potential of these treatments can be better appreciated. The aim of this article is to examine the role of HGF/c-Met signalling in in vitro and in vivo models of breast cancer, to describe the mechanisms of aberrant c-Met signalling in human tissues, and to give a brief overview of the anti-c-Met therapies currently being evaluated in breast cancer patients. We will show that the HGF/c-Met pathway is associated with breast cancer progression and suggest that there is a firm basis for continued development of anti-c-Met treatment, particularly for patients with basal-like and triple-negative breast cancer.

  12. Fibroblast growth factor 7 inhibits cholesterol 7{alpha}-hydroxylase gene expression in hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Zhichao [Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai (China); Yu, Xuemei [Department of Endocrinology, Fengxian Central Hospital, Shanghai (China); Wu, Weibin; Jia, Dongwei; Chen, Yinle; Ji, Lingling; Liu, Xijun; Peng, Xiaomin [Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai (China); Li, Yintao [Institute of Endocrinology and Diabetology, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai (China); Yang, Lili [Department of Endocrinology, Fengxian Central Hospital, Shanghai (China); Ruan, Yuanyuan; Gu, Jianxin [Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai (China); Ren, Shifang, E-mail: renshifang@fudan.edu.cn [Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai (China); Zhang, Songwen, E-mail: songwenzhang@fudan.edu.cn [Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai (China)

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer FGF7 strongly and rapidly down-regulates the expression of CYP7A1 in hepatocytes. Black-Right-Pointing-Pointer FGF7 suppresses the expression of CYP7A1 via FGFR2 and downstream JNK activation. Black-Right-Pointing-Pointer Blocking FGF7 abrogates HSC-induced inhibition of CYP7A1 expression in hepatocytes. -- Abstract: Cholesterol 7{alpha}-hydroxylase (CYP7A1) is the initial and rate-limiting enzyme for bile acid synthesis. Transcription of the CYP7A1 gene is regulated by bile acids, nuclear receptors and cytokines. Fibroblast growth factor 7 (FGF7) secreted from activated hepatic stellate cells (HSC) during chronic liver fibrosis regulates hepatocyte survival and liver regeneration. In the carbon tetrachloride (CCl{sub 4})-induced fibrotic mouse liver, we demonstrated that the expression of CYP7A1 was largely decreased while the expression of FGF7 was significantly increased. We further demonstrated that FGF7 inhibited CYP7A1 gene expression in hepatocytes. Knockdown study by short interfering RNA, kinase inhibition and phosphorylation assays revealed that the suppression of CYP7A1 expression by FGF7 was mediated by FGFR2 and its downstream JNK signaling cascade. The FGF7 neutralizing antibody restored CYP7A1 expression in Hep3B cells treated with conditioned medium from HSC. In summary, the data suggest that FGF7 is a novel regulator of CYP7A1 expression in hepatocytes and may prevent hepatocytes from accumulating toxic bile acids during liver injury and fibrosis.

  13. cMET in NSCLC: Can We Cut off the Head of the Hydra? From the Pathway to the Resistance

    Energy Technology Data Exchange (ETDEWEB)

    Van Der Steen, Nele [Center for Oncological Research Antwerp, University of Antwerp, Universiteitsplein 1, Wilrijk 2610 (Belgium); Pauwels, Patrick [Center for Oncological Research Antwerp, University of Antwerp, Universiteitsplein 1, Wilrijk 2610 (Belgium); Molecular Pathology Unit, Pathology Department, Antwerp University Hospital, Wilrijkstraat 10, Edegem 2650 (Belgium); Gil-Bazo, Ignacio [Department of Oncology, Clínica Universidad de Navarra, Pamplona 31008 (Spain); Castañon, Eduardo [Department of Oncology, Clínica Universidad de Navarra, Pamplona 31008 (Spain); Phase I-Early Clinical Trials Unit, Oncology Department, Antwerp University Hospital, Wilrijkstraat 10, Edegem 2650 (Belgium); Raez, Luis [Thoracic Oncology Program, Memorial Cancer Institute, Memorial Health Care System, Pembroke Pines, FL 33024 (United States); Cappuzzo, Federico [4Thoracic Oncology Program, Memorial Cancer Institute, Memorial Health Care System, Pembroke Pines, FL 33024 (United States); Rolfo, Christian, E-mail: Christian.Rolfo@uza.be [Center for Oncological Research Antwerp, University of Antwerp, Universiteitsplein 1, Wilrijk 2610 (Belgium); Phase I-Early Clinical Trials Unit, Oncology Department, Antwerp University Hospital, Wilrijkstraat 10, Edegem 2650 (Belgium)

    2015-03-25

    In the last decade, the tyrosine kinase receptor cMET, together with its ligand hepatocyte growth factor (HGF), has become a target in non-small cell lung cancer (NSCLC). Signalization via cMET stimulates several oncological processes amongst which are cell motility, invasion and metastasis. It also confers resistance against several currently used targeted therapies, e.g., epidermal growth factor receptor (EGFR) inhibitors. In this review, we will discuss the basic structure of cMET and the most important signaling pathways. We will also look into aberrations in the signaling and the effects thereof in cancer growth, with the focus on NSCLC. Finally, we will discuss the role of cMET as resistance mechanism.

  14. NK4, an antagonist of hepatocyte growth factor (HGF), inhibits growth of multiple myeloma cells: molecular targeting of angiogenic growth factor.

    Science.gov (United States)

    Du, Wenlin; Hattori, Yutaka; Yamada, Taketo; Matsumoto, Kunio; Nakamura, Toshikazu; Sagawa, Morihiko; Otsuki, Takemi; Niikura, Takako; Nukiwa, Toshihiro; Ikeda, Yasuo

    2007-04-01

    Hepatocyte growth factor (HGF) promotes cell growth and motility and also increases neovascularization. Multiple myeloma (MM) cells produce HGF, and the plasma concentration of HGF is significantly elevated in patients with clinically active MM, suggesting that HGF might play a role in the pathogenesis of MM. NK4, an antagonist of HGF, is structurally homologous to angiostatin, and our previous report showed that NK4 inhibited the proliferation of vascular endothelial cells induced by HGF stimulation. The purposes of this study were to elucidate the contribution of HGF to the growth of MM cells as well as to investigate the possibility of the therapeutic use of NK4. In vitro study showed that NK4 protein stabilized the growth of MM cell lines and regulated the activation of c-MET, ERK1/2, STAT3, and AKT-1. Recombinant adenovirus containing NK4 cDNA (AdCMV.NK4) was injected intramuscularly into Icr/scid mice bearing tumors derived from HGF-producing MM cells. AdCMV.NK4 significantly inhibited the growth of these tumors in vivo. Histologic examination revealed that AdCMV.NK4 induced apoptosis of MM cells, accompanied by a reduction in neovascularization in the tumors. Thus, NK4 inhibited the growth of MM cells via antiangiogenic as well as direct antitumor mechanisms. The molecular targeting of HGF by NK4 could be applied as a novel therapeutic approach to MM. PMID:17179234

  15. Development of antibody-based c-Met inhibitors for targeted cancer therapy

    Directory of Open Access Journals (Sweden)

    Lee D

    2015-02-01

    Full Text Available Dongheon Lee, Eun-Sil Sung, Jin-Hyung Ahn, Sungwon An, Jiwon Huh, Weon-Kyoo You Hanwha Chemical R&D Center, Biologics Business Unit, Daejeon, Republic of Korea Abstract: Signaling pathways mediated by receptor tyrosine kinases (RTKs and their ligands play important roles in the development and progression of human cancers, which makes RTK-mediated signaling pathways promising therapeutic targets in the treatment of cancer. Compared with small-molecule compounds, antibody-based therapeutics can more specifically recognize and bind to ligands and RTKs. Several antibody inhibitors of RTK-mediated signaling pathways, such as human epidermal growth factor receptor 2, vascular endothelial growth factor, epidermal growth factor receptor or vascular endothelial growth factor receptor 2, have been developed and are widely used to treat cancer patients. However, since the therapeutic options are still limited in terms of therapeutic efficacy and types of cancers that can be treated, efforts are being made to identify and evaluate novel RTK-mediated signaling pathways as targets for more efficacious cancer treatment. The hepatocyte growth factor/c-Met signaling pathway has come into the spotlight as a promising target for development of potent cancer therapeutic agents. Multiple antibody-based therapeutics targeting hepatocyte growth factor or c-Met are currently in preclinical or clinical development. This review focuses on the development of inhibitors of the hepatocyte growth factor/c-Met signaling pathway for cancer treatment, including critical issues in clinical development and future perspectives for antibody-based therapeutics. Keywords: hepatocyte growth factor, ligands, receptor tyrosine kinase, signaling pathway, therapeutic agent

  16. c-Met inhibitor SU11274 enhances the response of the prostate cancer cell line DU145 to ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Hongliang; Li, Xiaoying; Sun, Shaoqian [Department of Radiation Oncology, Peking University First Hospital, Peking University, Beijing (China); Gao, Xianshu, E-mail: xsgao777@hotmail.com [Department of Radiation Oncology, Peking University First Hospital, Peking University, Beijing (China); Zhou, Demin, E-mail: deminzhou@bjmu.edu.cn [State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing (China)

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer c-Met inhibition could significantly enhance the radiosensitivity of DU145 cells. Black-Right-Pointing-Pointer The mechanisms of the radiosensitization effect of c-Met inhibition on DU145 cells were also presented in this paper. Black-Right-Pointing-Pointer This is the first study demonstrating the effectiveness of c-Met inhibition on treating HRPC cells with radiotherapy. -- Abstract: Hormone-refractory prostate cancer shows substantial resistance to most conventional therapies including radiotherapy, constitutes a key impediment to curing patients with the disease. c-Met overexpression plays a key role in prostate cancer tumorigenesis and disease progression. Here, we demonstrate that c-Met inhibition by SU11274 could significantly suppress cell survival and proliferation as well as enhance the radiosensitivity of DU145 cells. The underlying mechanisms of the effects of SU11274 on DU145 cells may include the inhibition of c-Met signaling, depolarization of the mitochondrial membrane potential, impairment of DNA repair function, abrogation of cell cycle arrest, and enhancement of cell death. Our study is the first to show the effectiveness of combining c-Met inhibition with ionizing radiation to cure hormone-refractory prostate cancer.

  17. 6-Phosphogluconate dehydrogenase regulates tumor cell migration in vitro by regulating receptor tyrosine kinase c-Met

    Energy Technology Data Exchange (ETDEWEB)

    Chan, Barden, E-mail: cchan@bidmc.harvard.edu [Division of Interdisciplinary Medicine and Biotechnology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215 (United States); VanderLaan, Paul A. [Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215 (United States); Sukhatme, Vikas P. [Division of Interdisciplinary Medicine and Biotechnology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215 (United States)

    2013-09-20

    Highlights: •Expression of 6PGD positively correlates with advancing stage of lung carcinoma. •Knockdown of 6PGD by shRNA potently inhibits c-Met tyrosine phosphorylation. •Exogenous HGF fails to restore c-Met phosphorylation in cells with 6PGD knocked down. •6PGD knockdown results in inhibition of cell migration in vitro. •Constitutively active TPR-cMet significantly restores migration of cells without 6PGD. -- Abstract: 6-Phosphogluconate dehydrogenase (6PGD) is the third enzyme in the oxidative pentose phosphate pathway (PPP). Recently, we reported that knockdown of 6PGD inhibited lung tumor growth in vitro and in a xenograft model in mice. In this study, we continued to examine the functional role of 6PGD in cancer. We show that 6PGD expression positively correlates with advancing stage of lung carcinoma. In search of functional signals related to 6PGD, we discovered that knockdown of 6PGD significantly inhibited phosphorylation of c-Met at tyrosine residues known to be critical for activity. This downregulation of c-Met phosphorylation correlated with inhibition of cell migration in vitro. Overexpression of a constitutively active c-Met specifically rescued the migration but not proliferation phenotype of 6PGD knockdown. Therefore, 6PGD appears to be required for efficient c-Met signaling and migration of tumor cells in vitro.

  18. Soluble c-Met Is a Reliable and Sensitive Marker to Detect c-Met Expression Level in Lung Cancer

    OpenAIRE

    Huilai Lv; Baoen Shan; Ziqiang Tian; Yong Li; Yuefeng Zhang; Shiwang Wen

    2015-01-01

    c-Met has been demonstrated as an attractive target in lung cancer therapy. Current studies showed that detection of c-Met status in tumor is critical in Met-targeted therapy. However not all patients are suitable for tissue sample collection. It is important to discover novel surrogate markers to detect c-Met status. In the study, soluble c-Met (s-Met) in plasma from 146 Chinese lung cancer patients and 40 disease-free volunteers was measured by enzyme-linked immunosorbent. In parallel, expr...

  19. Inhibition of glycogen synthesis in rat hepatocytes by medium Zn/sup 2 +/

    Energy Technology Data Exchange (ETDEWEB)

    Rognstad, R.

    1984-07-31

    In hepatocytes from fasted rats, Zn/sup 2 +/ in the range from 0 to 500..mu..M has relatively minor effects on gluconeogenesis from most substrates, or on ureagenesis from NH/sub 3/. In hepatocytes from fed rats, Zn/sup 2 +/ does not affect glycogenolysis. In hepatocytes from fasted rats, in which glycogen is being actively synthesized using the substrate combination of glycose, lactate and glutamine (all 10mM), Zn/sup 2 +/ markedly inhibits glucogen synthesis, with total inhibition at 500..mu..M, and a half maximal effect in the range from 50 to 100..mu..M. Dipicolinate (pyridine 2,6-dicarboxylate), a zinc chelator, is about as effective as L-glutamine in activating glycogen synthesis with the substrate combination of dihydroxyacetone, lactate and glucose (all 10mM). This suggests the possible hypothesis that endogenous Zn/sup 2 +/ might control the rate of glycogen synthesis in vivo. However, alternate explanations such as metabolite accumulation are also possible, since dipicolinate causes inhibition of gluconeogenesis from L-lactate. 28 references, 3 tables.

  20. SF/HGF-c-Met autocrine and paracrine promote metastasis of hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Qian Xie; Kang-Da Liu; Mei-Yu Hu; Kang Zhou

    2001-01-01

    AIM: To explore the role of SF/HGF-Met autocrine and parscrine in metastasis of hepatocellular carcinoma (HCC). METHODS: SF/HGF and c-met transcription and protein expression in HCC were examined by RT-PCR and Western Blot in 4 HCC cell lines, including HepG2, Hep3B,SMMC7721 and MHCC-1, the last cell line had a higher potential of metastasis. Sf/hgf cDNA was transfected by the method of Lipofectin into SMMC7721. SF/HGF and c-met antibody were used to stimulate and block SF/HGF-c-met signal transduction. Cell morphology, mobility, and proliferation were respectively compared by microscopic observation, wound healing assay and cell growth curve. RESULTS: HCC malignancy appeared to be relative to its met-SF/HGF expression. In MHCC-1, c-met expression was much stronger than that in other cell lines with lower potential of metastasis and only SF/HGF autocrine existed in MHCC-1. After sf/hgf cDNA transfection or conditioned medium of MHCC-1 stimulation, SMMC7721 changed into elongated morphology, and the abilities of proliferation ( P < 0.05) and mobility increased. Such bio-activity could he blocked by c-met antibody ( P< 0.05). CONCLUSION: The system of SF/HGF-c-met autocrine and paracrine played an important role in development and metastasis potential of HCC. Inhibition of SF/HGF-c-met signal transduction system may reduce the growth and metastasis of HCC.

  1. Induction of c-Met Proto-Oncogene by Epstein-Barr Virus Latent Membrane Protein-1 and the Correlation with Cervical Lymph Node Metastasis of Nasopharyngeal Carcinoma

    OpenAIRE

    Horikawa, Toshiyuki; Sheen, Tzung-Shiahn; Takeshita, Hajime; Sato, Hiroshi; Furukawa, Mitsuru; Yoshizaki, Tomokazu

    2001-01-01

    Nasopharyngeal carcinoma (NPC) is distinctive in head and neck carcinomas for its close association with Epstein-Barr virus and its highly metastatic nature. Up-regulation of cell motility is essential for enhancement of metastatic potential. The expression of c-Met proto-oncogene, a high-affinity receptor for hepatocyte growth factor/scatter factor, has been reported to correlate with metastatic ability of the tumor cell. We observed close association of c-Met expression with cervical lymph ...

  2. Absence of tpr-met and expression of c-met in human gastric mucosa and carcinoma.

    Science.gov (United States)

    Heideman, D A; Snijders, P J; Bloemena, E; Meijer, C J; Offerhaus, G J; Meuwissen, S G; Gerritsen, W R; Craanen, M E

    2001-08-01

    The c-met proto-oncogene, encoding the hepatocyte growth factor receptor, can be activated by various mechanisms. These include, among others, gene amplification with concomitant overexpression and the tpr-met oncogenic rearrangement. In the case of gastric cancer, contradictory results on the presence of the tpr-met oncogenic rearrangement have been published. The current study aimed therefore to assess the prevalence of tpr-met expression in Caucasian gastric adenocarcinomas, to evaluate the importance of this oncogene in their carcinogenesis. In addition, the level of c-met expression was determined, to evaluate the role of this alternative mode of activation of the proto-oncogene. A series of Caucasian gastric adenocarcinomas (n=43) and normal gastric mucosal samples (n=14) was analysed for tpr-met and c-met expression. Expression of tpr-met mRNA in the samples was performed by two reverse transcriptase polymerase chain reaction (RT-PCR) assays, with excellent correlation. The specificity of both methods was confirmed by direct sequencing of the PCR products of the MNNG-HOS cell line, which is known to contain the rearrangement. The level of c-met expression was assessed using semi-quantitative RT-PCR assays and immunohistochemistry (IHC). None of the normal gastric mucosal or gastric adenocarcinoma samples expressed tpr-met mRNA, as determined by both RT-PCR assays. Seventy per cent of the adenocarcinomas showed overexpression of c-met, according to elevated c-met mRNA levels, compared with the expression level of normal gastric mucosa. A significant correlation was found between the level of c-met mRNA and protein expression. In conclusion, these results strongly suggest that tpr-met activation does not play a role in Caucasian gastric carcinogenesis, while overexpression of the c-met gene occurs in the majority of Caucasian gastric adenocarcinomas.

  3. Cytoskeletal inhibitors, anti-adhesion molecule antibodies, and lectins inhibit hepatocyte spheroid formation.

    Directory of Open Access Journals (Sweden)

    Nakamura M

    2002-02-01

    Full Text Available We investigated the role of cytoskeletons, adhesion molecules, membrane-glycosylations, and proteoglycans in forming the shape of adult rat hepatocyte spheroids. Isolated hepatocytes were cultured on dishes coated with chondroitin sulfate phosphatidyl ethanolamine (CS-PE. Spheroid-forming ability was observed after adding cytoskeletal inhibitors (cytochalasin D, colchicine, okadaic acid, mycalolide B, anti-adhesion molecule antibodies (anti-E-cadherin, anti-connexin 32, anti-zo-1, a glycosphingolipid synthetic inhibitor (N-butyldeoxynojirimycin, a proteoglycan synthetic inhibitor (p-nitrophenyl-beta-D-xylopyranoside, and several lectins. Localization of actin was studied using confocal microscopy after rhodamine-phalloidin staining. Adding cytoskeletal inhibitors on the initial day resulted in weakly clustered cell aggregates rather than smoothly formed spheroids. These effects disappeared at lower reagent concentrations. When reagents were added on day 3, after the formation of spheroids, only mycalolide B was associated with an irregular spheroid surface; the others had no effect. Adding the anti-E-cadherin, anti-connexin 32 on the initial day showed inhibition of spheroid formation, but anti-zo-1 and proteoglycan synthetic inhibitor had no effects. Among the several lectins, only Wheat Germ Agglutinin (WGA, Ricinus communis Agglutinin I (RCA-I, and Concanavalin A (ConA showed inhibition. These results suggest that cytoskeletal conformation and some adhesion molecules are necessary to form spheroids. Based on the interactions between lectins and hepatocytes in the present study, hepatocytes appear to contain an N-linked complex or N-linked hybrid glycosylated chains.

  4. Evaluation of c-Met, HGF, and HER-2 expressions in gastric carcinoma and their association with other clinicopathological factors

    Directory of Open Access Journals (Sweden)

    Yıldız Y

    2016-09-01

    Full Text Available Yetkin Yıldız,1 Cenk Sokmensuer,2 Suayib Yalcin1 1Department of Medical Oncology, 2Department of Pathology, Hacettepe University, Ankara, Turkey Background: Met and HER-2 are proto-oncogenes encoding receptor tyrosine kinase c-Met and HER-2, respectively. Hepatocyte growth factor (HGF is a ligand of c-Met. The frequency of c-Met, HGF, and HER-2 expressions in gastric cancer and their association with other clinicopathological factors have not been fully understood. Patients and methods: Patients with stage 1–4 disease were analyzed. Expressions of c-Met, HGF, and HER-2 were examined using immunohistochemistry. Results: A total of 143 patients, 97 males and 46 females, were included. C-Met scores were 3(+ in 31.5%, 2(+ in 27.3%, and 1(+ in 10.5% of the patients. There was no statistically significant difference in age, sex, tumor location, differentiation, Lauren classification, TNM staging, presence of distant metastasis, depth of tumor invasion (T, lymphovascular invasion, and survival between c-Met subgroups. Overall HGF positivity was 20.6%. HER-2 scores were 3(+ in 9.1%, 2(+ in 9.8%, and 1(+ in 16.1% of the patients. HER-2 overexpression was associated with better differentiation, intestinal subtype, and advanced stage. C-Met overexpressions were 84.6% in the HER-2-overexpression-positive group and 56.2% in the HER-2-overexpression-negative group. There were no statistically significant differences in survival between the high c-Met-expression-positive and -negative stage 3 and stage 4 patients and between the HGF-positive and -negative groups. The mean survival was 11.6±6.3 months in the HER-2-overexpression-positive stage 4 group and 11.9±6.8 months in the HER-2-overexpression-negative stage 4 group. There were no statistically significant differences in survival between the two groups. Conclusion: c-Met was not associated with any prognostic factors in gastric cancer. HER-2 was associated with better differentiation, intestinal

  5. cAMP prevents TNF-induced apoptosis through inhibiting DISC complex formation in rat hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharjee, Rajesh [Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213 (United States); Xiang, Wenpei [Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213 (United States); Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People' s Republic of China (China); Wang, Yinna [Vascular Medicine Institute, University of Pittsburgh School of Medicine, 10051-5A BST 3, 3501 Fifth Avenue, Pittsburgh, PA 15261 (United States); Zhang, Xiaoying [Department of Medicine/Endocrinology Division, University of Pittsburgh Medical Center, 200 Lothrop St., Pittsburgh, PA 15213 (United States); Billiar, Timothy R., E-mail: billiartr@upmc.edu [Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213 (United States)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer cAMP blocks cell death induced by TNF and actinomycin D in cultured hepatocytes. Black-Right-Pointing-Pointer cAMP blocks NF-{kappa}B activation induced by TNF and actinomycin D. Black-Right-Pointing-Pointer cAMP blocks DISC formation following TNF and actinomycin D exposure. Black-Right-Pointing-Pointer cAMP blocks TNF signaling at a proximal step. -- Abstract: Tumor necrosis factor {alpha} (TNF) is a pleiotropic proinflammatory cytokine that plays a role in immunity and the control of cell proliferation, cell differentiation, and apoptosis. The pleiotropic nature of TNF is due to the formation of different signaling complexes upon the binding of TNF to its receptor, TNF receptor type 1 (TNFR1). TNF induces apoptosis in various mammalian cells when the cells are co-treated with a transcription inhibitor like actinomycin D (ActD). When TNFR1 is activated, it recruits an adaptor protein, TNF receptor-associated protein with death domain (TRADD), through its cytoplasmic death effector domain (DED). TRADD, in turn, recruits other signaling proteins, including TNF receptor-associated protein 2 (TRAF2) and receptor-associated protein kinase (RIPK) 1, to form a complex. Subsequently, this complex combines with FADD and procaspase-8, converts into a death-inducing signaling complex (DISC) to induce apoptosis. Cyclic AMP (cAMP) is a second messenger that regulates various cellular processes such as cell proliferation, gene expression, and apoptosis. cAMP analogues are reported to act as anti-apoptotic agents in various cell types, including hepatocytes. We found that a cAMP analogue, dibutyryl cAMP (db-cAMP), inhibits TNF + ActD-induced apoptosis in rat hepatocytes. The protein kinase A (PKA) inhibitor KT-5720 reverses this inhibitory effect of cAMP on apoptosis. Cytoprotection by cAMP involves down-regulation of various apoptotic signal regulators like TRADD and FADD and inhibition of caspase-8 and caspase-3 cleavage. We also found

  6. Low c-Met expression levels are prognostic for and predict the benefits of temozolomide chemotherapy in malignant gliomas

    Science.gov (United States)

    Li, Ming-Yang; Yang, Pei; Liu, Yan-Wei; Zhang, Chuan-Bao; Wang, Kuan-Yu; Wang, Yin-Yan; Yao, Kun; Zhang, Wei; Qiu, Xiao-Guang; Li, Wen-Bin; Peng, Xiao-Xia; Wang, Yong-Zhi; Jiang, Tao

    2016-01-01

    Aberrant c-Met has been implicated in the development of many cancers. The objective of this study was to identify an unfavorable prognostic marker that might guide decisions regarding clinical treatment strategies for high-grade gliomas. C-Met expression was measured using immunohistochemistry in 783 gliomas, and we further analyzed c-Met mRNA levels using the Agilent Whole Genome mRNA Microarray in 286 frozen samples. In vitro, we performed cell migration and invasion assays. Cell sensitivity to temozolomide (TMZ) chemotherapy was determined using MTT assays. Both mRNA and protein levels of c-Met were significantly associated with tumor grade progression and inversely correlated with overall and progression-free survival in high-grade gliomas (all P < 0.0001). These findings were nearly consistent at the mRNA level across 3 independent cohorts. Multivariable analysis indicated that c-Met was an independent prognostic marker after adjusting for age, preoperative Karnofsky Performance Status (KPS) score, the extent of resection, radiotherapy, TMZ chemotherapy, and O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status. Further analysis in vitro revealed that downregulating the expression of c-Met dramatically inhibited cell migration and invasion capacities, enhanced sensitivity to TMZ chemotherapy in H4 and U87 glioma cells. Our results suggest that c-Met may serve as a potential predictive maker for clinical decision making. PMID:26879272

  7. Evaluation of c-Met, HGF, and HER-2 expressions in gastric carcinoma and their association with other clinicopathological factors

    Science.gov (United States)

    Yıldız, Yetkin; Sokmensuer, Cenk; Yalcin, Suayib

    2016-01-01

    Background Met and HER-2 are proto-oncogenes encoding receptor tyrosine kinase c-Met and HER-2, respectively. Hepatocyte growth factor (HGF) is a ligand of c-Met. The frequency of c-Met, HGF, and HER-2 expressions in gastric cancer and their association with other clinicopathological factors have not been fully understood. Patients and methods Patients with stage 1–4 disease were analyzed. Expressions of c-Met, HGF, and HER-2 were examined using immunohistochemistry. Results A total of 143 patients, 97 males and 46 females, were included. C-Met scores were 3(+) in 31.5%, 2(+) in 27.3%, and 1(+) in 10.5% of the patients. There was no statistically significant difference in age, sex, tumor location, differentiation, Lauren classification, TNM staging, presence of distant metastasis, depth of tumor invasion (T), lymphovascular invasion, and survival between c-Met subgroups. Overall HGF positivity was 20.6%. HER-2 scores were 3(+) in 9.1%, 2(+) in 9.8%, and 1(+) in 16.1% of the patients. HER-2 overexpression was associated with better differentiation, intestinal subtype, and advanced stage. C-Met overexpressions were 84.6% in the HER-2-overexpression-positive group and 56.2% in the HER-2-overexpression-negative group. There were no statistically significant differences in survival between the high c-Met-expression-positive and -negative stage 3 and stage 4 patients and between the HGF-positive and -negative groups. The mean survival was 11.6±6.3 months in the HER-2-overexpression-positive stage 4 group and 11.9±6.8 months in the HER-2-overexpression-negative stage 4 group. There were no statistically significant differences in survival between the two groups. Conclusion c-Met was not associated with any prognostic factors in gastric cancer. HER-2 was associated with better differentiation, intestinal subtype, advanced stage, and c-Met overexpression. PMID:27703380

  8. The c-Met receptor tyrosine kinase inhibitor MP470 radiosensitizes glioblastoma cells

    International Nuclear Information System (INIS)

    Glioblastoma multiforme (GBM) is resistant to current cytotoxic therapies, in part because of enhanced DNA repair. Activation of the receptor tyrosine kinase c-Met has been shown to protect cancer cells from DNA damage. We hypothesized that inhibiting c-Met would decrease this protection and thus sensitize resistant tumor cells to the effects of radiation therapy. Eight human GBM cell lines were screened for radiosensitivity to the small-molecule c-Met inhibitor MP470 with colony-count assays. Double-strand (ds) DNA breaks was quantified by using antibodies to gamma H2AX. Western blotting demonstrate expression of RAD51, glycogen synthase kinase (GSK)-3β, and other proteins. A murine xenograft tumor flank model was used for in vivo radiosensitization studies. MP470 reduced c-Met phosphorylation and enhanced radiation-induced cell kill by 0.4 logs in SF767 cells. Cells pretreated with MP470 had more ds DNA damage than cells treated with radiation alone. Mechanistically, MP470 was shown to inhibit dsDNA break repair and increase apoptosis. MP470 influences various survival and DNA repair related proteins such as pAKT, RAD51 and GSK3β. In vivo, the addition of MP470 to radiation resulted in a tumor-growth-delay enhancement ratio of 2.9 over radiation alone and extended survival time. GBM is a disease site where radiation is often used to address both macroscopic and microscopic disease. Despite attempts at dose escalation outcomes remain poor. MP470, a potent small-molecule tyrosine kinase inhibitor of c-Met, radiosensitized several GBM cell lines both in vitro and in vivo, and may help to improve outcomes for patients with GBM

  9. The indole alkaloid meleagrin, from the olive tree endophytic fungus Penicillium chrysogenum, as a novel lead for the control of c-Met-dependent breast cancer proliferation, migration and invasion.

    Science.gov (United States)

    Mady, Mohamed S; Mohyeldin, Mohamed M; Ebrahim, Hassan Y; Elsayed, Heba E; Houssen, Wael E; Haggag, Eman G; Soliman, Randa F; El Sayed, Khalid A

    2016-01-15

    Fungi of the genus Penicillium produce unique and chemically diverse biologically active secondary metabolites, including indole alkaloids. The role of dysregulated hepatocyte growth factor (HGF) and its receptor, c-Met, in the development and progression of breast carcinoma is documented. The goal of this work is to explore the chemistry and bioactivity of the secondary metabolites of the endophytic Penicillium chrysogenum cultured from the leaf of the olive tree Olea europea, collected in its natural habitat in Egypt. This fungal extract showed good inhibitory activities against the proliferation and migration of several human breast cancer lines. The CH2Cl2 extract of P. chrysogenum mycelia was subjected to bioguided chromatographic separation to afford three known indole alkaloids; meleagrin (1), roquefortine C (2) and DHTD (3). Meleagrin inhibited the growth of the human breast cancer cell lines MDA-MB-231, MDA-468, BT-474, SK BR-3, MCF7 and MCF7-dox, while similar treatment doses were found to have no effect on the growth and viability of the non-tumorigenic human mammary epithelial cells MCF10A. Meleagrin also showed excellent ATP competitive c-Met inhibitory activity in Z-Lyte assay, which was further confirmed via molecular docking studies and Western blot analysis. In addition, meleagrin treatment caused a dose-dependent inhibition of HGF-induced cell migration, and invasion of breast cancer cell lines. Meleagrin treatment potently suppressed the invasive triple negative breast tumor cell growth in an orthotopic athymic nude mice model, promoting this unique natural product from hit to a lead rank. The indole alkaloid meleagrin is a novel lead c-Met inhibitory entity useful for the control of c-Met-dependent metastatic and invasive breast malignancies. PMID:26692349

  10. Pantethine inhibits cholesterol and fatty acid syntheses and stimulates carbon dioxide formation in isolated rat hepatocytes.

    Science.gov (United States)

    Cighetti, G; Del Puppo, M; Paroni, R; Fiorica, E; Galli Kienle, M

    1987-02-01

    The effects of pantethine on cholesterol and fatty acid metabolism were investigated in isolated rat hepatocytes. Preincubation of the cells with pantethine induced a concentration-dependent decrease of the radioactivity incorporated into carbon dioxide and lipids in incubations with [2-14C]acetate. When pantethine and the labeled substrate were simultaneously added to the cell suspension, there was an enhancement of carbon dioxide radioactivity at short incubation time (5 min) whereas, at longer incubation time, values were comparable to those of controls; lipid radioactivity, instead, was dramatically reduced by pantethine even at short incubation time and decreased further during the incubation, being 23% of that of controls at 60 min. Analysis of the incubation medium showed that pantethine induced a concentration- and time-dependent release of acetate into the medium. Results of the effect of the acetate concentration on the incorporation of [2-14C]acetate radioactivity into CO2 and lipids in control hepatocytes allowed the conclusion that the above-described modifications induced by pantethine are only partially attributable to the dilution of the labeled substrate, and that catabolism of acetate to carbon dioxide is stimulated by the disulphide pantethine, whereas cholesterol and fatty acid syntheses are inhibited. PMID:3106549

  11. INHIBITION OF BILE ACID ACCUMULATION DECREASED THE EXCESSIVE HEPATOCYTE APOPTOSIS AND IMPROVED THE LIVER SECRETION FUNCTIONS ON OBSTRUCTIVE JAUNDICE PATIENTS

    Directory of Open Access Journals (Sweden)

    Akmal Taher

    2011-06-01

    Full Text Available Excessive hepatocyte apoptosis induced by bile acid accumulation occurred in severe obstructive jaundice, and impair the liver secretion function. The objective of this study is to determine whether the inhibition of bile acid accumulation through bile duct decompression affect the excessive hepatocyte apoptosis and caused improvement the liver secretion functions on human model. In this study we use a before and after study on severe obstructive jaundice patients due to extra hepatic bile duct tumor was decompressed. Bile duct decompression was performed as a model of the role of inhibition of bile acid accumulation inhibition bile acid accumulation and excessive hepatocyte apoptosis. Bile acid and marker of liver secretion functions were serially measured. Liver biopsy pre and post decompression was performed for Hepatocyte apoptosis pathologic examination by TUNEL fluorescing, which measured by 2 people in double blinded system. Total bile acid, and liver secretion functions were measured by automated chemistry analyzer. The result of this study shows that twenty one severe obstructive jaundice patients were included. After decompression the hepatocyte apoptosis index decreased from an average of 53.1 (SD 105 to 11.7 (SD 13.6 (p < 0.05. Average of bile acid serum decreased from 96.4 (SD 53.8 to 19.9 (SD 39.5 until 13.0 (SD 12.6 μmol/L (p < 0.05 Total ilirubin decreased from 20.0 (SD 8.9 to 13.3 (SD 5.0 until 6.2 (SD 4.0 mg/dL (p < 0.05, while the phosphates alkaline (ALP and γ-glutamil transpeptidase (γ-GT activities also decreased ignificantly. In conclusion, bile acids accumulation and excessive hepatocyte poptosis through bile duct decompression improve the liver secretion functions by inhibition mechanism.

  12. Histidine-rich stabilized polyplexes for cMet-directed tumor-targeted gene transfer

    Science.gov (United States)

    Kos, Petra; Lächelt, Ulrich; Herrmann, Annika; Mickler, Frauke Martina; Döblinger, Markus; He, Dongsheng; Krhač Levačić, Ana; Morys, Stephan; Bräuchle, Christoph; Wagner, Ernst

    2015-03-01

    Overexpression of the hepatocyte growth factor receptor/c-Met proto oncogene on the surface of a variety of tumor cells gives an opportunity to specifically target cancerous tissues. Herein, we report the first use of c-Met as receptor for non-viral tumor-targeted gene delivery. Sequence-defined oligomers comprising the c-Met binding peptide ligand cMBP2 for targeting, a monodisperse polyethylene glycol (PEG) for polyplex surface shielding, and various cationic (oligoethanamino) amide cores containing terminal cysteines for redox-sensitive polyplex stabilization, were assembled by solid-phase supported syntheses. The resulting oligomers exhibited a greatly enhanced cellular uptake and gene transfer over non-targeted control sequences, confirming the efficacy and target-specificity of the formed polyplexes. Implementation of endosomal escape-promoting histidines in the cationic core was required for gene expression without additional endosomolytic agent. The histidine-enriched polyplexes demonstrated stability in serum as well as receptor-specific gene transfer in vivo upon intratumoral injection. The co-formulation with an analogous PEG-free cationic oligomer led to a further compaction of pDNA polyplexes with an obvious change of shape as demonstrated by transmission electron microscopy. Such compaction was critically required for efficient intravenous gene delivery which resulted in greatly enhanced, cMBP2 ligand-dependent gene expression in the distant tumor.Overexpression of the hepatocyte growth factor receptor/c-Met proto oncogene on the surface of a variety of tumor cells gives an opportunity to specifically target cancerous tissues. Herein, we report the first use of c-Met as receptor for non-viral tumor-targeted gene delivery. Sequence-defined oligomers comprising the c-Met binding peptide ligand cMBP2 for targeting, a monodisperse polyethylene glycol (PEG) for polyplex surface shielding, and various cationic (oligoethanamino) amide cores containing

  13. Chinese Herbal Preparation Xuebijing Potently Inhibits Inflammasome Activation in Hepatocytes and Ameliorates Mouse Liver Ischemia-Reperfusion Injury.

    Directory of Open Access Journals (Sweden)

    Xiqiang Liu

    Full Text Available The Chinese herb preparation Xuebijing injection (XBJ has been widely used in the management of various septic disorders or inflammation-related conditions, however the molecular mechanism of its anti-inflammatory effect remains largely elusive. In the current study, we found that XBJ treatment potently ameliorated mouse hepatic ischemia-reperfusion (IR injury, manifested as decreased liver function tests (LDH, ALT, AST, improved inflammation and less hepatocyte apoptosis. Notably, XBJ markedly inhibited inflammasome activation and IL-1 production in mouse livers subjected to IRI, even in the absence of Kupffer cells, suggesting Kupffer cells are not necessary for hepatic inflammasome activation upon Redox-induced sterile inflammation. This finding led us to investigate the role of XBJ on hepatocyte apoptosis and inflammasome activation using an in vitro hydrogen peroxide (H2O2-triggered hepatocyte injury model. Our data clearly demonstrated that XBJ potently inhibited apoptosis, as well as caspase-1 cleavage and IL-1β production in a time- and dose-dependent manner in isolated hepatocytes, suggesting that in addition to its known modulatory effect on NF-κB-dependent inflammatory gene expression, it also has a direct impact on hepatocyte inflammasome activation. The current study not only deepens our understanding of how XBJ ameliorates inflammation and apoptosis, but also has immediate practical significance in many clinical situations such as partial hepatectomy, liver transplantation, etc.

  14. Use of Cassette Dosing in Sandwich-Cultured Rat and Human Hepatocytes to Identify Drugs that Inhibit Bile Acid Transport

    OpenAIRE

    Kristina K Wolf; Vora, Sapana; Webster, Lindsey O.; Generaux, Grant T.; Polli, Joseph W; Brouwer, Kim L.R.

    2009-01-01

    Hepatocellular accumulation of bile acids due to inhibition of the canalicular bile salt export pump (BSEP/ABCB11) is one proposed mechanism of drug-induced liver injury (DILI). Some hepatotoxic compounds also are potent inhibitors of bile acid uptake by Na+-dependent taurocholate cotransporting polypeptide (NTCP/SLC10A1). This study used a cassette dosing approach in rat and human sandwich-cultured hepatocytes (SCH) to determine whether known or suspected hepatotoxic drugs inhibit bile acid ...

  15. Weight loss reversed obesity-induced HGF/c-Met pathway and basal-like breast cancer progression

    Directory of Open Access Journals (Sweden)

    Sneha eSundaram

    2014-07-01

    Full Text Available Epidemiologic studies demonstrate that obesity is associated with an aggressive subtype of breast cancer called basal-like breast cancer (BBC. Using the C3(1-TAg murine model of BBC, we previously demonstrated that mice displayed an early onset of tumors when fed obesogenic diets in the adult window of susceptibility. Obesity was also shown to elevate mammary gland expression and activation of hepatocyte growth factor (HGF/c-Met compared to lean controls, a pro-tumorigenic pathway associated with BBC in patients. Epidemiologic studies estimate that weight loss could prevent a large proportion of BBC. We sought to investigate whether weight loss in adulthood prior to tumor onset would protect mice from accelerated tumorigenesis observed in obese mice. Using a life-long model of obesity, C3(1-TAg mice were weaned onto and maintained on an obesogenic high fat diet. Obese mice displayed significant elevations in tumor progression, but not latency or burden. Tumor progression was significantly reversed when obese mice were induced to lose weight by switching to a control low fat diet prior to tumor onset compared to mice maintained on obesogenic diet. It is likely that other factors regulated tumor progression, hence we investigated the HGF/c-Met pathway known to regulate tumorigenesis. Importantly, HGF/c-Met expression in normal mammary glands and c-Met in tumors was elevated with obesity and was significantly reversed with weight loss. Changes in tumor growth could not be explained by measures of HGF action including phospho-AKT or phospho-S6. Other mediators associated with oncogenesis such as hyperinsulinemia and a high leptin/adiponectin ratio were elevated by obesity and reduced with weight loss. In sum, weight loss significantly blunted the obesity-responsive pro-tumorigenic HGF/c-Met pathway and improved several metabolic risk factors associated with BBC, which together may have contributed to the dramatic reversal of obesity-driven tumor

  16. Signal transduction and downregulation of C-MET in HGF stimulated low and highly metastatic human osteosarcoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Husmann, Knut, E-mail: khusmann@research.balgrist.ch [Laboratory for Orthopedic Research, Department of Orthopedics, Balgrist University Hospital, University of Zurich, Zurich (Switzerland); Ducommun, Pascal [Laboratory for Orthopedic Research, Department of Orthopedics, Balgrist University Hospital, University of Zurich, Zurich (Switzerland); Division of Plastic Surgery and Hand Surgery, Department of Surgery, University Hospital Zurich, Zurich (Switzerland); Sabile, Adam A.; Pedersen, Else-Marie; Born, Walter; Fuchs, Bruno [Laboratory for Orthopedic Research, Department of Orthopedics, Balgrist University Hospital, University of Zurich, Zurich (Switzerland)

    2015-09-04

    The poor outcome of osteosarcoma (OS), particularly in patients with metastatic disease and a five-year survival rate of only 20%, asks for more effective therapeutic strategies targeting malignancy-promoting mechanisms. Dysregulation of C-MET, its ligand hepatocyte growth factor (HGF) and the fusion oncogene product TPR-MET, first identified in human MNNG-HOS OS cells, have been described as cancer-causing factors in human cancers. Here, the expression of these molecules at the mRNA and the protein level and of HGF-stimulated signaling and downregulation of C-MET was compared in the parental low metastatic HOS and MG63 cell lines and the respective highly metastatic MNNG-HOS and 143B and the MG63-M6 and MG63-M8 sublines. Interestingly, expression of TPR-MET was only observed in MNNG-HOS cells. HGF stimulated the phosphorylation of Akt and Erk1/2 in all cell lines investigated, but phospho-Stat3 remained at basal levels. Downregulation of HGF-stimulated Akt and Erk1/2 phosphorylation was much faster in the HGF expressing MG63-M8 cells than in HOS cells. Degradation of HGF-activated C-MET occurred predominantly through the proteasomal and to a lesser extent the lysosomal pathway in the cell lines investigated. Thus, HGF-stimulated Akt and Erk1/2 signaling as well as proteasomal degradation of HGF activated C-MET are potential therapeutic targets in OS. - Highlights: • Expression of TPR-MET was only observed in MNNG-HOS cells. • HGF stimulated the phosphorylation of Akt and Erk1/2 but not of Stat3 in osteosarcoma cell lines. • Degradation of HGF-activated C-MET occurred predominantly through the proteasomal pathway.

  17. Bile acids activate fibroblast growth factor 19 signaling in human hepatocytes to inhibit cholesterol 7α-hydroxylase gene expression

    OpenAIRE

    Song, Kwang-Hoon; Li, Tiangang; Owsley, Erika; Strom, Stephen; Chiang, John Y. L.

    2009-01-01

    Mouse fibroblast growth factor 15 (FGF15) and human ortholog FGF19 have been identified as the bile acid-induced intestinal factors that mediate bile acid feedback inhibition of cholesterol 7α-hydroxylase gene transcription in mouse liver. The mechanism underlying FGF15/FGF19 inhibition of bile acid synthesis in hepatocytes remains unclear. Chenodeoxycholic acid (CDCA) and a farnesoid X receptor (FXR)-specific agonist GW4064 strongly induced FGF19 but inhibited CYP7A1 mRNA levels in primary h...

  18. Inducing effects of hepatocyte growth factor on the expression of vascular endothelial growth factor in human colorectal carcinoma cells through MEK and PI3K signaling pathways

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yu-hua; WEI Wei; XU Hao; WANG Yan-yan; WU Wen-xi

    2007-01-01

    Background Vascular endothelial growth factor plays a key role in human colorectal carcinoma invasion and metastasis. However, the regulation mechanism remains unknown. Recent studies have shown that several cytokines can regulate the expression of vascular endothelial growth factor in tumor cells. In this study, we investigated whether hepatocyte growth factor can regulate the expression of vascular endothelial growth factor in colorectal carcinoma cells.Methods Hepatocyte growth factor and vascular endothelial growth factor in human serum were measured by ELISA.The mRNA level of vascular endothelial growth factor was analyzed by reverse transcription-PCR. Western blot assay was performed to evaluate levels of c-Met and several other proteins involved in the MAPK and PI3K signaling pathways in colorectal carcinoma cells.Results Serum hepatocyte growth factor and vascular endothelial growth factor were significantly increased in colorectal carcinoma subjects. In vitro extraneous hepatocyte growth factor markedly increased protein and mRNA levels of vascular endothelial growth factor in colorectal carcinoma cells. Hepatocyte growth factor induced phosphorylation of c-Met, ERK1/2 and AKT in a dose-dependent manner. Specific inhibitors on MEK and PI3K inhibited the hepatocyte growth factor-induced expression of vascular endothelial growth factor in colorectal carcinoma cells.Conclusion This present study indicates that hepatocyte growth factor upregulates the expression of vascular endothelial growth factor in colorectal carcinoma cells via the MEK/ERK and PI3K/AKT signaling pathways.

  19. YAP Inhibition Restores Hepatocyte Differentiation in Advanced HCC, Leading to Tumor Regression

    Directory of Open Access Journals (Sweden)

    Julien Fitamant

    2015-03-01

    Full Text Available Defective Hippo/YAP signaling in the liver results in tissue overgrowth and development of hepatocellular carcinoma (HCC. Here, we uncover mechanisms of YAP-mediated hepatocyte reprogramming and HCC pathogenesis. YAP functions as a rheostat in maintaining metabolic specialization, differentiation, and quiescence within the hepatocyte compartment. Increased or decreased YAP activity reprograms subsets of hepatocytes to different fates associated with deregulation of the HNF4A, CTNNB1, and E2F transcriptional programs that control hepatocyte quiescence and differentiation. Importantly, treatment with small interfering RNA-lipid nanoparticles (siRNA-LNPs targeting YAP restores hepatocyte differentiation and causes pronounced tumor regression in a genetically engineered mouse HCC model. Furthermore, YAP targets are enriched in an aggressive human HCC subtype characterized by a proliferative signature and absence of CTNNB1 mutations. Thus, our work reveals Hippo signaling as a key regulator of the positional identity of hepatocytes, supports targeting of YAP using siRNA-LNPs as a paradigm of differentiation-based therapy, and identifies an HCC subtype that is potentially responsive to this approach.

  20. EGFR is dispensable for c-Met-mediated proliferation and survival activities in mouse adult liver oval cells.

    Science.gov (United States)

    Martínez-Palacián, A; del Castillo, G; Herrera, B; Fernández, M; Roncero, C; Fabregat, I; Sánchez, A

    2012-02-01

    Liver progenitor cells rise as potential critical players in hepatic regeneration but also carcinogenesis. It is therefore mandatory to define the signals controlling their activation and expansion. Recently, by using a novel in vitro model of oval cell lines expressing a mutant tyrosine kinase-inactive form of c-Met we demonstrated that autocrine c-Met signalling plays an essential role in promoting oval cell survival. Here, we investigated the significance of the epidermal growth factor receptor (EGFR) signalling in oval cell proliferation and survival, as well as a potential functional crosstalk between the c-Met and the EGFR pathways. We found an autocrine activation of the EGFR-triggered pathway in Met(flx/flx) and Met(-/-) oval cells as judged by constitutive expression of the EGFR ligands, transforming growth factor-alpha (TGF-α) and heparin-binding EGF like growth factor (HB-EGF), and activation of EGFR. On the other hand, treatment with AG1478, a specific inhibitor of EGFR, effectively blocked endogenous and EGF-induced proliferation, while increased serum withdrawal and transforming growth factor-beta (TGF-β)-induced apoptosis. These results suggest that constitutively activated EGFR might promote oval cell proliferation and survival. We found that hepatocyte growth factor (HGF) does not transactivate EGFR nor EGF transactivates c-Met. Furthermore, treatment with AG1478 or EGFR gene silencing did not interfere with HGF-mediated activation of target signals, such as protein kinase B (AKT/PKB), and extracellular signal-regulated kinases 1/2 (ERK 1/2), nor did it have any effect on HGF-induced proliferative and antiapoptotic activities in Met(flx/flx) cells, showing that HGF does not require EGFR activation to mediate such responses. EGF induced proliferation and survival equally in Met(flx/flx) and Met(-/-) oval cells, proving that EGFR signalling does not depend on c-Met tyrosine kinase activity. Together, our results provide strong evidence that in

  1. WNT5A inhibits hepatocyte proliferation and concludes β-catenin signaling in liver regeneration.

    Science.gov (United States)

    Yang, Jing; Cusimano, Antonella; Monga, Jappmann K; Preziosi, Morgan E; Pullara, Filippo; Calero, Guillermo; Lang, Richard; Yamaguchi, Terry P; Nejak-Bowen, Kari N; Monga, Satdarshan P

    2015-08-01

    Activation of Wnt/β-catenin signaling during liver regeneration (LR) after partial hepatectomy (PH) is observed in several species. However, how this pathway is turned off when hepatocyte proliferation is no longer required is unknown. We assessed LR in liver-specific knockouts of Wntless (Wls-LKO), a protein required for Wnt secretion from a cell. When subjected to PH, Wls-LKO showed prolongation of hepatocyte proliferation for up to 4 days compared with littermate controls. This coincided with increased β-catenin-T-cell factor 4 interaction and cyclin-D1 expression. Wls-LKO showed decreased expression and secretion of inhibitory Wnt5a during LR. Wnt5a expression increased between 24 and 48 hours, and Frizzled-2 between 24 and 72 hours, after PH in normal mice. Treatment of primary mouse hepatocytes and liver tumor cells with Wnt5a led to a notable decrease in β-catenin-T-cell factor activity, cyclin-D1 expression, and cell proliferation. Intriguingly, Wnt5a-LKO did not display any prolongation of LR because of compensation by other cells. In addition, Wnt5a-LKO hepatocytes failed to respond to exogenous Wnt5a treatment in culture because of a compensatory decrease in Frizzled-2 expression. In conclusion, we demonstrate Wnt5a to be, by default, a negative regulator of β-catenin signaling and hepatocyte proliferation, both in vitro and in vivo. We also provide evidence that the Wnt5a/Frizzled-2 axis suppresses β-catenin signaling in hepatocytes in an autocrine manner, thereby contributing to timely conclusion of the LR process.

  2. Activation of c-Met and upregulation of CD44 expression are associated with the metastatic phenotype in the colorectal cancer liver metastasis model.

    Directory of Open Access Journals (Sweden)

    Victoria A Elliott

    Full Text Available BACKGROUND: Liver metastasis is the most common cause of death in patients with colorectal cancer. Despite extensive research into the biology of cancer progression, the molecular mechanisms that drive colorectal cancer metastasis are not well characterized. METHODS: HT29 LM1, HT29 LM2, HT29 LM3 cell lines were derived from the human colorectal cancer cell line HT29 following multiple rounds of in vivo selection in immunodeficient mice. RESULTS: CD44 expression, a transmembrane glycoprotein involved in cell-cell and cell-matrix adhesions, and cancer cells adhesion to endothelial cells was increased in all in vivo selected cell lines, with maximum CD44 expression and cancer cells adhesion to endothelial cells in the highly metastatic HT29 LM3 cell line. Activation of c-Met upon hepatocyte growth factor (HGF stimulation in the in vivo selected cell lines is CD44 independent. In vitro separation of CD44 high and low expression cells from HT29 LM3 cell line with FACS sorting confirmed that c-Met activation is CD44 independent upon hepatocyte growth factor stimulation. Furthermore, in vivo evaluation of CD44 low and high expressing HT29 LM3 cells demonstrated no difference in liver metastasis penetrance. CONCLUSIONS: Taken together, our findings indicate that the aggressive metastatic phenotype of in vivo selected cell lines is associated with overexpression of CD44 and activation of c-MET. We demonstrate that c-Met activation is CD44 independent upon hepatocyte growth factor stimulation and confirm that CD44 expression in HT29 LM3 cell line is not responsible for the increase in metastatic penetrance in HT29 LM3 cell line.

  3. Dynamic expression and localization of c-MET isoforms in the developing rat pancreas.

    Science.gov (United States)

    Wu, Yulong; Cheng, Mei; Shi, Zhen; Feng, Zhenqing; Guan, Xiaohong

    2014-01-01

    Pancreata from Sprague Dawley rats of different developmental stages were studied to determine the expression and cellular localization of different c-MET isoforms in the developing rat pancreas. Pancreatic mRNA and protein expression levels of c-MET at different developmental stages from embryo to adult were detected by reverse transcription-polymerase chain reaction and by western blotting. To identify the cellular localization of c-MET protein in the developing rat pancreas, double immunofluorescent staining was performed using antibodies for cell type-specific markers and for c-MET. The expression of two isoforms of c-MET (190 kDa and 170 kDa) coincided with the development of the pancreas. The 190 kDa isoform of c-MET is expressed during embryonic stages, and its expression is replaced by the expression of the 170 kDa isoform as the pancreas develops. Only the 170 kDa isoform is expressed in the adult rat pancreas. Throughout all stages of pancreatic development, c-MET is expressed by vimentin-positive cells. In contrast, c-MET staining was stronger in rat pancreata from newborn to adult stages and overlapped with insulin-positive beta-cells. The dynamic expression and localization of different c-MET isoforms in the rat pancreas during different developmental stages indicates that distinct c-MET isoform might be involved in different aspects of pancreatic development.

  4. Farnesoid X Receptor Inhibits the Transcriptional Activity of Carbohydrate Response Element Binding Protein in Human Hepatocytes

    OpenAIRE

    Caron, Sandrine; Huaman Samanez, Carolina; Dehondt, Hélène; Ploton, Maheul; Briand, Olivier; Lien, Fleur; Dorchies, Emilie; Dumont, Julie; Postic, Catherine; Cariou, Bertrand; Lefebvre, Philippe; Staels, Bart

    2013-01-01

    The glucose-activated transcription factor carbohydrate response element binding protein (ChREBP) induces the expression of hepatic glycolytic and lipogenic genes. The farnesoid X receptor (FXR) is a nuclear bile acid receptor controlling bile acid, lipid, and glucose homeostasis. FXR negatively regulates hepatic glycolysis and lipogenesis in mouse liver. The aim of this study was to determine whether FXR regulates the transcriptional activity of ChREBP in human hepatocytes and to unravel the...

  5. Inhibition of hepatocyte nuclear factor 4 transcriptional activity by the nuclear factor {kappa}B pathway

    OpenAIRE

    Nikolaidou-Neokosmidou, Varvara; Zannis, Vassilis I.; Kardassis, Dimitris

    2006-01-01

    Abstract Hepatocyte Nuclear Factor 4 (HNF-4) is a key regulator of liver specific gene expression in mammals. We have shown previously that the activity of the human apolipoprotein C-III (APOC3) promoter is positively regulated by the anti-inflammatory cytokine Transforming Growth Factor {beta} (TGF{beta}) and its effectors Smad3 and Smad4 proteins via physical and functional interactions between Smads and HNF-4. We now show that the pro-inflammatory cytokine Tumor Necrosis Factor ...

  6. Inhibition of bile salt transport by drugs associated with liver injury in primary hepatocytes from human, monkey, dog, rat, and mouse.

    Science.gov (United States)

    Zhang, Jie; He, Kan; Cai, Lining; Chen, Yu-Chuan; Yang, Yifan; Shi, Qin; Woolf, Thomas F; Ge, Weigong; Guo, Lei; Borlak, Jürgen; Tong, Weida

    2016-08-01

    Interference of bile salt transport is one of the underlying mechanisms for drug-induced liver injury (DILI). We developed a novel bile salt transport activity assay involving in situ biosynthesis of bile salts from their precursors in primary human, monkey, dog, rat, and mouse hepatocytes in suspension as well as LC-MS/MS determination of extracellular bile salts transported out of hepatocytes. Glycine- and taurine-conjugated bile acids were rapidly formed in hepatocytes and effectively transported into the extracellular medium. The bile salt formation and transport activities were time‒ and bile-acid-concentration‒dependent in primary human hepatocytes. The transport activity was inhibited by the bile salt export pump (BSEP) inhibitors ketoconazole, saquinavir, cyclosporine, and troglitazone. The assay was used to test 86 drugs for their potential to inhibit bile salt transport activity in human hepatocytes, which included 35 drugs associated with severe DILI (sDILI) and 51 with non-severe DILI (non-sDILI). Approximately 60% of the sDILI drugs showed potent inhibition (with IC50 values <50 μM), but only about 20% of the non-sDILI drugs showed this strength of inhibition in primary human hepatocytes and these drugs are associated only with cholestatic and mixed hepatocellular cholestatic (mixed) injuries. The sDILI drugs, which did not show substantial inhibition of bile salt transport activity, are likely to be associated with immune-mediated liver injury. Twenty-four drugs were also tested in monkey, dog, rat and mouse hepatocytes. Species differences in potency were observed with mouse being less sensitive than other species to inhibition of bile salt transport. In summary, a novel assay has been developed using hepatocytes in suspension from human and animal species that can be used to assess the potential for drugs and/or drug-derived metabolites to inhibit bile salt transport and/or formation activity. Drugs causing sDILI, except those by immune

  7. High levels of c-Met is associated with poor prognosis in glioblastoma

    DEFF Research Database (Denmark)

    Petterson, Stine Asferg; Dahlrot, Rikke Hedegaard; Hermansen, Simon Kjær;

    2015-01-01

    with WHO grade I, II, III and IV tumors were analyzed using immunohistochemical staining and advanced image analysis. Strong c-Met expression was found in tumor cells, blood vessels, and peri-necrotic areas. At the subcellular level, c-Met was identified in the cytoplasm and in the cell membrane......The tyrosine kinase receptor c-Met has been suggested to be involved in crucial parts of glioma biology like tumor stemness, growth and invasion. The aim of this study was to investigate the prognostic value of c-Met in a population-based glioma patient cohort. Tissue samples from 238 patients...

  8. Quantification of Drug-Induced Inhibition of Canalicular Cholyl-l-Lysyl-Fluorescein Excretion From Hepatocytes by High Content Cell Imaging.

    Science.gov (United States)

    Barber, Jane A; Stahl, Simone H; Summers, Claire; Barrett, Gillian; Park, B Kevin; Foster, John R; Kenna, J Gerald

    2015-11-01

    We describe the use of a commercially available high content cell imaging algorithm (Cellomics Arrayscan Spot Detector) to quantify biliary excretion of the fluorescent probe substrate cholyl-l-lysyl-fluorescein (CLF) from rat hepatocytes cultured in collagen/matrigel sandwich configuration and to explore inhibition of this process by a variety of test compounds. The method provided robust, reproducible data. Twenty-nine pharmaceuticals inhibited biliary CLF efflux from hepatocytes and a broad range of potencies of inhibition were observed (IC50 values ranged between Bsep), which suggests that the tested drugs inhibit both Bsep and Mrp2. Calculation of the ratios between the maximum human plasma concentrations of the drugs and their CLF efflux inhibition IC50 values raised the possibility that for many, but not all, of them the in vitro effects may be functionally significant in vivo and that Mrp2 inhibition might be a drug-induced liver injury (DILI) risk factor. These data indicate that imaging hepatocyte CLF inhibition is a promising new method for quantification of biliary efflux inhibition by drugs, which could aid assessment of compound-related DILI risk. PMID:26220638

  9. Cdc42 and phosphoinositide 3-kinase drive Rac-mediated actin polymerization downstream of c-Met in distinct and common pathways

    DEFF Research Database (Denmark)

    Bosse, Tanja; Ehinger, Julia; Czuchra, Aleksandra;

    2007-01-01

    Activation of c-Met, the hepatocyte growth factor (HGF)/scatter factor receptor induces reorganization of the actin cytoskeleton, which drives epithelial cell scattering and motility and is exploited by pathogenic Listeria monocytogenes to invade nonepithelial cells. However, the precise...... required the simultaneous inactivation of both Cdc42 and PI3-kinase signaling. Moreover, Cdc42 activation was fully independent of PI3-kinase activity, whereas the latter partly depended on Cdc42. Finally, Cdc42 function did not require its interaction with the actin nucleation-promoting factor N...

  10. Activation of the constitutive androstane receptor inhibits gluconeogenesis without affecting lipogenesis or fatty acid synthesis in human hepatocytes

    International Nuclear Information System (INIS)

    Objective: Accumulating evidence suggests that activation of mouse constitutive androstane receptor (mCAR) alleviates type 2 diabetes and obesity by inhibiting hepatic gluconeogenesis, lipogenesis, and fatty acid synthesis. However, the role of human (h) CAR in energy metabolism is largely unknown. The present study aims to investigate the effects of selective hCAR activators on hepatic energy metabolism in human primary hepatocytes (HPH). Methods: Ligand-based structure–activity models were used for virtual screening of the Specs database ( (www.specs.net)) followed by biological validation in cell-based luciferase assays. The effects of two novel hCAR activators (UM104 and UM145) on hepatic energy metabolism were evaluated in HPH. Results: Real-time PCR and Western blotting analyses reveal that activation of hCAR by UM104 and UM145 significantly repressed the expression of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, two pivotal gluconeogenic enzymes, while exerting negligible effects on the expression of genes associated with lipogenesis and fatty acid synthesis. Functional experiments show that UM104 and UM145 markedly inhibit hepatic synthesis of glucose but not triglycerides in HPH. In contrast, activation of mCAR by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, a selective mCAR activator, repressed the expression of genes associated with gluconeogenesis, lipogenesis, and fatty acid synthesis in mouse primary hepatocytes, which were consistent with previous observations in mouse model in vivo. Conclusion: Our findings uncover an important species difference between hCAR and mCAR in hepatic energy metabolism, where hCAR selectively inhibits gluconeogenesis without suppressing fatty acid synthesis. Implications: Such species selectivity should be considered when exploring CAR as a potential therapeutic target for metabolic disorders. - Highlights: • Novel hCAR activators were identified by computational and biological approaches. • The role

  11. Activation of the constitutive androstane receptor inhibits gluconeogenesis without affecting lipogenesis or fatty acid synthesis in human hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Lynch, Caitlin; Pan, Yongmei; Li, Linhao [Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201 (United States); Heyward, Scott; Moeller, Timothy [Bioreclamation In Vitro Technologies, Baltimore, MD 21227 (United States); Swaan, Peter W. [Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201 (United States); Wang, Hongbing, E-mail: hwang@rx.umaryland.edu [Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD 21201 (United States)

    2014-08-15

    Objective: Accumulating evidence suggests that activation of mouse constitutive androstane receptor (mCAR) alleviates type 2 diabetes and obesity by inhibiting hepatic gluconeogenesis, lipogenesis, and fatty acid synthesis. However, the role of human (h) CAR in energy metabolism is largely unknown. The present study aims to investigate the effects of selective hCAR activators on hepatic energy metabolism in human primary hepatocytes (HPH). Methods: Ligand-based structure–activity models were used for virtual screening of the Specs database ( (www.specs.net)) followed by biological validation in cell-based luciferase assays. The effects of two novel hCAR activators (UM104 and UM145) on hepatic energy metabolism were evaluated in HPH. Results: Real-time PCR and Western blotting analyses reveal that activation of hCAR by UM104 and UM145 significantly repressed the expression of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, two pivotal gluconeogenic enzymes, while exerting negligible effects on the expression of genes associated with lipogenesis and fatty acid synthesis. Functional experiments show that UM104 and UM145 markedly inhibit hepatic synthesis of glucose but not triglycerides in HPH. In contrast, activation of mCAR by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, a selective mCAR activator, repressed the expression of genes associated with gluconeogenesis, lipogenesis, and fatty acid synthesis in mouse primary hepatocytes, which were consistent with previous observations in mouse model in vivo. Conclusion: Our findings uncover an important species difference between hCAR and mCAR in hepatic energy metabolism, where hCAR selectively inhibits gluconeogenesis without suppressing fatty acid synthesis. Implications: Such species selectivity should be considered when exploring CAR as a potential therapeutic target for metabolic disorders. - Highlights: • Novel hCAR activators were identified by computational and biological approaches. • The role

  12. A Schisandra-Derived Compound Schizandronic Acid Inhibits Entry of Pan-HCV Genotypes into Human Hepatocytes

    Science.gov (United States)

    Qian, Xi-Jing; Zhang, Xiao-Lian; Zhao, Ping; Jin, Yong-Sheng; Chen, Hai-Sheng; Xu, Qing-Qiang; Ren, Hao; Zhu, Shi-Ying; Tang, Hai-Lin; Zhu, Yong-Zhe; Qi, Zhong-Tian

    2016-01-01

    Despite recent progress in the development of hepatitis C virus (HCV) inhibitors, cost-effective antiviral drugs, especially among the patients receiving liver transplantations, are still awaited. Schisandra is a traditional medicinal herb used to treat a range of liver disorders including hepatitis for thousands of years in China. To isolate the bioactive compounds of schisandra for the treatment of HCV infection, we screened a schisandra-extracts library and identified a tetracyclic triterpenoid, schizandronic acid (SZA), as a novel HCV entry inhibitor. Our findings suggested that SZA potently inhibited pan-HCV genotype entry into hepatoma cells and primary human hepatocytes without interfering virus binding on cell surface or internalization. However, virion-cell fusion process was impaired in the presence of SZA, along with the increased host membrane fluidity. We also found that SZA inhibited the spread of HCV to the neighboring cells, and combinations of SZA with interferon or telaprevir resulted in additive synergistic effect against HCV. Additionally, SZA diminished the establishment of HCV infection in vivo. The SZA target is different from conventional direct-acting antiviral agents, therefore, SZA is a potential therapeutic compound for the development of effective HCV entry inhibitors, especially for patients who need to prevent HCV reinfection during the course of liver transplantations. PMID:27252043

  13. The role of cMet in non-small cell lung cancer resistant to EGFR-inhibitors: did we really find the target?

    Science.gov (United States)

    Passiglia, Francesco; Van Der Steen, Nele; Raez, Luis; Pauwels, Patrick; Gil-Bazo, Ignacio; Santos, Edgardo; Santini, Daniele; Tesoriere, Giovanni; Russo, Antonio; Bronte, Giuseppe; Zwaenepoel, Karen; Cappuzzo, Federico; Rolfo, Christian

    2014-01-01

    The advent of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) represented the most important innovation in NSCLC treatment over the last years. However, despite a great initial activity, secondary mutations in the same target, or different alterations in other molecular pathways, inevitably occur, leading to the emergence of acquired resistance, in median within the first year of treatment. In this scenario, the mesenchymal-epidermal transition (cMET) tyrosine kinase receptor and its natural ligand, the hepatocyte growth factor (HGF), seem to play an important role. Indeed either the overexpression or the amplification of cMET, as well as the overexpression of the HGF, have been reported in a substantial subgroup of NSCLC patients resistant to EGFR-TKIs. Several cMET-inhibitors have been developed as potential therapeutic candidates, and are currently under investigation in clinical trials. These compounds include both monoclonal antibodies and TKIs, and most of them have been investigated as dual combinations including an anti-EGFR TKI, to improve the efficacy of the available treatments, and ultimately overcome acquired resistance to EGFR-inhibitors.

  14. c-Met overexpression in inflammatory breast carcinomas: automated quantification on tissue microarrays

    OpenAIRE

    Garcia, S.; Dalès, J-P; Jacquemier, J.; Charafe-Jauffret, E; Birnbaum, D; Andrac-Meyer, L; Lavaut, M-N; Allasia, C; Carpentier-Meunier, S; Bonnier, P.; Charpin-Taranger, C

    2007-01-01

    Inflammatory breast carcinoma (IBC) is a rare but aggressive tumour associated with poor outcome owing to early metastases. Increased expression of c-Met protein correlates with reduced survival and high metastatic risk in human cancers including breast carcinomas and is targetable by specific drugs, that could potentially improve the prognosis. In the present study, we compared c-Met expression in IBC (n=41) and non-IBC (n=480) immunohistochemically (Ventana Benchmark autostainer) in two tis...

  15. Obesity-mediated regulation of HGF/c-Met is associated with reduced basal-like breast cancer latency in parous mice.

    Directory of Open Access Journals (Sweden)

    Sneha Sundaram

    Full Text Available It is widely thought that pregnancy reduces breast cancer risk, but this lacks consideration of breast cancer subtypes. While a full term pregnancy reduces risk for estrogen receptor positive (ER+ and luminal breast cancers, parity is associated with increased risk of basal-like breast cancer (BBC subtype. Basal-like subtypes represent less than 10% of breast cancers and are highly aggressive, affecting primarily young, African American women. Our previous work demonstrated that high fat diet-induced obesity in nulliparous mice significantly blunted latency in C3(1-TAg mice, a model of BBC, potentially through the hepatocyte growth factor (HGF/c-Met oncogenic pathway. Experimental studies have examined parity and obesity individually, but to date, the joint effects of parity and obesity have not been studied. We investigated the role of obesity in parous mice on BBC. Parity alone dramatically blunted tumor latency compared to nulliparous controls with no effects on tumor number or growth, while obesity had only a minor role in further reducing latency. Obesity-associated metabolic mediators and hormones such as insulin, estrogen, and progesterone were not significantly regulated by obesity. Plasma IL-6 was also significantly elevated by obesity in parous mice. We have previously reported a potential role for stromal-derived hepatocyte growth factor (HGF via its cognate receptor c-Met in the etiology of obesity-induced BBC tumor onset and in both human and murine primary coculture models of BBC-aggressiveness. Obesity-associated c-Met concentrations were 2.5-fold greater in normal mammary glands of parous mice. Taken together, our studies demonstrate that, parity in C3(1-TAg mice dramatically reduced BBC latency compared to nulliparous mice. In parous mice, c-Met is regulated by obesity in unaffected mammary gland and is associated with tumor onset. C3(1-TAg mice recapitulate epidemiologic findings such that parity drives increased BBC risk and

  16. Leflunomide or A77 1726 protect from acetaminophen-induced cell injury through inhibition of JNK-mediated mitochondrial permeability transition in immortalized human hepatocytes

    International Nuclear Information System (INIS)

    Leflunomide, a disease-modifying anti-rheumatic drug, protects against T-cell-mediated liver injury by poorly understood mechanisms. The active metabolite of leflunomide, A77 1726 (teriflunomide) has been shown to inhibit stress-activated protein kinases (JNK pathway), which are key regulators of mitochondria-mediated cell death. Therefore, we hypothesized that leflunomide may protect from drugs that induce the mitochondrial permeability transition (mPT) by blocking the JNK signaling pathway. To this end, we exposed cultured immortalized human hepatocytes (HC-04) to the standard protoxicant drug acetaminophen (APAP), which induces CsA-sensitive mPT-mediated cell death. We determined the effects of leflunomide on the extent of APAP-induced hepatocyte injury and the upstream JNK-mediated mitochondrial signaling pathways. We found that leflunomide or A77 1726 concentration-dependently protected hepatocytes from APAP (1 mM)-induced mitochondrial permeabilization and lethal cell injury. This was not due to proximal inhibition of CYP-catalyzed APAP bioactivation to its thiol-reactive metabolite. Instead, we demonstrate that leflunomide (20 μM) inhibited the APAP-induced early (3 h) activation (phosphorylation) of JNK1/2, thus inhibiting phosphorylation of the anti-apoptotic protein Bcl-2 and preventing P-Bcl-2-mediated induction of the mPT. This greatly attenuated mitochondrial cytochrome c release, which we used as a marker for mitochondrial permeabilization. The specific JNK2 inhibitor SP600125 similarly protected from APAP-induced cell death. In conclusion, these findings are consistent with our hypothesis that leflunomide protects from protoxicant-induced hepatocyte injury by inhibiting JNK signaling and preventing mPT induction

  17. Hepatocyte Growth Factor from a Clinical Perspective: A Pancreatic Cancer Challenge

    Energy Technology Data Exchange (ETDEWEB)

    Rizwani, Wasia [Department of Biochemistry, Osmania University, Hyderabad, Telangana 500007 (India); Allen, Amanda E.; Trevino, Jose G., E-mail: Jose.Trevino@surgery.ufl.edu [Department of Surgery, University of Florida, 1600 SW Archer Rd, Rm 6175, P.O. Box 100109, Gainesville, FL 32610 (United States)

    2015-09-03

    Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States and incidence rates are rising. Both detection and treatment options for pancreatic cancer are limited, providing a less than 5% five-year survival advantage. The need for new biomarkers for early detection and treatment of pancreatic cancer demands the efficient translation of bench knowledge to provide clinical benefit. One source of therapeutic resistance is the pancreatic tumor microenvironment, which is characterized by desmoplasia and hypoxia making it less conducive to current therapies. A major factor regulating desmoplasia and subsequently promoting chemoresistance in pancreatic cancer is hepatocyte growth factor (HGF), the sole ligand for c-MET (mesenchymal-epithelial transition), an epithelial tyrosine kinase receptor. Binding of HGF to c-MET leads to receptor dimerization and autophosphorylation resulting in the activation of multiple cellular processes that support cancer progression. Inhibiting activation of c-MET in cancer cells, in combination with other approaches for reducing desmoplasia in the tumor microenvironment, might significantly improve the success of chemotherapy. Therefore, HGF makes a potent novel target for developing therapeutic strategies in combination with existing drugs for treating pancreatic adenocarcinoma. This review provides a comprehensive analysis of HGF and its promising potential as a chemotherapeutic target for pancreatic cancer.

  18. Hepatocyte Growth Factor from a Clinical Perspective: A Pancreatic Cancer Challenge

    International Nuclear Information System (INIS)

    Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States and incidence rates are rising. Both detection and treatment options for pancreatic cancer are limited, providing a less than 5% five-year survival advantage. The need for new biomarkers for early detection and treatment of pancreatic cancer demands the efficient translation of bench knowledge to provide clinical benefit. One source of therapeutic resistance is the pancreatic tumor microenvironment, which is characterized by desmoplasia and hypoxia making it less conducive to current therapies. A major factor regulating desmoplasia and subsequently promoting chemoresistance in pancreatic cancer is hepatocyte growth factor (HGF), the sole ligand for c-MET (mesenchymal-epithelial transition), an epithelial tyrosine kinase receptor. Binding of HGF to c-MET leads to receptor dimerization and autophosphorylation resulting in the activation of multiple cellular processes that support cancer progression. Inhibiting activation of c-MET in cancer cells, in combination with other approaches for reducing desmoplasia in the tumor microenvironment, might significantly improve the success of chemotherapy. Therefore, HGF makes a potent novel target for developing therapeutic strategies in combination with existing drugs for treating pancreatic adenocarcinoma. This review provides a comprehensive analysis of HGF and its promising potential as a chemotherapeutic target for pancreatic cancer

  19. Sequence-defined cMET/HGFR-targeted Polymers as Gene Delivery Vehicles for the Theranostic Sodium Iodide Symporter (NIS) Gene.

    Science.gov (United States)

    Urnauer, Sarah; Morys, Stephan; Krhac Levacic, Ana; Müller, Andrea M; Schug, Christina; Schmohl, Kathrin A; Schwenk, Nathalie; Zach, Christian; Carlsen, Janette; Bartenstein, Peter; Wagner, Ernst; Spitzweg, Christine

    2016-08-01

    The sodium iodide symporter (NIS) as well-characterized theranostic gene represents an outstanding tool to target different cancer types allowing noninvasive imaging of functional NIS expression and therapeutic radioiodide application. Based on its overexpression on the surface of most cancer types, the cMET/hepatocyte growth factor receptor serves as ideal target for tumor-selective gene delivery. Sequence-defined polymers as nonviral gene delivery vehicles comprising polyethylene glycol (PEG) and cationic (oligoethanoamino) amide cores coupled with a cMET-binding peptide (cMBP2) were complexed with NIS-DNA and tested for receptor-specificity, transduction efficiency, and therapeutic efficacy in hepatocellular cancer cells HuH7. In vitro iodide uptake studies demonstrated high transduction efficiency and cMET-specificity of NIS-encoding polyplexes (cMBP2-PEG-Stp/NIS) compared to polyplexes without targeting ligand (Ala-PEG-Stp/NIS) and without coding DNA (cMBP2-PEG-Stp/Antisense-NIS). Tumor recruitment and vector biodistribution were investigated in vivo in a subcutaneous xenograft mouse model showing high tumor-selective iodide accumulation in cMBP2-PEG-Stp/NIS-treated mice (6.6 ± 1.6% ID/g (123)I, biological half-life 3 hours) by (123)I-scintigraphy. Therapy studies with three cycles of polyplexes and (131)I application resulted in significant delay in tumor growth and prolonged survival. These data demonstrate the enormous potential of cMET-targeted sequence-defined polymers combined with the unique theranostic function of NIS allowing for optimized transfection efficiency while eliminating toxicity.

  20. Selective inhibition of liver X receptor α-mediated lipogenesis in primary hepatocytes by licochalcone A

    OpenAIRE

    Oh, Gyun-Sik; Lee, Gang Gu; Yoon, Jin; Oh, Won Keun; Kim, Seung-Whan

    2015-01-01

    Background Sterol regulatory element binding protein-1c (SREBP-1c) is a regulator of the lipogenic pathway and is transcriptionally activated by liver X receptor α (LXRα). This study aims to investigate phytochemicals inhibiting the autonomous transactivity of LXRα with potentials as SREBP-1c inhibitors. Licochalcone A (LicA) is a flavonoid isolated from licorice root of Glycyrrhiza plant. Methods The effects of 238 natural chemicals on autonomous transactivity of LXRα were determined by the ...

  1. Design, synthesis and biological evaluation of novel 4-phenoxy-6,7-disubstituted quinolines possessing (thio)semicarbazones as c-Met kinase inhibitors.

    Science.gov (United States)

    Zhai, Xin; Bao, Guanglong; Wang, Limei; Cheng, Mingke; Zhao, Meng; Zhao, Sijia; Zhou, Hongyang; Gong, Ping

    2016-03-15

    In continuing our efforts to identify small molecules able to inhibit c-Met kinase, three series of novel 6,7-disubstituted-4-phenoxyquinoline derivatives (23a-w, 26a-d and 30a-d) bearing (thio)semicarbazone scaffold were designed, synthesized and evaluated for their cytotoxicity. The biological data revealed that most compounds exhibited moderate-to-excellent activity against HT-29, MKN-45, A549 cancer cell lines and relative poor potency toward MDA-MB-231 cell as well as hardly any cytotoxicity in normal PBL cell. Eleven compounds were further examined for their inhibitory activity against c-Met kinase and three compounds (23h, 23n and 26a) demonstrated good inhibitory activity. This work resulted in the discovery of a potent c-Met inhibitor 23n, bearing 2-hydroxy-3-allylphenyl group at R(2) moiety, as a valuable lead molecule, which possessed remarkable cytotoxicity and high selectivity against A549 and HT-29 cell lines with IC50 values of 11 nM and 27 nM. Besides, it displayed excellent c-Met kinase inhibition on a single-digital nanomolar level (IC50=1.54 nM). Meanwhile, the results from preliminarily in vivo study reflected that compound 23n showed promising overall PK profiles, consistent with the efficacy in both MKN-45 and HT-29 tumor xenograft mice model. These results clearly indicated that compound 23n is a potent and highly selective c-Met inhibitor and its favorable in vitro and in vivo profiles warrant further investigation. PMID:26897090

  2. Sulforaphane- and phenethyl isothiocyanate-induced inhibition of aflatoxin B1-mediated genotoxicity in human hepatocytes: role of GSTM1 genotype and CYP3A4 gene expression.

    Science.gov (United States)

    Gross-Steinmeyer, Kerstin; Stapleton, Patricia L; Tracy, Julia H; Bammler, Theo K; Strom, Stephen C; Eaton, David L

    2010-08-01

    Primary cultures of human hepatocytes were used to investigate whether the dietary isothiocyanates, sulforaphane (SFN), and phenethyl isothiocyanate (PEITC) can reduce DNA adduct formation of the hepatocarcinogen aflatoxin B(1) (AFB). Following 48 h of pretreatment, 10 and 50 microM SFN greatly decreased AFB-DNA adduct levels, whereas 25muM PEITC decreased AFB-DNA adducts in some but not all hepatocyte preparations. Microarray and quantitative reverse transcriptase (RT)-PCR analyses of gene expression in SFN and PEITC-treated hepatocytes demonstrated that SFN greatly decreased cytochrome P450 (CYP) 3A4 mRNA but did not induce the expression of either glutathione S-transferase (GST) M1 or GSTT1. The protective effects of SFN required pretreatment; cotreatment of hepatocytes with SFN and AFB in the absence of pretreatment had no effect on AFB-DNA adduct formation. When AFB-DNA adduct formation was evaluated by GST genotype, the presence of one or two functional alleles of GSTM1 was associated with a 75% reduction in AFB-DNA adducts, compared with GSTM1 null. In conclusion, these results demonstrate that the inhibition of AFB-DNA adduct formation by SFN is dependent on changes in gene expression rather than direct inhibition of catalytic activity. Transcriptional repression of genes involved in AFB bioactivation (CYP3A4 and CYP1A2), but not transcriptional activation of GSTs, may be responsible for the protective effects of SFN. Although GSTM1 expression was not induced by SFN, the presence of a functional GSTM1 allele can afford substantial protection against AFB-DNA damage in human liver. The downregulation of CYP3A4 by SFN may have important implications for drug interactions. PMID:20442190

  3. Novel germline c-MET mutation in a family with hereditary papillary renal carcinoma

    DEFF Research Database (Denmark)

    Wadt, Karin; Gerdes, Anne-Marie; Hansen, Thomas V O;

    2012-01-01

    Hereditary papillary renal carcinoma (HPRC) is a highly penetrant hereditary renal cancer syndrome caused by germline missense mutations in the c-MET proto-oncogene. HPRC is clinically characterized by multiple bilateral papillary renal-cell carcinomas. Here we report a family with a novel missense...

  4. Cooperation between c-Met and focal adhesion kinase family members in medulloblastoma and implications for therapy

    OpenAIRE

    Guessous, Fadila; Yang, Yanzhi; Johnson, Elizabeth; Marcinkiewicz, Lukasz; Smith, Matthew; Zhang, Ying; Kofman, Alexander; Schiff, David; Christensen, James; Abounader, Roger

    2011-01-01

    We previously demonstrated the involvement of the tyrosine kinase receptor c-Met in medulloblastoma malignancy. The nonreceptor tyrosine kinases FAK and Pyk2, are key players in the progression of different cancers. However, their role in medulloblastoma malignancy is not well understood. In this study, using a protein array approach, we found that c-Met phosphorylates FAK and Pyk2 in medulloblastoma cells. We therefore studied the interactions between c-Met and FAK/Pyk2 and their implication...

  5. Cigarette smoking induces overexpression of c-Met receptor in microvessels of oral lichen planus

    OpenAIRE

    Kłosek, Sebastian K.; Sporny, Stanisław; Stasikowska-Kanicka, Olga; Kurnatowska, Anna J.

    2011-01-01

    Introduction Cigarette smoking is related to many pathological conditions; however, chemical substances affect the oral cavity first, so it is important to consider its influence on oral mucosa and oral potentially pre-malignant lesions. The aim of this study was to investigate the effect of smoking on microvessel density in oral lichen planus. Special emphasis was placed on examining the relationship between the expression of c-Met receptor in blood vessels and smoking habits. Material and m...

  6. Inhibition of preS1-hepatocyte interaction by an array of recombinant human antibodies from naturally recovered individuals

    DEFF Research Database (Denmark)

    Sankhyan, Anurag; Sharma, Chandresh; Dutta, Durgashree;

    2016-01-01

    binding signature, interacting with different amino acids within the preS1-peptide region. Ability to prevent binding of the preS1 protein (N-terminus 60a.a.) to HepG2 cells stably expressing hNTCP (HepG2-hNTCP-C4 cells), the HBV receptor on human hepatocytes was taken as a surrogate marker...

  7. The mutated human gene encoding hepatocyte nuclear factor 1β inhibits kidney formation in developing Xenopus embryos

    OpenAIRE

    Wild, Wiltrud; Pogge von Strandmann, Elke; Nastos, Aristotelis; Senkel, Sabine; Lingott-Frieg, Anja; Bulman, Michael; Bingham, Coralie; Ellard, Sian; Hattersley, Andrew T; Ryffel, Gerhart U.

    2000-01-01

    The transcription factor hepatocyte nuclear factor 1β (HNF1β) is a tissue-specific regulator that also plays an essential role in early development of vertebrates. In humans, four heterozygous mutations in the HNF1β gene have been identified that lead to early onset of diabetes and severe primary renal defects. The degree and type of renal defects seem to depend on the specific mutation. We show that the frameshift mutant P328L329fsdelCCTCT associated with nephron agenesis retains its DNA-bin...

  8. Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition

    Directory of Open Access Journals (Sweden)

    Laura Ordovás

    2015-11-01

    Full Text Available Tools for rapid and efficient transgenesis in “safe harbor” loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs. We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes.

  9. Expressions of estrogen receptor subtypes and c-met proto-oncogene in endometrial carcinoma and their correlation

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Objective To investigate the expressions of estrogen receptor(ER)subtypes and c-met proto-oncogene in human endometrial carcinomas and to assess the clinical significance of ER and c-met in this carcinoma.Methods Reverse transcription PCR(RT-PCR)was used to detect the expressions of ERα,ERβ and c-met proto-oncogene mRNA in 30 samples of endometrial carcinoma and 11 samples of normal endometrium.Results The expression of ERα in endometrial carcinoma(0.70±0.40)was significantly reduced in comparison to that i...

  10. Low c-Met expression levels are prognostic for and predict the benefits of temozolomide chemotherapy in malignant gliomas

    OpenAIRE

    Ming-Yang Li; Pei Yang; Yan-Wei Liu; Chuan-Bao Zhang; Kuan-Yu Wang; Yin-Yan Wang; Kun Yao; Wei Zhang; Xiao-Guang Qiu; Wen-Bin Li; Xiao-Xia Peng; Yong-Zhi Wang; Tao Jiang

    2016-01-01

    Aberrant c-Met has been implicated in the development of many cancers. The objective of this study was to identify an unfavorable prognostic marker that might guide decisions regarding clinical treatment strategies for high-grade gliomas. C-Met expression was measured using immunohistochemistry in 783 gliomas, and we further analyzed c-Met mRNA levels using the Agilent Whole Genome mRNA Microarray in 286 frozen samples. In vitro, we performed cell migration and invasion assays. Cell sensitivi...

  11. Inhibition of fatty acid synthesis in rat hepatocytes by exogenous polyunsaturated fatty acids is caused by lipid peroxidation

    DEFF Research Database (Denmark)

    Mikkelsen, L.; Hansen, Harald S.; Grunnet, N.;

    1993-01-01

    by the peroxidized PUFA. Arachidonic acid and eicosapentaenoic acid showed a dose- and time-dependent cytotoxicity. Two other antioxidants: 50 µM a-tocopherol acid succinate and 1 µM N,N'-diphenyl-1,4-phenylenediamine, both proved more efficient than a-tocopherol phosphate. There was a significant correlation......Rat hepatocyte long-term cultures were utilized to investigate the impact of different polyunsaturated fatty acids (PUFA) on the insulin-induced de novo fatty acid synthesis in vitro. The addition of 0.5 mM albumin-complexed oleic, linoleic, columbinic, arachidonic, eicosapentaenoic...... or docosahexaenoic acid resulted in a marked suppression of fatty acid synthesis. By evaluation of cell viability (determined as the leakage of lactate dehydrogenase (LDH)) it turned our, that the antioxidant used (50 µM a-tocopherol phosphate) had a low antioxidant activity, resulting in cytotoxic effects...

  12. Research Progress of HGF/c-MET Inhibitor in the Treatment 
of Non-small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Tao JIANG

    2015-04-01

    Full Text Available Molecular targeted therapy has become more and more important in the treatment of non-small cell lung cancer (NSCLC. HGF/c-MET plays the pivotal role in the growth, development and tolerance to epidermal growth factor receptor tyrosine kinase inhibitor of NSCLC. Moreover it has become another heat point in the molecular targeted therapy of NSCLC. c-MET amplification or high expression was deemed to another significant gene modification beyond EGFR and ALK. In the preclinical studies, HGF/c-MET inhibitors have showed the promising anti-tumor effect. Recently, some phase II/III clinical trials have proved that these inhibitors could improve the survival of patients with NSCLC. Hence we performed this review to elaborate the research progress of c-MET inhibitor in the treatment of NSCLC.

  13. Low c-Met expression levels are prognostic for and predict the benefits of temozolomide chemotherapy in malignant gliomas.

    Science.gov (United States)

    Li, Ming-Yang; Yang, Pei; Liu, Yan-Wei; Zhang, Chuan-Bao; Wang, Kuan-Yu; Wang, Yin-Yan; Yao, Kun; Zhang, Wei; Qiu, Xiao-Guang; Li, Wen-Bin; Peng, Xiao-Xia; Wang, Yong-Zhi; Jiang, Tao

    2016-01-01

    Aberrant c-Met has been implicated in the development of many cancers. The objective of this study was to identify an unfavorable prognostic marker that might guide decisions regarding clinical treatment strategies for high-grade gliomas. C-Met expression was measured using immunohistochemistry in 783 gliomas, and we further analyzed c-Met mRNA levels using the Agilent Whole Genome mRNA Microarray in 286 frozen samples. In vitro, we performed cell migration and invasion assays. Cell sensitivity to temozolomide (TMZ) chemotherapy was determined using MTT assays. Both mRNA and protein levels of c-Met were significantly associated with tumor grade progression and inversely correlated with overall and progression-free survival in high-grade gliomas (all P maker for clinical decision making. PMID:26879272

  14. Expression and Significance of CD44, CD47 and c-met in Ovarian Clear Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Huimin Wang

    2015-02-01

    Full Text Available Aims: The aim of the present study is to investigate the differential expression of CD44, CD47 and c-met in ovarian clear cell carcinoma (OCCC, the correlation in their expression and their relationship with the biological behavior of OCCC. Methods: We used immunohistochemistry to examine the expression of CD44, CD47 and c-met in OCCC (86 cases and investigated the effects of the expression and interaction of these molecules on the development of OCCC. Results: CD44, CD47 and c-met expression was significantly high in OCCC. Expression of CD44 and CD47 correlated with patient surgical stage, chemotherapy resistance and prognosis (all p < 0.05, and expression of c-met correlated with chemotherapy resistance and prognosis (all p < 0.05, but did not correlate with lymph node metastasis (all p > 0.05. The surgical stage, CD44, CD47 and c-met expression were independent risk factors for OCCC prognosis (all p < 0.05. Patients with low levels of CD44, CD47 and c-met showed better survival than those with high levels (all p < 0.05. There was a positive correlation between CD44 (or CD47 and c-met, as well as between CD44 and CD47 (the Spearman correlation coefficient rs was 0.783, 0.776 and 0.835, respectively, all p < 0.01. Additionally, pairwise correlation analysis of these three markers shows that the high expression of CD44/CD47, CD44/c-met and CD47/c-met were correlated with patient surgical stage, chemotherapy resistance and prognosis (all p < 0.05, but did not correlate with lymph node metastasis (all p > 0.05. Conclusions: Expression of CD44, CD47 and c-met was upregulated in OCCC and pairwise correlation. CD44, CD47 and c-met may have synergistic effects on the development of OCCC and are prognostic factors for ovarian cancer.

  15. Hepatocyte and keratinocyte growth factors and their receptors in human lung emphysema

    Directory of Open Access Journals (Sweden)

    Marchal Joëlle

    2005-10-01

    Full Text Available Abstract Background Hepatocyte and keratinocyte growth factors are key growth factors in the process of alveolar repair. We hypothesized that excessive alveolar destruction observed in lung emphysema involves impaired expression of hepatocyte and keratinocyte growth factors or their respective receptors, c-met and keratinocyte growth factor receptor. The aim of our study was to compare the expression of hepatocyte and keratinocyte growth factors and their receptors in lung samples from 3 groups of patients: emphysema; smokers without emphysema and non-smokers without emphysema. Methods Hepatocyte and keratinocyte growth factor proteins were analysed by immunoassay and western blot; mRNA expression was measured by real time quantitative polymerase chain reaction. Results Hepatocyte and keratinocyte growth factors, c-met and keratinocyte growth factor receptor mRNA levels were similar in emphysema and non-emphysema patients. Hepatocyte growth factor mRNA correlated negatively with FEV1 and the FEV1/FVC ratio both in emphysema patients and in smokers with or without emphysema. Hepatocyte and keratinocyte growth factor protein concentrations were similar in all patients' groups. Conclusion The expression of hepatocyte and keratinocyte growth factors and their receptors is preserved in patients with lung emphysema as compared to patients without emphysema. Hepatocyte growth factor mRNA correlates with the severity of airflow obstruction in smokers.

  16. Fullerene inhibits benzo(a)pyrene Efflux from Cyprinus carpio hepatocytes by affecting cell membrane fluidity and P-glycoprotein expression.

    Science.gov (United States)

    Chen, Qiqing; Hu, Xialin; Wang, Rui; Yuan, Jin; Yin, Daqiang

    2016-05-01

    P-Glycoprotein (P-gp) can protect cells by pumping out toxic compounds, and has been found widely expressed in fish tissues. Here, we illustrate the P-gp efflux ability for benzo(a)pyrene (BaP) in the hepatocytes of common carp (Cyprinus carpio) after exposing to fullerene aqueous suspension (nC60). The results revealed that nC60 increased the membrane fluidity by decreasing the ratio of saturated to unsaturated fatty acids, and increased the cholesterol contents. These findings, combined with 10-38% and 70-75% down-regulation of P-gp mRNA and protein respectively, suggested that nC60 caused inhibition on P-gp efflux transport system. Therefore, we further investigated the cellular efflux ability for BaP. Results showed unequivocally that nC60 is a potent P-gp inhibitor. The retaining BaP amounts after efflux were elevated by 1.7-2.8 fold during the 10 day exposure. Meanwhile, 5mg/L humic acid (one of the important fractions of natural organic matter, which is ubiquitous in aquatic environment) alleviated the nC60 damage to hepatocytes in terms of oxidative damage, cholesterol increment, and P-gp content reduction; and finally attenuated the suppressed P-gp efflux ability. Collectively, this study provides the first evidence of nC60 toxicity to P-gp functionality in fish and illustrates the possible mechanism of the suppressed P-gp efflux ability for BaP.

  17. Molecular modeling studies and synthesis of novel quinoxaline derivatives with potential anticancer activity as inhibitors of c-Met kinase.

    Science.gov (United States)

    Abbas, Hebat-Allah S; Al-Marhabi, Aisha R; Eissa, Sally I; Ammar, Yousry A

    2015-10-15

    In an effort to develop potent anti-cancer agents, we have synthesized some substituted quinoxaline derivatives. Reaction of 6-bromo-3-methylquinoxalin-2(1H)-one 1 with aromatic aldehydes furnished the styryl derivatives 2a-e. Alkylation of 1 with ethyl chloroacetate produced the N-alkyl derivatives 3. Hydrazinolysis of the ester derivative 3 with hydrazine hydrate afforded the hydrazide derivative 4. In addition, chlorination of 1 with phosphorus oxychloride afforded the 2-chloro derivative 5 which was used as a key intermediate for the synthesis of substituted quinoxaline derivatives 6-8, N-pyrazole derivative 9, tetrazolo[1,5-a]quinoxaline derivative 10 and Schiff base derivatives 13, 15 via reaction with several nucleophiles reagents. Docking methodologies were used to predict their binding conformation to explain the differences of their tested biological activities. All the tested compounds were screened in vitro for their cytotoxic effect on three tumor cell lines. Some new quinoxaline derivatives were studied as inhibitors of c-Met kinase, a receptor associated with high tumor grade and poor prognosis in a number of human cancers. Compounds 2e, 4, 7a, 12a, 12b and 13 showed the highest binding affinity with CDOCKER energy score, while showed the lowest IC50 values against three types of cancer cell lines. It is worth to mention that, compounds 2e, 7a, 12b and 13 showed comparable inhibition activity to the reference drug, while compounds 4 and 12a showed a more potent inhibition activity than Doxorubicin.

  18. Convergent RANK- and c-Met-mediated signaling components predict survival of patients with prostate cancer: an interracial comparative study.

    Directory of Open Access Journals (Sweden)

    Peizhen Hu

    Full Text Available We reported (PLoS One 6 (12:e28670, 2011 that the activation of c-Met signaling in RANKL-overexpressing bone metastatic LNCaP cell and xenograft models increased expression of RANK, RANKL, c-Met, and phosphorylated c-Met, and mediated downstream signaling. We confirmed the significance of the RANK-mediated signaling network in castration resistant clinical human prostate cancer (PC tissues. In this report, we used a multispectral quantum dot labeling technique to label six RANK and c-Met convergent signaling pathway mediators simultaneously in formalin fixed paraffin embedded (FFPE tissue specimens, quantify the intensity of each expression at the sub-cellular level, and investigated their potential utility as predictors of patient survival in Caucasian-American, African-American and Chinese men. We found that RANKL and neuropilin-1 (NRP-1 expression predicts survival of Caucasian-Americans with PC. A Gleason score ≥ 8 combined with nuclear p-c-Met expression predicts survival in African-American PC patients. Neuropilin-1, p-NF-κB p65 and VEGF are predictors for the overall survival of Chinese men with PC. These results collectively support interracial differences in cell signaling networks that can predict the survival of PC patients.

  19. Activation of p21CIP1/WAF1 gene expression and inhibition of cell proliferation by overexpression of hepatocyte nuclear factor-4α

    International Nuclear Information System (INIS)

    The F9 murine embryonal carcinoma cell line provides an attractive system for studying epithelial differentiation and antiproliferative processes. We have recently established F9 cells expressing doxycycline-inducible hepatocyte nuclear factor (HNF)-4α and shown that HNF-4α triggers the gene expression of tight-junction molecules, occludin, claudin-6, and claudin-7, as well as formation of functional tight junctions and polarized epithelial morphology (Exp. Cell Res. 286, [2003] 288). Since these events were very similar to those induced by retinoids, we investigated whether HNF-4α, like retinoid receptors, was involved in the control of cell proliferation. We herein show that HNF-4α up-regulates expression of the p21 gene, but not the p15, p16, p18, p19, or p27 gene, in a p53-independent manner, and inhibits cell growth in F9 cells. Similar results were observed in rat lung endothelial cells, in which expression of HNF-4α is conditionally induced by doxycycline. Furthermore, we demonstrate, by reporter assay, that HNF-4α significantly elevates the transcriptional activity of the p21 promoter. Since, HNF-4α is expressed not only in the liver but also in organs containing epithelial cells, such as kidney, intestine, pancreas, and stomach, it might also play critical roles in the regulation of epithelial morphogenesis and proliferation in these organs

  20. Multiplexed quantum dot labeling of activated c-Met signaling in castration-resistant human prostate cancer.

    Directory of Open Access Journals (Sweden)

    Peizhen Hu

    Full Text Available The potential application of multiplexed quantum dot labeling (MQDL for cancer detection and prognosis and monitoring therapeutic responses has attracted the interests of bioengineers, pathologists and cancer biologists. Many published studies claim that MQDL is effective for cancer biomarker detection and useful in cancer diagnosis and prognosis, these studies have not been standardized against quantitative biochemical and molecular determinations. In the present study, we used a molecularly characterized human prostate cancer cell model exhibiting activated c-Met signaling with epithelial to mesenchymal transition (EMT and lethal metastatic progression to bone and soft tissues as the gold standard, and compared the c-Met cell signaling network in this model, in clinical human prostate cancer tissue specimens and in a castration-resistant human prostate cancer xenograft model. We observed c-Met signaling network activation, manifested by increased phosphorylated c-Met in all three. The downstream survival signaling network was mediated by NF-κB and Mcl-1 and EMT was driven by receptor activator of NF-κB ligand (RANKL, at the single cell level in clinical prostate cancer specimens and the xenograft model. Results were confirmed by real-time RT-PCR and western blots in a human prostate cancer cell model. MQDL is a powerful tool for assessing biomarker expression and it offers molecular insights into cancer progression at both the cell and tissue level with high degree of sensitivity.

  1. Multiplexed quantum dot labeling of activated c-Met signaling in castration-resistant human prostate cancer.

    Science.gov (United States)

    Hu, Peizhen; Chu, Gina C-Y; Zhu, Guodong; Yang, Hua; Luthringer, Daniel; Prins, Gail; Habib, Fouad; Wang, Yuzhuo; Wang, Ruoxiang; Chung, Leland W K; Zhau, Haiyen E

    2011-01-01

    The potential application of multiplexed quantum dot labeling (MQDL) for cancer detection and prognosis and monitoring therapeutic responses has attracted the interests of bioengineers, pathologists and cancer biologists. Many published studies claim that MQDL is effective for cancer biomarker detection and useful in cancer diagnosis and prognosis, these studies have not been standardized against quantitative biochemical and molecular determinations. In the present study, we used a molecularly characterized human prostate cancer cell model exhibiting activated c-Met signaling with epithelial to mesenchymal transition (EMT) and lethal metastatic progression to bone and soft tissues as the gold standard, and compared the c-Met cell signaling network in this model, in clinical human prostate cancer tissue specimens and in a castration-resistant human prostate cancer xenograft model. We observed c-Met signaling network activation, manifested by increased phosphorylated c-Met in all three. The downstream survival signaling network was mediated by NF-κB and Mcl-1 and EMT was driven by receptor activator of NF-κB ligand (RANKL), at the single cell level in clinical prostate cancer specimens and the xenograft model. Results were confirmed by real-time RT-PCR and western blots in a human prostate cancer cell model. MQDL is a powerful tool for assessing biomarker expression and it offers molecular insights into cancer progression at both the cell and tissue level with high degree of sensitivity. PMID:22205960

  2. Beta 1-integrin–c-Met cooperation reveals an inside-in survival signalling on autophagy-related endomembranes

    Science.gov (United States)

    Barrow-McGee, Rachel; Kishi, Naoki; Joffre, Carine; Ménard, Ludovic; Hervieu, Alexia; Bakhouche, Bakhouche A.; Noval, Alejandro J.; Mai, Anja; Guzmán, Camilo; Robert-Masson, Luisa; Iturrioz, Xavier; Hulit, James; Brennan, Caroline H.; Hart, Ian R.; Parker, Peter J.; Ivaska, Johanna; Kermorgant, Stéphanie

    2016-01-01

    Receptor tyrosine kinases (RTKs) and integrins cooperate to stimulate cell migration and tumour metastasis. Here we report that an integrin influences signalling of an RTK, c-Met, from inside the cell, to promote anchorage-independent cell survival. Thus, c-Met and β1-integrin co-internalize and become progressively recruited on LC3B-positive ‘autophagy-related endomembranes' (ARE). In cells growing in suspension, β1-integrin promotes sustained c-Met-dependent ERK1/2 phosphorylation on ARE. This signalling is dependent on ATG5 and Beclin1 but not on ATG13, suggesting ARE belong to a non-canonical autophagy pathway. This β1-integrin-dependent c-Met-sustained signalling on ARE supports anchorage-independent cell survival and growth, tumorigenesis, invasion and lung colonization in vivo. RTK–integrin cooperation has been assumed to occur at the plasma membrane requiring integrin ‘inside-out' or ‘outside-in' signalling. Our results report a novel mode of integrin–RTK cooperation, which we term ‘inside-in signalling'. Targeting integrin signalling in addition to adhesion may have relevance for cancer therapy. PMID:27336951

  3. Comparative PET/CT study with 11C-MET and 18F-FDG for diagnosing Glioma

    International Nuclear Information System (INIS)

    In this paper, we investigate the diagnostic value of 11C-methionine (MET) positron emission tomography/computed tomography (PET/CT) for brain gliomas, and compare the results to 18F-fluorodeoxyglucose. Forty-four patients with suspected gliomas were examined with 11C-MET and 18F-FDG PET/CT. 18F-FDG and 11C-MET PET/CT images were compared and evaluated by visual and semiquantitative analysis. The accuracy of 11C-MET and 18F-FDG PET/CT for detecting gliomas were 88.6% and 65.9%, respectively. Semiquantitative analysis showed that the 26 gliomas had higher mean ± SD T/NGmax ratio on 11C-MET PET/CT than on 18F-FDG PET/CT(1.95±0.52 vs. 0.90±0.27, t=9.101, P11C-MET had a higher sensitivity than 18F-FDG (83.3% vs.33.3%, χ2 =4.16, P18F-FDG in the sensitivity for high-grade gliomas(100% vs. 64.3%, χ2=3.20, P>0.05). The difference was no significant, too, between high-and low-grade gliomas, compared by 11C-MET T/NGmax ratio (2.07±0.51 vs. 1.81±0.52, t=1.302, P=0.205). 18F-FDG T/NGmax ratio in high-grade gliomas was significantly higher than that in low-grade gliomas (1.03±0.30 vs. 0.75±0.11, t=3.198, P=0.004). It is concluded that 11C-MET PET/CT is more accurate than 18F-FDG PET/CT for detecting and delineating gliomas, especially for low-grade gliomas, and it can play a complement role to 18F-FDG in tumor grading. (authors)

  4. Activation of RAS/ERK alone is insufficient to inhibit RXRα function and deplete retinoic acid in hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Ai-Guo, E-mail: wangaiguotl@hotmail.com; Song, Ya-Nan; Chen, Jun; Li, Hui-Ling; Dong, Jian-Yi; Cui, Hai-Peng; Yao, Liang; Li, Xue-Feng; Gao, Wen-Ting; Qiu, Ze-Wen; Wang, Fu-Jin; Wang, Jing-Yu, E-mail: wangjingyus@163.com

    2014-09-26

    Highlights: • The activation of RAS/ERK is insufficient to inhibit RXRα function and deplete RA. • The retinoid metabolism-related genes are down-regulated by ras oncogene. • The atRA has no effect on preventing hepatic tumorigenesis or curing the developed hepatic nodules. - Abstract: Activation of RAS/ERK signaling pathway, depletion of retinoid, and phosphorylation of retinoid X receptor alpha (RXRα) are frequent events found in liver tumors and thought to play important roles in hepatic tumorigenesis. However, the relationships among them still remained to be elucidated. By exploring the transgenic mouse model of hepatic tumorigenesis induced by liver-specific expression of H-ras12V oncogene, the activation of RAS/ERK, the mRNA expression levels of retinoid metabolism-related genes, the contents of retinoid metabolites, and phosphorylation of RXRα were determined. RAS/ERK signaling pathway was gradually and significantly activated in hepatic tumor adjacent normal liver tissues (P) and hepatic tumor tissues (T) of H-ras12V transgenic mice compared with normal liver tissues (Wt) of wild type mice. On the contrary, the mRNA expression levels of retinoid metabolism-related genes were significantly reduced in P and T compared with Wt. Interestingly, the retinoid metabolites 9-cis-retinoic acid (9cRA) and all-trans-retinoic acid (atRA), the well known ligands for nuclear transcription factor RXR and retinoic acid receptor (RAR), were significantly decreased only in T compared with Wt and P, although the oxidized polar metabolite of atRA, 4-keto-all-trans-retinoic-acid (4-keto-RA) was significantly decreased in both P and T compared with Wt. To our surprise, the functions of RXRα were significantly blocked only in T compared with Wt and P. Namely, the total protein levels of RXRα were significantly reduced and the phosphorylation levels of RXRα were significantly increased only in T compared with Wt and P. Treatment of H-ras12V transgenic mice at 5-week

  5. Ochratoxin A activates opposing c-MET/PI3K/Akt and MAPK/ERK 1-2 pathways in human proximal tubule HK-2 cells.

    Science.gov (United States)

    Özcan, Zeynep; Gül, Gizem; Yaman, Ibrahim

    2015-08-01

    Ochratoxin A (OTA) is a mycotoxin produced as a secondary metabolite by filamentous fungi, such as Aspergillus and Penicillium. Because OTA is a common contaminant of food and feeds, humans and animals are frequently exposed to OTA in daily life. It has been classified as a carcinogen in rodents and a possible carcinogen in humans. OTA has been shown to deregulate a variety of different signal transduction pathways in a cell type- and dosage-depending manner resulting in contrasting physiological effects, such as survival or cell death. While the ERK1-2 and JNK/SAPK MAPK pathways are major targets, knowledge about their role in OTA-mediated cell survival and death is fragmented. Similarly, the contribution of the PI3K/Akt pathway to the carcinogenic effect of OTA in proximal tubule cells has not been elucidated in detail. In this study, we demonstrated that OTA induced sustained activation of the PI3K/Akt and MEK/ERK1-2 signaling pathways in a dose- and time-dependent manner in HK-2 cells. Chemical inhibition of ERK1-2 activation or overexpression of dominant-negative and kinase-dead MEK1 leads to increased cell viability and decreased apoptosis in OTA-treated cells. Blockage of PI3K/Akt with Wortmannin aggravated the negative effect of OTA on cell viability and increased the levels of apoptosis. Moreover, we identified the c-MET proto-oncogene as an upstream receptor tyrosine kinase responsible for OTA-induced activation of PI3K/Akt signaling in HK-2 cells. Our data suggest that OTA may potentiate carcinogenesis by sustained activation of c-MET/PI3K/Akt signaling through suppression of apoptosis induced by MEK/ERK1-2 activation in damaged renal proximal tubule epithelial cells.

  6. Ochratoxin A activates opposing c-MET/PI3K/Akt and MAPK/ERK 1-2 pathways in human proximal tubule HK-2 cells.

    Science.gov (United States)

    Özcan, Zeynep; Gül, Gizem; Yaman, Ibrahim

    2015-08-01

    Ochratoxin A (OTA) is a mycotoxin produced as a secondary metabolite by filamentous fungi, such as Aspergillus and Penicillium. Because OTA is a common contaminant of food and feeds, humans and animals are frequently exposed to OTA in daily life. It has been classified as a carcinogen in rodents and a possible carcinogen in humans. OTA has been shown to deregulate a variety of different signal transduction pathways in a cell type- and dosage-depending manner resulting in contrasting physiological effects, such as survival or cell death. While the ERK1-2 and JNK/SAPK MAPK pathways are major targets, knowledge about their role in OTA-mediated cell survival and death is fragmented. Similarly, the contribution of the PI3K/Akt pathway to the carcinogenic effect of OTA in proximal tubule cells has not been elucidated in detail. In this study, we demonstrated that OTA induced sustained activation of the PI3K/Akt and MEK/ERK1-2 signaling pathways in a dose- and time-dependent manner in HK-2 cells. Chemical inhibition of ERK1-2 activation or overexpression of dominant-negative and kinase-dead MEK1 leads to increased cell viability and decreased apoptosis in OTA-treated cells. Blockage of PI3K/Akt with Wortmannin aggravated the negative effect of OTA on cell viability and increased the levels of apoptosis. Moreover, we identified the c-MET proto-oncogene as an upstream receptor tyrosine kinase responsible for OTA-induced activation of PI3K/Akt signaling in HK-2 cells. Our data suggest that OTA may potentiate carcinogenesis by sustained activation of c-MET/PI3K/Akt signaling through suppression of apoptosis induced by MEK/ERK1-2 activation in damaged renal proximal tubule epithelial cells. PMID:25002221

  7. From AR to c-Met: Androgen deprivation leads to a signaling pathway switch in prostate cancer cells

    OpenAIRE

    Liu, Tiancheng; Mendes, Desiree E.; Berkman, Clifford E.

    2013-01-01

    Elucidating the role of androgen deprivation in the transition from androgen-dependence to independence may enable the development of more specific therapeutic strategies against prostate cancer. Our previous in vitro model was employed to further assess the effects of continuous androgen-deprivation on prostate cancer cells (LNCaP) with respect to both androgen receptor (AR) and c-Met expression. The results indicated that long-term androgen deprivation resulted in a signaling pathway switch...

  8. c-Met Expression Is a Marker of Poor Prognosis in Patients With Locally Advanced Head and Neck Squamous Cell Carcinoma Treated With Chemoradiation

    Energy Technology Data Exchange (ETDEWEB)

    Baschnagel, Andrew M. [Department of Radiation Oncology, William Beaumont Hospital, Royal Oak, Michigan (United States); Williams, Lindsay [Department of Pathology, William Beaumont Hospital, Royal Oak, Michigan (United States); Hanna, Alaa; Chen, Peter Y.; Krauss, Daniel J. [Department of Radiation Oncology, William Beaumont Hospital, Royal Oak, Michigan (United States); Pruetz, Barbara L. [Beaumont BioBank, William Beaumont Hospital, Royal Oak, Michigan (United States); Akervall, Jan [Beaumont BioBank, William Beaumont Hospital, Royal Oak, Michigan (United States); Department of Otolaryngology, William Beaumont Hospital, Royal Oak, Michigan (United States); Wilson, George D., E-mail: George.Wilson@Beaumont.edu [Department of Radiation Oncology, William Beaumont Hospital, Royal Oak, Michigan (United States); Beaumont BioBank, William Beaumont Hospital, Royal Oak, Michigan (United States)

    2014-03-01

    Purpose: To examine the prognostic significance of c-Met expression in relation to p16 and epidermal growth factor receptor (EGFR) in patients with locally advanced head and neck squamous cell carcinoma (HNSCC) treated with definitive concurrent chemoradiation. Methods and Materials: Archival tissue from 107 HNSCC patients treated with chemoradiation was retrieved, and a tissue microarray was assembled. Immunohistochemical staining of c-Met, p16, and EGFR was performed. c-Met expression was correlated with p16, EGFR, clinical characteristics, and clinical endpoints including locoregional control (LRC), distant metastasis (DM), disease-free survival (DFS), and overall survival (OS). Results: Fifty-one percent of patients were positive for p16, and 53% were positive for EGFR. Both p16-negative (P≤.001) and EGFR-positive (P=.019) status predicted for worse DFS. Ninety-three percent of patients stained positive for c-Met. Patients were divided into low (0, 1, or 2+ intensity) or high (3+ intensity) c-Met expression. On univariate analysis, high c-Met expression predicted for worse LRC (hazard ratio [HR] 2.27; 95% CI, 1.08-4.77; P=.031), DM (HR 4.41; 95% CI, 1.56-12.45; P=.005), DFS (HR 3.00; 95% CI, 1.68-5.38; P<.001), and OS (HR 4.35; 95% CI, 2.13-8.88; P<.001). On multivariate analysis, after adjustment for site, T stage, smoking history, and EGFR status, only high c-Met expression (P=.011) and negative p16 status (P=.003) predicted for worse DFS. High c-Met expression was predictive of worse DFS in both EGFR-positive (P=.032) and -negative (P=.008) patients. In the p16-negative patients, those with high c-Met expression had worse DFS (P=.036) than did those with low c-Met expression. c-Met expression was not associated with any outcome in the p16-positive patients. Conclusions: c-Met is expressed in the majority of locally advanced HNSCC cases, and high c-Met expression predicts for worse clinical outcomes. High c-Met expression predicted for worse DFS in p16

  9. Synthesis and biological evaluation of 4-(2-fluorophenoxy)-3,3'-bipyridine derivatives as potential c-met inhibitors.

    Science.gov (United States)

    Zhao, Sijia; Zhang, Yu; Zhou, Hongyang; Xi, Shuancheng; Zou, Bin; Bao, Guanglong; Wang, Limei; Wang, Jiao; Zeng, Tianfang; Gong, Ping; Zhai, Xin

    2016-09-14

    Six series of novel 4-(2-fluorophenoxy)-3,3'-bipyridine derivatives conjugated with aza-aryl formamide/amine scaffords were designed and synthesized through a structure-based molecular hybridization approach. The target compounds were evaluated for c-Met kinase inhibitory activities and cytotoxicity against four cancer cell lines (HT-29, A549, MKN-45 and MDA-MB-231) in vitro. Most compounds exhibited moderate to excellent potency, and the most promising candidate 26c (c-Met kinase IC50 = 8.2 nM) showed a 4.7-fold increase in cytotoxicity against c-Met-addicted MKN-45 cell line in vitro (IC50 = 3 nM), superior to that of Foretinib (IC50 = 23 nM). The preliminary structure-activity relationship indicated that a 1H-benzo [e] [1,3,4]thiadiazine-3-carboxamide-4,4-dioxide moiety as linker contributed to the antitumor potency. PMID:27187857

  10. A comparison of consistency of detecting c-MET gene amplification in peripheral blood and tumor tissue of nonsmall cell lung cancer patients

    Directory of Open Access Journals (Sweden)

    Daobao Chen

    2015-01-01

    Conclusion: The consistency of the c-MET gene amplification in peripheral blood and tissue is high. c-MET gene amplification of peripheral blood could be used for clinical diagnosis and treatment in cases when tissue specimen is hard to get.

  11. Matriptase is required for the active form of hepatocyte growth factor induced Met, focal adhesion kinase and protein kinase B activation on neural stem/progenitor cell motility.

    Science.gov (United States)

    Fang, Jung-Da; Lee, Sheau-Ling

    2014-07-01

    Hepatocyte growth factor (HGF) is a chemoattractant and inducer for neural stem/progenitor (NS/P) cell migration. Although the type II transmembrane serine protease, matriptase (MTP) is an activator of the latent HGF, MTP is indispensable on NS/P cell motility induced by the active form of HGF. This suggests that MTP's action on NS/P cell motility involves mechanisms other than proteolytic activation of HGF. In the present study, we investigate the role of MTP in HGF-stimulated signaling events. Using specific inhibitors of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) or focal adhesion kinase (FAK), we demonstrated that in NS/P cells HGF-activated c-Met induces PI3k-Akt signaling which then leads to FAK activation. This signaling pathway ultimately induces MMP2 expression and NS/P cell motility. Knocking down of MTP in NS/P cells with specific siRNA impaired HGF-stimulation of c-Met, Akt and FAK activation, blocked HGF-induced production of MMP2 and inhibited HGF-stimulated NS/P cell motility. MTP-knockdown NS/P cells cultured in the presence of recombinant protein of MTP protease domain or transfected with the full-length wild-type but not the protease-defected MTP restored HGF-responsive events in NS/P cells. In addition to functioning as HGF activator, our data revealed novel function of MTP on HGF-stimulated c-Met signaling activation.

  12. Overexpression of c-met in the early stage of pancreatic carcinogenesis; altered expression is not sufficient for progression from chronic pancreatitis to pancreatic cancer

    Institute of Scientific and Technical Information of China (English)

    Jun Yu; Eishi Nagai; Masao Tanaka; Kenoki Ohuchida; Kazuhiro Mizumoto; Nami Ishikawa; Yasuhiro Ogura; Daisuke Yamada; Takuya Egami; Hayato Fujita; Seiji Ohashi

    2006-01-01

    AIM: To investigate c-met expression during early pancreatic carcinogenesis.METHODS: We used 46 bulk tissues and 36 microdissected samples, including normal pancreas, chronic pancreatitis, and pancreatic cancer, for quantitative real time reverse transcription-polymerase chain reaction.RESULTS: In bulk tissue analyses, pancreatic cancer tissues expressed significantly higher levels of c-met than did chronic pancreatitis and normal pancreas tissues.c-met levels did not differ between chronic pancreatitis and normal pancreas tissues. In microdissection-based analyses, c-met was expressed at higher levels in microdissected pancreatic cancer cells and pancreatitisaffected epithelial cells than in normal ductal epithelial cells (both, P < 0.01). Interestingly, pancreatitis-affected epithelial cells expressed levels of c-met similar to those of pancreatic cancer cells.CONCLUSION: Overexpression of c-met occurs during the early stage of pancreatic carcinogenesis, and a single alteration of c-met expression is not sufficient for progression of chronic pancreatitis-affected epithelial cells to pancreatic cancer cells.

  13. Farnesoid X receptor inhibits the transcriptional activity of carbohydrate response element binding protein in human hepatocytes. : Transrepression of ChREBP by FXR

    OpenAIRE

    Caron, Sandrine; Samanez, Carolina Huaman; Dehondt, Hélène; Ploton, Maheul; Briand, Olivier; Lien, Fleur; Dorchies, Emilie; Dumont, Julie; Postic, Catherine; Cariou, Bertrand; Lefebvre, Philippe; Staels, Bart

    2013-01-01

    The glucose-activated transcription factor carbohydrate response element binding protein (ChREBP) induces the expression of hepatic glycolytic and lipogenic genes. The farnesoid X receptor (FXR) is a nuclear bile acid receptor controlling bile acid, lipid, and glucose homeostasis. FXR negatively regulates hepatic glycolysis and lipogenesis in mouse liver. The aim of this study was to determine whether FXR regulates the transcriptional activity of ChREBP in human hepatocytes and to unravel the...

  14. Curcumin inhibits iron overload-induced hepatocytic apoptosis and nuclear factor-κB activity%姜黄素对过量铁导致的肝细胞凋亡及NF-κB活性的影响

    Institute of Scientific and Technical Information of China (English)

    钱晶晶; 翟旭光; 牛明辉; 周倩; 周亚军

    2012-01-01

    Objective Iron is an essential micronutrient for human beings but its overload induces various diseases of liver,the main body storage site for iron,such as liver fibrosis.Curcumin is a natural polyphenol derived from turmeric and has been used widely.Its pharmacological action has attracted great attention in recent years.The apoptosis of rat cultured hepatocytes was induced by FeNTA (ferric nitrilotriacetate)-induced Iron overload.The present study was to examine the effect of curcumin at low concentrations on FeNTA-induced apoptosis of hepatocytes and elucidate the underlying mechanisms.Methods After the incubation of hepatocytes with 100 μmol/L FeNTA in the presence or absence of 1 - 10 μmmol/L of curcumin,a series of analyses were performed,including the analyses of hepatocytic apoptosis,the expressions of proteins relating with the regulations of cell apoptosis,caspase-3 activity,the production of reactive oxygen species (ROS) and nuclesr factor NF-κB activity.Results Curcumin reduced the FeNTA-induced hepatocytic apoptosis by 46.65% and significantly down-regulated the protein levels of Bcl-2 and Bcl-XL In contrast,it had no effect on the protein levels of Bax and Bad.The curcumin treatment reduced FeNTA-caused production of ROS and caspase-3 activity by 45.01% and 59.71%respectively.And the NF-κB activity was also inhibited.Conclusion Curcumin at low concentrations reduces iron overload-caused hepatocytic apoptosis and NF-κB activity,the key regulatory transcription factor for the inflammation-related gene expression in cultured hepatocyte.%目的 利用次氮基三乙酸铁(ferric nitrilotriacetate,FeNTA)引起肝细胞的铁过载,诱导培养的大鼠正常肝细胞凋亡,观察低浓度姜黄素对铁过量诱导的肝细胞凋亡的影响及其机制.方法 FeNTA加姜黄素与培养的肝细胞孵育,检测肝细胞凋亡,凋亡相关蛋白的表达,caspase-3活性,自由基的产生,核转录因子κB(NF-κB)活性.结果 10 μmol/L的姜

  15. Gene signatures derived from a c-MET-driven liver cancer mouse model predict survival of patients with hepatocellular carcinoma.

    Directory of Open Access Journals (Sweden)

    Irena Ivanovska

    Full Text Available Biomarkers derived from gene expression profiling data may have a high false-positive rate and must be rigorously validated using independent clinical data sets, which are not always available. Although animal model systems could provide alternative data sets to formulate hypotheses and limit the number of signatures to be tested in clinical samples, the predictive power of such an approach is not yet proven. The present study aims to analyze the molecular signatures of liver cancer in a c-MET-transgenic mouse model and investigate its prognostic relevance to human hepatocellular carcinoma (HCC. Tissue samples were obtained from tumor (TU, adjacent non-tumor (AN and distant normal (DN liver in Tet-operator regulated (TRE human c-MET transgenic mice (n = 21 as well as from a Chinese cohort of 272 HBV- and 9 HCV-associated HCC patients. Whole genome microarray expression profiling was conducted in Affymetrix gene expression chips, and prognostic significances of gene expression signatures were evaluated across the two species. Our data revealed parallels between mouse and human liver tumors, including down-regulation of metabolic pathways and up-regulation of cell cycle processes. The mouse tumors were most similar to a subset of patient samples characterized by activation of the Wnt pathway, but distinctive in the p53 pathway signals. Of potential clinical utility, we identified a set of genes that were down regulated in both mouse tumors and human HCC having significant predictive power on overall and disease-free survival, which were highly enriched for metabolic functions. In conclusions, this study provides evidence that a disease model can serve as a possible platform for generating hypotheses to be tested in human tissues and highlights an efficient method for generating biomarker signatures before extensive clinical trials have been initiated.

  16. Relationship between pathological characteristics of prostate cancer and MACC1, c-Met, Apaf-1 as well as Caspase-9 expression in tumor tissue

    Institute of Scientific and Technical Information of China (English)

    Hang-Yu Ai; Xue-De Qiu

    2016-01-01

    Objective:To study the MACC1, c-Met, Apaf-1 and Caspase-9 expression in prostate cancer tissue and their relationship with the pathological characteristics of tumor.Methods:Prostate cancer and benign prostatic hyperplasia patients who received surgical treatment in our hospital from May 2015 to March 2016 were selected as the research subjects, prostate cancer tissue and benign prostatic hyperplasia tissue were collected during surgery to determine MACC1, c-Met, Apaf-1 and Caspase-9 expression, and serum specimens were collected to determine miR-let7i, -32, -128, -196a and -218 expression levels.Results: mRNA content of MACC1 and c-Met in prostate cancer tissue were significantly higher than those in benign prostatic hyperplasia tissue while mRNA content of Apaf-1 and Caspase-9 were significantly lower than those in benign prostatic hyperplasia tissue, and the higher the Gleason grading and the higher the Whitmore-Prout staging, the higher the mRNA content of MACC1 and c-Met in prostate cancer tissue and the lower the mRNA content of Apaf-1 and Caspase-9; serum miR-32, miR-128 and miR-196a expression levels in prostate cancer patients were significantly higher than those in patients with benign prostatic hyperplasia and negatively correlated with the mRNA content of Apaf-1 and Caspase-9, and the expression levels of miR-let7i and miR-218 were significantly lower than those in patients with benign prostatic hyperplasia and negatively correlated with MACC1 and c-Met.Conclusion: High MACC1 and c-Met expression and low Caspase-9 and Apaf-1 expression are related to the occurrence and progression of prostate cancer, and the MACC1, c-Met, Apaf-1 and Caspase-9 expression in prostate cancer tissue are regulated by miRNAs.

  17. Hepatic Stellate Cell Coculture Enables Sorafenib Resistance in Huh7 Cells through HGF/c-Met/Akt and Jak2/Stat3 Pathways

    Directory of Open Access Journals (Sweden)

    Weibo Chen

    2014-01-01

    Full Text Available Purpose. Tumor microenvironment confers drug resistance to kinase inhibitors by increasing RKT ligand levels that result in the activation of cell-survival signaling including PI3K and MAPK signals. We assessed whether HSC-LX2 coculture conferred sorafenib resistance in Huh7 and revealed the mechanism underlying the drug resistance. Experimental Design. The effect of LX2 on sorafenib resistance was determined by coculture system with Huh7 cells. The rescue function of LX2 supernatants was assessed by MTT assay and fluorescence microscopy. The underlying mechanism was tested by administration of pathway inhibitors and manifested by Western blotting. Results. LX2 coculture significantly induced sorafenib resistance in Huh7 by activating p-Akt that led to reactivation of p-ERK. LX2 secreted HGF into the culture medium that triggered drug resistance, and exogenous HGF could also induce sorafenib resistance. The inhibition of p-Akt blocked sorafenib resistance caused by LX2 coculture. Increased phosphorylation of Jak2 and Stat3 was also detected in LX2 cocultured Huh7 cells. The Jak inhibitor tofacitinib reversed sorafenib resistance by blocking Jak2 and Stat3 activation. The combined administration of sorafenib and p-Stat3 inhibitor S3I-201 augmented induced apoptosis even in the presence of sorafenib resistance. Conclusions. HSC-LX2 coculture induced sorafenib resistance in Huh7 through multiple pathways: HGF/c-Met/Akt pathway and Jak2/Stat3 pathway. A combined administration of sorafenib and S3I-201 was able to augment sorafenib-induced apoptosis even in the presence of LX2 coculture.

  18. Underexpression of miR-34a in hepatocellular carcinoma and its contribution towards enhancement of proliferating inhibitory effects of agents targeting c-MET.

    Directory of Open Access Journals (Sweden)

    Yiwu Dang

    Full Text Available Aberrant expression of microRNA-34a (miR-34a has been reported to be involved in the tumorigenesis and progression of various classes of malignancies. However, its role in hepatocellular carcinoma (HCC has not been completely clarified. In the current study, we have investigated the clinical significance and the in vitro contribution of miR-34a on biological functions of human HCCs. miR-34a expression in eighty-three cases of HCC formalin-fixed paraffin-embedded (FFPE tissues decreased significantly compared to that in the adjacent liver tissues (P<0.01, as detected by real-time quantitative RT-PCR (RT-qPCR. miR-34a expression in the groups of TNM stage I and II, without metastasis and without portal vein tumor embolus, was significantly higher than that of their corresponding groups (P<0.05. In functional experiments, miR-34a mimic suppressed cell growth, migration and invasion, meanwhile it increased cellular apoptosis and caspase activity in HCC cells. miR-34a mimic also reduced phospho-ERK1/2 and phospho-stat5 signaling. In addition, miR-34a mimic enhanced the effect of cell proliferation inhibition and caspase activity induction of agents targeting c-MET (siRNAs and small molecular inhibitor su11274. In conclusion, miR-34a may act as a tumor suppressor miRNA of HCC. The strategies to increase miR-34a level might be a critical targeted therapy for HCC in future.

  19. Hepatocytes as Immunological Agents.

    Science.gov (United States)

    Crispe, Ian N

    2016-01-01

    Hepatocytes are targeted for infection by a number of major human pathogens, including hepatitis B virus, hepatitis C virus, and malaria. However, hepatocytes are also immunological agents in their own right. In systemic immunity, they are central in the acute-phase response, which floods the circulation with defensive proteins during diverse stresses, including ischemia, physical trauma, and sepsis. Hepatocytes express a variety of innate immune receptors and, when challenged with pathogen- or damage-associated molecular patterns, can deliver cell-autonomous innate immune responses that may result in host defense or in immunopathology. Important human pathogens have evolved mechanisms to subvert these responses. Finally, hepatocytes talk directly to T cells, resulting in a bias toward immune tolerance. PMID:26685314

  20. Induction and inhibition of cytochrome P450 1A1 and ethoxyresorufin-O-deethylation activity by polybrominated diphenyl ethers (PBDEs) in cynomolgus monkey primary hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Peters, L.; Sanderson, J.T.; Berg, M. van den [Utrecht Univ. (Netherlands). Inst. for Risk Assessment Sciences; Bergman, A. [Stockholm Univ. (Sweden). Dept. of Environmental Chemistry

    2004-09-15

    Brominated flame retardants (BFRs) make up for 39% of the worldwide flame-retardants market. One groups of BFR, Polybrominated diphenylethers (PBDEs) are used as additive flameretardants in plastic materials, paints, and textile fabrics. Some PBDEs have been found to be lipophilic and persistent, and consequently bioaccumulate. Recently, levels of some PBDEs have been increasing in fish, wildlife, and in human tissue. The structural similarity of certain PBDE congeners to other polyhalogenated aromatic hydrocarbons such as polychlorinated biphenyls (PCBs) has raised concerns that these compounds might act as agonists for the aryl hydrocarbon receptor (AhR). If some of these PBDEs were to act as Ah receptor agonists, they would warrant inclusion in the toxic equivalence factor (TEF) concept. CYP1A1 is a cytochrome P450 (CYP) enzyme that is involved in phase 1 biotransformation of xenobiotics and endogenous compounds such as estrogens. Many CYP enzymes detoxify xenobiotics or bioactivate xenobiotics to reactive intermediates. Although CYP1A1 is expressed in all mammals, there are differences in expression levels among species and tissues. To study the possible dioxin-like effects of environmentally most relevant PBDEs (BDE47, 77, 99, 100, 153, 154, 183, 209), the Ah receptor-mediated induction CYP1A1 was studied in cynomolgus monkey (Macaca fascicularis) primary hepatocytes. CYP 1A1 is the major enzyme that catalyses the deethylation of 7-ethoxyresorufin to resorufin. This ethoxyresorufin-Odeethylation (EROD) activity was used as a marker for CYP1A1 activity.

  1. Effects of quercetin on hepatocyte stimulating factor production by mouse peritoneal macrophases

    Institute of Scientific and Technical Information of China (English)

    周斌; 张俊平; 刘宏; 殷明; 钱定华

    2003-01-01

    Objective: To study the effects of quercetin on hepatocyte stimulating factor production from mouse peritoneal macrophages. Methods: Hepatocyte stimulating factor was evaluated by the amount of fibrinogen synthesized in Hep3B cells. Interleukin-6 activity was measured by B9 cell proliferation methyl thiazolyl tetrazolium colorimetric method. Hep3B cell supernatant fibrinogen was quantitated with ELISA. Results: LPS induced the synthesis of hepatocyte stimulating factor in mouse peritoneal macrophages, and hepatocyte stimulating factor promotes the synthesis of fibrinogen from Hep3B cells. Quercetin(5 to 40 μmol/ L)inhibited the synthesis of hepatocyte stimulating factor stimulated by LPS. Quercetin(5 to 20 μmol/ L) inhibited release of interleukin-6 from mouse peritoneal macrophages induced by 0.5 g/ L fibrin fibrinogen degradation products. Conclusion: Quercetin inhibits the synthesis of hepatocyte stimulating factor in macrophages.

  2. Basal efflux of bile acids contributes to drug-induced bile acid-dependent hepatocyte toxicity in rat sandwich-cultured hepatocytes.

    Science.gov (United States)

    Susukida, Takeshi; Sekine, Shuichi; Ogimura, Eiichiro; Aoki, Shigeki; Oizumi, Kumiko; Horie, Toshiharu; Ito, Kousei

    2015-10-01

    The bile salt export pump (BSEP or Bsep) functions as an apical transporter to eliminate bile acids (BAs) from hepatocytes into the bile. BSEP or Bsep inhibitors engender BA retention, suggested as an underlying mechanism of cholestatic drug-induced liver injury. We previously reported a method to evaluate BSEP-mediated BA-dependent hepatocyte toxicity by using sandwich-cultured hepatocytes (SCHs). However, basal efflux transporters, including multidrug resistance-associated proteins (MRP or Mrp) 3 and 4, also participate in BA efflux. This study examined the contribution of basal efflux transporters to BA-dependent hepatocyte toxicity in rat SCHs. The apical efflux of [(3)H]taurocholic acid (TC) was potently inhibited by 10 μM cyclosporine A (CsA), with later inhibition of basal [(3)H]TC efflux, while MK571 simultaneously inhibited both apical and basal [(3)H]TC efflux. CsA-induced BA-dependent hepatocyte toxicity was 30% at most at 10 μM CsA and ∼60% at 50 μM, while MK571 exacerbated hepatocyte toxicity at concentrations of ≥50 μM. Quinidine inhibited only basal [(3)H]TC efflux and showed BA-dependent hepatocyte toxicity in rat SCHs. Hence, inhibition of basal efflux transporters as well as Bsep may precipitate BA-dependent hepatocyte toxicity in rat SCHs. PMID:26055650

  3. Indolo[3,2-b]carbazole inhibits gap junctional intercellular communication in rat primary hepatocytes and acts as a potential tumor promoter

    DEFF Research Database (Denmark)

    Herrmann, Susan; Seidelin, Michel; Bisgaard, Hanne Cathrine;

    2002-01-01

    environment of the stomach after intake of I3C, has a similar structure to, and shares biological effects with, the well-known tumor promoter 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD). Therefore, we hypothesized that ICZ could be responsible for the potential tumor-promoting activity of I3C. The aim of the....... Significant inhibition was observed after 8 and 12 h of treatment with 1 and 0.1 µM ICZ, respectively. Maximum GJIC inhibition (cell–cell communication only 5% of control values) was observed after 24–48 h of ICZ treatment. Continued exposure to 1 µM ICZ suppressed GJIC until ~120 h. Both ICZ and TCDD...

  4. 抑制脂筏对肝细胞生长因子受体介导的信号跨膜转导作用的影响%Effects of lipid rafts on signal transmembrane transduction mediated by c-Met

    Institute of Scientific and Technical Information of China (English)

    王蕾; 赵玉峰; 李亚丽; 徐跃飞; 夏泉; 马克里

    2008-01-01

    Objective To study the effects of lipid rafts on cell signal transmembrane transduction mediated by c-Met. Methods After HepG2Cells were treated with MβCD to disrupt the lipid rafts and were treated with artificial recombination hepatocyte growth factor to activate c-Met, the activities of PLCγ1/PKC, P13K/Akt and MAPK signaling pathways in HepG2 cells were analyzed using Western blot. Results (1) After disruption of lipid rafts with MβCD, phosphorylation of PLCγ1 decreased by 35% (P=0.022); the content of PLCγ in the cytoplasm increased by 1.75 fold (P=0.017); PLCγ1 conjugated with membrane decreased by 30% (P=0.037). (2) The content of PKB in the cytosol decreased by 38% (P=0.028), and the phosphorylation level of PKB conjugated with membrane decreased by 14% (P=0.041). At the same time, PDK translocation from cytosol to the plasma membrane and its activation were inhibited by treatment with MβCD. (3) Treatment with MβCD had no significant effect on ErK/MAPK, p38/MAPK and JNK/MAPK signaling pathways. Conclusion Disruption of lipid rafts with MβCD inhibits the activation of PLCγ1/PKC and PI3K/PKB signaling pathways by HGF/cMet, but has no effect on MAPK signaling pathway.%目的 探讨脂筏在肝细胞生长因子受体介导的信号跨膜转导中的作用.方法 采用甲基-β-环糊精(MβCD)处理HepG2细胞,以干扰脂筏的形成,然后加入人工重组肝细胞生长因子以活化其受体.采用Western blot技术及计算机扫描定量分析分别检测MβCD处理组和对照组细胞磷脂酶Cγ1(PLCγ1)/二脂酰甘油/蛋白激酶C信号通路、磷脂酰肌醇-3-激酶/磷脂酰肌醇依赖的蛋白激酶/蛋白激酶B信号通路和有丝分裂原激活的蛋白激酶(MAPK)信号通路活性的变化.结果 (1)用MβCD处理细胞后,细胞PLCγ1磷酸化程度降低,为对照组的65%(P=0.022);胞浆中PLCγ1含量增加,为对照组的1.75倍(P=0.017);膜结合的PLCγ1减少,为对照组的70%(P=0.037).(2)用MβCD处理细胞

  5. Design, Synthesis, and Biological Evaluation of 4-Phenoxyquinoline Derivatives Containing Benzo[d]thiazole-2-yl Urea as c-Met Kinase Inhibitors.

    Science.gov (United States)

    Lei, Hongrui; Hu, Gang; Wang, Yu; Han, Pei; Liu, Zijian; Zhao, Yanfang; Gong, Ping

    2016-08-01

    A series of novel 4-phenoxyquinoline derivatives containing the benzo[d]thiazole-2-yl urea moiety were synthesized and evaluated for their cytotoxicity against the HT-29, MKN-45, and H460 cell lines. The structures of the target compounds were confirmed by (1) H NMR and MS spectra. Most of them showed moderate to excellent potency against the three tested cell lines. Especially, compound 23 was identified a promising agent (c-Met IC50  = 17.6 nM), showing the most potent anticancer activities with IC50 values of 0.18, 0.06, and 0.01 µM against the HT-29, MKN-45, and H460 cell lines, respectively. The docking results of 23 with the c-Met kinase model 3LQ8 showed a specific binding mode between the ligand and the target protein.

  6. Lysophosphatidic acid transactivates both c-Met and epidermal growth factor receptor, and induces cyclooxygenase-2 expression in human colon cancer LoVo cells

    Institute of Scientific and Technical Information of China (English)

    Dai Shida; Joji Kitayama; Hironori Yamaguchi; Hiroharu Yamashita; Ken Mori; Toshiaki Watanabe; Hirokazu Nagawa

    2005-01-01

    AIM: To examine whether lysophosphatidic acid (LPA)induces phosphorylation of c-Met and epidermal growth factor receptor (EGFR), both of which have been proposed as prognostic markers of colorectal cancer, and whether LPA induces cyclooxygenase-2 (COX-2) expression in human colon cancer cells.METHODS: Using a human colon cancer cell line, LoVo cells, we performed immunoprecipitation analysis,followed by Western blot analysis. We also examined whether LPA induced COX-2 expression, by Western blot analysis.RESULTS: Immunoprecipitation analysis revealed that 10 μmol/L LPA induced tyrosine phosphorylation of c-Met and EGFR in LoVo cells within a few minutes. We found that c-Met tyrosine phosphorylation induced by LPA was not attenuated by pertussis toxin or a matrix metalloproteinase inhibitor, in marked contrast to the results for EGFR. In addition, 0.2-40 μmol/L LPA induced COX-2 expression in a dose-dependent manner.CONCLUSION: Our results suggest that LPA acts upstream of various receptor tyrosine kinases (RTKs) and COX-2,and thus may act as a potent stimulator of colorectal cancer.

  7. Evaluation of clinial usefulness of [sup 11]C-methionine positron emission tomography ([sup 11]C-MET-PET) as a tool for liver functional imaging

    Energy Technology Data Exchange (ETDEWEB)

    Enomoto, Kazuo; Matsui, Yoshifumi; Okazumi, Shinichi (Chiba Univ. (Japan). School of Medicine) (and others)

    1994-03-01

    We studied [sup 11]C-MET-PET in 17 clinical cases, 10 patients with obstructive jaundice and 7 normal volunteers, and analyzed its efficacy for the evaluation of hepatic functional reserve in major hepatectomy candidates. Differential absorption ratio (DAR) of [sup 11]C was compared to the hepatic protein synthesis rate (HPS), which is measured as the incorporation rate of [sup 3]H-labeled leucine in protein fraction, using needle biopsied liver specimen obtained from each hepatic segment. In the cases of normal liver function, DAR was well correlated with HPS. Also in jaundice cases with two exceptions, low HPS segment was demonstrated as low DAR segment. Consequently, MET-PET images could clearly provide functional liver imaging. After injection of [sup 11]C-MET, the increase in rate of radioactivity of [sup 11]C in plasma protein fraction was higher in jaundice cases than in normal volunteers, which is in accord with the results of our former study that cholestatic liver has accelerated protein synthesis rate. In summary, since [sup 11]C-MET-PET could demonstrate liver functional imaging, it might be a possible tool for liver function assessment in major hepatectomy candidates. (author).

  8. Phase II study evaluating 2 dosing schedules of oral foretinib (GSK1363089, cMET/VEGFR2 inhibitor, in patients with metastatic gastric cancer.

    Directory of Open Access Journals (Sweden)

    Manish A Shah

    Full Text Available PURPOSE: The receptors for hepatocyte and vascular endothelial cell growth factors (MET and VEGFR2, respectively are critical oncogenic mediators in gastric adenocarcinoma. The purpose is to examine the safety and efficacy of foretinib, an oral multikinase inhibitor targeting MET, RON, AXL, TIE-2, and VEGFR2 receptors, for the treatment of metastatic gastric adenocarcinoma. PATIENTS AND METHODS: Foretinib safety and tolerability, and objective response rate (ORR were evaluated in patients using intermittent (240 mg/day, for 5 days every 2 weeks or daily (80 mg/day dosing schedules. Thirty evaluable patients were required to achieve alpha = 0.10 and beta = 0.2 to test the alternative hypothesis that single-agent foretinib would result in an ORR of ≥ 25%. Up to 10 additional patients could be enrolled to ensure at least eight with MET amplification. Correlative studies included tumor MET amplification, MET signaling, pharmacokinetics and plasma biomarkers of foretinib activity. RESULTS: From March 2007 until October 2009, 74 patients were enrolled; 74% male; median age, 61 years (range, 25-88; 93% had received prior therapy. Best response was stable disease (SD in 10 (23% patients receiving intermittent dosing and five (20% receiving daily dosing; SD duration was 1.9-7.2 months (median 3.2 months. Of 67 patients with tumor samples, 3 had MET amplification, one of whom had SD. Treatment-related adverse events occurred in 91% of patients. Rates of hypertension (35% vs. 15% and elevated aspartate aminotransferase (23% vs. 8% were higher with intermittent dosing. In both patients with high baseline tumor phospho-MET (pMET, the pMET:total MET protein ratio decreased with foretinib treatment. CONCLUSION: These results indicate that few gastric carcinomas are driven solely by MET and VEGFR2, and underscore the diverse molecular oncogenesis of this disease. Despite evidence of MET inhibition by foretinib, single-agent foretinib lacked efficacy in

  9. Schisandrin B inhibits cell growth and induces cellular apoptosis and autophagy in mouse hepatocytes and macrophages: implications for its hepatotoxicity

    Directory of Open Access Journals (Sweden)

    Zhang Y

    2015-04-01

    Kip1 and checkpoint kinase 1. Furthermore, Sch B markedly increased the apoptosis of AML-12 and RAW 264.7 cells with a decrease in the expression of B-cell lymphoma-extra-large and (Bcl-xl B-cell lymphoma 2 (Bcl-2, but an increase in the expression of B-cell lymphoma 2-associated X protein (Bax. Sch B promoted the cleavage of caspase 3 and poly-adenosine diphosphate-ribose polymerase (PARP in both cell lines. Additionally, Sch B significantly induced autophagy of AML-12 and RAW 264.7 cells. Sch B inhibited the activation of phosphatidylinositol 3-kinase (PI3K/protein kinase B (Akt/mammalian target of rapamycin (mTOR signaling pathway, as indicated by their altered phosphorylation, contributing to the proautophagic effect of Sch B. Taken together, our findings show that the inducing effects of Sch B on cell cycle arrest, apoptosis, and autophagy may contribute to its liver toxic effects, which might provide a clue for the investigation of the molecular toxic targets and underlying mechanisms for Sch B-induced hepatotoxicity in herbal consumers. More studies are warranted to fully delineate the underlying mechanisms, efficacy, and safety of Sch B for clinical use.Keywords: herbal medicine, liver toxicity, mTOR, Bcl-2, AML-12 cell, RAW 264.7 cell

  10. Autocrine-controlled formation and function of tissue-like aggregates by primary hepatocytes in micropatterned hydrogel arrays.

    Science.gov (United States)

    Williams, Courtney M; Mehta, Geeta; Peyton, Shelly R; Zeiger, Adam S; Van Vliet, Krystyn J; Griffith, Linda G

    2011-04-01

    The liver carries out a variety of essential functions regulated in part by autocrine signaling, including hepatocyte-produced growth factors and extracellular matrix (ECM). The local concentrations of autocrine factors are governed by a balance between receptor-mediated binding at the cell surface and diffusion into the local matrix and are thus expected to be influenced by the dimensionality of the cell culture environment. To investigate the role of growth factor and ECM-modulated autocrine signaling in maintaining appropriate primary hepatocyte survival, metabolic functions, and polarity, we created three-dimensional cultures of defined geometry using micropatterned semisynthetic polyethylene glycol-fibrinogen hydrogels to provide a mechanically compliant, nonadhesive material platform that could be modified by cell-secreted factors. We found that in the absence of exogenous peptide growth factors or ECM, hepatocytes retain the epidermal growth factor (EGF) receptor ligands (EGF and transforming growth factor-α) and the proto-oncogenic mesenchymal epithelial transition factor (c-MET) ligand hepatocyte growth factor (HGF), along with fibronectin. Further, hepatocytes cultured in this three-dimensional microenvironment maintained high levels of liver-specific functions over the 10-day culture period. Function-blocking inhibitors of α5β1 or EGF receptor dramatically reduced cell viability and function, suggesting that signaling by both these receptors is needed for in vitro survival and function of hepatocytes in the absence of other exogenous signals. PMID:21121876

  11. Novel role for endogenous hepatocyte growth factor in the pathogenesis of intracranial aneurysms.

    Science.gov (United States)

    Peña-Silva, Ricardo A; Chalouhi, Nohra; Wegman-Points, Lauren; Ali, Muhammad; Mitchell, Ian; Pierce, Gary L; Chu, Yi; Ballas, Zuhair K; Heistad, Donald; Hasan, David

    2015-03-01

    Inflammation plays a key role in formation and rupture of intracranial aneurysms. Because hepatocyte growth factor (HGF) protects against vascular inflammation, we sought to assess the role of endogenous HGF in the pathogenesis of intracranial aneurysms. Circulating HGF concentrations in blood samples drawn from the lumen of human intracranial aneurysms or femoral arteries were compared in 16 patients. Tissue from superficial temporal arteries and ruptured or unruptured intracranial aneurysms collected from patients undergoing clipping (n=10) were immunostained with antibodies to HGF and its receptor c-Met. Intracranial aneurysms were induced in mice treated with PF-04217903 (a c-Met antagonist) or vehicle. Expression of inflammatory molecules was also measured in cultured human endothelial, smooth muscle cells and monocytes treated with lipopolysaccharides in presence or absence of HGF and PF-04217903. We found that HGF concentrations were significantly higher in blood collected from human intracranial aneurysms (1076±656 pg/mL) than in femoral arteries (196±436 pg/mL; P0.05), but significantly increased the prevalence of subarachnoid hemorrhage and decreased survival in mice (Psignaling through c-Met may decrease inflammation in endothelial cells and protect against intracranial aneurysm rupture. PMID:25510828

  12. The expression of microRNA-34a is inversely correlated with c-MET and CDK6 and has a prognostic significance in lung adenocarcinoma patients.

    Science.gov (United States)

    Hong, Ji Hyung; Roh, Kang San; Suh, Sung-Suk; Lee, Sukchan; Sung, Sook Whan; Park, Jae Kil; Byun, Jae Ho; Kang, Jin Hyoung

    2015-12-01

    We aimed to establish whether the expression of microRNA-34a (miR-34a) is correlated with that of c-MET and G1 phase regulators such as cyclin dependent kinase (CDK) 4, CDK6, and cyclin D (CCND) 1 in non-small cell lung cancer (NSCLC), and whether a relationship exists between miR-34a expression and both clinicopathologic factors and recurrence-free survival (RFS). For 58 samples archived from NSCLC patients, we measured the expression of miR-34a and c-MET, CDK4/6, and CCND1 by quantitative RT-PCR and assessed the relationship between miR-34a expression, clinicopathological factors, and RFS. The expression of miR-34a was significantly lower in squamous cell tumors (P adenocarcinoma patients with high miR-34a expression than in those with low miR-34a expression (55.6 vs. 21.6 months; P = 0.020). With univariate analysis, statistically significant prognostic factors for RFS in adenocarcinoma patients were miR-34a expression (Relative risk (RR), 8.14; P = 0.049), TNM stage (RR, 13.55; P = 0.001), LN metastasis (RR, 4.19; P = 0.043), and the presence of lymphatic invasion (RR, 7.05; P = 0.015). In multivariate analysis, only miR-34a was prognostic for RFS (RR, 11.5; P = 0.027). miR-34a expression was inversely correlated with that of c-MET and CDK6 in NSCLC, and had prognostic significance for RFS, especially in adenocarcinoma patients.

  13. Bile acid formation in primary human hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Curt Einarsson; Ewa Ellis; Anna Abrahamsson; Bo-G6ran Ericzon; Ingemar Bj rkhem; Magnus Axelson

    2000-01-01

    AIM To evaluate a culture system for bile acid formation in primary human hepatocytes in comparison with HepG2 cells. METHODS Hepatocytes were isolated from normal human liver tissue and were cultured in serum-free William's E medium. The medium was collected and renewed every 24 h. Bile acids and their precursors in media were finally analysed by gas chromatography-mass spectrometry. RESULTS Cholic acid ( CA ) andchenodeoxycholic acid (CDCA) conjugated with glycine or taurine accounted for 70% and 25% of total steroids. A third of CDCA was also conjugated with sulphuric acid. Dexamathasone and thyroid hormorm alone or in combination did not significantly effect bile acid formation. The addition of cyclosporin A (10 μmol/L) inhibited the synthesis of CA and CDCA by about 13% and 30%, respectively. CONCLUSION Isolated human hepatocytes in primary culture behave as in the intact liver by converting cholesterol to conjugated CA and CDCA. This is in contrast to cultured HepG2 cells, which release large amounts of bile acid precursors and unconjugated bile acids into the medium.

  14. Culture of cryopreserved rat hepatocyte

    Institute of Scientific and Technical Information of China (English)

    Haitao Yin; Gaojun Teng; Lifeng Wang; Baorui Liu; Xiaoping Qian

    2006-01-01

    Objective: To study the method of cryopreserving rat hepatocytes and double collagen gel culture measurement after its cryopreservation. Methods: Rat hepatocytes, isolated by two-step perfusion with collagenase using an extra corporeal perfusion apparatus, were cryopreserved in double collagen gel with culture medium added by epidermal growth factor(EGF).The expression of cell function and cellular morphology were examined during culture. Results: The hepatocytes cryopreserved in double collagen gel concluding EGF showed good morphology and biological characteristics. After thawing, the MTT metabolism and protein synthesis of hepatocytes in sandwich ± EGF groups were better than those in control group. And the morphology and function of hepatocytes in sandwich group was better than that in EGF group(P < 0.05). Conclusion: Double collagen gel culture can keep hepatocyte's activities. Thawed hepatocytes can be cultivated with collagenous matrix, which provides an environment that more closely resembles that in vivo and maintain the expression of certain liver-specific function of hepatocytes.

  15. Lung adenocarcinoma harboring concomitant SPTBN1-ALK fusion, c-Met overexpression, and HER-2 amplification with inherent resistance to crizotinib, chemotherapy, and radiotherapy.

    Science.gov (United States)

    Gu, Fei-Fei; Zhang, Yong; Liu, Yang-Yang; Hong, Xiao-Hua; Liang, Jin-Yan; Tong, Fan; Yang, Jing-Song; Liu, Li

    2016-01-01

    Crizotinib is a multi-targeted tyrosine kinase inhibitor (TKI) with activity against mesenchymal-epithelial transition factor (MET) and anaplastic lymphoma kinase (ALK). However, the concomitant oncogenic drivers may affect the sensitivity of crizotinib. Herein, we present a 69-year-old never-smoker Chinese male with advanced lung adenocarcinoma harboring concomitant spectrin beta non-erythrocytic 1 (SPTBN1)-ALK fusion, c-Met overexpression, and human epidermal growth factor receptor-2 (HER-2) amplification with inherent resistance to crizotinib, chemotherapy, and radiotherapy. Although the patient received timely and comprehensive treatment, the overall survival was only 8 months. Therefore, c-Met overexpression, HER-2 gene amplification, and SPTBN1-ALK gene fusion can coexist in lung adenocarcinoma and may become a potential biomarker of cancer refractory to crizotinib, chemotherapy, and radiotherapy as well as of a relatively poor prognosis. In addition, the novel SPTBN1-ALK fusion gene may become a potential target for anti-tumor therapy. PMID:27496196

  16. Molecular cytotoxic mechanisms of chlorpromazine in isolated rat hepatocytes.

    Science.gov (United States)

    MacAllister, Stephanie L; Young, Cheryl; Guzdek, Anna; Zhidkov, Nickholas; O'Brien, Peter J

    2013-01-01

    Chlorpromazine (CPZ), a member of the largest class of first-generation antipsychotic agents, is known to cause hepatotoxicity in the form of cholestasis and hepatocellular necrosis in some patients. The mechanism of CPZ hepatotoxicity is unclear, but is thought to result from reactive metabolite formation. The goal of this research was to assess potential cytotoxic mechanisms of CPZ using the accelerated cytotoxicity mechanism screening (ACMS) technique with freshly isolated rat hepatocytes. This study identified CPZ cytotoxicity and inhibition of mitochondrial membrane potential (MMP) to be concentration-dependent. Furthermore, inhibition of cytochrome P450s (CYPs), including CYP2D1 and 1A2, delayed CPZ cytotoxicity, suggesting a role for CYP activation of CPZ to a toxic metabolite(s) in this model. Metabolism studies also demonstrated glucuronide and glutathione (GSH) requirement for CPZ detoxification in hepatocytes. Inactivating the 2-electron reduction pathway, NAD(P)H quinone oxidoreductase (NQO1), caused a significant increase in hepatocyte susceptibility to CPZ, indicating quinoneimine contribution to CPZ cytotoxicity. Nontoxic concentrations of peroxidase/H(2)O(2) (inflammatory model) increased cytotoxicity in CPZ-treated hepatocytes and caused additional mitochondrial toxicity. Inflammation further depleted GSH and increased oxidized glutathione (GSSG) levels. Results suggest activation of CPZ to reactive metabolites by 2 pathways in hepatocytes: (i) a CYP-catalyzed quinoneimine pathway, and (ii) a peroxidase-catalyzed oxidation of CPZ to CPZ radicals.

  17. Mechanism of c-Met and EGFR tyrosine kinase inhibitor resistance through epithelial mesenchymal transition in non-small cell lung cancer.

    Science.gov (United States)

    Rastogi, Ichwaku; Rajanna, Supriya; Webb, Andrew; Chhabra, Gagan; Foster, Brad; Webb, Brian; Puri, Neelu

    2016-09-01

    According to currently available estimates from Cancer Research UK, 14.1 million new lung cancer cases were diagnosed and a staggering 8.2 million people worldwide died from lung cancer in 2012. EGFR and c-Met are two tyrosine kinase receptors most commonly overexpressed or mutated in Non-small Cell Lung Cancer (NSCLC) resulting in increased proliferation and survival of lung cancer cells. Tyrosine kinase inhibitors (TKIs), such as erlotinib, approved by the FDA as first/second line therapy for NSCLC patients have limited clinical efficacy due to acquired resistance. In this manuscript, we investigate and discuss the role of epithelial mesenchymal transition (EMT) in the development of resistance against EGFR and c-Met TKIs in NSCLC. Our findings show that Zeb-1, a transcriptional repressor of E-Cadherin, is upregulated in TKI-resistant cells causing EMT. We observed that TKI-resistant cells have increased gene and protein expression of EMT related proteins such as Vimentin, N-Cadherin, β-Catenin and Zeb-1, while expression of E-Cadherin, an important cell adhesion molecule, was suppressed. We also confirmed that TKI-resistant cells display mesenchymal cell type morphology, and have upregulation of β-Catenin which may regulate expression of Zeb-1, a transcriptional repressor of E-Cadherin in TKI-resistant NSCLC cells. Finally, we show that down-regulating Zeb-1 by inducing miR-200a or β-Catenin siRNA can increase drug sensitivity of TKI-resistant cells. PMID:27396618

  18. Irradiation leads to sensitization of hepatocytes to TNF-α-mediated apoptosis by upregulation of IκB expression

    OpenAIRE

    Gürleyen, Hakan; Christiansen, Hans; Tello, Khodr; Dudas, Joszef; Hermann, Robert; Rave-Fränk, Margret; Clemens F. Hess; Ramadori, Giuliano; Saile, Bernhard

    2008-01-01

    This study aimed to reveal the pathophysiological signalling responsible for radiation-induced sensitization of hepatocytes to TNF-α-mediated apoptosis. IκB was upregulated in irradiated hepatocytes. Administration of IκB antisense oligonucleotides prior to irradiation inhibited occurrence of apoptosis after TNF-α administration. Caspases-8, -9 and -3 activities were increased in irradiated hepatocytes and downregulation of apoptosis by IκB antisense oligonucleotides was mediated by suppressi...

  19. Genetic abolishment of hepatocyte proliferation activates hepatic stem cells.

    Directory of Open Access Journals (Sweden)

    Yoko Endo

    Full Text Available Quiescent hepatic stem cells (HSCs can be activated when hepatocyte proliferation is compromised. Chemical injury rodent models have been widely used to study the localization, biomarkers, and signaling pathways in HSCs, but these models usually exhibit severe promiscuous toxicity and fail to distinguish damaged and non-damaged cells. Our goal is to establish new animal models to overcome these limitations, thereby providing new insights into HSC biology and application. We generated mutant mice with constitutive or inducible deletion of Damaged DNA Binding protein 1 (DDB1, an E3 ubiquitin ligase, in hepatocytes. We characterized the molecular mechanism underlying the compensatory activation and the properties of oval cells (OCs by methods of mouse genetics, immuno-staining, cell transplantation and gene expression profiling. We show that deletion of DDB1 abolishes self-renewal capacity of mouse hepatocytes in vivo, leading to compensatory activation and proliferation of DDB1-expressing OCs. Partially restoring proliferation of DDB1-deficient hepatocytes by ablation of p21, a substrate of DDB1 E3 ligase, alleviates OC proliferation. Purified OCs express both hepatocyte and cholangiocyte markers, form colonies in vitro, and differentiate to hepatocytes after transplantation. Importantly, the DDB1 mutant mice exhibit very minor liver damage, compared to a chemical injury model. Microarray analysis reveals several previously unrecognized markers, including Reelin, enriched in oval cells. Here we report a genetic model in which irreversible inhibition of hepatocyte duplication results in HSC-driven liver regeneration. The DDB1 mutant mice can be broadly applied to studies of HSC differentiation, HSC niche and HSCs as origin of liver cancer.

  20. Current status of hepatocyte xenotransplantation.

    Science.gov (United States)

    Meier, Raphael P H; Navarro-Alvarez, Nalu; Morel, Philippe; Schuurman, Henk-Jan; Strom, Stephen; Bühler, Leo H

    2015-11-01

    The treatment of acute liver failure, a condition with high mortality, comprises optimal clinical care, and in severe cases liver transplantation. However, there are limitations in availability of organ donors. Hepatocyte transplantation is a promising alternative that could fill the medical need, in particular as the bridge to liver transplantation. Encapsulated porcine hepatocytes represent an unlimited source that could function as a bioreactor requiring minimal immunosuppression. Besides patients with acute liver failure, patients with alcoholic hepatitis who are unresponsive to a short course of corticosteroids are a target for hepatocyte transplantation. In this review we present an overview of the innate immune barriers in hepatocyte xenotransplantation, including the role of complement and natural antibodies; the role of phagocytic cells and ligands like CD47 in the regulation of phagocytic cells; and the role of Natural Killer cells. We present also some illustrations of physiological species incompatibilities in hepatocyte xenotransplantation, such as incompatibilities in the coagulation system. An overview of the methodology for cell microencapsulation is presented, followed by proof-of-concept studies in rodent and nonhuman primate models of fulminant liver failure: these studies document the efficacy of microencapsulated porcine hepatocytes which warrants progress towards clinical application. Lastly, we present an outline of a provisional clinical trial, that upon completion of preclinical work could start within the upcoming 2-3 years.

  1. The Mechanism of Gefitinib Resistance Induced by Hepatocyte Growth Factor 
in Sensitive Non-small Cell Lung Cancer Cells in Vitro

    Directory of Open Access Journals (Sweden)

    Xianglan XUAN

    2013-01-01

    Full Text Available Background and objective Previous studies have reported that Met might be related to gefitinib resistance in non-small cell lung cancer (NSCLC. The present study aims to explore the mechanism of hepatocyte growth factor (HGF-induced gefitinib resistance in different gene types of sensitive NSCLC in vitro. Methods The PC-9 and H292 cell lines were chosen and induced by HGF. The cell survival was measured using MTT assay, the cell cycle distribution was measured using PI assay, and cell apoptosis with an Annexin V-PE assay, respectively. The c-Met and p-Met protein expression was determined via Western blot analysis. Results Gefitinib inhibited the growth of PC-9 and H292 cells in a dose-dependent manner. The concentration-survival curves of both cell lines shifted to the right when induced with HGF. HGF did not affect PC-9 and H292 cell proliferation. The cell also had a higher cell survival rate when treated with HGF and gefitinib compared with that under gefitinib alone (P<0.05. The apoptotic rate and cell cycle progression showed no significant difference between the HG and G group (P>0.05. HGF stimulated Met phosphorylation in the PC-9 and H292 cells. Gefitinib inhibited the HGF-induced Met phosphorylation in PC-9 cells, but not in H292 cells. Conclusion HGF induces gefitinib resistance in PC-9 and H292 cells. HGF-induced Met phosphorylation may be an important mechanism of gefitinib resistance in sensitive NSCLC.

  2. Endotoxin-stimulated Rat Hepatic Stellate Cells Induce Autophagy in Hepatocytes as a Survival Mechanism.

    Science.gov (United States)

    Dangi, Anil; Huang, Chao; Tandon, Ashish; Stolz, Donna; Wu, Tong; Gandhi, Chandrashekhar R

    2016-01-01

    Bacterial lipopolysaccharide (LPS)-stimulated hepatic stellate cells (HSCs) produce many cytokines including IFNβ, TNFα, and IL6, strongly inhibit DNA synthesis, but induce apoptosis of a small number of hepatocytes. In vivo administration of LPS (up to 10 mg/mL) causes modest inflammation and weight loss in rats but not mortality. We determined whether LPS-stimulated HSCs instigate mechanisms of hepatocyte survival. Rats received 10 mg/kg LPS (i.p.) and determinations were made at 6 h. In vitro, HSCs were treated with 100 ng/mL LPS till 24 h. The medium was transferred to hepatocytes, and determinations were made at 0-12 h. Controls were HSC-conditioned medium or medium-containing LPS. LPS treatment of rats caused autophagy in hepatocytes, a physiological process for clearance of undesirable material including injured or damaged organelles. This was accompanied by activation of c-Jun NH2 terminal kinase (JNK) and apoptosis of ~4-5% of hepatocytes. In vitro, LPS-conditioned HSC medium (LPS/HSC) induced autophagy in hepatocytes but apoptosis of only ~10% of hepatocytes. While LPS/HSC stimulated activation of JNK (associated with cell death), it also activated NFkB and ERK1/2 (associated with cell survival). LPS-stimulated HSCs produced IFNβ, and LPS/HSC-induced autophagy in hepatocytes and their apoptosis were significantly inhibited by anti-IFNβ antibody. Blockade of autophagy, on the other hand, strongly augmented hepatocyte apoptosis. While LPS-stimulated HSCs cause apoptosis of a subpopulation of hepatocytes by producing IFNβ, they also induce cell survival mechanisms, which may be of critical importance in resistance to liver injury during endotoxemia.

  3. Influence of colchicine and vinblastine on the intracellular migration of secretory and membrane glycoproteins: III. Inhibition of intracellular migration of membrane glycoproteins in rat intestinal columnar cells and hepatocytes as visualized by light and electron-microscope radioautography after 3H-fucose injection

    International Nuclear Information System (INIS)

    In the present work, the effects of these drugs on migration of membrane glycoproteins have been examined at the ultrastructural level in duodenal villous columnar cells and hepatocytes. Young (40 gm) rats were given a single intravenous injection of colchicine (4.0 mg) or vinblastine (2.0 mg). At 10 min after colchicine and 30 min after vinblastine administration, the rats were injected with 3H-fucose. Control rats received 3H-fucose only. All rats were sacrificed 90 min after 3H-fucose injection and their tissues processed for radioautography. In duodenal villous columnar cells, 3H-fucose labeling of the apical plasma membrane was reduced by 51% after colchicine and by 67% after vinblastine treatment; but there was little change in labeling of the lateral plasma membrane. Labeling of the Golgi apparatus increased. This suggests that labeled glycoproteins destined for the apical plasma membrane were inhibited from leaving the Golgi region, while migration to the lateral plasma membrane was not impaired. In hepatocytes, labeling of the sinusoidal plasma membrane was reduced by 83% after colchicine and by 85% after vinblastine treatment. Labeling of the lateral plasma membrane also decreased, although not so dramatically. Labeling of the Golgi apparatus and neighboring secretory vesicles increased. This indicates that the drugs inhibited migration of membrane glycoproteins from the Golgi region to the various portions of the plasma membrane. Accumulation of secretory vesicles at the sinusoidal front suggests that exocytosis may also have been partially inhibited. In both cell types, microtubules almost completely disappeared after drug treatment. Microtubules may, therefore, be necessary for intracellular transport of membrane glycoproteins, although the possibility of a direct action of these drugs on Golgi or plasma membranes must also be considered

  4. CYP2E1-dependent hepatotoxicity and oxidative damage after ethanol administration in human primary hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Lie-Gang Liu; Hong Yan; Ping Yao; Wen Zhang; Li-Jun Zou; Fang-Fang Song; Ke Li; Xiu-Fa Sun

    2005-01-01

    AIM: To observe the relationship between ethanol-induced oxidative damage in human primary cultured hepatocytes and cytochrome P450 2E1 (CYP2E1) activity, in order to address if inhibition of CYP2E1 could attenuate ethanol-induced cellular damage.METHODS: The dose-dependent (25-100 mmol/L) and time-dependent (0-24 h) exposures of primary human cultured hepatocytes to ethanol were carried out. CYP2E1 activity and protein expression were detected by spectrophotometer and Western blot analysis respectively.Hepatotoxicity was investigated by determination of lactate dehydrogenase (LDH) and aspartate transaminase (AST) level in hepatocyte culture supernatants, as well as the intracellular formation of malondialdehyde (MDA).RESULTS: A dose-and time-dependent response between ethanol exposure and CYP2E1 activity in human hepatocytes was demonstrated. Moreover, there was a time-dependent increase of CYP2E1 protein after 100 mmol/L ethanol exposure. Meanwhile, ethanol exposure of hepatocytes caused a time-dependent increase of ceilular MDA level, LDH, and AST activities in supernatants.Furthermore, the inhibitor of CYP2E1, diallyl sulfide (DAS) could partly attenuate the increases of MDA, LDH, and AST in human hepatocytes.CONCLUSION: A positive relationship between ethanol-induced oxidative aamage in human primary cultured hepatocytes and CYP2E1 activity was exhibited, and the inhibition of CYP2E1 could partly attenuate ethanol-induced oxidative damage.

  5. Overexpression of NK2 inhibits liver regeneration after partial hepatectomy in mice

    Institute of Scientific and Technical Information of China (English)

    Toshiyuki Otsuka; Hitoshi Takagi; Glenn Merlino; Masatomo Mori; Norio Horiguchi; Daisuke Kanda; Takashi Kosone; Yuichi Yamazaki; Kazuhisa Yuasa; Naondo Sohara; Satoru Kakizaki; Ken Sato

    2005-01-01

    AIM: To investigate the in vivo effects of NK2 on liver regeneration after partial hepatectomy (PH). METHODS: Survival after PH was observed with 21 NK2 transgenic mice and 23 wild-type (WT) mice over 10 d. Liver regeneration was analyzed using histology and immunohistochemistry. Expressions of genes were analyzed using Northern blot analysis, immunoprecipitation and immunoblotting, and reverse transcriptase polymerase chain reaction assay. KaplanMeier method and the log-rank test were used for ahalyzing the survival after PH. Differences in the resultsof immunohistochemistry and percentage of liver regeneration was determined by the Student's t-test. RESULTS: More than half of NK2 transgenic mice died within 48 h after PH. After PH, increased deposition of small lipid droplets in hepatocytes was evident and hepatic proliferation was inhibited in NK2 transgenic mice. The hepatic expression and kinase activity of HGF receptor, c-Met, were unchanged among WT mice and NK2 transgenic mice after PH. The expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in liver tissues were prolonged in NK2 transgenic mice that died after PH.CONCLUSION: Our findings indicate that overexpression of NK2 inhibits liver regeneration after PH.

  6. Response of porcine hepatocytes in primary culture to plasma from severe viral hepatitis patients

    Institute of Scientific and Technical Information of China (English)

    Yong-Bo Cheng; Ying-Jie Wang; Shi-Chang Zhang; Jun Liu; Zhi Chen; Jia-Jia Li

    2005-01-01

    AIM: To observe the effects of plasma from patients with severe viral hepatitis (SVHP) on the growth and metabolism of porcine hepatocytes and the clinical efficiency of bioartificial liver device.METHODS: Hepatocytes were isolated from male porcines by collagenase perfusion. The synthesis of DNA and total protein, leakages of AST and LDH, changes in glutathione (GSH), catalase and morphology of porcine hepatocytes exposed to SVHP were investigated to indicate the effect of plasma from patients with severe hepatitis on the growth, injury, detoxification, and morphology of porcine hepatocytes.RESULTS: The synthesis of DNA and protein was inhibited in the medium containing 100% SVHP compared to the controls. The leakages of LDH and AST increased in porcine hepatocytes following exposure to 100% SVHP for 5 h. The difference between 100% SVHP and 10% newborn calf serum (NCS) was significant in t-test (LDH: t = 24.552, P = 0.001; AST: t = 4.169, P =0.014). After exposure to SVHP for 24 h, alterations in GSH status were significant (F = 2.746, P<0.05) between porcine hepatocytes in 100% SVHP and 10% NCS, but no alteration occurred in the culture medium after 48 h (F = 4.378, P<0.05). A similar profile was observed in catalase activity. Many round vacuoles were observed in porcine hepatocytes cultured in SVHP. The membranes of these cells became indistinct and almost all the cells died on d 5.CONCLUSION: Plasma from patients with severe hepatitis inhibits the growth, injures membrane, disturbs GSH homeostasis and induces morphological changes of porcine hepatocytes. It is suggested that SVHP should be pretreated to reduce the toxin load and improve the performance of porcine hepatocytes in extracorporeal liver-support devices.

  7. Instant Blood-Mediated Inflammatory Reaction in Hepatocyte Transplantation: Current Status and Future Perspectives.

    Science.gov (United States)

    Lee, Charlotte A; Dhawan, Anil; Smith, Richard A; Mitry, Ragai R; Fitzpatrick, Emer

    2016-01-01

    Hepatocyte transplantation (HT) is emerging as a promising alternative to orthotopic liver transplantation (OLT) in patients with certain liver-based metabolic disease and acute liver failure. Hepatocytes are generally infused into the portal venous system, from which they migrate into the liver cell plates of the native organ. One of the major hurdles to the sustained success of this therapy is early cell loss, with up to 70% of hepatocytes lost immediately following infusion. This is largely thought to be due to the instant blood-mediated inflammatory reaction (IBMIR), resulting in the activation of complement and coagulation pathways. Transplanted hepatocytes produce and release tissue factor (TF), which activates the coagulation pathway, leading to the formation of thrombin and fibrin clots. Thrombin can further activate a number of complement proteins, leading to the activation of the membrane attack complex (MAC) and subsequent hepatocyte cell death. Inflammatory cells including granulocytes, monocytes, Kupffer cells, and natural killer (NK) cells have been shown to cluster around transplanted hepatocytes, leading to their rapid clearance shortly after transplantation. Current research aims to improve cell engraftment and prevent early cell loss. This has been proven successful in vitro using pharmacological interventions such as melagatran, low-molecular-weight dextran sulphate, and N-acetylcysteine (NAC). Effective inhibition of IBMIR would significantly improve hepatocyte engraftment, proliferation, and function, providing successful treatment for patients with liver-based metabolic diseases. PMID:26996786

  8. SIRT1 Disruption in Human Fetal Hepatocytes Leads to Increased Accumulation of Glucose and Lipids.

    Directory of Open Access Journals (Sweden)

    Takamasa Tobita

    Full Text Available There are unprecedented epidemics of obesity, such as type II diabetes and non-alcoholic fatty liver diseases (NAFLD in developed countries. A concerning percentage of American children are being affected by obesity and NAFLD. Studies have suggested that the maternal environment in utero might play a role in the development of these diseases later in life. In this study, we documented that inhibiting SIRT1 signaling in human fetal hepatocytes rapidly led to an increase in intracellular glucose and lipids levels. More importantly, both de novo lipogenesis and gluconeogenesis related genes were upregulated upon SIRT1 inhibition. The AKT/FOXO1 pathway, a major negative regulator of gluconeogenesis, was decreased in the human fetal hepatocytes inhibited for SIRT1, consistent with the higher level of gluconeogenesis. These results indicate that SIRT1 is an important regulator of lipid and carbohydrate metabolisms within human fetal hepatocytes, acting as an adaptive transcriptional response to environmental changes.

  9. SIRT1 Disruption in Human Fetal Hepatocytes Leads to Increased Accumulation of Glucose and Lipids

    Science.gov (United States)

    Tobita, Takamasa; Guzman-Lepe, Jorge; Takeishi, Kazuki; Nakao, Toshimasa; Wang, Yang; Meng, Fanying; Deng, Chu-Xia; Collin de l’Hortet, Alexandra; Soto-Gutierrez, Alejandro

    2016-01-01

    There are unprecedented epidemics of obesity, such as type II diabetes and non-alcoholic fatty liver diseases (NAFLD) in developed countries. A concerning percentage of American children are being affected by obesity and NAFLD. Studies have suggested that the maternal environment in utero might play a role in the development of these diseases later in life. In this study, we documented that inhibiting SIRT1 signaling in human fetal hepatocytes rapidly led to an increase in intracellular glucose and lipids levels. More importantly, both de novo lipogenesis and gluconeogenesis related genes were upregulated upon SIRT1 inhibition. The AKT/FOXO1 pathway, a major negative regulator of gluconeogenesis, was decreased in the human fetal hepatocytes inhibited for SIRT1, consistent with the higher level of gluconeogenesis. These results indicate that SIRT1 is an important regulator of lipid and carbohydrate metabolisms within human fetal hepatocytes, acting as an adaptive transcriptional response to environmental changes. PMID:26890260

  10. Preparation and property analysis of a hepatocyte targeting pH-sensitive liposome

    Institute of Scientific and Technical Information of China (English)

    Si-Yuan Wen; Xiao-Hong Wang; Li Lin; Wei Guan; Sheng-Qi Wang

    2004-01-01

    AIM: To develop a hepatocyte targeting pH-sensitive liposome for drug delivery based on active targeting technology mediated by asialoglycoprotein receptors.METHODS: Four types of targeting molecules with galactose residue were synthesized and mixed with pH-sensitive lipids DC-chol/DOPE to prepare liposome with integrated property of hepatocyte specificity and pH sensitivity. Liposome 18-gal was selected with the best transfection activity through cellular uptake experiment. Property analysis was made through experiments of competitive inhibition of receptors,red blood cell hemolysis,in vitro cytotoxicity test by MTS assay and mediation of inhibitory effects of antisense phosphorothioate ODN on gene expression, etc.RESULTS: Liposome L8-gal had the desired properties of hepatocyte specificity, pH sensitivity, low cytotoxicity, and high transfection efficiency.CONCLUSION: Liposome 18-gal can be further developed as a potential hepatocyte- targeting delivery system.

  11. Expressions and significances of HGF and c-Met in the malignant transformation of nasal inverted papilloma%HGF、c-Met鼻内翻性乳头瘤恶变中的表达变化及其意义

    Institute of Scientific and Technical Information of China (English)

    段晓辉; 于艳芳; 张艳丽; 王延臣; 裴志萍; 康震; 乔国勇

    2012-01-01

    目的:分析肝细胞生长因子(HGF)与其受体(c-Met)在不同期鼻内翻性乳头瘤中的表达变化,探讨其在鼻内翻性乳头瘤恶变中的作用.方法:选择我院2005年1月至2010年12月间确诊为鼻内翻性乳头状瘤的55例及鼻腔恶性乳头状瘤30例作为实验对象,所有患者均行内镜下切除病变组织,对手术切除组织采用免疫组织化学方法SP法检测HGF、c-Met的表达,并统计其染色得分,比较鼻内翻性乳头瘤组织、癌旁组织与恶性组织的HGF、c-Met表达.分析不同分型的鼻内翻性乳头瘤的HGF、c-Met的表达变化.结果:HGF、c-Met在鼻内翻性乳头瘤组织、癌旁组织与恶性组织中的表达变化一致,均为恶性组织高于鼻内翻性乳头瘤组织与癌旁组织,癌旁组织的表达最低.鼻内翻性乳头瘤的T1期HGF表达明显低于T3期(P<0.05),T4期(P<0.01),c-Met表达T1期明显低于T3、T4期(P<0.01).结论:HGF、c-Met在鼻内翻性乳头瘤中的表达与其不同分期有关,HGF、c-Met可能促进鼻内翻性乳头的恶变.%Objective To analysis the expressions of HGF and c-Met in different stages of intranasal inverted papilloma and try to explore the effects of HGF and c-Met in the malignant transformation of nasal inverted papilloma. Methods 30 cases with confirmed malignant papilloma and 55 cases with nasal inverted papilloma in our hospital from Jan 2005 to Dec 2010 were received as experimental objects. All the patients underwent endoscopic removal of diseased tissue. The expressions of HGF and c-Met in nasal inverted papilloma tissue, adjacent tissues and malignant tissues were determined by immunohistochemical method, and the stain score of each sample was calculated. The expressions of HGF and c-Met in different stages of nasal inverted papilloma were analyzed. Results The staining scores of HGF and c-Met in malignant tissue were higher than those in nasal inverted papilloma tissues and the adjacent tissues, the expressions

  12. Inhibitory effects of berberine on ion channels of rat hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Fang Wang; Hong-Yi Zhou; Gang Zhao; Li-Ying Fu; Lan Cheng; Jian-Guo Chen; Wei-Xing Yao

    2004-01-01

    AIM: To examine the effects of berberine, an isoquinoline alkaloid with a long history used as a tonic remedy for liver and heart, on ion channels of isolated rat hepatocytes.METHODS: Tight-seal whole-cell patch-clamp techniques were performed to investigate the effects of berberine on the delayed outward potassium currents (IK), inward rectifier potassium currents (IK1) and Ca2+ release-activated Ca2+currents (ICRAC) in enzymatically isolated rat hepatocytes.RESULTS: Berberine 1-300 nmol/L reduced IK in a concentration dependent manner with EC50 of 38.86±5.37 μmol/L and nH of 0.82±0.05 (n = 8). When the bath solution was changed to tetraethylammonium (TEA) 8 mmol/L, IK was inhibited.Berberine 30 μmol/L reduced IK at all examined membrane potentials, especially at potentials positive to +60 mV (n = 8,P<0.05 or P<0.01 vs control). Berberine had mild inhibitory effects on IK1 in rat hepatocytes. Berberine 1-300 μmol/L also inhibited ICRAC in a concentration-dependent fashion.The fitting parameters were EC50 = 47.20±10.86 μmol/L,nH = 0.71±0.09 (n = 8). The peak value of ICRAC in the Ⅰ-Ⅴrelationship was decreased by berberine 30 μmol/L at potential negative to -80 mV (n = 8, P<0.05 vscontrol). But the reverse potential of ICRAC occurred at voltage 0 mV in all cells.CONCLUSION: Berberine has inhibitory effects on potassium and calcium currents in isolated rat hepatocytes, which may be involved in hepatoprotection.

  13. Hepatocyte Growth Factor Effects on Mesenchymal Stem Cells Derived from Human Arteries: A Novel Strategy to Accelerate Vascular Ulcer Wound Healing

    Directory of Open Access Journals (Sweden)

    Sabrina Valente

    2016-01-01

    Full Text Available Vascular ulcers are a serious complication of peripheral vascular disease, especially in diabetics. Several approaches to treat the wounds are proposed but they show poor outcomes and require long healing times. Hepatocyte Growth Factor/Scatter Factor (HGF/SF is a pleiotropic cytokine exerting many biological activities through the c-Met receptor. This study was aimed at verifying whether HGF/SF influences proliferation, migration, and angiogenesis on mesenchymal stem cells isolated from human arteries (hVW-MSCs. hVW-MSCs were exposed to NIBSC HGF/SF (2.5, 5, 10, and 70 ng/mL from 6 hrs to 7 days. HGF and c-MET mRNA and protein expression, cell proliferation (Alamar Blue and Ki–67 assay, migration (scratch and transwell assays, and angiogenesis (Matrigel were investigated. hVW-MSCs displayed stemness features and expressed HGF and c-MET. HGF/SF did not increase hVW-MSC proliferation, whereas it enhanced the cell migration, the formation of capillary-like structures, and the expression of angiogenic markers (vWF, CD31, and KDR. The HGF/SF effects on hVW-MSC migration and angiogenic potential are of great interest to accelerate wound healing process. Local delivery of HGF/SF could therefore improve the healing of unresponsive vascular ulcers.

  14. Hepatocyte Growth Factor Effects on Mesenchymal Stem Cells Derived from Human Arteries: A Novel Strategy to Accelerate Vascular Ulcer Wound Healing.

    Science.gov (United States)

    Valente, Sabrina; Ciavarella, Carmen; Pasanisi, Emanuela; Ricci, Francesca; Stella, Andrea; Pasquinelli, Gianandrea

    2016-01-01

    Vascular ulcers are a serious complication of peripheral vascular disease, especially in diabetics. Several approaches to treat the wounds are proposed but they show poor outcomes and require long healing times. Hepatocyte Growth Factor/Scatter Factor (HGF/SF) is a pleiotropic cytokine exerting many biological activities through the c-Met receptor. This study was aimed at verifying whether HGF/SF influences proliferation, migration, and angiogenesis on mesenchymal stem cells isolated from human arteries (hVW-MSCs). hVW-MSCs were exposed to NIBSC HGF/SF (2.5, 5, 10, and 70 ng/mL) from 6 hrs to 7 days. HGF and c-MET mRNA and protein expression, cell proliferation (Alamar Blue and Ki-67 assay), migration (scratch and transwell assays), and angiogenesis (Matrigel) were investigated. hVW-MSCs displayed stemness features and expressed HGF and c-MET. HGF/SF did not increase hVW-MSC proliferation, whereas it enhanced the cell migration, the formation of capillary-like structures, and the expression of angiogenic markers (vWF, CD31, and KDR). The HGF/SF effects on hVW-MSC migration and angiogenic potential are of great interest to accelerate wound healing process. Local delivery of HGF/SF could therefore improve the healing of unresponsive vascular ulcers.

  15. A novel herbal formulation "LiverCare" differentially regulates primary rat hepatocyte and hepatocarcinoma cell proliferation in vitro.

    Science.gov (United States)

    Vidyashankar, Satyakumar; Varma, Sandeep R; Azeemudin, Mohammed; Godavarthi, Ashok; Krishna, Nandakumar S; Patki, Pralhad Sadashiv

    2011-09-01

    Hepatocyte growth factor (HGF) plays an important role in hepatocyte proliferation. HGF expression is regulated by various signaling molecules and nuclear receptors. In the present study, LiverCare(®) (LC), a novel polyherbal formulation (The Himalaya Drug Company, Bangalore, India), was evaluated for its efficacy, using co-cultures of primary rat hepatocytes-non-parenchymal cells (NPCs) and human hepatocellular carcinoma cells (HepG2). The rate of primary hepatocyte co-culture proliferation was significantly and dose-dependently increased by LC as determined by [(3)H]thymidine incorporation into newly synthesized DNA and cell proliferation assay. LC also increased HGF expression in primary hepatocyte co-culture. Albumin and urea content remained constant during proliferation of hepatocyte co-cultures in the presence of LC with decreased activity of alanine aminotransferase. It is interesting that LC inhibited incorporation of [(3)H]thymidine into DNA in HepG2 cells. LC enhanced peroxisome proliferator-activated receptor-α expression during hepatocyte proliferation, whereas tumor necrosis factor-α expression remained unaffected. In conclusion, our study clearly showed that LC differentially regulates primary rat hepatocytes and human hepatocarcinoma cell proliferation. LC may be a promising candidate for treating degenerative liver diseases by enhancing liver regeneration. PMID:21812649

  16. CM2 antigen, a potential novel molecule participating in glucuronide transport on rat hepatocyte canalicular membrane

    Directory of Open Access Journals (Sweden)

    L. Wang

    2012-06-01

    Full Text Available The polarized molecules predominately distributing at hepatocyte canalicular surface play a vital role in disclosing the process of bile formation and etiopathogenisis of cholestatic live diseases. Therefore, it is important to find novel polarized molecules on hepatocyte canalicular membrane. In the present study, canalicular membrane vesicles (CMVs isolated from rat hepatocyte by density gradient centrifugation were used as immunogens to produce hybridoma and 46 strains of monoclonal antibodies (mAb against CMVs were obtained. With a series of morphological assay methods, including immunohistochemistry, immunofluorescence and immuno-electron microscope, the antigens recognized by canalicular mAb1 (CM1 and canalicular mAb2 (CM2 were confirmed to predominately distribute at hepatocyte canalicular membrane. Transport activity assay revealed that CM2 could inhibit ATP-dependent E217βG uptake of rat hepatocyte CMVs. Meanwhile, Western blotting analysis showed that the molecular mass of CM2 antigen was approximately 110kDa, which was much less than Mr 180kDa of multidrug resistance-associated protein 2 (MRP2 involved in glucuronide transport. These data indicated that CM2 antigen might be a potential novel molecule participating in glucuronide transport on the hepatocyte canalicular membrane.

  17. Expression of CCR5 and c-Met in Breast Cancer and Its Significance%乳腺癌中CCR5和c-Met的表达及意义

    Institute of Scientific and Technical Information of China (English)

    张玉文; 王丹; 刘亚

    2014-01-01

    Objective To study the signiifcance of CCR5 and c-Met expression in breast cancer.Methods The expression of CCR5 and c-Met in 45 cases of breast cancer was analyzed by using immunohistochemisty. Results CCR5 was expressed in 55.6% of cancerous breast tissue (25/45), whereas it was only expressed in 8.9% of normal breast tissue (4/45). Signiifcant difference was noted between the expression levels (P<0.01). c-Met was expressed in 51% of cancerous breast tissue (23/45), but only 6.6% (3/45) of normal tissue. The observed difference in expression level of c-Met was also statistically signiifcant (P<0.01). On the other hand, 44.4% (20/45) of breast cancer patients with lymph node metastasis showed co-expression of both c-Met and CCR5, compared to 22.2% (10/45) in the normal breast tissue. Our ifndings demonstrate signiifcant associations between the expression of c-Met, CCR5 and lymph node metastasis in breast cancer patients. Conclusion CCR5 and c-Met expression was associated with poor prognosis of breast cancer patients, which could be predict the prognosis of breast cancer.%目的:探讨乳腺癌组织中CCR5和c-Met的表达及其意义。方法采用免疫组化SP法检测45例乳腺癌中CCR5和c-Met的表达。结果乳腺癌组织CCR5阳性率55.6%(25/45),正常乳腺组织中CCR5表达率8.9%(4/45),两种组织阳性表达率差异显著(P<0.01),c-Met的阳性表达率为51%(23/45),正常乳腺组织中c-Met表达率6.6%(3/45),两种组织阳性表达率差异显著(P<0.01)。在伴有淋巴结转移的乳腺癌中c-Met和CCR5的共同阳性率44.4%(20/45),无淋巴结转移的乳腺癌中阳性表达率为22.2%(10/45)。CCR5和c-Met的表达和乳腺癌的淋巴结转移成正相关。结论CCR5和c-Met与提示乳腺癌的转移预后因素有关,可作为预测乳腺癌转移的参考指标之一。

  18. Combination Lopinavir and Ritonavir Alter Exogenous and Endogenous Bile Acid Disposition in Sandwich-Cultured Rat Hepatocytes

    OpenAIRE

    Griffin, LaToya M.; Watkins, Paul B.; Perry, Cassandra H.; Robert L St Claire; Brouwer, Kim L.R.

    2013-01-01

    Inhibition of the bile salt export pump (BSEP) can cause intracellular accumulation of bile acids and is a risk factor for drug-induced liver injury in humans. Antiretroviral protease inhibitors lopinavir (LPV) and ritonavir (RTV) are reported BSEP inhibitors. However, the consequences of LPV and RTV, alone and combined (LPV/r), on hepatocyte viability, bile acid transport, and endogenous bile acid disposition in rat hepatocytes have not been examined. The effect of LPV, RTV, and LPV/r on cel...

  19. Effect of sodium citrate plasma on growth and function of hepatocytes

    Institute of Scientific and Technical Information of China (English)

    CHENG Yong-bo; WANG Ying-jie; CHEN Zhi; ZHANG Shi-chang; CHEN Guo-zhi; LEI Juan

    2006-01-01

    Objective:To observe the effect of sodium citrate plasma (scP) on the growth and function of hepatocytes. Methods: HepG2, fetus and porcine hepatocytes were cultured. The viability, cell cycle and apoptosis, the leakage of LDH, AFP, total protein, glutathione and the changes on morphology of hepatocytes exposured to scP were investigated. Results: (1)Cultured in 10%, 30%, 50%, 100% scP for 24 h,the viability of HepG2 cells was inhibited (F=40. 108, P=0. 001). After 48 h, nearly all cells died except10% scP group. (2)Exposured to scP for 24 h,the percentage of S phase of the cell cycle was significantly increased and apoptosis was also significantly increased compared to control cultures. (3) The leakages of LDH were increased in the HepG2, fetus and porcine hepatocytes following exposure to scP for 5 h. (4)The synthesis of AFP in fetus and porcine hepatocytes were inhibited in medium containing 10% scP for 3d (t values were 8.1902, 5. 1034 separately, P<0. 01). Exposure of HepG2 cells to scP within 24 h resulted in a decrease in the total protein synthesis and a increase inthe GSH content. (5)Most of HepG2, fetus and porcine hepatocytes died in all except 10% scP groups after 24 h exposed to scP. Conclusion :scP can damage hepatocytes, which results from citric acid and sodium citrate contained in the fluid of blood maintenance.

  20. Uptake of albumin nanoparticle surface modified with glycyrrhizin by primary cultured rat hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Sheng-Jun Mao; Shi-Xiang Hou; Ru He; Liang-Ke Zhang; Da-Peng Wei; Yue-Qi Bi; Hui Jin

    2005-01-01

    AIM: To investigate the uptake difference between bovine serum albumin nanoparticle (BSA-NP) and bovine serum albumin nanoparticles with their surface modified byglycyrrhizin (BSA-NP-GL) and to develop a novel hepatocyte targeting BSA-NP-GL based on active targeting technology mediated by specific binding site of GL on rat cellular membrane. METHODS: Calcein loaded bovine serum albumin nanoparticles (Cal-BSA-NP) were prepared by desolvation process. Glycyrrhizin was conjugated to the surface reactive amino groups (SRAG) of Cal-BSA-NP by sodium periodate oxidization, which resulted in calcein-loaded bovine serum albumin nanoparticles with their surface modified by glycyrrhizin (Cal-BSA-NP-GL). The morphology of the two types of prepared nanoparticles (NP) was observed by transmission electron microscopy. The diameter of NP was measured with a laser particle size analyzer. The interaction between Cal-BSA-NP-GL and primary cultured hepatocytes was studied through cellular uptake experiments. The uptake amount of Cal-BSA-NPGL and Cal-BSA-NP by rat hepatocytes was determinedby fluorospectrophotometry. Uptake characteristics were investigated through experiments of competitive inhibition of specific binding site of GL. RESULTS: Both Cal-BSA-NP-GL and Cal-BSA-NP had regular spherical surfaces. The average diameter of CalBSA-NP-GL and Cal-BSA-NP was 77 and 79 nm respectively. The uptake amount of the two NP by hepatocytes reached its maximum at 2 h after incubation. The uptake amount of Cal-BSA-NP-GL by rat hepatocytes was 4.43-fold higher than that of Cal-BSA-NP. There was a significant difference in the uptake of Cal-BSA-NP-GL and Cal-BSA-NP by hepatocytes (P<0.01). The uptake of Cal-BSA-NP-GL was inhibited when GL was added previously to isolated rat hepatocytes, and the uptake of Cal-BSA-NP was not affected by GL.CONCLUSION: A binding site of GL is present on the surface of rat hepatocytes, BSA-NP-GL may be internalized via this site by hepatocytes and can be used as

  1. Insulin-induced CARM1 upregulation facilitates hepatocyte proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Yeom, Chul-gon; Kim, Dong-il; Park, Min-jung; Choi, Joo-hee [College of Veterinary Medicine, Chonnam National University, Gwangju 500-757 (Korea, Republic of); Jeong, Jieun; Wi, Anjin; Park, Whoashig [Jeollanamdo Forest Resources Research Institute, Naju 520-833 (Korea, Republic of); Han, Ho-jae [College of Veterinary Medicine, Seoul National University, Seoul 151-741 (Korea, Republic of); Park, Soo-hyun, E-mail: parksh@chonnam.ac.kr [College of Veterinary Medicine, Chonnam National University, Gwangju 500-757 (Korea, Republic of)

    2015-06-05

    Previously, we reported that CARM1 undergoes ubiquitination-dependent degradation in renal podocytes. It was also reported that CARM1 is necessary for fasting-induced hepatic gluconeogenesis. Based on these reports, we hypothesized that treatment with insulin, a hormone typically present under the ‘fed’ condition, would inhibit gluconeogenesis via CARM1 degradation. HepG2 cells, AML-12 cells, and rat primary hepatocytes were treated with insulin to confirm CARM1 downregulation. Surprisingly, insulin treatment increased CARM1 expression in all cell types examined. Furthermore, treatment with insulin increased histone 3 methylation at arginine 17 and 26 in HepG2 cells. To elucidate the role of insulin-induced CARM1 upregulation, the HA-CARM1 plasmid was transfected into HepG2 cells. CARM1 overexpression did not increase the expression of lipogenic proteins generally increased by insulin signaling. Moreover, CARM1 knockdown did not influence insulin sensitivity. Insulin is known to facilitate hepatic proliferation. Like insulin, CARM1 overexpression increased CDK2 and CDK4 expression. In addition, CARM1 knockdown reduced the number of insulin-induced G2/M phase cells. Moreover, GFP-CARM1 overexpression increased the number of G2/M phase cells. Based on these results, we concluded that insulin-induced CARM1 upregulation facilitates hepatocyte proliferation. These observations indicate that CARM1 plays an important role in liver pathophysiology. - Highlights: • Insulin treatment increases CARM1 expression in hepatocytes. • CARM1 overexpression does not increase the expression of lipogenic proteins. • CARM1 knockdown does not influence insulin sensitivity. • Insulin-induced CARM1 upregulation facilitates hepatocyte proliferation.

  2. 18F-FDG与11C-MET PET-CT显像对磁共振成像中环形强化脑肿瘤和非肿瘤性病变的鉴别诊断%Value of 18F-FDG and 11C-MET PET-CT in differentiation of brain ringlike-enhanced neoplastic and non-neoplastic lesions on MRI hnaging

    Institute of Scientific and Technical Information of China (English)

    蔡莉; 高硕; 李大成; 李彦生; 陈秋松; 邢喜玲

    2009-01-01

    Objective To evaluate the value of 18F-FDG and 11C-MET PET-CT scan in differentiation of brain ringiike-enhanced lesions on MRI imaging. Methods Forty-one brain ringlike-enhanced lesions on MRI imaging including 30 brain tumors and 11 non-neoplastic lesions confirmed pathologically or clinically underwent 18F-FDG and 11C-MET PET-CT brain scan. Among them, 15 patients who were suspected to have brain metastasis received body scan by 18F-FDG PET-CT. Both images were analyzed visually and semi-quantitatively. Results Visual analysis: for brain tumors the diagnostic sensitivity, specificity and accuracy of 18F-FDG PET-CT was 53.3%, 72.7%, 58.5%, versus 96.7%, 90.9%, 95.1% of 11C-MET PET-CT, respectively. All the primary foci in 9 patients with brain metastases were detected by body 18F-FDG PET-CT scan. Semiquantitative analysis: There was a significant difference in the uptake between highly differentiated malignant and poorly differentiated tumors as well as non-neoplastic lesions for both tracers (P0.05). Conclusion Both 18F-FDG and 11C-MET PET-CT are useful in differentiation of brain ringlike-enhanced lesions on MRI imaging. 11C-MET PET-CT is more helpful than 18F-FDG PET-CT in differential diagnosis of low-grade neoplastic from non-neoplastic lesions. Combination of 18F-FDG and 11C-MET PET-CT scans can improve the accuracy of differential diagnosis for brain ringlike-enhanced lesions on MRI imaging.%目的 探讨18F-FDG与11C-MET PET-CT显像对常规磁共振成像(MRI)不易鉴别的环形强化脑肿瘤与非肿瘤性病变的鉴别诊断价值.方法 对41例病史及临床体征不典型、MRI上呈相似明显环形强化的脑内病变患者,分别行18F-FDG及11C-MET PET-CT头部扫描,其中15例不除外脑转移瘤者加行18F-FDG PET-CT体部扫描,并对扫描结果进行视觉分析和半定量分析.经病理或临床证实为肿瘤性病变30例,非肿瘤性病变11例.结果 视觉分析结果显示,18F-FDG PET-CT正确诊断肿瘤性病变16

  3. Clinical applications of hepatocyte transplantation

    Institute of Scientific and Technical Information of China (English)

    Giada Pietrosi; Giovanni Battista Vizzini; Salvatore Gruttadauria; Bruno Gridelli

    2009-01-01

    The shortage of organ donors is a problem worldwide, with approximately 15% of adult patients with lifethreatening liver diseases dying while on the waiting list. The use of cell transplantation for liver disease is an attempt to correct metabolic defects, or to support liver function as a bridge to liver transplantation and, as such, has raised a number of expectations. Most of the available studies briefly reported here focus on adult hepatocyte transplantation (HT), and the results are neither reproducible nor comparable, because the means of infusion, amount of injected cells and clinical variability differ among the studies. To better understand the specific role of HT in the management of end-stage liver disease, it is important that controlled studies, designed on the principles of evidence-based medicine, be done in order to guarantee the reproducibility of results.

  4. Primary monolayer culture of adult mouse hepatocytes

    International Nuclear Information System (INIS)

    Primary monolayer cultures of adult mouse hepatocytes isolated by collagenase perfusion of the liver in situ were exposed to 2 hepatotropic viruses, an avian influenza A virus adapted to grow in mouse liver in vivo and a herpes simplex type I virus. Influenza virus infection led to lysis of individual hepatocytes and total monolayer destruction within 18 to 120 hours after infection according to the virus dose used. Virus replication was evidenced by assaying hepatocyte supernates for hemagglutinin and infectivity, immunofluorescent staining and by electron microscopy. Herpes virus infection resulted in polykaryocyte formation followed by nuclear pycnosis and cell lysis. Virus replication was assayed by titration of supernate infectivity. (auth.)

  5. Role of hepatocyte nuclear factor 4α (HNF4α) in cell proliferation and cancer.

    Science.gov (United States)

    Walesky, Chad; Apte, Udayan

    2015-01-01

    Hepatocyte nuclear factor 4α (HNF4α) is an orphan nuclear receptor commonly known as the master regulator of hepatic differentiation, owing to the large number of hepatocyte-specific genes it regulates. Whereas the role of HNF4α in hepatocyte differentiation is well recognized and extensively studied, its role in regulation of cell proliferation is relatively less known. Recent studies have revealed that HNF4α inhibits proliferation not only of hepatocytes but also cells in colon and kidney. Further, a growing number of studies have demonstrated that inhibition or loss of HNF4α promotes tumorigenesis in the liver and colon, and reexpression of HNF4α results in decreased cancer growth. Studies using tissue-specific conditional knockout mice, knock-in studies, and combinatorial bioinformatics of RNA/ChIP-sequencing data indicate that the mechanisms of HNF4α-mediated inhibition of cell proliferation are multifold, involving epigenetic repression of promitogenic genes, significant cross talk with other cell cycle regulators including c-Myc and cyclin D1, and regulation of miRNAs. Furthermore, studies indicate that posttranslational modifications of HNF4α may change its activity and may be at the core of its dual role as a differentiation factor and repressor of proliferation. This review summarizes recent findings on the role of HNF4α in cell proliferation and highlights the newly understood function of this old receptor.

  6. On the thyroid hormone-induced increase in respiratory capacity of isolated rat hepatocytes.

    Science.gov (United States)

    Gregory, R B; Berry, M N

    1991-12-01

    The respiratory capacities of hepatocytes, derived from hypothyroid, euthyroid and hyperthyroid rats, have been compared by measuring rates of oxygen uptake and by titrating components of the respiratory chain with specific inhibitors. Thyroid hormone increased the maximal rate of substrate-stimulated respiration and also increased the degree of ionophore-stimulated oxygen uptake. In titration experiments, similar concentrations of oligomycin or antimycin were required for maximal inhibition of respiration regardless of thyroid state, suggesting that the changes in respiratory capacity were not the result of variation in the amounts of ATP synthase or cytochrome b. However, less rotenone was required for maximal inhibition of respiration in the hypothyroid state than in cells from euthyroid or hyperthyroid rats, implying that hepatocytes from hypothyroid animals contain less NADH dehydrogenase. The concentration of carboxyatractyloside necessary for maximal inhibition of respiration was 100 microM in hepatocytes from hypothyroid rats, but 200 microM and 300 microM in hepatocytes from euthyroid and hyperthyroid rats, respectively, indicating a possible correlation between levels of thyroid hormone and the amount or activity of adenine nucleotide translocase. The increased capacity for coupled respiration in response to thyroid hormone is not associated with an increase in the components of the electron transport chain or ATP synthase, but correlates with an increased activity of adenine nucleotide translocase. PMID:1751550

  7. Metformin protects rat hepatocytes against bile acid-induced apoptosis.

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    Titia E Woudenberg-Vrenken

    Full Text Available BACKGROUND: Metformin is used in the treatment of Diabetes Mellitus type II and improves liver function in patients with non-alcoholic fatty liver disease (NAFLD. Metformin activates AMP-activated protein kinase (AMPK, the cellular energy sensor that is sensitive to changes in the AMP/ATP-ratio. AMPK is an inhibitor of mammalian target of rapamycin (mTOR. Both AMPK and mTOR are able to modulate cell death. AIM: To evaluate the effects of metformin on hepatocyte cell death. METHODS: Apoptotic cell death was induced in primary rat hepatocytes using either the bile acid glycochenodeoxycholic acid (GCDCA or TNFα in combination with actinomycin D (actD. AMPK, mTOR and phosphoinositide-3 kinase (PI3K/Akt were inhibited using pharmacological inhibitors. Apoptosis and necrosis were quantified by caspase activation, acridine orange staining and Sytox green staining respectively. RESULTS: Metformin dose-dependently reduces GCDCA-induced apoptosis, even when added 2 hours after GCDCA, without increasing necrotic cell death. Metformin does not protect against TNFα/ActD-induced apoptosis. The protective effect of metformin is dependent on an intact PI3-kinase/Akt pathway, but does not require AMPK/mTOR-signaling. Metformin does not inhibit NF-κB activation. CONCLUSION: Metformin protects against bile acid-induced apoptosis and could be considered in the treatment of chronic liver diseases accompanied by inflammation.

  8. The DNA replication licensing factor miniature chromosome maintenance 7 is essential for RNA splicing of epidermal growth factor receptor, c-Met, and platelet-derived growth factor receptor.

    Science.gov (United States)

    Chen, Zhang-Hui; Yu, Yan P; Michalopoulos, George; Nelson, Joel; Luo, Jian-Hua

    2015-01-16

    Miniature chromosome maintenance 7 (MCM7) is an essential component of DNA replication licensing complex. Recent studies indicate that MCM7 is amplified and overexpressed in a variety of human malignancies. In this report, we show that MCM7 binds SF3B3. The binding motif is located in the N terminus (amino acids 221-248) of MCM7. Knockdown of MCM7 or SF3B3 significantly increased unspliced RNA of epidermal growth factor receptor, platelet-derived growth factor receptor, and c-Met. A dramatic drop of reporter gene expression of the oxytocin exon 1-intron-exon 2-EGFP construct was also identified in SF3B3 and MCM7 knockdown PC3 and DU145 cells. The MCM7 or SF3B3 depleted cell extract failed to splice reporter RNA in in vitro RNA splicing analyses. Knockdown of SF3B3 and MCM7 leads to an increase of cell death of both PC3 and DU145 cells. Such cell death induction is partially rescued by expressing spliced c-Met. To our knowledge, this is the first report suggesting that MCM7 is a critical RNA splicing factor, thus giving significant new insight into the oncogenic activity of this protein.

  9. Parvovirus B19-Induced Apoptosis of Hepatocytes

    OpenAIRE

    Poole, Brian D.; Karetnyi, Yuory V.; Naides, Stanley J.

    2004-01-01

    Parvovirus B19 (B19 virus) can persist in multiple tissues and has been implicated in a variety of diseases, including acute fulminant liver failure. The mechanism by which B19 virus induces liver failure remains unknown. Hepatocytes are nonpermissive for B19 virus replication. We previously reported that acute fulminant liver failure associated with B19 virus infection was characterized by hepatocellular dropout. We inoculated both primary hepatocytes and the hepatocellular carcinoma cell li...

  10. 5α-Reductase Type 2 Regulates Glucocorticoid Action and Metabolic Phenotype in Human Hepatocytes.

    Science.gov (United States)

    Nasiri, Maryam; Nikolaou, Nikolaos; Parajes, Silvia; Krone, Nils P; Valsamakis, George; Mastorakos, George; Hughes, Beverly; Taylor, Angela; Bujalska, Iwona J; Gathercole, Laura L; Tomlinson, Jeremy W

    2015-08-01

    Glucocorticoids and androgens have both been implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD); androgen deficiency in males, androgen excess in females, and glucocorticoid excess in both sexes are associated with NAFLD. Glucocorticoid and androgen action are regulated at a prereceptor level by the enzyme 5α-reductase type 2 (SRD5A2), which inactivates glucocorticoids to their dihydrometabolites and converts T to DHT. We have therefore explored the role of androgens and glucocorticoids and their metabolism by SRD5A2 upon lipid homeostasis in human hepatocytes. In both primary human hepatocytes and human hepatoma cell lines, glucocorticoids decreased de novo lipogenesis in a dose-dependent manner. Whereas androgen treatment (T and DHT) increased lipogenesis in cell lines and in primary cultures of human hepatocytes from female donors, it was without effect in primary hepatocyte cultures from men. SRD5A2 overexpression reduced the effects of cortisol to suppress lipogenesis and this effect was lost following transfection with an inactive mutant construct. Conversely, pharmacological inhibition using the 5α-reductase inhibitors finasteride and dutasteride augmented cortisol action. We have demonstrated that manipulation of SRD5A2 activity can regulate lipogenesis in human hepatocytes in vitro. This may have significant clinical implications for those patients prescribed 5α-reductase inhibitors, in particular augmenting the actions of glucocorticoids to modulate hepatic lipid flux. PMID:25974403

  11. Evaluation of Azathioprine-Induced Cytotoxicity in an In Vitro Rat Hepatocyte System

    Directory of Open Access Journals (Sweden)

    Abdullah Al Maruf

    2014-01-01

    Full Text Available Azathioprine (AZA is widely used in clinical practice for preventing graft rejection in organ transplantations and various autoimmune and dermatological diseases with documented unpredictable hepatotoxicity. The potential molecular cytotoxic mechanisms of AZA towards isolated rat hepatocytes were investigated in this study using “Accelerated Cytotoxicity Mechanism Screening” techniques. The concentration of AZA required to cause 50% cytotoxicity in 2 hrs at 37°C was found to be 400 μM. A significant increase in AZA-induced cytotoxicity and reactive oxygen species (ROS formation was observed when glutathione- (GSH- depleted hepatocytes were used. The addition of N-acetylcysteine decreased cytotoxicity and ROS formation. Xanthine oxidase inhibition by allopurinol decreased AZA-induced cytotoxicity, ROS, and hydrogen peroxide (H2O2 formation and increased % mitochondrial membrane potential (MMP. Addition of N-acetylcysteine and allopurinol together caused nearly complete cytoprotection against AZA-induced hepatocyte death. TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl, a known ROS scavenger and a superoxide dismutase mimic, and antioxidants, like DPPD (N,N′-diphenyl-p-phenylenediamine, Trolox (a water soluble vitamin E analogue, and mesna (2-mercaptoethanesulfonate, also decreased hepatocyte death and ROS formation. Results from this study suggest that AZA-induced cytotoxicity in isolated rat hepatocytes may be partly due to ROS formation and GSH depletion that resulted in oxidative stress and mitochondrial injury.

  12. Evaluation of azathioprine-induced cytotoxicity in an in vitro rat hepatocyte system.

    Science.gov (United States)

    Al Maruf, Abdullah; Wan, Luke; O'Brien, Peter J

    2014-01-01

    Azathioprine (AZA) is widely used in clinical practice for preventing graft rejection in organ transplantations and various autoimmune and dermatological diseases with documented unpredictable hepatotoxicity. The potential molecular cytotoxic mechanisms of AZA towards isolated rat hepatocytes were investigated in this study using "Accelerated Cytotoxicity Mechanism Screening" techniques. The concentration of AZA required to cause 50% cytotoxicity in 2 hrs at 37°C was found to be 400 μM. A significant increase in AZA-induced cytotoxicity and reactive oxygen species (ROS) formation was observed when glutathione- (GSH-) depleted hepatocytes were used. The addition of N-acetylcysteine decreased cytotoxicity and ROS formation. Xanthine oxidase inhibition by allopurinol decreased AZA-induced cytotoxicity, ROS, and hydrogen peroxide (H2O2) formation and increased % mitochondrial membrane potential (MMP). Addition of N-acetylcysteine and allopurinol together caused nearly complete cytoprotection against AZA-induced hepatocyte death. TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl), a known ROS scavenger and a superoxide dismutase mimic, and antioxidants, like DPPD (N,N'-diphenyl-p-phenylenediamine), Trolox (a water soluble vitamin E analogue), and mesna (2-mercaptoethanesulfonate), also decreased hepatocyte death and ROS formation. Results from this study suggest that AZA-induced cytotoxicity in isolated rat hepatocytes may be partly due to ROS formation and GSH depletion that resulted in oxidative stress and mitochondrial injury. PMID:25101277

  13. Citotoxicity of Fipronil on Hepatocytes Isolated from Rat and Effects of Its Biotransformation

    Directory of Open Access Journals (Sweden)

    Marieli Guelfi

    2015-12-01

    Full Text Available ABSTRACT The aim of this study was to characterize the mechanism of toxicity of fipronil on hepatocytes isolated from the rat and the effect of its biotransformation on the toxicological potential. The toxicity of fipronil was assessed by monitoring the oxygen consumption and mitochondrial membrane potential, intracellular ATP concentration, Ca2+ homeostasis and cell viability. The cell viability was evaluated by trypan blue exclusion in hepatocytes that were isolated from the normal rats and by the release of the enzymes alanine transaminase and aspartate transaminase in hepatocytes that were isolated from the normal rats or proadifen-pretreated rats. Fipronil reduced mitochondrial respiration in the cells that were energized with glutamate plus malate in a dose-dependent manner and dissipated the mitochondrial membrane potential that was accompanied by a reduction in ATP concentration and a disruption of intracellular Ca2+ homeostasis. The cell viability was affected by fipronil with higher potency in hepatocytes that were isolated from the normal rats, which indicated that the metabolism of this insecticide increased its toxicological potential. The results of this study indicated that the toxicity of fipronil to the hepatocytes was related to the inhibition of mitochondrial activity, which led to decreased ATP synthesis and a consequent alteration in intracellular Ca2+ homeostasis and ultimately resulted in cell death.

  14. Hepatitis C-induced hepatocyte apoptosis following liver transplantation is enhanced by immunosuppressive agents.

    Science.gov (United States)

    Lim, E J; Chin, R; Nachbur, U; Silke, J; Jia, Z; Angus, P W; Torresi, J

    2016-09-01

    In recurrent hepatitis C (HCV) post-liver transplantation (OLT), the combination of immunosuppressants and HCV is postulated to increase hepatocyte apoptosis and liver fibrosis. We evaluated hepatocyte apoptosis within the liver tissue of patients with postOLT HCV recurrence compared to HCV-negative individuals and correlated these findings with the effects of immunosuppressants on HCV-induced cell death and its inhibition in primary mouse hepatocytes (PMoH). Liver biopsies from patients with and without HCV were evaluated by immunohistochemistry for markers of apoptosis M30 CytoDEATH (M30) and cleaved PARP (clPARP). PMoH from C57BL/6 mice were infected with recombinant adenoviruses (rAdHCV) that expressed HCV proteins in hepatocytes. Infected cells were treated with cyclosporine, tacrolimus, sirolimus and/or MMF with or without pan-caspase inhibitor Q-VD-Oph. Cell viability and apoptosis were evaluated using crystal violet assays and Western immunoblots probed for cleaved caspase-3 (clCas3) and clPARP. Both M30 and clPARP were increased in the liver biopsies of patients with postOLT HCV recurrence compared to HCV-negative individuals. Treatment of rAdHCV-infected PMoH with cyclosporine, tacrolimus or sirolimus reduced cell viability and increased clCas3 and clPARP compared to rAdHCV infection alone. Addition of MMF to cyclosporine, tacrolimus or sirolimus further reduced cell viability and increased clCas3 and clPARP. Q-VD-Oph improved cell viability in HCV-infected PMoH treated with immunosuppressants alone and in combination and reduced clCas3 and clPARP by approximately 90%. Immunosuppressive agents, especially in combination, enhanced apoptosis in HCV-infected hepatocytes. The finding that Q-VD-Oph reversed hepatocyte death suggests that treatments utilizing apoptosis inhibition might reduce liver injury in postOLT HCV recurrence. PMID:27167351

  15. Selective insulin resistance in hepatocyte senescence

    International Nuclear Information System (INIS)

    Insulin resistance has been described in association with chronic liver disease for decades. Hepatocyte senescence has been demonstrated in chronic liver disease and as many as 80% of hepatocytes show a senescent phenotype in advanced liver disease. The aim of this study was to understand the role of hepatocyte senescence in the development of insulin resistance. Senescence was induced in HepG2 cells via oxidative stress. The insulin metabolic pathway was studied in control and senescent cells following insulin stimulation. GLUT2 and GLUT4 expressions were studied in HepG2 cells and human liver tissue. Further, GLUT2 and GLUT4 expressions were studied in three independent chronic liver disease cohorts. Signalling impairment distal to Akt in phosphorylation of AS160 and FoxO1 was evident in senescent HepG2 cells. Persistent nuclear localisation of FoxO1 was demonstrated in senescent cells despite insulin stimulation. Increased GLUT4 and decreased GLUT2 expressions were evident in senescent cells, human cirrhotic liver tissue and publically available liver disease datasets. Changes in GLUT expressions were associated with a poor clinical prognosis. In conclusion, selective insulin resistance is evident in senescent HepG2 cells and changes in GLUT expressions can be used as surrogate markers of hepatocyte senescence. - Highlights: • Senescent hepatocytes demonstrate selective insulin resistance. • GLUT changes act as markers of hepatocyte senescence and have prognostic value. • Study offers insight into long noticed intimacy of cirrhosis and insulin resistance

  16. Selective insulin resistance in hepatocyte senescence

    Energy Technology Data Exchange (ETDEWEB)

    Aravinthan, Aloysious [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom); Challis, Benjamin [Institute of Metabolic Sciences, University of Cambridge, Cambridge (United Kingdom); Shannon, Nicholas [Cancer Research UK Cambridge Institute, Cambridge (United Kingdom); Hoare, Matthew [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom); Cancer Research UK Cambridge Institute, Cambridge (United Kingdom); Heaney, Judith [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom); Foundation for Liver Research, Institute of Hepatology, London (United Kingdom); Alexander, Graeme J.M., E-mail: gja1000@doctors.org.uk [Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Cambridge (United Kingdom)

    2015-02-01

    Insulin resistance has been described in association with chronic liver disease for decades. Hepatocyte senescence has been demonstrated in chronic liver disease and as many as 80% of hepatocytes show a senescent phenotype in advanced liver disease. The aim of this study was to understand the role of hepatocyte senescence in the development of insulin resistance. Senescence was induced in HepG2 cells via oxidative stress. The insulin metabolic pathway was studied in control and senescent cells following insulin stimulation. GLUT2 and GLUT4 expressions were studied in HepG2 cells and human liver tissue. Further, GLUT2 and GLUT4 expressions were studied in three independent chronic liver disease cohorts. Signalling impairment distal to Akt in phosphorylation of AS160 and FoxO1 was evident in senescent HepG2 cells. Persistent nuclear localisation of FoxO1 was demonstrated in senescent cells despite insulin stimulation. Increased GLUT4 and decreased GLUT2 expressions were evident in senescent cells, human cirrhotic liver tissue and publically available liver disease datasets. Changes in GLUT expressions were associated with a poor clinical prognosis. In conclusion, selective insulin resistance is evident in senescent HepG2 cells and changes in GLUT expressions can be used as surrogate markers of hepatocyte senescence. - Highlights: • Senescent hepatocytes demonstrate selective insulin resistance. • GLUT changes act as markers of hepatocyte senescence and have prognostic value. • Study offers insight into long noticed intimacy of cirrhosis and insulin resistance.

  17. On the mechanism of metformin-inhibited hepatic gluconeogenesis

    Institute of Scientific and Technical Information of China (English)

    唐红菊

    2014-01-01

    Objective To investigate the underlying molecular mechanisms of metformin in inhibiting hepatic gluconeogenesis.Methods Primary hepatocytes of mice were isolated by a modified version of the collagenase method.The effect of metformin on glucose production in primary hepatocytes was detected by glucose oxidation method.The

  18. Rat hepatocytes weighted gene co-expression network analysis identifies specific modules and hub genes related to liver regeneration after partial hepatectomy.

    Directory of Open Access Journals (Sweden)

    Yun Zhou

    Full Text Available The recovery of liver mass is mainly mediated by proliferation of hepatocytes after 2/3 partial hepatectomy (PH in rats. Studying the gene expression profiles of hepatocytes after 2/3 PH will be helpful to investigate the molecular mechanisms of liver regeneration (LR. We report here the first application of weighted gene co-expression network analysis (WGCNA to analyze the biological implications of gene expression changes associated with LR. WGCNA identifies 12 specific gene modules and some hub genes from hepatocytes genome-scale microarray data in rat LR. The results suggest that upregulated MCM5 may promote hepatocytes proliferation during LR; BCL3 may play an important role by activating or inhibiting NF-kB pathway; MAPK9 may play a permissible role in DNA replication by p38 MAPK inactivation in hepatocytes proliferation stage. Thus, WGCNA can provide novel insight into understanding the molecular mechanisms of LR.

  19. Directed random walks and constraint programming reveal active pathways in hepatocyte growth factor signaling.

    Science.gov (United States)

    Kittas, Aristotelis; Delobelle, Aurélien; Schmitt, Sabrina; Breuhahn, Kai; Guziolowski, Carito; Grabe, Niels

    2016-01-01

    An effective means to analyze mRNA expression data is to take advantage of established knowledge from pathway databases, using methods such as pathway-enrichment analyses. However, pathway databases are not case-specific and expression data could be used to infer gene-regulation patterns in the context of specific pathways. In addition, canonical pathways may not always describe the signaling mechanisms properly, because interactions can frequently occur between genes in different pathways. Relatively few methods have been proposed to date for generating and analyzing such networks, preserving the causality between gene interactions and reasoning over the qualitative logic of regulatory effects. We present an algorithm (MCWalk) integrated with a logic programming approach, to discover subgraphs in large-scale signaling networks by random walks in a fully automated pipeline. As an exemplary application, we uncover the signal transduction mechanisms in a gene interaction network describing hepatocyte growth factor-stimulated cell migration and proliferation from gene-expression measured with microarray and RT-qPCR using in-house perturbation experiments in a keratinocyte-fibroblast co-culture. The resulting subgraphs illustrate possible associations of hepatocyte growth factor receptor c-Met nodes, differentially expressed genes and cellular states. Using perturbation experiments and Answer Set programming, we are able to select those which are more consistent with the experimental data. We discover key regulator nodes by measuring the frequency with which they are traversed when connecting signaling between receptors and significantly regulated genes and predict their expression-shift consistently with the measured data. The Java implementation of MCWalk is publicly available under the MIT license at: https://bitbucket.org/akittas/biosubg.

  20. Modulation of Insulin Sensitivity of Hepatocytes by the Pharmacological Downregulation of Phospholipase D

    Directory of Open Access Journals (Sweden)

    Nataliya A. Babenko

    2015-01-01

    Full Text Available Background. The role of phospholipase D (PLD as a positive modulator of glucose uptake activation by insulin in muscle and adipose cells has been demonstrated. The role of PLD in the regulation of glucose metabolism by insulin in the primary hepatocytes has been determined in this study. Methods. For this purpose, we studied effects of inhibitors of PLD on glucose uptake and glycogen synthesis stimulation by insulin. To determine the PLD activity, the method based on determination of products of transphosphatidylation reaction, phosphatidylethanol or phosphatidylbutanol, was used. Results. Inhibition of PLD by a general antagonist (1-butanol or specific inhibitor, halopemide, or N-hexanoylsphingosine, or by cellular ceramides accumulated in doxorubicin-treated hepatocytes decreased insulin-stimulated glucose metabolism. Doxorubicin-induced hepatocytes resistance to insulin action could be abolished by inhibition of ceramide production. Halopemide could nullify this effect. Addition of propranolol, as well as inhibitors of phosphatidylinositol 3-kinase (PI3-kinase (wortmannin, LY294002 or suppressors of Akt phosphorylation/activity, luteolin-7-O-glucoside or apigenin-7-O-glucoside, to the culture media could block cell response to insulin action. Conclusion. PLD plays an important role in the insulin signaling in the hepatocytes. PLD is activated downstream of PI3-kinase and Akt and is highly sensitive to ceramide content in the liver cells.

  1. Conserved balance of hepatocyte nuclear DNA content in mononuclear and binuclear hepatocyte populations during the course of chronic viral hepatitis

    Institute of Scientific and Technical Information of China (English)

    Hidenori Toyoda; Takashi Kumada; Olivier Bregerie; Christian Brechot; Chantal Desdouets

    2006-01-01

    AIM: To analyze the percentages of hepatocytes with increased nuclear DNA content, i.e., tetraploid (4n) and octoploid (8n) nuclei, and then compared mononuclear and binuclear hepatocyte populations:METHODS: The percentages of mononuclear diploid(2n), 4n, and 8n hepatocytes and those of binuclear 2× 2n, 2 × 4n, and 2 × 8n hepatocytes were determined with a method that can simultaneously measure hepatocyte nuclear DNA content and binuclearity in 62patients with chronic hepatitis B or C. The percentage of 4n and 8n hepatocytes in the mononuclear hepatocyte population was compared with the percentage of 2 ×4n and 2 × 8n hepatocytes in the binuclear hepatocyte population.RESULTS: The percentages of 4n and 8n hepatocytes in mononuclear hepatocytes and 2 × 4n and 2 × 8n hepatocytes in binuclear hepatocytes were similar,regardless of the activity or fibrosis grade of chronic hepatitis and regardless of the infecting virus.CONCLUSION: The distribution of nuclear DNA content within mononuclear and binuclear hepatocyte populations was conserved during the course of chronic viral hepatitis.

  2. Transcriptome of extracellular vesicles released by hepatocytes.

    Directory of Open Access Journals (Sweden)

    Felix Royo

    Full Text Available The discovery that the cells communicate through emission of vesicles has opened new opportunities for better understanding of physiological and pathological mechanisms. This discovery also provides a novel source for non-invasive disease biomarker research. Our group has previously reported that hepatocytes release extracellular vesicles with protein content reflecting the cell-type of origin. Here, we show that the extracellular vesicles released by hepatocytes also carry RNA. We report the messenger RNA composition of extracellular vesicles released in two non-tumoral hepatic models: primary culture of rat hepatocytes and a progenitor cell line obtained from a mouse foetal liver. We describe different subpopulations of extracellular vesicles with different densities and protein and RNA content. We also show that the RNA cargo of extracellular vesicles released by primary hepatocytes can be transferred to rat liver stellate-like cells and promote their activation. Finally, we provide in vitro and in vivo evidence that liver-damaging drugs galactosamine, acetaminophen, and diclofenac modify the RNA content of these vesicles. To summarize, we show that the extracellular vesicles secreted by hepatocytes contain various RNAs. These vesicles, likely to be involved in the activation of stellate cells, might become a new source for non-invasive identification of the liver toxicity markers.

  3. Angiotensin II Induces C-Reactive Protein Expression via AT1-ROS-MAPK-NF-κB Signal Pathway in Hepatocytes

    Directory of Open Access Journals (Sweden)

    Jingjing Zhao

    2013-09-01

    Full Text Available Background: C-reactive protein (CRP participates in development of inflammatory diseases. Hepatocytes are a major contributor of circulating CRP. Although angiotensin II (Ang II is known to evoke inflammatory response, it remains unknown whether Ang II induces CRP expression in hepatocytes. The present study observed effect of Ang II on CRP expression and the related signal pathway in hepatocytes. Methods: mRNA and protein expressions in human hepatocytes were determined with RT-PCR and Western blot respectively. Reactive oxygen species (ROS was measured using a fluorescence probe. CRP in liver and serum of rats was determined by immunohistochemistry and ELISA respectively. Results: Ang II induced mRNA and protein expression of CRP in hepatocytes and increased CRP production in liver and CRP level in serum. Losartan reduced Ang II- induced CRP expression in hepatocytes. Losartan and thenoyltrifluoroacetone decreased Ang II-stimulated ROS production. N-acetylcysteine antagonized Ang II-induced CRP expression. Losartan and N-acetylcysteine inhibited Ang II-activated ERK1/2. Unlike ERK1/2, only losartan inhibited Ang II-activated JNK. Furthermore, pyrrolidine dithiocarbamate abolished Ang II-induced CRP expression. Conclusion: Ang II has ability to induce CRP expression in hepatocytes in vitro and in vivo through AT1 receptor followed by ROS, MAPK and NF-κB signal pathway.

  4. Cell biology is different in small volumes: endogenous signals shape phenotype of primary hepatocytes cultured in microfluidic channels

    Science.gov (United States)

    Haque, Amranul; Gheibi, Pantea; Gao, Yandong; Foster, Elena; Son, Kyung Jin; You, Jungmok; Stybayeva, Gulnaz; Patel, Dipali; Revzin, Alexander

    2016-01-01

    The approaches for maintaining hepatocytes in vitro are aimed at recapitulating aspects of the native liver microenvironment through the use of co-cultures, surface coatings and 3D spheroids. This study highlights the effects of spatial confinement-a less studied component of the in vivo microenvironment. We demonstrate that hepatocytes cultured in low-volume microfluidic channels (microchambers) retain differentiated hepatic phenotype for 21 days whereas cells cultured in regular culture plates under identical conditions de-differentiate after 7 days. Careful consideration of nutrient delivery and oxygen tension suggested that these factors could not solely account for enhanced cell function in microchambers. Through a series of experiments involving microfluidic chambers of various heights and inhibition of key molecular pathways, we confirmed that phenotype of hepatocytes in small volumes was shaped by endogenous signals, both hepato-inductive growth factors (GFs) such as hepatocyte growth factor (HGF) and hepato-disruptive GFs such as transforming growth factor (TGF)-β1. Hepatocytes are not generally thought of as significant producers of GFs–this role is typically assigned to nonparenchymal cells of the liver. Our study demonstrates that, in an appropriate microenvironment, hepatocytes produce hepato-inductive and pro-fibrogenic signals at the levels sufficient to shape their phenotype and function. PMID:27681582

  5. Profile of hepatocyte apoptosis and bile lakes before and after bile duct decompression in severe obstructive jaundice patients

    Institute of Scientific and Technical Information of China (English)

    ToarJMLalisang; RadenSjamsuhidajat; NurjatiCSiregar; AkmalTaher

    2010-01-01

    BACKGROUND: Excessive hepatocyte apoptosis and bile lakes in severe obstructive jaundice might impair liver functions. Although decompression of the bile duct has been reported to improve liver functions in animal studies, the mechanism of obstruction differs from that in humans. This study aimed to determine the profiles of hepatocyte apoptosis and bile lakes following bile duct decompression in patients with severe obstructive jaundice in the clinical setting. METHODS: We conducted a "before and after study" on severe obstructive jaundice patients as a model of inhibition of the excessive process by bile duct decompression. Specimens of liver biopsies were taken before and after decompression of the bile duct and then stained by terminal deoxynucleotide transferase-mediated dUTP nick end-labeling (TUNEL) to identify hepatocyte apoptosis and by hematoxilin-eosin (HE) to identify bile lakes. All measurements were independently done by 2 observers. RESULTS: Twenty-one severe obstructive jaundice patients were included. In all patients, excessive hepatocyte apoptosis and bile lakes were apparent. After decompression, the hepatocyte apoptosis index decreased from 53.1 (SD 105) to 11.7 (SD 13.6) (P CONCLUSION: Bile duct decompression improves hepatocyte apoptosis and bile lakes in cases of severe obstructive jaundice, similar to the findings in animal studies.

  6. Resveratrol differentially regulates NAMPT and SIRT1 in Hepatocarcinoma cells and primary human hepatocytes.

    Directory of Open Access Journals (Sweden)

    Susanne Schuster

    Full Text Available Resveratrol is reported to possess chemotherapeutic properties in several cancers. In this study, we wanted to investigate the molecular mechanisms of resveratrol-induced cell cycle arrest and apoptosis as well as the impact of resveratrol on NAMPT and SIRT1 protein function and asked whether there are differences in hepatocarcinoma cells (HepG2, Hep3B cells and non-cancerous primary human hepatocytes. We found a lower basal NAMPT mRNA and protein expression in hepatocarcinoma cells compared to primary hepatocytes. In contrast, SIRT1 was significantly higher expressed in hepatocarcinoma cells than in primary hepatocytes. Resveratrol induced cell cycle arrest in the S- and G2/M- phase and apoptosis was mediated by activation of p53 and caspase-3 in HepG2 cells. In contrast to primary hepatocytes, resveratrol treated HepG2 cells showed a reduction of NAMPT enzymatic activity and increased p53 acetylation (K382. Resveratrol induced NAMPT release from HepG2 cells which was associated with increased NAMPT mRNA expression. This effect was absent in primary hepatocytes where resveratrol was shown to function as NAMPT and SIRT1 activator. SIRT1 inhibition by EX527 resembled resveratrol effects on HepG2 cells. Furthermore, a SIRT1 overexpression significantly decreased both p53 hyperacetylation and resveratrol-induced NAMPT release as well as S-phase arrest in HepG2 cells. We could show that NAMPT and SIRT1 are differentially regulated by resveratrol in hepatocarcinoma cells and primary hepatocytes and that resveratrol did not act as a SIRT1 activator in hepatocarcinoma cells.

  7. Increased Production of Sonic Hedgehog by Ballooned Hepatocytes

    OpenAIRE

    Rangwala, Fatima; Cynthia D Guy; Lu, Jiuyi; SUZUKI, Ayako; Burchette, James L.; Abdelmalek, Manal F; Chen, Wei; Diehl, Anna Mae

    2011-01-01

    Ballooned hepatocytes distinguish nonalcoholic steatohepatitis (NASH) from steatosis. Such cells contain dilated endoplasmic reticulum and ubiquitin aggregates, characteristics of endoplasmic reticulum stress. Hepatocyte ballooning increases risk for fibrosis in NASH, suggesting ballooned hepatocytes release pro-fibrogenic factors. Hedgehog ligands function as pro-fibrogenic factors in liver diseases, but mechanisms for Hedgehog ligand production remain poorly understood. We evaluated the hyp...

  8. Metabolism of cysteine by cyteinesulfinate-independent pathway(s) in rat hepatocytes

    International Nuclear Information System (INIS)

    The metabolism of cysteine (CYS) and that of cysteinesulfinate (CSA) were studied in freshly isolated hepatocytes from fed rats. In incubations of rat hepatocytes with either 1 or 25 mM CSA, over 90% of the 14CO2 formed from [1-14C]CSA could be accounted for by production of hypotaurine plus taurine. In similar incubations with 1 or 25 mM CYS, only 4% of 14CO2 evolution from [1-14C]CYS could be accounted for by production of hypotaurine plus taurine. Addition of unlabeled CSA inhibited recovery of label from [1-14C]CYS as 14CO2 by 33%. Metabolism of CYS and of CSA were affected differently by addition of α-ketoglutarate, a cosubstrate for transamination, or of propargylglycine, an inhibitor of cystathionase activity. These data suggest that a substantial proportion of CYS is catabolized by CSA-independent pathways in the rat hepatocyte. Although addition of α-ketoglutarate to incubations of hepatocytes with CSA resulted in a marked increase in CSA catabolism via the transamination pathway, addition of keto acids to incubation systems had little or no effect on production of any metabolite from CYS. Thus, CYS transamination does not appear to be a major pathway of CYS metabolism in the hepatocyte. Inhibition of cystathionase with propargylglycine reduced both 14CO2 production from [1-14C]CYS and ammonia plus urea nitrogen production from CYS by about 50%; CSA catabolism was not affected. Thus, cleavage of cyst(e)ine by cystathionase may be an important physiological pathway for CYS catabolism in the liver

  9. Metabolism of cysteine by cyteinesulfinate-independent pathway(s) in rat hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Stipanuk, M.H.; De La Rosa, J.; Drake, M.R.

    1986-05-01

    The metabolism of cysteine (CYS) and that of cysteinesulfinate (CSA) were studied in freshly isolated hepatocytes from fed rats. In incubations of rat hepatocytes with either 1 or 25 mM CSA, over 90% of the /sup 14/CO/sub 2/ formed from (1-/sup 14/C)CSA could be accounted for by production of hypotaurine plus taurine. In similar incubations with 1 or 25 mM CYS, only 4% of /sup 14/CO/sub 2/ evolution from (1-/sup 14/C)CYS could be accounted for by production of hypotaurine plus taurine. Addition of unlabeled CSA inhibited recovery of label from (1-/sup 14/C)CYS as /sup 14/CO/sub 2/ by 33%. Metabolism of CYS and of CSA were affected differently by addition of ..cap alpha..-ketoglutarate, a cosubstrate for transamination, or of propargylglycine, an inhibitor of cystathionase activity. These data suggest that a substantial proportion of CYS is catabolized by CSA-independent pathways in the rat hepatocyte. Although addition of ..cap alpha..-ketoglutarate to incubations of hepatocytes with CSA resulted in a marked increase in CSA catabolism via the transamination pathway, addition of keto acids to incubation systems had little or no effect on production of any metabolite from CYS. Thus, CYS transamination does not appear to be a major pathway of CYS metabolism in the hepatocyte. Inhibition of cystathionase with propargylglycine reduced both /sup 14/CO/sub 2/ production from (1-/sup 14/C)CYS and ammonia plus urea nitrogen production from CYS by about 50%; CSA catabolism was not affected. Thus, cleavage of cyst(e)ine by cystathionase may be an important physiological pathway for CYS catabolism in the liver.

  10. Hepatocyte growth factor in lung repair and pulmonary fibrosis

    Institute of Scientific and Technical Information of China (English)

    Ronald Allan M PANGANIBAN; Regina M DAY

    2011-01-01

    Pulmonary remodeling is characterized by the permanent and progressive loss of the normal alveolar architecture, especially the loss of alveolar epithelial and endothelial cells, persistent proliferation of activated fibroblasts, or myoflbroblasts, and alteration of extracellular matrix. Hepatocyte growth factor (HGF) is a pleiotropic factor, which induces cellular motility, survival, proliferation, and morphogenesis, depending upon the cell type. In the adult, HGF has been demonstrated to play a critical role in tissue repair, including in the lung. Administration of HGF protein or ectopic expression of HGF has been demonstrated in animal models of pulmonary fibrosis to induce normal tissue repair and to prevent fibrotic remodeling. HGF-induced inhibition of fibrotic remodeling may occur via multiple direct and indirect mechanisms including the induction of cell survival and proliferation of pulmonary epithelial and endothelial cells, and the reduction of myofibroblast accumulation.

  11. SIRT1 Disruption in Human Fetal Hepatocytes Leads to Increased Accumulation of Glucose and Lipids

    OpenAIRE

    Tobita, Takamasa; Guzman-Lepe, Jorge; Takeishi, Kazuki; Nakao, Toshimasa; Wang, Yang; Meng, Fanying; Deng, Chu-Xia; Collin de l’Hortet, Alexandra; Soto-Gutierrez, Alejandro

    2016-01-01

    There are unprecedented epidemics of obesity, such as type II diabetes and non-alcoholic fatty liver diseases (NAFLD) in developed countries. A concerning percentage of American children are being affected by obesity and NAFLD. Studies have suggested that the maternal environment in utero might play a role in the development of these diseases later in life. In this study, we documented that inhibiting SIRT1 signaling in human fetal hepatocytes rapidly led to an increase in intracellular gluco...

  12. Stimulation of glycogen synthesis and lipogenesis by glutamine in isolated rat hepatocytes.

    OpenAIRE

    Lavoinne, A; Baquet, A.; Hue, Louis

    1987-01-01

    Glutamine stimulated glycogen synthesis and lactate production in hepatocytes from overnight-fasted normal and diabetic rats. The effect, which was half-maximal with about 3 mM-glutamine, depended on glucose concentration and was maximal below 10 mM-glucose. beta-2-Aminobicyclo[2.2.1.]heptane-2-carboxylic acid, an analogue of leucine, stimulated glutaminase flux, but inhibited the stimulation of glycogen synthesis by glutamine. Various purine analogues and inhibitors of purine synthesis were ...

  13. Hepatocyte Growth Factor Reduces Free Cholesterol-Mediated Lipotoxicity in Primary Hepatocytes by Countering Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Mayra Domínguez-Pérez

    2016-01-01

    Full Text Available Cholesterol overload in the liver has shown toxic effects by inducing the aggravation of nonalcoholic fatty liver disease to steatohepatitis and sensitizing to damage. Although the mechanism of damage is complex, it has been demonstrated that oxidative stress plays a prominent role in the process. In addition, we have proved that hepatocyte growth factor induces an antioxidant response in hepatic cells; in the present work we aimed to figure out the protective effect of this growth factor in hepatocytes overloaded with free cholesterol. Hepatocytes from mice fed with a high-cholesterol diet were treated or not with HGF, reactive oxygen species present in cholesterol overloaded hepatocytes significantly decreased, and this effect was particularly associated with the increase in glutathione and related enzymes, such as γ-gamma glutamyl cysteine synthetase, GSH peroxidase, and GSH-S-transferase. Our data clearly indicate that HGF displays an antioxidant response by inducing the glutathione-related protection system.

  14. Hepatocyte Growth Factor Reduces Free Cholesterol-Mediated Lipotoxicity in Primary Hepatocytes by Countering Oxidative Stress

    Science.gov (United States)

    Domínguez-Pérez, Mayra; Nuño-Lámbarri, Natalia; Clavijo-Cornejo, Denise; Luna-López, Armando; Souza, Verónica; Bucio, Leticia; Miranda, Roxana U.; Muñoz, Linda; Gomez-Quiroz, Luis Enrique; Uribe-Carvajal, Salvador; Gutiérrez-Ruiz, María Concepción

    2016-01-01

    Cholesterol overload in the liver has shown toxic effects by inducing the aggravation of nonalcoholic fatty liver disease to steatohepatitis and sensitizing to damage. Although the mechanism of damage is complex, it has been demonstrated that oxidative stress plays a prominent role in the process. In addition, we have proved that hepatocyte growth factor induces an antioxidant response in hepatic cells; in the present work we aimed to figure out the protective effect of this growth factor in hepatocytes overloaded with free cholesterol. Hepatocytes from mice fed with a high-cholesterol diet were treated or not with HGF, reactive oxygen species present in cholesterol overloaded hepatocytes significantly decreased, and this effect was particularly associated with the increase in glutathione and related enzymes, such as γ-gamma glutamyl cysteine synthetase, GSH peroxidase, and GSH-S-transferase. Our data clearly indicate that HGF displays an antioxidant response by inducing the glutathione-related protection system. PMID:27143995

  15. Effects of dehydroepiandrosterone (DHEA) on glucose metabolism in isolated hepatocytes from Zucker rats

    International Nuclear Information System (INIS)

    DHEA has been shown to competitively inhibit the pentose phosphate shunt (PPS) enzyme glucose-6-phosphate dehydrogenase (G6PD) when added in vitro to supernatants or homogenates prepared from mammalian tissues. However, no consistent effect on G6PD activity has been determined in tissue removed from DHEA-treated rats. To explore the effects of DHEA on PPS, glucose utilization was measured in hepatocytes from lean and obese male Zucker rats (8 wks of age) following 1 wk of DHEA treatment (0.6% in diet). Incubation of isolated hepatocytes from treated lean Zucker rats with either [1-14C] glucose or [6-14C] glucose resulted in significant decreases in CO2 production and total glucose utilization. DHEA-lean rats also had lowered fat pad weights. In obese rats, there was no effect of 1 wk of treatment on either glucose metabolism or fat pad weight. The calculated percent contribution of the PPS to glucose metabolism in hepatocytes was not changed for either DHEA-lean or obese rats when compared to control rats. In conclusion, 1 wk of DHEA treatment lowered overall glucose metabolism in hepatocytes of lean Zucker rats, but did not selectively affect the PPS. The lack of an effect of short-term treatment in obese rats may be due to differences in their metabolism or storage/release of DHEA in tissues in comparison to lean rats

  16. Recruitment of stem cells by hepatocyte growth factor via intracoronary gene transfection in the postinfarction heart failure

    Institute of Scientific and Technical Information of China (English)

    YANG; ZhiJian; WANG; Wei; MA; DongChao; ZHANG; YouRong; WANG; LianSheng; ZHANG; YuQing; XU; ShunLin; CHEN; Bo; MIAO; DengShun; CAO; KeJiang

    2007-01-01

    We aim to study the amelioration effect of adenovirus5-mediated human hepatocyte growth factor gene transfer on postinfarction heart failure in swine model. Twelve Suzhong young swine were randomly divided into 2 groups of 6 pigs each: Ad5-HGF group and mock-vector Ad5 group. Four weeks after ligation of the left anterior descending coronary artery, Ad5-HGF was intracoronarily transferred into the myocardium. Simultaneously, gate cardiac perfusion imaging was performed to evaluate the heart function. Three weeks later, gate cardiac perfusion imaging was performed again, then the hearts were removed and sectioned for immunohistochemical examination to illustrate the effects of Ad5-HGF on infarcted myocardium. The expression of HGF was examined by ELISA. The results were: (1) compared with the mock-vector Ad5 group, high expression of human HGF was observed in the myocardium of Ad5-HGF group; (2) in the Ad5-HGF group, the number of CD117+ cells co-expressing c-Met per mm2 was significantly larger; (3) the improvement in LVEF was greater in the Ad5-HGF group than in the mock-vector Ad5 group. We concluded that: (1) high expression of human HGF was observed in the myocardium through intracoronary gene transfection; (2) HGF can improve the mobilization of CD117+/c-Met+ stem cells into ischemic myocardium. The amelioration effect of HGF on postinfarction heart failure could not be limited to stimulating angiogenesis, anti-apoptosis, anti-fibrosis, but was also involved in the recruitment of stem cells into myocardium.

  17. Roles of hepatocyte nuclear factors in hepatitis B virus infection.

    Science.gov (United States)

    Kim, Doo Hyun; Kang, Hong Seok; Kim, Kyun-Hwan

    2016-08-21

    Approximately 350 million people are estimated to be persistently infected with hepatitis B virus (HBV) worldwide. HBV maintains persistent infection by employing covalently closed circular DNA (cccDNA), a template for all HBV RNAs. Chronic hepatitis B (CHB) patients are currently treated with nucleos(t)ide analogs such as lamivudine, adefovir, entecavir, and tenofovir. However, these treatments rarely cure CHB because they are unable to inhibit cccDNA transcription and inhibit only a late stage in the HBV life cycle (the reverse transcription step in the nucleocapsid). Therefore, an understanding of the factors regulating cccDNA transcription is required to stop this process. Among numerous factors, hepatocyte nuclear factors (HNFs) play the most important roles in cccDNA transcription, especially in the generation of viral genomic RNA, a template for HBV replication. Therefore, proper control of HNF function could lead to the inhibition of HBV replication. In this review, we summarize and discuss the current understanding of the roles of HNFs in the HBV life cycle and the upstream factors that regulate HNFs. This knowledge will enable the identification of new therapeutic targets to cure CHB. PMID:27610013

  18. Fipronil induces CYP isoforms and cytotoxicity in human hepatocytes.

    Science.gov (United States)

    Das, Parikshit C; Cao, Yan; Cherrington, Nathan; Hodgson, Ernest; Rose, Randy L

    2006-12-15

    Recent studies have demonstrated the potential of pesticides to either inhibit or induce xenobiotic metabolizing enzymes in humans. Exposure of human hepatocytes to doses of fipronil (5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl) sulfinyl]-1H-pyrazole-3-carbonitrile) ranging from 0.1 to 25 microM resulted in a dose dependent increase in CYP1A1 mRNA expression (3.5 to approximately 55-fold) as measured by the branched DNA assay. In a similar manner, CYP3A4 mRNA expression was also induced (10-30-fold), although at the higher doses induction returned to near control levels. CYP2B6 and 3A5 were also induced by fipronil, although at lower levels (2-3-fold). Confirmation of bDNA results were sought through western blotting and/or enzyme activity assays. Western blots using CYP3A4 antibody demonstrated a dose responsive increase from 0.5 to 1 microM followed by decreasing responses at higher concentrations. Similar increases and decreases were observed in CYP3A4-specific activity levels as measured using 6beta-hydroxytestosterone formation following incubation with testosterone. Likewise, activity levels for a CYP1A1-specific substrate, luciferin CEE, demonstrated that CYP1A1 enzyme activities were maximally induced by 1 microM fipronil followed by dramatically declining activity measurements at 10 and 25 microM. Cytotoxic effects of fipronil and fipronil sulfone were examined using the adenylate kinase and the trypan blue exclusion assays in HepG2 cells and human hepatocytes. The results indicate both that HepG2 cells and primary human hepatocytes are sensitive to the cytotoxic effects of fipronil. The maximum induction of adenylate kinase was ca. 3-fold greater than the respective controls in HepG2 and 6-10-fold in the case of primary hepatocytes. A significant time- and dose-dependent induction of adenylate kinase activity in HepG2 cells was noted from 0.1 to 12.5 microM fipronil followed by decreasing activities at 25 and 50 microM. For

  19. Determination of a novel anticancer c-Met inhibitor LS-177 in rat plasma and tissues with a validated UPLC-MS/MS method: application to pharmacokinetics and tissue distribution study.

    Science.gov (United States)

    Ju, Ping; Liu, Zhenzhen; Jiang, Yu; Zhao, Simin; Zhang, Lunhui; Zhang, Yuanyuan; Gu, Liqiang; Tang, Xing; Bi, Kaishun; Chen, Xiaohui

    2015-07-01

    LS-177 is a novel small-molecule kinase inhibitor employed to interrupt the c-Met signaling pathway. A rapid and sensitive ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for determination of LS-177 in rat plasma and tissues. The biosamples were extracted by liquid-liquid extraction with methyl tert-butyl ether and separated on a C18 column (50 × 4.6 mm, 2.6 µm) using a gradient elution mobile phase consisting of acetonitrile-0.1% formic acid water. Under the optimal conditions, the selectivity of the method was satisfactory with no endogenous interference. The intraday and interday precisions (relative standard deviation) were LS-177 was stable during the preparation and analytical processes. The UPLC-MS/MS method was successfully applied to pharmacokinetic and tissue distribution studies. The results indicated that there was no significant drug accumulation after multiple-dose oral administration of LS-177. The tissue distribution study exhibited significant higher uptakes of LS-177 in stomach, intestine, lung and liver among all of the tissues. The results in pharmacokinetics and tissue distribution may provide a meaningful basis for clinical application.

  20. Duck hepatitis B virus covalently closed circular DNA appears to survive hepatocyte mitosis in the growing liver

    Energy Technology Data Exchange (ETDEWEB)

    Reaiche-Miller, Georget Y.; Thorpe, Michael; Low, Huey Chi; Qiao, Qiao; Scougall, Catherine A. [School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA 5005 (Australia); Mason, William S.; Litwin, Samuel [Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111 (United States); Jilbert, Allison R., E-mail: allison.jilbert@adelaide.edu.au [School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA 5005 (Australia)

    2013-11-15

    Nucleos(t)ide analogues that inhibit hepatitis B virus (HBV) DNA replication are typically used as monotherapy for chronically infected patients. Treatment with a nucleos(t)ide analogue eliminates most HBV DNA replication intermediates and produces a gradual decline in levels of covalently closed circular DNA (cccDNA), the template for viral RNA synthesis. It remains uncertain if levels of cccDNA decline primarily through hepatocyte death, or if loss also occurs during hepatocyte mitosis. To determine if cccDNA survives mitosis, growing ducklings infected with duck hepatitis B virus (DHBV) were treated with the nucleoside analogue, Entecavir. Viremia was suppressed at least 10{sup 5}-fold, during a period when average liver mass increased 23-fold. Analysis of the data suggested that if cccDNA synthesis was completely inhibited, at least 49% of cccDNA survived hepatocyte mitosis. However, there was a large duck-to-duck variation in cccDNA levels, suggesting that low level cccDNA synthesis may contribute to this apparent survival through mitosis. - Highlights: • The hepatitis B virus nuclear template is covalently closed circular DNA (cccDNA). • cccDNA was studied during liver growth in duck hepatitis B virus infected ducks. • Virus DNA replication and new cccDNA synthesis were inhibited with Entecavir. • At least 49% of cccDNA appeared to survive hepatocyte mitosis. • Low level virus DNA synthesis may contribute to survival of cccDNA through mitosis.

  1. Duck hepatitis B virus covalently closed circular DNA appears to survive hepatocyte mitosis in the growing liver

    International Nuclear Information System (INIS)

    Nucleos(t)ide analogues that inhibit hepatitis B virus (HBV) DNA replication are typically used as monotherapy for chronically infected patients. Treatment with a nucleos(t)ide analogue eliminates most HBV DNA replication intermediates and produces a gradual decline in levels of covalently closed circular DNA (cccDNA), the template for viral RNA synthesis. It remains uncertain if levels of cccDNA decline primarily through hepatocyte death, or if loss also occurs during hepatocyte mitosis. To determine if cccDNA survives mitosis, growing ducklings infected with duck hepatitis B virus (DHBV) were treated with the nucleoside analogue, Entecavir. Viremia was suppressed at least 105-fold, during a period when average liver mass increased 23-fold. Analysis of the data suggested that if cccDNA synthesis was completely inhibited, at least 49% of cccDNA survived hepatocyte mitosis. However, there was a large duck-to-duck variation in cccDNA levels, suggesting that low level cccDNA synthesis may contribute to this apparent survival through mitosis. - Highlights: • The hepatitis B virus nuclear template is covalently closed circular DNA (cccDNA). • cccDNA was studied during liver growth in duck hepatitis B virus infected ducks. • Virus DNA replication and new cccDNA synthesis were inhibited with Entecavir. • At least 49% of cccDNA appeared to survive hepatocyte mitosis. • Low level virus DNA synthesis may contribute to survival of cccDNA through mitosis

  2. Autocrine expression of hepatocyte growth factor and its cytoprotective effect on hepatocyte poisoning

    Institute of Scientific and Technical Information of China (English)

    Yong He; Jun Zhou; Ke-Feng Dou; Yong Chen; Qing-Guo Yan; Hai-Min Li

    2004-01-01

    AIM: To construct pEGFP-hepatocyte growth factor (HGF)expression vector,the to detect its expression in transfected human hepatocytes, and to investigate the influence of autocrine HGF expression on the proliferative potential and cytoprotective effects in human hepatocytes.METHODS: Human HGF cDNA was ligated to the pEGFP vector.Recombinant plasmid was transfected into human hepatocyte line QZG with liposome. Expression of HGF protein was observed by fluorescence microscopy and immunohistochemistry. Hepatic cells were collected 24, 48, and 72 h after transfection to detect the number of [3H]-TdR uptake in DNA. DNA synthesis was observed by using PCNA stain immunohistochemistry.Acute liver cell damage was induced by carbon tetrachloride. Cytoprotective effect was observed by examining the survival rate of hepatocytes and leakage of intracellular alanine transaminase (ALT) and potassium ions.RESULTS: HGF identification of pEGFP-HGF by enzyme digestion showed that HGF fragment was cloned into BamH I and Sa/I sites of pEGFP-N3. Expression of GFP in transfected hepatocytes was observed with fluorescence microscopy.The [3H]-TdR uptake became 7 times as many as in the control group 96 h after transfection. After HGF transfection,the survival rate of hepatocytes poisoned by CCl4 significantly increased (83% vs 61%, P<0.05), and the leakage of intracellular alanine transaminase and potassium ions decreased(586 nkat/L vs1089 nkat/L, P<0.01; and 5.59 mmol/L vs6.02 mmol/L, P<0.01 respectively). Culture of transfected hepatic cells promoted the proliferation of other nontransfected cells.CONCLUSION: Transfected HGF is expressed in hepatic cells and has the activity of promoting cell division and protecting hepatic cells against poisoning.

  3. 乳腺浸润性导管癌患者癌组织c-Met蛋白的表达及病理探讨%C-Met protein expression of cancer tissue of patients with breast inifltrating ductal carcinoma and its pathological study

    Institute of Scientific and Technical Information of China (English)

    王晓娟

    2016-01-01

    Objective to analyze and investigate c-Met protein expression of cancer tissue of patients with breast infiltrating ductal carcinoma and its pathological significance.Methods randomly choose clinical pathological specimens of 140 patients with breast infiltrating ductal carcinoma in our hospital from January 2015 to December 2015 as research objects, detect c-met protein expression of breast cancer tissue, and analyze relations between c-Met protein expression and TNM staging, tumor pathological grading, lymph node metastasis and tumor size.Results there were 60 cases positive c-Met protein, accounting for 42.86%; 80 cases negative c-Met protein, accounting for 57.14%. C-met protein expression positive rate in pathological grade I, II, and III and different tumor sizes showed no significant difference (P> 0.05); expression positive rate at TNM stage III to IV and breast cancer tissue with lymph node metastasis was significantly higher than that in TNM stage I-II and breast cancer tissue without lymph node metastasis, and data difference showed statistical significance (P0.05);在TNM分期III~IV期及有淋巴结转移的乳腺癌组织中表达阳性率明显高于TNM分期I~II期无淋巴转移的乳腺癌组织,且数据均差异有统计学意义(P<0.05)。结论 c-Met蛋白的表达在乳腺癌的转移以及侵袭中具有不可忽视的作用,可以采用干预手段或者是检测c-Met蛋白表达来进行对乳腺癌其发生、发展情况和患者的预后做判断。

  4. Biotransformation and cytotoxic effects of hydroxychavicol, an intermediate of safrole metabolism, in isolated rat hepatocytes.

    Science.gov (United States)

    Nakagawa, Yoshio; Suzuki, Toshinari; Nakajima, Kazuo; Ishii, Hidemi; Ogata, Akio

    2009-06-15

    The biotransformation and cytotoxic effects of hydroxychavicol (HC; 1-allyl-3,4-dihydroxybenzene), which is a catecholic component in piper betel leaf and a major intermediary metabolite of safrole in rats and humans, was studied in freshly isolated rat hepatocytes. The exposure of hepatocytes to HC caused not only concentration (0.25-1.0mM)- and time (0-3h)-dependent cell death accompanied by the loss of cellular ATP, adenine nucleotide pools, reduced glutathione, and protein thiols, but also the accumulation of glutathione disulfide and malondialdehyde, indicating lipid peroxidation. At a concentration of 1mM, the cytotoxic effects of safrole were less than those of HC. The loss of mitochondrial membrane potential and generation of oxygen radical species assayed using 2',7'-dichlorodihydrofluoresein diacetate (DCFH-DA) in hepatocytes treated with HC were greater than those with safrole. HC at a weakly toxic level (0.25 and/or 0.50mM) was metabolized to monoglucuronide, monosulfate, and monoglutathione conjugates, which were identified by mass spectra and/or (1)H nuclear magnetic resonance spectra. The amounts of sulfate rather than glucuronide or glutathione conjugate predominantly increased, accompanied by a loss of the parent compound, with time. In hepatocytes pretreated with either diethyl maleate or salicylamide, HC-induced cytotoxicity was enhanced, accompanied by a decrease in the formation of these conjugates and by the inhibition of HC loss. Taken collectively, our results indicate that (a) mitochondria are target organelles for HC, which elicits cytotoxicity through mitochondrial failure related to mitochondrial membrane potential at an early stage and subsequently lipid peroxidation through oxidative stress at a later stage; (b) the onset of cytotoxicity depends on the initial and residual concentrations of HC rather than those of its metabolites; (c) the toxicity of HC is greater than that of safrole, suggesting the participation of a catecholic

  5. Assessing the therapeutic potential of lab-made hepatocytes.

    Science.gov (United States)

    Rezvani, Milad; Grimm, Andrew A; Willenbring, Holger

    2016-07-01

    Hepatocyte transplantation has potential as a bridge or even alternative to whole-organ liver transplantation. Because donor livers are scarce, realizing this potential requires the development of alternative cell sources. To be therapeutically effective, surrogate hepatocytes must replicate the complex function and ability to proliferate of primary human hepatocytes. Ideally, they are also autologous to eliminate the need for immune suppression, which can have severe side effects and may not be sufficient to prevent rejection long term. In the past decade, several methods have been developed to generate hepatocytes from other readily and safely accessible somatic cells. These lab-made hepatocytes show promise in animal models of liver diseases, supporting the feasibility of autologous liver cell therapies. Here, we review recent preclinical studies exemplifying different types of lab-made hepatocytes that can potentially be used in autologous liver cell therapies. To define the therapeutic efficacy of current lab-made hepatocytes, we compare them to primary human hepatocytes, focusing on engraftment efficiency and posttransplant proliferation and function. In addition to summarizing published results, we discuss animal models and assays effective in assessing therapeutic efficacy. This analysis underscores the therapeutic potential of current lab-made hepatocytes, but also highlights deficiencies and uncertainties that need to be addressed in future studies aimed at developing liver cell therapies with lab-made hepatocytes. (Hepatology 2016;64:287-294). PMID:27014802

  6. Experimental hepatocyte xenotransplantation--a comprehensive review of the literature.

    Science.gov (United States)

    Zhou, Huidong; Liu, Hong; Ezzelarab, Mohamed; Schmelzer, Eva; Wang, Yi; Gerlach, Jörg; Gridelli, Bruno; Cooper, David K C

    2015-01-01

    Hepatocyte transplantation (Tx) is a potential therapy for certain diseases of the liver, including hepatic failure. However, there is a limited supply of human livers as a source of cells and, after isolation, human hepatocytes can be difficult to expand in culture, limiting the number available for Tx. Hepatocytes from other species, for example, the pig, have therefore emerged as a potential alternative source. We searched the literature through the end of 2014 to assess the current status of experimental research into hepatocyte xenoTx. The literature search identified 51 reports of in vivo cross-species Tx of hepatocytes in a variety of experimental models. Most studies investigated the Tx of human (n = 23) or pig (n = 19) hepatocytes. No studies explored hepatocytes from genetically engineered pigs. The spleen was the most common site of Tx (n = 23), followed by the liver (through the portal vein [n = 6]) and peritoneal cavity (n = 19). In 47 studies (92%), there was evidence of hepatocyte engraftment and function across a species barrier. The data provided by this literature search strengthen the hypothesis that xenoTx of hepatocytes is feasible and potentially successful as a clinical therapy for certain liver diseases, including hepatic failure. By excluding vascular structures, hepatocytes isolated from genetically engineered pig livers may address some of the immunological problems of xenoTx.

  7. Experimental hepatocyte xenotransplantation--a comprehensive review of the literature.

    Science.gov (United States)

    Zhou, Huidong; Liu, Hong; Ezzelarab, Mohamed; Schmelzer, Eva; Wang, Yi; Gerlach, Jörg; Gridelli, Bruno; Cooper, David K C

    2015-01-01

    Hepatocyte transplantation (Tx) is a potential therapy for certain diseases of the liver, including hepatic failure. However, there is a limited supply of human livers as a source of cells and, after isolation, human hepatocytes can be difficult to expand in culture, limiting the number available for Tx. Hepatocytes from other species, for example, the pig, have therefore emerged as a potential alternative source. We searched the literature through the end of 2014 to assess the current status of experimental research into hepatocyte xenoTx. The literature search identified 51 reports of in vivo cross-species Tx of hepatocytes in a variety of experimental models. Most studies investigated the Tx of human (n = 23) or pig (n = 19) hepatocytes. No studies explored hepatocytes from genetically engineered pigs. The spleen was the most common site of Tx (n = 23), followed by the liver (through the portal vein [n = 6]) and peritoneal cavity (n = 19). In 47 studies (92%), there was evidence of hepatocyte engraftment and function across a species barrier. The data provided by this literature search strengthen the hypothesis that xenoTx of hepatocytes is feasible and potentially successful as a clinical therapy for certain liver diseases, including hepatic failure. By excluding vascular structures, hepatocytes isolated from genetically engineered pig livers may address some of the immunological problems of xenoTx. PMID:25950141

  8. Optimized Hepatocyte-Like Cells with Functional Drug Transporters Directly-Reprogrammed from Mouse Fibroblasts and their Potential in Drug Disposition and Toxicology

    Directory of Open Access Journals (Sweden)

    Zhi-Tao Wu

    2016-05-01

    Full Text Available Background/Aims: To develop a suitable hepatocyte-like cell model that could be a substitute for primary hepatocytes with essential transporter expression and functions. Induced hepatocyte-like (iHep cells directly reprogrammed from mice fibroblast cells were fully characterized. Methods: Naïve iHep cells were transfected with nuclear hepatocyte factor 4 alpha (Hnf4α and treated with selected small molecules. Sandwich cultured configuration was applied. The mRNA and protein expression of transporters were determined by Real Time PCR and confocal. The functional transporters were estimated by drug biliary excretion measurement. The inhibition of bile acid efflux transporters by cholestatic drugs were assessed. Results: The expression and function of p-glycoprotein (P-gp, bile salt efflux pump (Bsep, multidrug resistance-associated protein 2 (Mrp2, Na+-dependent taurocholate cotransporting polypeptide (Ntcp, and organic anion transporter polypedtides (Oatps in iHep cells were significantly improved after transfection of hepatocyte nuclear factor 4 alpha (Hnf4α and treatment with selected inducers. In vitro intrinsic biliary clearances (CLb,int of optimized iHep cells for rosuvastatin, methotrexate, d8-TCA (deuterium-labeled sodium taurocholate acid and DPDPE ([D-Pen2,5] enkephalin hydrate correlated well with that of sandwich-cultured primary mouse hepatocytes (SCMHs (r2 = 0.984. Cholestatic drugs were evaluated and the results were compared well with primary mice hepatocytes. Conclusion: The optimized iHep cells expressed functional drug transporters and were comparable to primary mice hepatocytes. This study suggested direct reprogramming could provide a potential alternative to primary hepatocytes for drug candidate hepatobiliary disposition and hepatotoxicity screening.

  9. Iron regulation by hepatocytes and free radicals

    OpenAIRE

    Takami, Taro; Sakaida, Isao

    2011-01-01

    Iron is an essential metallic microelement for life. However, iron overload is toxic. The liver serves an important role as a storehouse for iron in the body. About 20–25 mg of iron is required each day for hemoglobin synthesis. To maintain iron homeostasis, transferrin and transferrin receptors are primarily involved in the uptake of iron into hepatocytes, ferritin in its storage, and ferroportin in its export. Moreover, hepcidin controls ferroportin and plays a central role in the iron meta...

  10. Inducible nitric-oxide synthase attenuates vasopressin-dependent Ca2+ signaling in rat hepatocytes.

    Science.gov (United States)

    Patel, Sandip; Gaspers, Lawrence D; Boucherie, Sylviane; Memin, Elisabeth; Stellato, Kerri Anne; Guillon, Gilles; Combettes, Laurent; Thomas, Andrew P

    2002-09-13

    Increases in both Ca(2+) and nitric oxide levels are vital for a variety of cellular processes; however, the interaction between these two crucial messengers is not fully understood. Here, we demonstrate that expression of inducible nitric-oxide synthase in hepatocytes, in response to inflammatory mediators, dramatically attenuates Ca(2+) signaling by the inositol 1,4,5-trisphosphate-forming hormone, vasopressin. The inhibitory effects of induction were reversed by nitric oxide inhibitors and mimicked by prolonged cyclic GMP elevation. Induction was without effect on Ca(2+) signals in response to AlF(4)(-) or inositol 1,4,5-trisphosphate, indicating that phospholipase C activation and release of Ca(2+) from inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores were not targets for nitric oxide inhibition. Vasopressin receptor levels, however, were dramatically reduced in induced cultures. Our data provide a possible mechanism for hepatocyte dysfunction during chronic inflammation. PMID:12097323

  11. Role of the transsulfuration pathway and of cystathionase activity in the formation of cysteine and sulfate from methionine in rat hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Rao, A.M.; Stipanuk, M.H. (Cornell Univ., Ithaca, NY (United States))

    1990-02-26

    Hepatic cystathionase activity in premature infants has been reported to be about 23% of mature levels. The possibility that premature infants require dietary cysteine because of a limited capacity for methionine transsulfuration has been suggested. To assess the extent to which low hepatic cystathionase levels affect methionine flux to cysteine in hepatocytes, the effect of inhibition of cystathionase activity with propargylglycine on the metabolism of L-({sup 35}S)methionine was determined in studies with freshly isolated rat hepatocytes. Cystathionase activity was inhibited by 25%, 42%, 63% and 76% (maximal inhibition) by treatment of hepatocytes with 2.5 {mu}M, 0.01 mM, 0.02 mM and 2 mM propargylglycine, respectively. Inhibition of cystathionase activity up to 63% had no significant effect of ({sup 35}S)glutathione, {sup 35}SO{sub 4}{sup {minus}2}S, or ({sup 35}S)cysteine formation from ({sup 35}S)methionine. However, maximal inhibition of cystathionase markedly inhibited the metabolism of ({sup 35}S)methionine to ({sup 35}S)glutathione by 93%, to {sup 35}SO{sub 4}{sup {minus}2} by 88% and to ({sup 35}S)methionine to ({sup 35}S)cystathionine accumulation in these incubation systems was 60-times control. These results with isolated rat hepatocytes suggest that the presence of less than 25% of the mature level of cystathionase activity may limit cysteine synthesis by the methionine transsulfuration pathway.

  12. Role of the transsulfuration pathway and of cystathionase activity in the formation of cysteine and sulfate from methionine in rat hepatocytes

    International Nuclear Information System (INIS)

    Hepatic cystathionase activity in premature infants has been reported to be about 23% of mature levels. The possibility that premature infants require dietary cysteine because of a limited capacity for methionine transsulfuration has been suggested. To assess the extent to which low hepatic cystathionase levels affect methionine flux to cysteine in hepatocytes, the effect of inhibition of cystathionase activity with propargylglycine on the metabolism of L-[35S]methionine was determined in studies with freshly isolated rat hepatocytes. Cystathionase activity was inhibited by 25%, 42%, 63% and 76% (maximal inhibition) by treatment of hepatocytes with 2.5 μM, 0.01 mM, 0.02 mM and 2 mM propargylglycine, respectively. Inhibition of cystathionase activity up to 63% had no significant effect of [35S]glutathione, 35SO4-2S, or [35S]cysteine formation from [35S]methionine. However, maximal inhibition of cystathionase markedly inhibited the metabolism of [35S]methionine to [35S]glutathione by 93%, to 35SO4-2 by 88% and to [35S]methionine to [35S]cystathionine accumulation in these incubation systems was 60-times control. These results with isolated rat hepatocytes suggest that the presence of less than 25% of the mature level of cystathionase activity may limit cysteine synthesis by the methionine transsulfuration pathway

  13. Heme oxygenase-1 prevents non-alcoholic steatohepatitis through suppressing hepatocyte apoptosis in mice

    Directory of Open Access Journals (Sweden)

    Fu Na

    2010-10-01

    Full Text Available Abstract Objective Heme oxygenase-1 (HO-1, the rate-limiting enzyme in heme catabolism, has been reported to have potential antioxidant properties. However, the role of HO-1 on hepatocyte apoptosis remains unclear. We aim to elucidate the effects of HO-1 on oxidative stress related hepatocellular apoptosis in nutritional steatohepatitis in mice. Methods C57BL/6J mice were fed with methionine-choline deficient (MCD diet for four weeks to induce hepatic steatohepatitis. HO-1 chemical inducer (hemin, HO-1 chemical inhibitor zinc protoporphyrin IX (ZnPP-IX and/or adenovirus carrying HO-1 gene (Ad-HO-1 were administered to mice, respectively. Hepatocyte apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL assay, the mRNA and protein expression of apoptosis related genes were assayed by quantitative real-time PCR and Western blot. Results Hepatocyte signs of oxidative related apoptotic injury were presented in mice fed with MCD diet for 4 weeks. Induction of HO-1 by hemin or Ad-HO-1 significantly attenuated the severity of liver histology, which was associated with decreased hepatic lipid peroxidation content, reduced number of apoptotic cells by TUNEL staining, down-regulated expression of pro-apoptosis related genes including Fas/FasL, Bax, caspase-3 and caspase-9, reduced expression of cytochrome p4502E1 (CYP2E1, inhibited cytochrome c (Cyt-c release, and up-regulated expression of anti-apoptosis gene Bcl-2. Whereas, inhibition of HO-1 by ZnPP-IX caused oxidative stress related hepatic injury, which concomitant with increased number of TUNEL positive cells and up-regulated expression of pro-apoptosis related genes. Conclusions The present study provided evidences for the protective role of HO-1 in preventing nutritional steatohepatitis through suppressing hepatocyte apoptosis in mice.

  14. Primary hepatocyte culture in collagen gel mixture and collagen sandwich

    Institute of Scientific and Technical Information of China (English)

    Ying-Jie Wang; Hong-Ling Liu; Hai-Tao Guo; Hong-Wei Wen; Jun Liu

    2004-01-01

    AIM: To explore the methods of hepatocytes culture in a collagen gel mixture or between double layers of collagen sandwich configuration and to examine the functional and cytomorphological characteristics of cultured hepatocytes.METHODS: A two-step collagenase perfusion technique was used to isolate the hepatocytes from Wistar rats or newborn Chinese experimental piglets. The isolated hepatocytes were cultured in a collagen gel mixture or between double layers of collagen sandwich configuration respectively. The former was that rat hepatocytes were mixed with type I rat tail collagen solution till gelled, and the medium was added onto the gel. The latter was that swine hepatocytes were seeded on a plate precoated with collagen gel for 24 h, then another layer of collagen gel was overlaid, resulting in a sandwich configuration. The cytomorphological characteristics, albumin secretion, and LDH-release of the hepatocytes cultured in these two models were examined.RESULTS: Freshly isolated rat hepatocytes were successfully mixed and fixed in collagen gel, and cultured in the gel condition. During the culture period, the urea synthesized and secreted by rat hepatocytes was detected throughout the period. Likewise, newborn experimental piglet hepatocytes were successfully fixed between the double layers of collagen gel, forming a sandwich configuration.Within a week of culture, the albumin secreted by swine hepatocytes was detected by SDS/PAGE analysis. The typical cytomorphological characteristics of the hepatocytes cultured by the above two culture models were found under a phasecontrast microscope. There was little LDH-release during the culture period.CONCLUSION: Both collagen gel mixture and double layers of collagen sandwich configuration can provide cultural conditions much closer to in vivoenvironment, and are helpful for maintaining specific hepatic fiJnctions and cytomorphological characteristics. A collagen gel mixture culture may be more eligible for the

  15. Deficiency of G1 regulators P53, P21Cip1 and/or pRb decreases hepatocyte sensitivity to TGFβ cell cycle arrest

    Directory of Open Access Journals (Sweden)

    Harrison David J

    2007-11-01

    Full Text Available Abstract Background TGFβ is critical to control hepatocyte proliferation by inducing G1-growth arrest through multiple pathways leading to inhibition of E2F transcription activity. The retinoblastoma protein pRb is a key controller of E2F activity and G1/S transition which can be inhibited in viral hepatitis. It is not known whether the impairment of pRb would alter the growth inhibitory potential of TGFβ in disease. We asked how Rb-deficiency would affect responses to TGFβ-induced cell cycle arrest. Results Primary hepatocytes isolated from Rb-floxed mice were infected with an adenovirus expressing CRE-recombinase to delete the Rb gene. In control cells treatment with TGFβ prevented cells to enter S phase via decreased cMYC activity, activation of P16INK4A and P21Cip and reduction of E2F activity. In Rb-null hepatocytes, cMYC activity decreased slightly but P16INK4A was not activated and the great majority of cells continued cycling. Rb is therefore central to TGFβ-induced cell cycle arrest in hepatocytes. However some Rb-null hepatocytes remained sensitive to TGFβ-induced cell cycle arrest. As these hepatocytes expressed very high levels of P21Cip1 and P53 we investigated whether these proteins regulate pRb-independent signaling to cell cycle arrest by evaluating the consequences of disruption of p53 and p21Cip1. Hepatocytes deficient in p53 or p21Cip1 showed diminished growth inhibition by TGFβ. Double deficiency had a similar impact showing that in cells containing functional pRb; P21Cip and P53 work through the same pathway to regulate G1/S in response to TGFβ. In Rb-deficient cells however, p53 but not p21Cip deficiency had an additive effect highlighting a pRb-independent-P53-dependent effector pathway of inhibition of E2F activity. Conclusion The present results show that otherwise genetically normal hepatocytes with disabled p53, p21Cip1 or Rb genes respond less well to the antiproliferative effects of TGFβ. As the function of

  16. Hepatocytes isolated from neoplastic liver-immunomagnetic purging as a new source for transplantation

    OpenAIRE

    Gunasegaram, Aravin; Akhter, Javed; Yao, Peng; Johnson, Loreena A; Riodan, Stephen M; Morris, David L

    2008-01-01

    AIM: To investigate whether hepatocytes isolated from macroscopically normal liver during hepatic resection for neoplasia could provide a novel source of healthy hepatocytes, including the development of reliable protocols for malignant cells removal from the hepatocyte preparation.

  17. Differences in the Epigenetic Regulation of Cytochrome P450 Genes between Human Embryonic Stem Cell-Derived Hepatocytes and Primary Hepatocytes.

    Directory of Open Access Journals (Sweden)

    Han-Jin Park

    Full Text Available Human pluripotent stem cell-derived hepatocytes have the potential to provide in vitro model systems for drug discovery and hepatotoxicity testing. However, these cells are currently unsuitable for drug toxicity and efficacy testing because of their limited expression of genes encoding drug-metabolizing enzymes, especially cytochrome P450 (CYP enzymes. Transcript levels of major CYP genes were much lower in human embryonic stem cell-derived hepatocytes (hESC-Hep than in human primary hepatocytes (hPH. To verify the mechanism underlying this reduced expression of CYP genes, including CYP1A1, CYP1A2, CYP1B1, CYP2D6, and CYP2E1, we investigated their epigenetic regulation in terms of DNA methylation and histone modifications in hESC-Hep and hPH. CpG islands of CYP genes were hypermethylated in hESC-Hep, whereas they had an open chromatin structure, as represented by hypomethylation of CpG sites and permissive histone modifications, in hPH. Inhibition of DNA methyltransferases (DNMTs during hepatic maturation induced demethylation of the CpG sites of CYP1A1 and CYP1A2, leading to the up-regulation of their transcription. Combinatorial inhibition of DNMTs and histone deacetylases (HDACs increased the transcript levels of CYP1A1, CYP1A2, CYP1B1, and CYP2D6. Our findings suggest that limited expression of CYP genes in hESC-Hep is modulated by epigenetic regulatory factors such as DNMTs and HDACs.

  18. Hepatic Stellate Cell-Derived Microvesicles Prevent Hepatocytes from Injury Induced by APAP/H2O2

    Directory of Open Access Journals (Sweden)

    Renwei Huang

    2016-01-01

    Full Text Available Hepatic stellate cells (HSCs, previously described for liver-specific mesenchymal stem cells (MSCs, appear to contribute to liver regeneration. Microvesicles (MVs are nanoscale membrane fragments, which can regulate target cell function by transferring contents from their parent cells. The aim of this study was to investigate the effect of HSC-derived MVs on xenobiotic-induced liver injury. Rat and human hepatocytes, BRL-3A and HL-7702, were used to build hepatocytes injury models by n-acetyl-p-aminophenol n-(APAP or H2O2 treatment. MVs were prepared from human and rat HSCs, LX-2, and HST-T6 and, respectively, added to injured BRL-3A and HL-7702 hepatocytes. MTT assay was utilized to determine cell proliferation. Cell apoptosis was analyzed by flow cytometry and hoechst33258 staining. Western blot was used for analyzing the expression of activated caspase-3. Liver injury indicators, alanine aminotransferase (ALT, aspartate aminotransferase (AST, and lactate dehydrogenase (LDH in culture medium were also assessed. Results showed that (1 HSC-MVs derived from LX-2 and HST-T6 were positive to CD90 and annexin V surface markers; (2 HSC-MVs dose-dependently improved the viability of hepatocytes in both injury models; (3 HSC-MVs dose-dependently inhibited the APAP/H2O2 induced hepatocytes apoptosis and activated caspase-3 expression and leakage of LDH, ALT, and AST. Our results demonstrate that HSC-derived MVs protect hepatocytes from toxicant-induced injury.

  19. Novel regulation of Ski protein stability and endosomal sorting by actin cytoskeleton dynamics in hepatocytes.

    Science.gov (United States)

    Vázquez-Victorio, Genaro; Caligaris, Cassandre; Del Valle-Espinosa, Eugenio; Sosa-Garrocho, Marcela; González-Arenas, Nelly R; Reyes-Cruz, Guadalupe; Briones-Orta, Marco A; Macías-Silva, Marina

    2015-02-13

    TGF-β-induced antimitotic signals are highly regulated during cell proliferation under normal and pathological conditions, such as liver regeneration and cancer. Up-regulation of the transcriptional cofactors Ski and SnoN during liver regeneration may favor hepatocyte proliferation by inhibiting TGF-β signals. In this study, we found a novel mechanism that regulates Ski protein stability through TGF-β and G protein-coupled receptor (GPCR) signaling. Ski protein is distributed between the nucleus and cytoplasm of normal hepatocytes, and the molecular mechanisms controlling Ski protein stability involve the participation of actin cytoskeleton dynamics. Cytoplasmic Ski is partially associated with actin and localized in cholesterol-rich vesicles. Ski protein stability is decreased by TGF-β/Smads, GPCR/Rho signals, and actin polymerization, whereas GPCR/cAMP signals and actin depolymerization promote Ski protein stability. In conclusion, TGF-β and GPCR signals differentially regulate Ski protein stability and sorting in hepatocytes, and this cross-talk may occur during liver regeneration.

  20. Mechanism of radiation-induced diacylglycerol production in primary cultured rat hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Nakajima, Tetsuo; Yukawa, Osami [National Inst. of Radiological Sciences, Chiba (Japan)

    1999-06-01

    Protein kinase C (PKC) is known to be a key enzyme in radiation-induced signal transduction pathways. We have previously demonstrated that {gamma}-irradiation induces PKC activation and translocation from cytosol to membranes as a consequence of membrane lipid peroxidation in cultured rat hepatocytes (Int. J. Radiat. Biol. 70, 473-480, 1996). The present study was undertaken to investigate production of diacylglycerol, an endogenous activator of PKC, following {gamma}-irradiation of hepatocytes. Diacylglycerol content increased 3 min after irradiation, then decreased at 15 min and increased again at 30 min, indicating a biphasic pattern. This result implies participation of diacylglycerol in the radiation-induced activation of PKC in hepatocytes. In order to clarify the mechanism of the initial process of radiation-induced diacylglycerol production, the effects of reactive oxygens were investigated. Treatment of cells with hydroxyl radical, a major oxygen radical produced by radiation, induced diacylglycerol production without any change in the content of phosphatidylcholine, showing a peak at 1 min after treatment. No change in the diacylglycerol content was observed at that time by hydrogen peroxide treatment. Furthermore, the diacylglycerol production by hydroxyl radical was inhibited by pretreatment with neomycin sulfate, a phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitor. These results suggest that radiation exerts PI-PLC activation through hydroxyl radical generation, followed by diacylglycerol production and PKC activation. (author)

  1. Effects of retrorsine on mouse hepatocyte proliferation after liver injury

    Institute of Scientific and Technical Information of China (English)

    Xiao-Fei Zhou; Qian Wang; Jian-Xin Chu; Ai-Lian Liu

    2006-01-01

    AIM: To study the effect of retrorsine on mouse hepatocyte proliferation.METHODS: Mice and rats were treated respectively with two injections of retrorsine (as retrosine-treated group) or saline (as non-treated group) at 2 wk intervals.They received a single injection of carbon tetrachloride (CCl4) 4 wk later. On d 0, 1, 2, 3, 4, 6, 15 after CCl4 administration, the animals were killed and their livers were excised. Hematoxylin and eosin (HE) staining and Ki-67 antibody immunohistochemical analysis of liver samples were used to evaluate the pathological changes and hepatocyte proliferation.RESULTS: In rats treated with retrorsine and CCl4, the liver displayed obvious megalocytosis, proliferation of mild bile duct, small hepatocyte-forming nodule, which were not found in liver samples from non-treated group.However, in mice treated with retrorsine combined with CCl4, the liver displayed hepatocyte degeneration and necrosis in perivenous areas. There was no obvious difference between retrorsine-treated group and nontreated group. Ki-67 immunohistochemical analysis showed that in rats treated with retrorsine, the positive hepatocytes mainly found in small hepatocyte nodules,were obviously less than those in non-treated group. The mice treated with retrorsine showed that the number of Ki-67 positive hepatocytes was very high and more than that in non-treated group.CONCLUSION: Retrorsine has no effect on mouse hepatocyte proliferation.

  2. Scavenger receptor BI boosts hepatocyte permissiveness to Plasmodium infection.

    NARCIS (Netherlands)

    Yalaoui, S.; Huby, T.; Franetich, J.F.; Gego, A.; Rametti, A.; Moreau, M.; Collet, X.; Siau, A.; Gemert, G.J.A. van; Sauerwein, R.W.; Luty, A.J.F.; Vaillant, J.C.; Hannoun, L.; Chapman, J.; Mazier, D.; Froissard, P.

    2008-01-01

    Infection of hepatocytes by Plasmodium falciparum sporozoites requires the host tetraspanin CD81. CD81 is also predicted to be a coreceptor, along with scavenger receptor BI (SR-BI), for hepatitis C virus. Using SR-BI-knockout, SR-BI-hypomorphic and SR-BI-transgenic primary hepatocytes, as well as s

  3. Mechanisms of the statins cytotoxicity in freshly isolated rat hepatocytes.

    Science.gov (United States)

    Abdoli, Narges; Heidari, Reza; Azarmi, Yadollah; Eghbal, Mohammad Ali

    2013-06-01

    Statins are potent drugs, used as lipid-lowering agents in cardiovascular diseases. Hepatotoxicity is one of the serious adverse effects of statins, and the exact mechanism of hepatotoxicity is not yet clear. In this study, the cytotoxic effects of the most commonly used statins, that is, atorvastatin, lovastatin, and simvastatin toward isolated rat hepatocytes, were evaluated. Markers, such as cell death, reactive oxygen species (ROS) formation, lipid peroxidation, mitochondrial membrane potential, and the amount of reduced and oxidized glutathione in the statin-treated hepatocytes, were investigated. It was found that the statins caused cytotoxicity toward rat hepatocytes dose dependently. An elevation in ROS formation, accompanied by a significant amount of lipid peroxidation and mitochondrial depolarization, was observed. Cellular glutathione reservoirs were decreased, and a significant amount of oxidized glutathione was formed. This study suggests that the adverse effect of statins toward hepatocytes is mediated through oxidative stress and the hepatocytes mitochondria play an important role in the statin-induced toxicity.

  4. Dichloroacetate Stimulates Glycogen Accumulation in Primary Hepatocytes through an Insulin-Independent Mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Lingohr, Melissa K.(Washington State University); Bull, Richard J.(SELF-EMPLOYED CONSULTANTS); Kato-Weinstein, Junko (UNIVERSITY PROGRAMS); Thrall, Brian D.(BATTELLE (PACIFIC NW LAB))

    2002-01-01

    Dichloroacetate (DCA), a by-product of water chlorination, causes liver cancer in B6C3F1 mice. A hallmark response observed in mice exposed to carcinogenic doses of DCA is an accumulation of hepatic glycogen content. To distinguish whether the in vivo glycogenic effect of DCA was dependent on insulin and insulin signaling proteins, experiments were conducted in isolated hepatocytes where insulin concentrations could be controlled. In hepatocytes isolated from male B6C3F1 mice, DCA increased glycogen levels in a dose-related manner, independently of insulin. The accumulation of hepatocellular glycogen induced by DCA was not the result of decreased glycogenolysis, since DCA had no effect on the rate of glucagon-stimulated glycogen breakdown. Glycogen accumulation caused by DCA treatment was not hindered by inhibitors of extracellular-regulated protein kinase kinase (Erk1/2 kinase or MEK) or p70 kDa S6 protein kinase (p70(S6K)), but was completely blocked by the phosphatidylinositol 3-kinase (PI3K) inhibitors, LY294002 and wortmannin. Similarly, insulin-stimulated glycogen deposition was not influenced by the Erk1/2 kinase inhibitor, PD098509, or the p70(S6K) inhibitor, rapamycin. Unlike DCA-stimulated glycogen deposition, PI3K-inhibition only partially blocked the glycogenic effect of insulin. DCA did not cause phosphorylation of the downstream PI3K target protein, protein kinase B (PKB/Akt). The phosphorylation of PKB/Akt did not correlate to insulin-stimulated glycogenesis either. Similar to insulin, DCA in the medium decreased IR expression in isolated hepatocytes. The results indicate DCA increases hepatocellular glycogen accumulation through a PI3K-dependent mechanism that does not involve PKB/Akt and is, at least in part, different from the classical insulin-stimulated glycogenesis pathway. Somewhat surprisingly, insulin-stimulated glycogenesis also appears not to involve PKB/Akt in isolated murine hepatocytes.

  5. Microarray analyses and molecular profiling of steatosis induction in immortalized human hepatocytes.

    Science.gov (United States)

    De Gottardi, Andrea; Vinciguerra, Manlio; Sgroi, Antonino; Moukil, Moulay; Ravier-Dall'Antonia, Florence; Pazienza, Valerio; Pugnale, Paolo; Foti, Michelangelo; Hadengue, Antoine

    2007-08-01

    Hepatic steatosis is an important risk factor for the development of inflammation, fibrosis and impaired liver regeneration. The factors regulating lipid accumulation and driving hepatic steatosis toward inflammation, fibrosis and impaired regeneration are largely unknown. The aim of this study was to identify major alterations in gene expression occurring in steatotic hepatocytes, and to analyze how these changes impact cellular processes associated with steatosis. Microarray gene chips and RT-PCR were performed to analyze changes in gene expression induced in fatty human immortalized hepatocytes after treatment with 50 muM oleic acid for 7 days. Lipid metabolism and triglyceride accumulation in these cells was examined by Oil-Red-O staining, thin-layer chromatography (TLC) and immunofluorescence. Caspase 3 activity, BrdU incorporation and trypan blue exclusion were used to study apoptosis, proliferation and cell viability. Finally, quantitative analysis of signalling induced by insulin was performed by Western blot. Characterization of steatosis in three hepatocyte-derived cell lines indicated that the immortalized human hepatocytes (IHH) line was the most appropriate cell line for this study. Gene expression analysis showed significant alterations in the transcription of two major classes of genes involved either in cholesterol and fatty acid biosynthesis, as well as lipid export, or in apoptosis and cell proliferation. Such changes were functionally relevant, since TLC indicated that synthesis and accumulation of triglycerides were increased in steatotic cells, while synthesis of cholesterol and fatty acids were decreased. Lipid accumulation in IHH was associated with an increased apoptosis and an inhibition of cell proliferation and viability. No detectable changes in genes associated with insulin resistance were observed in steatotic cells, but signalling induced by insulin was more efficient in steatotic IHH as compared to control cells. We conclude that IHH

  6. Reactive nitrogen species in acetaminophen-induced mitochondrial damage and toxicity in mouse hepatocytes.

    Science.gov (United States)

    Burke, Angela S; MacMillan-Crow, Lee Ann; Hinson, Jack A

    2010-07-19

    Acetaminophen (APAP) toxicity in primary mouse hepatocytes occurs in two phases. The initial phase (0-2 h) occurs with metabolism to N-acetyl-p-benzoquinoneimine which depletes glutathione, and covalently binds to proteins, but little toxicity is observed. Subsequent washing of hepatocytes to remove APAP and reincubating in media alone (2-5 h) results in toxicity. We previously reported that the reincubation phase occurs with mitochondrial permeability transition (MPT) and increased oxidative stress (dichlorodihydrofluorescein fluorescence) (DCFH(2)). Since DCFH(2) may be oxidized by multiple oxidative mechanisms, we investigated the role of reactive nitrogen species (RNS) leading to 3-nitrotyrosine in proteins by ELISA and by immunoblots. Incubation of APAP with hepatocytes for 2 h did not result in toxicity or protein nitration; however, washing hepatocytes and reincubating in media alone (2-5 h) resulted in protein nitration which correlated with toxicity. Inclusion of the MPT inhibitor, cyclosporine A, in the reincubation media eliminated toxicity and protein nitration. The general nitric oxide synthase (NOS) inhibitor L-NMMA and the neuronal NOS (NOS1) inhibitor, 7-nitroindazole, added in the reincubation media decreased toxicity and protein nitration; however, neither the inducible NOS (NOS2) inhibitors L-NIL (N6-(1-iminoethyl)-L-lysine) nor SAIT (S-(2-aminoethyl)isothiourea) decreased protein nitration or toxicity. The RNS scavengers, N-acetylcysteine, and high concentrations of APAP, added in the reincubation phase decreased toxicity and protein nitration. 7-Nitroindazole and cyclosporine A inhibited the APAP-induced loss of mitochondrial membrane potential when added in the reincubation phase. The data indicate a role for RNS in APAP induced toxicity.

  7. Metabolic activation capacity by primary hepatocytes expands the applicability of the embryonic stem cell test as alternative to experimental animal testing.

    Science.gov (United States)

    Hettwer, Michael; Reis-Fernandes, Marcos A; Iken, Marcus; Ott, Michael; Steinberg, Pablo; Nau, Heinz

    2010-08-01

    The murine embryonic stem cell test (EST) represents a validated alternative method for in vivo embryotoxicity testing. In the present study, primary hepatocytes were combined with the EST by a preincubation approach to improve its predictivity on bioactivation caused teratogenicity. As substances the well-known proteratogens cyclophosphamide (CPA) and valpromide (VPD) were used. The embryotoxic potential of CPA was detected by a strong decrease of the resulting ID(50)-concentration (50% inhibition of ES cell differentiation) after incubation with murine hepatocytes. Interspecies variation in metabolism was detected by testing VPD. After incubation of VPD with murine hepatocytes no inhibition of ES cell differentiation was observed, since hardly any teratogenic VPD metabolites were formed. In contrast, with human hepatocytes a significant conversion of VPD into the teratogen valproic acid (VPA) was observed. In summary we developed a co-culture approach for embryotoxicity testing, whereby the test compounds were incubated with hepatocytes and the supernatant was added to the ES cell culture to obtain a dose dependency of the preincubated test substances. PMID:20132877

  8. Cold Preservation of Human Adult Hepatocytes for Liver Cell Therapy.

    Science.gov (United States)

    Duret, Cedric; Moreno, Daniel; Balasiddaiah, Anangi; Roux, Solene; Briolotti, Phillipe; Raulet, Edith; Herrero, Astrid; Ramet, Helene; Biron-Andreani, Christine; Gerbal-Chaloin, Sabine; Ramos, Jeanne; Navarro, Francis; Hardwigsen, Jean; Maurel, Patrick; Aldabe, Rafael; Daujat-Chavanieu, Martine

    2015-01-01

    Hepatocyte transplantation is a promising alternative therapy for the treatment of hepatic failure, hepatocellular deficiency, and genetic metabolic disorders. Hypothermic preservation of isolated human hepatocytes is potentially a simple and convenient strategy to provide on-demand hepatocytes in sufficient quantity and of the quality required for biotherapy. In this study, first we assessed how cold storage in three clinically safe preservative solutions (UW, HTS-FRS, and IGL-1) affects the viability and in vitro functionality of human hepatocytes. Then we evaluated whether such cold-preserved human hepatocytes could engraft and repopulate damaged livers in a mouse model of liver failure. Human hepatocytes showed comparable viabilities after cold preservation in the three solutions. The ability of fresh and cold-stored hepatocytes to attach to a collagen substratum and to synthesize and secrete albumin, coagulation factor VII, and urea in the medium after 3 days in culture was also equally preserved. Cold-stored hepatocytes were then transplanted in the spleen of immunodeficient mice previously infected with adenoviruses containing a thymidine kinase construct and treated with a single dose of ganciclovir to induce liver injury. Engraftment and liver repopulation were monitored over time by measuring the blood level of human albumin and by assessing the expression of specific human hepatic mRNAs and proteins in the recipient livers by RT-PCR and immunohistochemistry, respectively. Our findings show that cold-stored human hepatocytes in IGL-1 and HTS-FRS preservative solutions can survive, engraft, and proliferate in a damaged mouse liver. These results demonstrate the usefulness of human hepatocyte hypothermic preservation for cell transplantation. PMID:25622096

  9. Protective role of morin, a flavonoid, against high glucose induced oxidative stress mediated apoptosis in primary rat hepatocytes.

    Directory of Open Access Journals (Sweden)

    Radhika Kapoor

    Full Text Available Apoptosis is an early event of liver damage in diabetes and oxidative stress has been linked to accelerate the apoptosis in hepatocytes. Therefore, the compounds that can scavenge ROS may confer regulatory effects on high-glucose induced apoptosis. In the present study, primary rat hepatocytes were exposed to high concentration (40 mM of glucose. At this concentration decreased cell viability and enhanced ROS generation was observed. Depleted antioxidant status of hepatocytes under high glucose stress was also observed as evident from transcriptional level and activities of antioxidant enzymes. Further, mitochondrial depolarisation was accompanied by the loss of mitochondrial integrity and altered expression of Bax and Bcl-2. Increased translocation of apoptotic proteins like AIF (Apoptosis inducing factor & Endo-G (endonuclease-G from its resident place mitochondria to nucleus was also observed. Cyt-c residing in the inter-membrane space of mitochondria also translocated to cytoplasm. These apoptotic proteins initiated caspase activation, DNA fragmentation, chromatin condensation, increased apoptotic DNA content in glucose treated hepatocytes, suggesting mitochondria mediated apoptotic mode of cell death. Morin, a dietary flavonoid from Psidium guajava was effective in increasing the cell viability and decreasing the ROS level. It maintained mitochondrial integrity, inhibited release of apoptotic proteins from mitochondria, prevented DNA fragmentation, chromatin condensation and hypodiploid DNA upon exposure to high glucose. This study confirms the capacity of dietary flavonoid Morin in regulating apoptosis induced by high glucose via mitochondrial mediated pathway through intervention of oxidative stress.

  10. Concanavalin A/IFN-gamma triggers autophagy-related necrotic hepatocyte death through IRGM1-mediated lysosomal membrane disruption.

    Directory of Open Access Journals (Sweden)

    Chih-Peng Chang

    Full Text Available Interferon-gamma (IFN-γ, a potent Th1 cytokine with multiple biological functions, can induce autophagy to enhance the clearance of the invading microorganism or cause cell death. We have reported that Concanavalin A (Con A can cause autophagic cell death in hepatocytes and induce both T cell-dependent and -independent acute hepatitis in immunocompetent and immunodeficient mice, respectively. Although IFN-γ is known to enhance liver injury in Con A-induced hepatitis, its role in autophagy-related hepatocyte death is not clear. In this study we report that IFN-γ can enhance Con A-induced autophagic flux and cell death in hepatoma cell lines. A necrotic cell death with increased lysosomal membrane permeabilization (LMP is observed in Con A-treated hepatoma cells in the presence of IFN-γ. Cathepsin B and L were released from lysosomes to cause cell death. Furthermore, IFN-γ induces immunity related GTPase family M member 1(IRGM1 translocation to lysosomes and prolongs its activity in Con A-treated hepatoma cells. Knockdown of IRGM1 inhibits the IFN-γ/Con A-induced LMP change and cell death. Furthermore, IFN-γ(-/- mice are resistant to Con A-induced autophagy-associated necrotic hepatocyte death. We conclude that IFN-γ enhances Con A-induced autophagic flux and causes an IRGM1-dependent lysosome-mediated necrotic cell death in hepatocytes.

  11. Protective effects of pLNCX-SOD gene transfection on hepatocyte injury induced by obstructive jaundice in rats

    Institute of Scientific and Technical Information of China (English)

    Peng Gong; Zhong-Yu Wang; Hong-Jiang Wang

    2007-01-01

    BACKGROUND: Hydrophobic bile acids lead to the generation of oxygen free radicals in mitochondria. Accordingly, this study is to investigate if gene delivery of superoxide dismutase (SOD) will reduce hepatocyte injury caused by experimental cholestasis. METHODS:The recombinant of pLNCX-SOD gene packaged with lipofection of AMINE was transfected into hepatocytes in vitro, which stably expressed the SOD gene. RESULTS:After transfection, hepatocytes enhanced the protective effect against injury to bile and the toxicity of serum in obstructive jaundice. The inhibition of bile at the concentration of 2% (v∶v, bile: DMEM 1∶50) decreased obviously from (78.80±12.35)% to (43.35±9.69)% in 12 hours, from (82.55±11.27)% to (-26.64±7.66)% in 24 hours, and from (83.83±18.69)% to (-19.27±14.38)% in 48 hours, compared with that of the untransfected cells (P CONCLUSION:Hepatocytes transfected with the pLNCX-SOD gene could obviously be resistant to the toxicity of bile and serum from rat with obstructive jaundice.

  12. Peroxisome proliferator-activated receptor alpha acts as a mediator of endoplasmic reticulum stress-induced hepatocyte apoptosis in acute liver failure

    Directory of Open Access Journals (Sweden)

    Li Zhang

    2016-07-01

    Full Text Available Peroxisome proliferator-activated receptor α (PPARα is a key regulator to ameliorate liver injury in cases of acute liver failure (ALF. However, its regulatory mechanisms remain largely undetermined. Endoplasmic reticulum stress (ER stress plays an important role in a number of liver diseases. This study aimed to investigate whether PPARα activation inhibits ER stress-induced hepatocyte apoptosis, thereby protecting against ALF. In a murine model of D-galactosamine (D-GalN- and lipopolysaccharide (LPS-induced ALF, Wy-14643 was administered to activate PPARα, and 4-phenylbutyric acid (4-PBA was administered to attenuate ER stress. PPARα activation ameliorated liver injury, because pre-administration of its specific inducer, Wy-14643, reduced the serum aminotransferase levels and preserved liver architecture compared with that of controls. The protective effect of PPARα activation resulted from the suppression of ER stress-induced hepatocyte apoptosis. Indeed, (1 PPARα activation decreased the expression of glucose-regulated protein 78 (Grp78, Grp94 and C/EBP-homologous protein (CHOP in vivo; (2 the liver protection by 4-PBA resulted from the induction of PPARα expression, as 4-PBA pre-treatment promoted upregulation of PPARα, and inhibition of PPARα by small interfering RNA (siRNA treatment reversed liver protection and increased hepatocyte apoptosis; (3 in vitro PPARα activation by Wy-14643 decreased hepatocyte apoptosis induced by severe ER stress, and PPARα inhibition by siRNA treatment decreased the hepatocyte survival induced by mild ER stress. Here, we demonstrate that PPARα activation contributes to liver protection and decreases hepatocyte apoptosis in ALF, particularly through regulating ER stress. Therefore, targeting PPARα could be a potential therapeutic strategy to ameliorate ALF.

  13. Raman spectroscopic identification of normal and malignant hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Jianyu Guo; Bing Du; Min Qian; Weiying Cai; Zugeng Wang; Zhenrong Sun

    2009-01-01

    Raman spectroscopy has strong potential for providing non-invasion diagnosis of cancers. In this paper, micro-Raman spectroscopy is used to diagnose one most common liver cancer, hepatocellular carcinoma (HCC). The statistical analyzes, including i-test, principal component analysis (PCA), and linear discrim inant analysis (LDA), are performed on the Raman spectra of malignant and normal hepatocytes. The t-test-LDA results show that the 786- and 1004-cm-1 bands of the malignant and normal hepatocytes are significantly different, and PCA-LDA results show an overall accuracy of 100% for the Raman spectro scopic identification of normal and malignant hepatocytes in our experiment.

  14. Hepatoprotective Activity of Water Extracts from Chaga Medicinal Mushroom, Inonotus obliquus (Higher Basidiomycetes) Against Tert-Butyl Hydroperoxide-Induced Oxidative Liver Injury in Primary Cultured Rat Hepatocytes.

    Science.gov (United States)

    Hong, Ki Bae; Noh, Dong Ouk; Park, Yooheon; Suh, Hyung Joo

    2015-01-01

    We examined the hepatoprotective activity of Inonotus obliquus water extract (IO-W) against tert-butyl hydroperoxide (t-BHP)-induced oxidative liver injury in the primary cultured rat hepatocyte. The 50% radical scavenging concentrations (SC50s) of IO-W for radical-scavenging activity against 2,2'-azino-bis-(3-ethylbenzothi- azoline-6-sulfonic acid) (ABTS) and 1,1-diphenyl-2-picryl-hydrazyl (DPPH) were 5.19 mg/mL and 0.39 mg/mL, respectively. IO-W pretreatment to the primary cultured hepatocytes significantly (p100 µg/ mL). In conclusion, this study demonstrates that IO-W exhibited hepatoprotective activity against t-BHP-induced oxidative liver injury in the primary cultured hepatocyte probably via its abilities of quenching free radicals, inhibiting the leakage of ALT, AST, and LDH, and decreasing MDA formation. PMID:26853962

  15. Evaluation of the endothelin receptor antagonists ambrisentan, bosentan, macitentan, and sitaxsentan as hepatobiliary transporter inhibitors and substrates in sandwich-cultured human hepatocytes.

    Directory of Open Access Journals (Sweden)

    Eve-Irene Lepist

    Full Text Available BACKGROUND: Inhibition of the transporter-mediated hepatobiliary elimination of bile salts is a putative mechanism for liver toxicity observed with some endothelin receptor antagonists (ERAs. METHODS: Sandwich-cultured human hepatocytes were used to study the hepatobiliary distribution and accumulation of exogenous taurocholate, ERAs and endogenous bile acids. The molecular mechanisms for findings in hepatocytes or clinical observations were further explored using either vesicular assays (efflux transporters or transfected cell-lines (uptake transporters. Inhibition constants (IC50 were measured for the human hepatobiliary transporters bile salt export pump (BSEP, sodium taurocholate cotransporting polypeptide (NTCP, multidrug resistance protein 2 (MRP2, P-glycoprotein (Pgp, breast cancer resistance protein (BCRP, organic anion-transporting polypeptide 1B1 (OATP1B1 and OATP1B3. RESULTS: The ERAs showed dose-dependent reductions in exogenous taurocholate cellular accumulation in human hepatocytes, with macitentan having the greatest effect. Consistent with their effects on bile acids, the ERAs inhibited bile transporters. IC50 values for OATP1B1 and OATP1B3 ranged from 2 µM for macitentan to 47 µM for ambrisentan. Macitentan and bosentan also inhibited NTCP with IC50 values of 10 and 36 µM, respectively. Similar to previously reported findings with sitaxsentan, BSEP inhibition was observed for bosentan and macitentan with IC50 values of 42 and 12 µM, respectively. In contrast, ambrisentan showed little or no inhibition of these transporters. Other transporters tested were weakly inhibited by the ERAs. Accumulation in hepatocytes was also a factor in the effects on bile transport. Macitentan demonstrated the greatest accumulation in human hepatocytes (∼100x followed by sitaxsentan (∼40x, bosentan (∼20x and ambrisentan (∼2x. CONCLUSIONS: Significant differences in the inhibition of hepatic transporters were observed between the

  16. The effect of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR on fatty acid oxidation in hepatocytes isolated from neonatal piglets

    Directory of Open Access Journals (Sweden)

    Xi Lin

    2012-10-01

    Full Text Available Abstract In the present study, the effect of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR on long-chain fatty acid oxidation by hepatocytes isolated from suckled neonatal pig liver (a low ketogenic and lipogenic tissue was tested. Incubation of hepatocytes with AICAR (0.5 mM in the presence of 1 mM of carnitine and 10 mM of glucose for 1 hour at 37°C had no significant effect on total [1-14C]-palmitate (0.5 mM oxidation (14CO2 and 14C-Acid soluble products (ASP. Consistent with the fatty acid oxidation, carnitine palmitoyltransferase I activity and inhibition of its activity by malonyl-CoA (10 μM assayed in cell homogenate also remained constant. However, addition of AICAR to the hepatocytes decreased 14CO2 production by 18% compared to control (p 2 caused a significant difference in distribution of oxidative products between 14CO2 and 14C-ASP (p 14C-ASP. Addition of insulin to hepatocyte incubations with AICAR did not change the oxidative product distribution between CO2 and ASP, but further promoted ACC activity. The increased ACC activity was 70% higher than in the control group when citrate was absent in the reaction medium and was 30% higher when citrate was present in the medium. Our results suggest that AICAR may affect the distribution of metabolic products from fatty acid oxidation by changing ACC activity in hepatocyte isolated from suckled neonatal piglets; however, the basis for the increase in ACC activity elicited by AICAR is not apparent.

  17. Modeling human liver biology using stem cell-derived hepatocytes

    OpenAIRE

    Sun, Pingnan; Zhou, XiaoLing; Farnworth, Sarah; Arvind H Patel; Hay, David C.

    2013-01-01

    Stem cell-derived hepatocytes represent promising models to study human liver biology and disease. This concise review discusses the recent progresses in the field, with a focus on human liver disease, drug metabolism and virus infection.

  18. Modeling Human Liver Biology Using Stem Cell-Derived Hepatocytes

    OpenAIRE

    Arvind H Patel; Hay, David C.; Farnworth, Sarah L.; Pingnan Sun; Xiaoling Zhou

    2013-01-01

    Stem cell-derived hepatocytes represent promising models to study human liver biology and disease. This concise review discusses the recent progresses in the field, with a focus on human liver disease, drug metabolism and virus infection.

  19. JNK1-dependent PUMA Expression Contributes to Hepatocyte Lipoapoptosis*

    OpenAIRE

    Cazanave, Sophie C; Mott, Justin L.; Elmi, Nafisa A.; Bronk, Steven F.; Werneburg, Nathan W.; Akazawa, Yuko; Kahraman, Alisan; Garrison, Sean P; Zambetti, Gerard P.; Charlton, Michael R.; Gores, Gregory J.

    2009-01-01

    Free fatty acids (FFA) induce hepatocyte lipoapoptosis by a c-Jun N-terminal kinase (JNK)-dependent mechanism. However, the cellular processes by which JNK engages the core apoptotic machinery during lipotoxicity, especially activation of BH3-only proteins, remain incompletely understood. Thus, our aim was to determine whether JNK mediates induction of BH3-only proteins during hepatocyte lipoapoptosis. The saturated FFA palmitate, but not the monounsaturated FFA oleate, induces an increase in...

  20. Effects of Aronia melanocarpa Fruit Juice on Isolated Rat Hepatocytes

    OpenAIRE

    Magdalena Kondeva-Burdina; Stefka Valcheva-Kuzmanova; Tsvetelina Markova; Mitka Mitcheva; Anna Belcheva

    2015-01-01

    Background: Aronia melanocarpa (Michx.) Elliot fruits are very rich in polyphenols – procyanidins, flavonoids, and phenolic acids. Objective: On rat hepatocytes, isolated by two-stepped collagenase perfusion, we investigated the effect of A. melanocarpa fruit juice (AMFJ) in two models of liver toxicity caused by (i) metabolic bioactivation of carbon tetrachloride (CCl4), and (ii) tert-butyl hydroperoxide (t-BuOOH)-induced oxidative stress. Materials and Methods: Isolated rat hepatocytes are ...

  1. The use of pig hepatocytes for biotransformation and toxicity studies.

    OpenAIRE

    Hoogenboom, L.A.P.

    1991-01-01

    The three main objectives of this study were, (1) to investigate the possibility to isolate viable hepatocytes from liver samples of pigs, (2) to study their use for biotransformation and toxicity studies, and (3) to demonstrate the value of this model, in particular in the field of residue toxicology.The main result from this study was the fact that hepatocytes can be successfully isolated from samples of pig livers obtained at the slaughterhouse, and that the cells retained a high viability...

  2. Effects of betaine on ethanol-stimulated secretion of IGF-Ⅰ and IGFBP-1 in rat primary hepatocytes: involvement of p42/44 MAPK activation

    Institute of Scientific and Technical Information of China (English)

    Myeong Soo Lee; Myung-Sunny Kim; Soo Young Park; Chang-Won Kang

    2006-01-01

    AIM: To evaluate the effects of betaine on the ethanolinduced secretion of IGF-Ⅰ and IGFBP-1 using radioimmunoassay and Western blotting, respectively, in primary cultured rat hepatocytes.METHODS: Hepatocytes isolated from male Sprague-Dawley rats were incubated with various concentrations of ethanol and PD98059 procedures. The hepatocytes were also treated with different doses of betaine (10-5,10-4, and 10-3 mol/L). We measured IGF-Ⅰ and IGFBP-1 using radioimmunoassay and Western blotting, respectively.RESULTS: The ethanol-induced inhibition of IGF-Ⅰ secretion was attenuated by betaine in a concentration-dependent manner in primary cultured rat hepatocytes. At 10-3 mol/L, betaine significantly increased IGF-Ⅰ secretion but decreased IGFBP-1 secretion. In addition, p42/44 mitogen-activated protein kinase (MAPK) activity was accelerated significantly from 10 min to 5 h after treatment with 10-3 mol/L betaine. Furthermore, the changes in IGF-1 and IGFBP-1 secretion resulting from the increased betaine-induced p42/44 MAPK activity in primary cultured rat hepatocytes was blocked by treatment with the MAPK inhibitor PD98059. Betaine treatment blocked the ethanol-induced inhibition of IGF-Ⅰ secretion and p42/44 MAPK activity, and the ethanol-induced increase in IGFBP-1 secretion.CONCLUSION: Betaine modulates the secretion of IGF-Ⅰ and IGFBP-1 via the activation of p42/44 MAPK in primary cultured rat hepatocytes. Betaine also alters the MAPK activations induced by ethanol.

  3. alpha-Amanitin induced apoptosis in primary cultured dog hepatocytes.

    Directory of Open Access Journals (Sweden)

    Adam Szelag

    2010-06-01

    Full Text Available Amatoxin poisoning is caused by mushroom species belonging to the genera Amanita, Galerina and Lepiota with the majority of lethal mushroom exposures attributable to Amanita phalloides. High mortality rate in intoxications with these mushrooms is principally a result of the acute liver failure following significant hepatocyte damage due to hepatocellular uptake of amatoxins. A wide variety of amatoxins have been isolated; however, alpha-amanitin (alpha-AMA appears to be the primary toxin. Studies in vitro and in vivo suggest that alpha-AMA does not only cause hepatocyte necrosis, but also may lead to apoptotic cell death. The objective of this study was to evaluate the complex hepatocyte apoptosis in alpha-AMA cytotoxicity. All experiments were performed on primary cultured canine hepatocytes. The cells were incubated for 12 h with alpha-AMA at a final concentration of 1, 5, 10 and 20 microM. Viability test (MTT assay, apoptosis evaluation (TUNEL reaction, detection of DNA laddering and electron microscopy were performed at 6 and 12 h of exposure to alpha-AMA. There was a clear correlation between hepatocyte viability, concentration of alpha-AMA and time of exposure to this toxin. The decline in cultured dog hepatocyte viability during the exposure to alpha-AMA is most likely preceded by enhanced cellular apoptosis. Our results demonstrate that apoptosis might contribute to pathogenesis of the severe liver injury in the course of amanitin intoxication, particularly during the early phase of poisoning.

  4. Protective effect of N-acetylcysteine against lipopolysaccharide injury in hepatocytes of neonatal mice

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The present study provides supportive evidence for the effective prevention and treatment of lipopolysaccharide(LPS)-induced hepatocyte injury in neonatal mice by N-acetylcysteine(NAC).Hepatocytes of neonatal mice were obtained through collagenase digestion of the liver.The hepatocytes were treated either with LPS (10 μg/mL)alone or with NAC(5 mmol/L)for 1 h before the addition of LPS(10 μg/mL).After LPS treatment,12 wells of the cultured hepatocytes and supernatants were harvested at 0,6,and 12 h,respectively.Levels of alanine aminotransferase(ALT)and nitric oxide(NO)in the supernatant were biochemically quantified and reverse transcription-polymerase chain reaction(RT-PCR)was performed to detect the expression of inducible nitric oxide synthase(iNOS)mRNA after different treatments.At 0 h following the treatment of primary cultured hepatocytes with LPS,the levels of ALT,NO and iNOS mRNA in the supernatant were(21.1±4.78)u/L,(1.6±0.31)μmol/L and 0.17±0.023,respectively;at 6 h,(59.8±8.59)u/L,(6.6±0.81)μmol/L and 0.71±0.091;and at 12 h,(89.6±15.30)u/L,(7.8±1.01)μmol/L,and 0.71±0.097.The levels of ALT,NO and iNOS mRNA at 6 and 12 h increased significantly,compared to those at 0 h(P<0.01).In contrast to LPS treatment alone,pretreatment with NAC before LPS addition significantly reduced the levels of ALT,NO and iNOS mRNA in the supernatant at 6 h to(40.8±7.30)u/L,(3.2±0.71)μmol/L,and 0.41±0.060;and at 12 h to(55.4±5.48)u/L,(4.0±0.71)μmol/L,and 0.40±0.067,respectively (P<0.01).However,the levels of ALT,NO and iNOS mRNA at 0 h did not change significantly with both treatment approaches.NAC has protective effects in hepatocytes of neonatal mice against LPS-induced injury as shown by the reduced levels of ALT,NO and iNOS mRNA when primary hepatocytes were treated with NAC prior to LPS stimulation.We postulate that NAC exhibits its protective function by inhibiting LPS-induced transcription of iNOS,resulting in decreased levels of NO.

  5. Force spectroscopy of hepatocytic extracellular matrix components

    Energy Technology Data Exchange (ETDEWEB)

    Yongsunthon, R., E-mail: YongsuntR@Corning.com [Corning Incorporated, SP-FR-01, R1S32D, Corning, NY 14831 (United States); Baker, W.A.; Bryhan, M.D.; Baker, D.E.; Chang, T.; Petzold, O.N.; Walczak, W.J.; Liu, J.; Faris, R.A.; Senaratne, W.; Seeley, L.A.; Youngman, R.E. [Corning Incorporated, SP-FR-01, R1S32D, Corning, NY 14831 (United States)

    2009-07-15

    We present atomic force microscopy and force spectroscopy data of live hepatocytes (HEPG2/C3A liver cell line) grown in Eagle's Minimum Essential Medium, a complex solution of salts and amino acids commonly used for cell culture. Contact-mode imaging and force spectroscopy of this system allowed correlation of cell morphology and extracellular matrix (ECM) properties with substrate properties. Force spectroscopy analysis of cellular 'footprints' indicated that the cells secrete large polymers (e.g., 3.5 {mu}m contour length and estimated MW 1000 kDa) onto their substrate surface. Although definitive identification of the polymers has not yet been achieved, fluorescent-labeled antibody staining has specified the presence of ECM proteins such as collagen and laminin in the cellular footprints. The stretched polymers appear to be much larger than single molecules of known ECM components, such as collagen and heparan sulfate proteoglycan, thus suggesting that the cells create larger entangled, macromolecular structures from smaller components. There is strong evidence which suggests that the composition of the ECM is greatly influenced by the hydrophobicity of the substrate surface, with preferential production and/or adsorption of larger macromolecules on hydrophobic surfaces.

  6. Membrane barrier of a porcine hepatocyte bioartificial liver.

    Science.gov (United States)

    Nyberg, Scott L; Yagi, Toshikazu; Matsushita, Takakazu; Hardin, Joseph; Grande, Joseph P; Gibson, Lawrence E; Platt, Jeffrey L

    2003-03-01

    Pores in the membrane of a bioartificial liver (BAL) allow it to function as a semipermeable barrier between its contents (i.e., liver cells) and components of the recipient's immune system. This study is designed to assess the influence of pore size on immune response to a BAL containing porcine hepatocytes. Sixteen healthy dogs were divided into four groups (four dogs per group) based on pore size of the BAL membrane and level of exposure to porcine hepatocytes. Group 1 dogs were administered porcine hepatocytes by intraperitoneal injection and served as positive controls. Group 2 dogs were exposed to porcine hepatocytes in a large-pore (200-nm) BAL, and group 3 dogs were exposed to porcine hepatocytes in a small-pore (10-nm) BAL. Group 4 dogs were exposed to a no-cell (unloaded) BAL and served as negative controls. Intraperitoneal injection of hepatocytes or 3 hours of BAL hemoperfusion was performed day 0 and 3 weeks later on day 21. Biochemical, humoral, and cellular measures of immune response were collected until day 44. The initiation of BAL hemoperfusion was associated with a rapid decline in CH(50) levels of complement and transient neutropenia and thrombocytopenia during all BAL exposures. Xenoreactive antibody response to BAL was increased by use of membranes with large pores and secondary exposures. Skin testing on day 42 showed a delayed-type hypersensitivity response to porcine hepatocytes that also correlated with level of previous antigen exposure. BAL treatment was associated with both immediate and elicited immunologic responses. The immediate response was transient and not influenced by membrane pore size, whereas elicited responses were influenced by pore size of the BAL during previous exposures. PMID:12619028

  7. Keratin 8 modulation of desmoplakin deposition at desmosomes in hepatocytes.

    Science.gov (United States)

    Loranger, Anne; Gilbert, Stéphane; Brouard, Jean-Simon; Magin, Thomas M; Marceau, Normand

    2006-12-10

    Keratins, the intermediate filament proteins of epithelial cells, connect to desmosomes, the cell-cell adhesion structures at the surface membrane. The building elements of desmosomes include desmoglein and desmocollin, which provide the actual cell adhesive properties, and desmoplakins, which anchor the keratin intermediate filaments to desmosomes. In the work reported here, we address the role of keratin 8 in modulating desmoplakin deposition at surface membrane in mouse hepatocytes. The experimental approach is based on the use of keratin 8- and keratin 18-null mouse hepatocytes as cell models. In wild-type mouse hepatocytes, desmoplakin is aligned with desmoglein and keratin 8 at the surface membrane. In keratin 8-null hepatocytes, the intermediate filament loss leads to alterations in desmoplakin distribution at the surface membrane, but not of desmoglein. Intriguingly, a significant proportion of keratin 18-null hepatocytes express keratin 8 at the surface membrane, associated with a proper desmoplakin alignment with desmoglein at desmosomes. A Triton treatment of the monolayer reveals that most of the desmoplakin present in either wild-type, keratin 8- or keratin 18-null hepatocytes is insoluble. Deletion analysis of keratin 8 further suggests that the recovery of desmoplakin alignment requires the keratin 8 rod domain. In addition, similarly to other works revealing a key role of desmoplakin phosphorylation on its interaction with intermediate filaments, we find that the phosphorylation status of the keratin 8 head domain affects desmoplakin distribution at desmosomes. Together, the data indicate that a proper alignment/deposition of desmoplakin with keratins and desmoglein in hepatocytes requires keratin 8, through a reciprocal phosphoserine-dependent process.

  8. Novel Cytochrome P450 Reaction Phenotyping for Low-Clearance Compounds Using the Hepatocyte Relay Method.

    Science.gov (United States)

    Yang, Xin; Atkinson, Karen; Di, Li

    2016-03-01

    A novel cytochrome P450 (P450) reaction phenotyping method for low-clearance compounds has been developed for eight P450 enzymes (CYP1A2, 2B6, 2D6, 2C8, 2C9, 2C19, 3A, and 3A4) and pan-cytochrome using the hepatocyte relay approach. Selective mechanism-based inhibitors were used to inactivate the individual P450 enzymes during preincubation, and inactivators were removed from the incubation before adding substrates to minimize reversible inhibition and maximize inhibitor specificity. The inhibitors were quite selective for specific P450 isoforms using the following inhibitor concentrations and preincubation times: furafylline (1 µM, 15 minutes) for CYP1A2, phencyclidine (20 µM, 15 minutes) for 2B6, paroxetine (1.8 µM, 15 minutes) for CYP2D6, gemfibrozil glucuronide (100 µM, 30 minutes) for 2C8, tienilic acid (15 µM, 30 minutes) for 2C9, esomeprazole (8 µM, 15 minutes) for 2C19, troleandomycin (25 µM, 15 minutes) for 3A4/5, CYP3cide (2 µM, 15 minutes) for 3A4, and 1-aminobenzotriazole (1 mM, 30 minutes) supplemented with tienilic acid (15 µM, 30 minutes) for pan-cytochrome. The inhibitors were successfully applied to the hepatocyte relay method in a 48-well format for P450 reaction phenotyping of low-clearance compounds. This novel method provides a new approach for determining the fraction metabolized of low-turnover compounds that are otherwise challenging with the traditional methods, such as chemical inhibitors with human liver microsomes and hepatocytes or human recombinant P450 enzymes. PMID:26700955

  9. The effect of human milk on DNA synthesis of neonatal rat hepatocytes in primary culture.

    Science.gov (United States)

    Kohno, Y; Shiraki, K; Mura, T

    1991-03-01

    We studied the effect of human milk on DNA synthesis of neonatal hepatocytes to elucidate the physiologic role of human milk in growth of the liver. Neonatal hepatocytes were isolated from 5-d-old rats and cultured in serum-free medium. Human milk stimulated DNA synthesis of these hepatocytes in a concentration-dependent manner. The stimulatory activity of 7.5% (vol/vol) human milk plus 0.1 mumol/L insulin was five times that of control and was almost the same as that of 20 micrograms/L human epidermal growth factor (hEGF) plus insulin. The effect of human milk was additive with treatment with hEGF and insulin. The milk associated with prolonged jaundice of infants was significantly more active than the milk that was not associated with jaundice, although the concentration of hEGF was not different between the two types of milk. The mitogenic activity of milk was heat-labile, inactivated by DTT and stable after treatment with trypsin. Three peaks of the activity were detected in milk by gel filtration and the fraction containing proteins of molecular weight between 36,000 and 76,000 showed the highest activity. Anti-hEGF antibody did not inhibit this activity completely. These results suggested the presence of mitogens other than hEGF or a more active form of hEGF in human milk. The milk associated with breast-milk jaundice exerts a different influence on cell growth and may affect maturation of the liver function related to bilirubin metabolism. The mitogenic activity of milk might be important for growth and development of the liver in infants.

  10. Bone morphogenetic protein-2 is a negative regulator of hepatocyte proliferation downregulated in the regenerating liver

    Institute of Scientific and Technical Information of China (English)

    Cui-Ping Xu; Wen-Min Ji; Gijs R van den Brink; Maikel P Peppelenbosch

    2006-01-01

    AIM: To characterize the expression and dynamic changes of bone morphogenetic protein (BMP)-2 in hepatocytes in the regenerating liver in rats after partial hepatectomy (PH), and examine the effects of BMP-2 on proliferation of human Huh7 hepatoma cells.METHODS: Fifty-four adult male Wistar rats were randomly divided into three groups: A normal control (NC) group, a partial hepatectomized (PH) group and a sham operated (SO) group. To study the effect of liver regeneration on BMP-2 expression, rats were sacrificed before and at different time points after PH or the sham intervention (6, 12, 24 and 48 h). For each time point, six rats were used in parallel. Expression and distribution of BMP-2 protein were determined in regenerating liver tissue by Western blot analysis and immunohistochemistry. Effects of BMP-2 on cell proliferation of human Huh7 hepatoma cell line were assessed using an MTT assay.RESULTS: In the normal liver strong BMP-2 expression was observed around the central and portal veins. The expression of BMP-2 decreased rapidly as measured by both immunohistochemistry and Western blot analysis.This decrease was at a maximum of 3.22 fold after 12 h and returned to normal levels at 48 h after PH. No significant changes in BMP-2 immunoreactivity were observed in the SO group. BMP-2 inhibited serum induced Huh7 cell proliferation.CONCLUSION: BMP-2 is expressed in normal adult rat liver and negatively regulates hepatocyte proliferation.The observed down regulation of BMP-2 following partial hepatectomy suggests that such down regulation may be necessary for hepatocyte proliferation.

  11. Suppression of bile acid synthesis by thyroid hormone in primary human hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Ewa Cristine Siljevik Ellis

    2006-01-01

    AIM: It is known that thyroid hormones alter the bile acid metabolism in humans, however the effect on individual enzymes has been difficult to elucidate.This is mainly due to the lack of human liver cell lines producing bile acids. We used cultures of primary human hepatocytes to study the effects of triiodothyronine (T3) on bile acid synthesis.METHODS: Primary hepatocytes were isolated from liver tissue obtained from three different patients undergoing liver resection due to underlying malignancy. The hepatocytes were cultured under serum-free conditions and treated from d 1 to d 5 with culture containing 0.1-1000 nmol/L of T3. Bile acid formation and mRNA levels of key enzymes were analysed.RESULTS: The lowest concentration of T3 decreased cholic acid (CA) formation to 43%-53% of controls and chenodeoxycholic acid (CDCA) to 52%-75% of controls on d 5. The highest dose further decreased CA formation to 16%-48% of controls while CDCA formation remained at 50%-117% of controls. Expression of mRNA levels of cholesterol 7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8B1) dose-dependently decreased.Sterol 27-hydroxylase (CYP27A1) levels also decreased,but not to the same extent.CONCLUSION: T3 dose-dependently decreased total bile acid formation in parallel with decreased expression of CYP7A1 and CYP8B1. CA formation is inhibited to a higher degree than CDCA, resulting in a marked decrease in the CA/CDCA ratio.

  12. Honokiol activates the LKB1–AMPK signaling pathway and attenuates the lipid accumulation in hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Min Suk; Kim, Jung Hwan; Kim, Hye Jung; Chang, Ki Churl; Park, Sang Won, E-mail: parksw@gnu.ac.kr

    2015-04-15

    Honokiol is a bioactive neolignan compound isolated from the species of Magnolia. This study was designed to elucidate the cellular mechanism by which honokiol alleviates the development of non-alcoholic steatosis. HepG2 cells were treated with honokiol for 1 h, and then exposed to 1 mM free fatty acid (FFA) for 24 h to simulate non-alcoholic steatosis in vitro. C57BL/6 mice were fed with a high-fat diet for 28 days, and honokiol (10 mg/kg/day) was daily treated. Honokiol concentration-dependently attenuated intracellular fat overloading and triglyceride (TG) accumulation in FFA-exposed HepG2 cells. These effects were blocked by pretreatment with an AMP-activated protein kinase (AMPK) inhibitor. Honokiol significantly inhibited sterol regulatory element-binding protein-1c (SREBP-1c) maturation and the induction of lipogenic proteins, stearoyl-CoA desaturase-1 (SCD-1) and fatty acid synthase (FAS) in FFA-exposed HepG2 cells, but these effects were blocked by pretreatment of an AMPK inhibitor. Honokiol induced AMPK phosphorylation and subsequent acetyl-CoA carboxylase (ACC) phosphorylation, which were inhibited by genetic deletion of liver kinase B1 (LKB1). Honokiol stimulated LKB1 phosphorylation, and genetic deletion of LKB1 blocked the effect of honokiol on SREBP-1c maturation and the induction of SCD-1 and FAS proteins in FFA-exposed HepG2 cells. Honokiol attenuated the increases in hepatic TG and lipogenic protein levels and fat accumulation in the mice fed with high-fat diet, while significantly induced LKB1 and AMPK phosphorylation. Taken together, our findings suggest that honokiol has an anti-lipogenic effect in hepatocytes, and this effect may be mediated by the LKB1–AMPK signaling pathway, which induces ACC phosphorylation and inhibits SREBP-1c maturation in hepatocytes. - Highlights: • Honokiol attenuates lipid accumulation induced by free fatty acid in hepatocyte. • Honokiol inhibits the increase in lipogenic enzyme levels induced by free fatty

  13. Automated detection of hepatotoxic compounds in human hepatocytes using HepaRG cells and image-based analysis of mitochondrial dysfunction with JC-1 dye

    International Nuclear Information System (INIS)

    , transporters expression and activity were similar to a mixed HepaRG population. → Long term differentiation period before HepaRG hepatocyte isolation influence this establishment. → These conditions were settled to develop a hepatoxicity test using automated-imaging analysis. → JC-1 detects MPT, an early event of cell death, in altered HepaRG hepatocytes when efflux of this dye by MDR1 is inhibited.

  14. The endocannabinoid N-arachidonoyl dopamine (NADA) selectively induces oxidative stress-mediated cell death in hepatic stellate cells but not in hepatocytes.

    Science.gov (United States)

    Wojtalla, Alexandra; Herweck, Frank; Granzow, Michaela; Klein, Sabine; Trebicka, Jonel; Huss, Sebastian; Lerner, Raissa; Lutz, Beat; Schildberg, Frank Alexander; Knolle, Percy Alexander; Sauerbruch, Tilman; Singer, Manfred Vincenz; Zimmer, Andreas; Siegmund, Sören Volker

    2012-04-15

    The endocannabinoid system is a crucial regulator of hepatic fibrogenesis. We have previously shown that the endocannabinoid anandamide (AEA) is a lipid mediator that blocks proliferation and induces death in hepatic stellate cells (HSCs), the main fibrogenic cell type in the liver, but not in hepatocytes. However, the effects of other endocannabinoids such as N-arachidonoyl dopamine (NADA) have not yet been investigated. The NADA-synthesizing enzyme tyrosine hydroxylase was mainly expressed in sympathetic neurons in portal tracts. Its expression pattern stayed unchanged in normal or fibrotic liver. NADA dose dependently induced cell death in culture-activated primary murine or human HSCs after 2-4 h, starting from 5 μM. Despite caspase 3 cleavage, NADA-mediated cell death showed typical features of necrosis, including ATP depletion. Although the cannabinoid receptors CB1, CB2, or transient receptor potential cation channel subfamily V, member 1 were expressed in HSCs, their pharmacological or genetic blockade failed to inhibit NADA-mediated death, indicating a cannabinoid-receptor-independent mechanism. Interestingly, membrane cholesterol depletion with methyl-β-cyclodextrin inhibited AEA- but not NADA-induced death. NADA significantly induced reactive oxygen species formation in HSCs. The antioxidant glutathione (GSH) significantly decreased NADA-induced cell death. Similar to AEA, primary hepatocytes were highly resistant against NADA-induced death. Resistance to NADA in hepatocytes was due to high levels of GSH, since GSH depletion significantly increased NADA-induced death. Moreover, high expression of the AEA-degrading enzyme fatty acid amide hydrolase (FAAH) in hepatocytes also conferred resistance towards NADA-induced death, since pharmacological or genetic FAAH inhibition significantly augmented hepatocyte death. Thus the selective induction of cell death in HSCs proposes NADA as a novel antifibrogenic mediator.

  15. COMMD1-deficient dogs accumulate copper in hepatocytes and provide a good model for chronic hepatitis and fibrosis.

    Directory of Open Access Journals (Sweden)

    Robert P Favier

    Full Text Available New therapeutic concepts developed in rodent models should ideally be evaluated in large animal models prior to human clinical application. COMMD1-deficiency in dogs leads to hepatic copper accumulation and chronic hepatitis representing a Wilson's disease like phenotype. Detailed understanding of the pathogenesis and time course of this animal model is required to test its feasibility as a large animal model for chronic hepatitis. In addition to mouse models, true longitudinal studies are possible due to the size of these dogs permitting detailed analysis of the sequence of events from initial insult to final cirrhosis. Therefore, liver biopsies were taken each half year from five new born COMMD1-deficient dogs over a period of 42 months. Biopsies were used for H&E, reticulin, and rubeanic acid (copper staining. Immunohistochemistry was performed on hepatic stellate cell (HSC activation marker (alpha-smooth muscle actin, α-SMA, proliferation (Ki67, apoptosis (caspase-3, and bile duct and liver progenitor cell (LPC markers keratin (K 19 and 7. Quantitative RT-PCR and Western Blots were performed on gene products involved in the regenerative and fibrotic pathways. Maximum copper accumulation was reached at 12 months of age, which coincided with the first signs of hepatitis. HSCs were activated (α-SMA from 18 months onwards, with increasing reticulin deposition and hepatocytic proliferation in later stages. Hepatitis and caspase-3 activity (first noticed at 18 months increased over time. Both HGF and TGF-β1 gene expression peaked at 24 months, and thereafter decreased gradually. Both STAT3 and c-MET showed an increased time-dependent activation. Smad2/3 phosphorylation, indicative for fibrogenesis, was present at all time-points. COMMD1-deficient dogs develop chronic liver disease and cirrhosis comparable to human chronic hepatitis, although at much higher pace. Therefore they represent a genetically-defined large animal model to test clinical

  16. PNPLA3 mediates hepatocyte triacylglycerol remodeling.

    Science.gov (United States)

    Ruhanen, Hanna; Perttilä, Julia; Hölttä-Vuori, Maarit; Zhou, You; Yki-Järvinen, Hannele; Ikonen, Elina; Käkelä, Reijo; Olkkonen, Vesa M

    2014-04-01

    The I148M substitution in patatin-like phospholipase domain containing 3 (PNPLA3(I148M)) determines a genetic form of nonalcoholic fatty liver disease. To elucidate the mode of PNPLA3 action in human hepatocytes, we studied effects of WT PNPLA3 (PNPLA3(WT)) and PNPLA3(I148M) on HuH7 cell lipidome after [(13)C]glycerol labeling, cellular turnover of oleic acid labeled with 17 deuterium atoms ([D17]oleic acid) in triacylglycerols (TAGs), and subcellular distribution of the protein variants. PNPLA3(I148M) induced a net accumulation of unlabeled TAGs, but not newly synthesized total [(13)C]TAGs. Principal component analysis (PCA) revealed that both PNPLA3(WT) and PNPLA3(I148M) induced a relative enrichment of TAGs with saturated FAs or MUFAs, with concurrent enrichment of polyunsaturated phosphatidylcholines. PNPLA3(WT) associated in PCA with newly synthesized [(13)C]TAGs, particularly 52:1 and 50:1, while PNPLA3(I148M) associated with similar preexisting TAGs. PNPLA3(WT) overexpression resulted in increased [D17]oleic acid labeling of TAGs during 24 h, and after longer incubations their turnover was accelerated, effects not detected with PNPLA3(I148M). PNPLA3(I148M) localized more extensively to lipid droplets (LDs) than PNPLA3(WT), suggesting that the substitution alters distribution of PNPLA3 between LDs and endoplasmic reticulum/cytosol. This study reveals a function of PNPLA3 in FA-selective TAG remodeling, resulting in increased TAG saturation. A defect in TAG remodeling activity likely contributes to the TAG accumulation observed in cells expressing PNPLA3(I148M). PMID:24511104

  17. Fluid shear stress modulation of hepatocyte-like cell function.

    Science.gov (United States)

    Rashidi, Hassan; Alhaque, Sharmin; Szkolnicka, Dagmara; Flint, Oliver; Hay, David C

    2016-07-01

    Freshly isolated human adult hepatocytes are considered to be the gold standard tool for in vitro studies. However, primary hepatocyte scarcity, cell cycle arrest and the rapid loss of cell phenotype limit their widespread deployment. Human embryonic stem cells and induced pluripotent stem cells provide renewable sources of hepatocyte-like cells (HLCs). Despite the use of various differentiation methodologies, HLCs like primary human hepatocytes exhibit unstable phenotype in culture. It has been shown that the functional capacity can be improved by adding back elements of human physiology, such as cell co-culture or through the use of natural and/or synthetic surfaces. In this study, the effect of fluid shear stress on HLC performance was investigated. We studied two important liver functions, cytochrome P450 drug metabolism and serum protein secretion, in static cultures and those exposed to fluid shear stress. Our study demonstrates that fluid shear stress improved Cyp1A2 activity by approximately fivefold. This was paralleled by an approximate ninefold increase in sensitivity to a drug, primarily metabolised by Cyp2D6. In addition to metabolic capacity, fluid shear stress also improved hepatocyte phenotype with an approximate fourfold reduction in the secretion of a foetal marker, alpha-fetoprotein. We believe these studies highlight the importance of introducing physiologic cues in cell-based models to improve somatic cell phenotype. PMID:26979076

  18. A Comprehensive Analysis of Plasmodium Circumsporozoite Protein Binding to Hepatocytes.

    Science.gov (United States)

    Zhao, Jinghua; Bhanot, Purnima; Hu, Junjie; Wang, Qian

    2016-01-01

    Circumsporozoite protein (CSP) is the dominant protein on the surface of Plasmodium sporozoites and plays a critical role in the invasion by sporozoites of hepatocytes. Contacts between CSP and heparin sulfate proteoglycans (HSPGs) lead to the attachment of sporozoites to hepatocytes and trigger signaling events in the parasite that promote invasion of hepatocytes. The precise sequence elements in CSP that bind HSPGs have not been identified. We performed a systematic in vitro analysis to dissect the association between Plasmodium falciparum CSP (PfCSP) and hepatocytes. We demonstrate that interactions between PfCSP and heparin or a cultured hepatoma cell line, HepG2, are mediated primarily by a lysine-rich site in the amino terminus of PfCSP. Importantly, the carboxyl terminus of PfCSP facilitates heparin-binding by the amino-terminus but does not interact directly with heparin. These findings provide insights into how CSP recognizes hepatocytes and useful information for further functional studies of CSP. PMID:27560376

  19. Epidermal growth factor-stimulated protein phosphorylation in rat hepatocytes

    International Nuclear Information System (INIS)

    Epidermal growth factor (EGF) causes a 6-fold increase in the phosphorylation state of a cytosolic protein (pp36, M/sub r/ = 36,000, pI = 5.5) in hepatocytes isolated from fasted, male, Wistar rats. Stimulation of 32P incorporation is observed as early as 1 min following treatment of hepatocytes with EGF and is still present at 30 min after exposure to the growth factor. The phosphate incorporated into pp36 in response to EGF is located predominantly in serine but not tyrosine residues. Phosphorylation of pp36 does not occur in response to insulin or to agents which specifically activate the cAMP-dependent protein kinase (S/sub p/ -cAMPS), protein kinase C (PMA) or Ca2+/calmodulin-dependent protein kinases (A23187) in these cells. Prior treatment of hepatocytes with the cAMP analog, S/sub p/-cAMPS, or ADP-ribosylation of N/sub i/, the inhibitory GTP-binding protein of the adenylate cyclase complex, does not prevent EGF-stimulated phosphorylation of pp36. However, as seen in other cell types, pretreatment of hepatocytes with PMA abolishes all EGF-mediated responses including phosphorylation of pp36. These results suggest that EGP specifically activates an uncharacterized, serine protein kinase in hepatocytes that is distal to the intrinsic EGF receptor tyrosine protein kinase. The rapid activation of this kinase suggests that it may play an important role in the early response of the cell to EGF

  20. Hepatitis C virus suppresses Hepatocyte Nuclear Factor 4 alpha, a key regulator of hepatocellular carcinoma.

    Science.gov (United States)

    Vallianou, Ioanna; Dafou, Dimitra; Vassilaki, Niki; Mavromara, Penelope; Hadzopoulou-Cladaras, Margarita

    2016-09-01

    Hepatitis C Virus (HCV) infection presents with a disturbed lipid profile and can evolve to hepatic steatosis and hepatocellular carcinoma (HCC). Hepatocyte Nuclear Factor 4 alpha (HNF4α) is the most abundant transcription factor in the liver, a key regulator of hepatic lipid metabolism and a critical determinant of Epithelial to Mesenchymal Transition and hepatic development. We have previously shown that transient inhibition of HNF4α initiates transformation of immortalized hepatocytes through a feedback loop consisting of miR-24, IL6 receptor (IL6R), STAT3, miR-124 and miR-629, suggesting a central role of HNF4α in HCC. However, the role of HNF4α in Hepatitis C Virus (HCV)-related hepatocarcinoma has not been evaluated and remains controversial. In this study, we provide strong evidence suggesting that HCV downregulates HNF4α expression at both transcriptional and translational levels. The observed decrease of HNF4α expression correlated with the downregulation of its downstream targets, HNF1α and MTP. Ectopic overexpression of HCV proteins also exhibited an inhibitory effect on HNF4α levels. The inhibition of HNF4α expression by HCV appeared to be mediated at transcriptional level as HCV proteins suppressed HNF4α gene promoter activity. HCV also up-regulated IL6R, activated STAT3 protein phosphorylation and altered the expression of acute phase genes. Furthermore, as HCV triggered the loss of HNF4α a consequent change of miR-24, miR-629 or miR-124 was observed. Our findings demonstrated that HCV-related HCC could be mediated through HNF4α-microRNA deregulation implying a possible role of HNF4α in HCV hepatocarcinogenesis. HCV inhibition of HNF4α could be sustained to promote HCC. PMID:27477312

  1. Fatty acids-stress attenuates gluconeogenesis induction and glucose production in primary hepatocytes

    Directory of Open Access Journals (Sweden)

    Budick-Harmelin Noga

    2012-07-01

    Full Text Available Abstract Background Hepatic gluconeogenesis tightly controls blood glucose levels in healthy individuals, yet disorders of fatty acids (FAs oxidation are characterized by hypoglycemia. We studied the ability of free-FAs to directly inhibit gluconeogenesis, as a novel mechanism that elucidates the hypoglycemic effect of FAs oxidation defects. Methods Primary rat hepatocytes were pre-treated with FAs prior to gluconeogenic stimuli with glucagon or dexamethasone and cAMP. Results Pre-treatment with 1 mM FAs (mixture of 2:1 oleate:palmitate for 1 hour prior to gluconeogenic induction, significantly decreases the induced expression of the gluconeogenic genes phosphoenolpyruvate carboxykinase (PEPCK and glucose-6-phosphatase (G6pase as well as the induced glucose production by the cells. The inhibitory effect of FAs upon gluconeogenesis is abolished when pre-treatment is elongated to 18 hours, allowing clearance of FAs into triglycerides by the cells. Replacement of palmitate with the non-metabolic fatty acid 2-bromopalmitate inhibits esterification of FAs into triglycerides. Accordingly, the increased exposure to unesterified-FAs allows their inhibitory effect to be extended even when pre-treatment is elongated to 18 hours. Similar changes were caused by FAs to the induction of peroxisome-proliferator-activated receptor-γ coactivator 1α (PGC1α expression, indicating this transcriptional coactivator as the mediating link of the effect. This inhibitory effect of FAs upon gluconeogenic induction is shown to involve reduced activation of cAMP response element-binding (CREB transcription factor. Conclusions The present results demonstrate that free-FAs directly inhibit the induced gluconeogenic response in hepatocytes. Hence, high levels of free-FAs may attenuate hepatic gluconeogenesis, and liver glucose output.

  2. In vitro culture of functionally active buffalo hepatocytes isolated by using a simplified manual perfusion method.

    Directory of Open Access Journals (Sweden)

    Santanu Panda

    Full Text Available In farm animals, there is no suitable cell line available to understand liver-specific functions. This has limited our understanding of liver function and metabolism in farm animals. Culturing and maintenance of functionally active hepatocytes is difficult, since they survive no more than few days. Establishing primary culture of hepatocytes can help in studying cellular metabolism, drug toxicity, hepatocyte specific gene function and regulation. Here we provide a simple in vitro method for isolation and short-term culture of functionally active buffalo hepatocytes.Buffalo hepatocytes were isolated from caudate lobes by using manual enzymatic perfusion and mechanical disruption of liver tissue. Hepatocyte yield was (5.3 ± 0.66×107 cells per gram of liver tissue with a viability of 82.3 ± 3.5%. Freshly isolated hepatocytes were spherical with well contrasted border. After 24 hours of seeding onto fibroblast feeder layer and different extracellular matrices like dry collagen, matrigel and sandwich collagen coated plates, hepatocytes formed confluent monolayer with frequent clusters. Cultured hepatocytes exhibited typical cuboidal and polygonal shape with restored cellular polarity. Cells expressed hepatocyte-specific marker genes or proteins like albumin, hepatocyte nuclear factor 4α, glucose-6-phosphatase, tyrosine aminotransferase, cytochromes, cytokeratin and α1-antitrypsin. Hepatocytes could be immunostained with anti-cytokeratins, anti-albumin and anti α1-antitrypsin antibodies. Abundant lipid droplets were detected in the cytosol of hepatocytes using oil red stain. In vitro cultured hepatocytes could be grown for five days and maintained for up to nine days on buffalo skin fibroblast feeder layer. Cultured hepatocytes were viable for functional studies.We developed a convenient and cost effective technique for hepatocytes isolation for short-term culture that exhibited morphological and functional characteristics of active hepatocytes

  3. Chemokine Receptors, CXCR1 and CXCR2, Differentially Regulate Exosome Release in Hepatocytes.

    Science.gov (United States)

    Nojima, Hiroyuki; Konishi, Takanori; Freeman, Christopher M; Schuster, Rebecca M; Japtok, Lukasz; Kleuser, Burkhard; Edwards, Michael J; Gulbins, Erich; Lentsch, Alex B

    2016-01-01

    Exosomes are small membrane vesicles released by different cell types, including hepatocytes, that play important roles in intercellular communication. We have previously demonstrated that hepatocyte-derived exosomes contain the synthetic machinery to form sphingosine-1-phosphate (S1P) in target hepatocytes resulting in proliferation and liver regeneration after ischemia/reperfusion (I/R) injury. We also demonstrated that the chemokine receptors, CXCR1 and CXCR2, regulate liver recovery and regeneration after I/R injury. In the current study, we sought to determine if the regulatory effects of CXCR1 and CXCR2 on liver recovery and regeneration might occur via altered release of hepatocyte exosomes. We found that hepatocyte release of exosomes was dependent upon CXCR1 and CXCR2. CXCR1-deficient hepatocytes produced fewer exosomes, whereas CXCR2-deficient hepatocytes produced more exosomes compared to their wild-type controls. In CXCR2-deficient hepatocytes, there was increased activity of neutral sphingomyelinase (Nsm) and intracellular ceramide. CXCR1-deficient hepatocytes had no alterations in Nsm activity or ceramide production. Interestingly, exosomes from CXCR1-deficient hepatocytes had no effect on hepatocyte proliferation, due to a lack of neutral ceramidase and sphingosine kinase. The data demonstrate that CXCR1 and CXCR2 regulate hepatocyte exosome release. The mechanism utilized by CXCR1 remains elusive, but CXCR2 appears to modulate Nsm activity and resultant production of ceramide to control exosome release. CXCR1 is required for packaging of enzymes into exosomes that mediate their hepatocyte proliferative effect. PMID:27551720

  4. The effects of human umbilical cord perivascular cells on rat hepatocyte structure and functional polarity.

    Science.gov (United States)

    Gómez-Aristizábal, Alejandro; Davies, John Edward

    2013-06-01

    Hepatocyte culture is a useful tool for the study of their biology and the development of bioartificial livers. However, many challenges have to be overcome since hepatocytes rapidly lose their normal phenotype in vitro. We have recently demonstrated that human umbilical cord perivascular cells (HUCPVCs) are able to provide support to hepatocytes. In the present study we go further into exploring the effects that HUCPVCs have in the functional polarization, and both the internal and external organization, of hepatocytes. Also, we investigate HUCPVC-hepatocyte crosstalk by tracking both the effects of HUCPVCs on hepatocyte transcription factors and those of hepatocytes on the expression of hepatotrophic factors in HUCPVCs. Our results show that HUCPVCs maintain the functional polarity of hepatocytes ex vivo, as judged by the secretion of fluorescein into bile canaliculi, for at least 40 days. Transmission electron microscopy revealed that hepatocytes in coculture organize in an organoid-like structure embedded in extracellular matrix surrounded by HUCPVCs. In coculture, hepatocytes displayed a higher expression of C/EBPα, implicated in maintenance of the mature hepatocyte phenotype, and HUCPVCs upregulated hepatocyte growth factor and Jagged1 indicating that these genes may play important roles in HUCPVC-hepatocyte interactions.

  5. Gelsolin-mediated activation of PI3K/Akt pathway is crucial for hepatocyte growth factor-induced cell scattering in gastric carcinoma.

    Science.gov (United States)

    Huang, Baohua; Deng, Shuo; Loo, Ser Yue; Datta, Arpita; Yap, Yan Lin; Yan, Benedict; Ooi, Chia Huey; Dinh, Thuy Duong; Zhuo, Jingli; Tochhawng, Lalchhandami; Gopinadhan, Suma; Jegadeesan, Tamilarasi; Tan, Patrick; Salto-Tellez, Manuel; Yong, Wei Peng; Soong, Richie; Yeoh, Khay Guan; Goh, Yaw Chong; Lobie, Peter E; Yang, Henry; Kumar, Alan Prem; Maciver, Sutherland K; So, Jimmy B Y; Yap, Celestial T

    2016-05-01

    In gastric cancer (GC), the main subtypes (diffuse and intestinal types) differ in pathological characteristics, with diffuse GC exhibiting early disseminative and invasive behaviour. A distinctive feature of diffuse GC is loss of intercellular adhesion. Although widely attributed to mutations in the CDH1 gene encoding E-cadherin, a significant percentage of diffuse GC do not harbor CDH1 mutations. We found that the expression of the actin-modulating cytoskeletal protein, gelsolin, is significantly higher in diffuse-type compared to intestinal-type GCs, using immunohistochemical and microarray analysis. Furthermore, in GCs with wild-type CDH1, gelsolin expression correlated inversely with CDH1 gene expression. Downregulating gelsolin using siRNA in GC cells enhanced intercellular adhesion and E-cadherin expression, and reduced invasive capacity. Interestingly, hepatocyte growth factor (HGF) induced increased gelsolin expression, and gelsolin was essential for HGF-medicated cell scattering and E-cadherin transcriptional repression through Snail, Twist and Zeb2. The HGF-dependent effect on E-cadherin was found to be mediated by interactions between gelsolin and PI3K-Akt signaling. This study reveals for the first time a function of gelsolin in the HGF/cMet oncogenic pathway, which leads to E-cadherin repression and cell scattering in gastric cancer. Our study highlights gelsolin as an important pro-disseminative factor contributing to the aggressive phenotype of diffuse GC.

  6. Gelsolin-mediated activation of PI3K/Akt pathway is crucial for hepatocyte growth factor-induced cell scattering in gastric carcinoma

    Science.gov (United States)

    Huang, Baohua; Deng, Shuo; Loo, Ser Yue; Datta, Arpita; Yap, Yan Lin; Yan, Benedict; Ooi, Chia Huey; Dinh, Thuy Duong; Zhuo, Jingli; Tochhawng, Lalchhandami; Gopinadhan, Suma; Jegadeesan, Tamilarasi; Tan, Patrick; Salto-Tellez, Manuel; Yong, Wei Peng; Soong, Richie; Yeoh, Khay Guan; Goh, Yaw Chong; Lobie, Peter E.; Yang, Henry; Kumar, Alan Prem; Maciver, Sutherland K.; So, Jimmy B.Y.; Yap, Celestial T.

    2016-01-01

    In gastric cancer (GC), the main subtypes (diffuse and intestinal types) differ in pathological characteristics, with diffuse GC exhibiting early disseminative and invasive behaviour. A distinctive feature of diffuse GC is loss of intercellular adhesion. Although widely attributed to mutations in the CDH1 gene encoding E-cadherin, a significant percentage of diffuse GC do not harbor CDH1 mutations. We found that the expression of the actin-modulating cytoskeletal protein, gelsolin, is significantly higher in diffuse-type compared to intestinal-type GCs, using immunohistochemical and microarray analysis. Furthermore, in GCs with wild-type CDH1, gelsolin expression correlated inversely with CDH1 gene expression. Downregulating gelsolin using siRNA in GC cells enhanced intercellular adhesion and E-cadherin expression, and reduced invasive capacity. Interestingly, hepatocyte growth factor (HGF) induced increased gelsolin expression, and gelsolin was essential for HGF-medicated cell scattering and E-cadherin transcriptional repression through Snail, Twist and Zeb2. The HGF-dependent effect on E-cadherin was found to be mediated by interactions between gelsolin and PI3K-Akt signaling. This study reveals for the first time a function of gelsolin in the HGF/cMet oncogenic pathway, which leads to E-cadherin repression and cell scattering in gastric cancer. Our study highlights gelsolin as an important pro-disseminative factor contributing to the aggressive phenotype of diffuse GC. PMID:27058427

  7. Resistin induces insulin resistance, but does not affect glucose output in rat-derived hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Feng LIU; Xiao-qing PAN; Mei GUO; Rong-hua CHEN; Xi-rong GUO; Tao YANG; Bin WANG; Min ZHANG; Nan GU; Jie QIU; Hong-qi FAN; Chun-mei ZHANG; Li FEI

    2008-01-01

    Aim: The aim of the present study was to observe the effects of resistin on insulin sensitivity and glucose output in rat-derived hepatocytes. Methods: The rat hepatoma cell line H4IIE was cultured and stimulated with resistin; supernant glucose and glycogen content were detected. The insulin receptor substrate (IRS)-1 and IRS-2, protein kinase B/Akt, glycogen synthase kinase-3β (GSK-3β), the suppressor of cytokine signaling 3 (SOCS-3) protein content, as well as the phosphorylation status were assessed by Western blotting. Specific antisense oligodeoxynucleotides directed against SOCS-3 were used to knockdown SOCS-3. Results: Resistin induced insulin resistance, but did not affect glucose output in rat hepatoma cell line H4IIE. Resistin attenuated multiple effects of insulin, including insulin-stimulated glycogen synthesis and phosphorylation of IRS, pro-tein kinase B/Akt, as well as GSK-3β. Resistin treatment markedly induced the gene and protein expression of SOCS-3, a known inhibitor of insulin signaling. Furthermore, a specific antisense oligodeoxynucleotide directed against SOCS-3 treatment prevented resistin from antagonizing insulin action. Conclusion: The major function of resistin on liver is to induce insulin resistance. SOCS-3 induc-tion may contribute to the resistin-mediated inhibition of insulin signaling in H4IIE hepatocytes.

  8. Differential expression of TRPM7 in rat hepatoma and embryonic and adult hepatocytes.

    Science.gov (United States)

    Lam, D Hung; Grant, Caroline E; Hill, Ceredwyn E

    2012-04-01

    TRPM7 channels are implicated in cellular survival, proliferation, and differentiation. However, a profile of TRPM7 activity in a specific cell type has not been determined from embryonic to terminally differentiated state. Here, we characterized TRPM7 expression in a spectrum of rat liver cells at different developmental stages. Using the whole-cell patch clamp technique, TRPM7-like Na(+) currents were identified in RLC-18 cells, a differentiated, proliferating hepatocellular line derived from day 17 embryonic rat liver. Currents were outwardly rectifying, enhanced in divalent-free solutions, and inhibited by intracellular Mg(2+). Reverse transcription - polymerase chain reaction (RT-PCR) revealed that RLC-18 cells express both TRPM6 and TRPM7. However, mean currents were reduced almost 80% by 1 mmol/L 2-aminoethoxyphenylborate (2-APB) and were abolished in RLC-18 cells heterologously expressing a dominant negative TRPM7 construct, suggesting that TRPM7 is the major current carrier in these cells. Functional comparison showed that relative to terminally differentiated adult rat hepatocytes, currents were 1.8 and 3.9 times higher in, respectively, RLC-18 and WIF-B cells, a rat hepatoma - human fibroblast cross. Our results demonstrate that plasma membrane TRPM7 channels are more highly expressed in proliferating cells as compared with terminally differentiated and nondividing rat hepatocytes and suggest that downregulation of this channel is associated with hepatocellular differentiation. PMID:22429021

  9. Effect of Liver Damage and Hyperbaric Oxygenation on Glutamine Synthetase of Hepatocytes.

    Science.gov (United States)

    Savilov, P N; Yakovlev, V N

    2016-01-01

    Activity of glutamine synthetase in the hepatocytes of healthy animals and animals with chronic CCl4-induced hepatitis was studied on white mature female rats after liver resection (15-20% of organ weight) and hyperbaric oxygenation (3 atm, 50 min, 3 times). Surgically operated left and non-operated middle lobes of the liver were analyzed on day 3 after liver resection and exposure to hyperbaric oxygenation. On day 65 of CCl4 poisoning, activity of glutamine synthetase decreased in both lobes and did not recover on day 3 after toxin cessation. Liver resection under conditions of CCl4-induced hepatitis restored reduced activity of glutamine synthetase in both liver lobes to the normal level. In healthy rats, the increase in glutamine synthetase activity after liver resection was found only in the middle lobe of the liver. Hyperbaric oxygenation enhanced the stimulatory effect of liver resection on glutamine synthetase activity in hepatocytes during chronic CCl4-induced hepatitis. In healthy animals with liver resection, activity of glutamine synthetase did not change after hyperbaric oxygenation, while normally oxygenation inhibited glutamine synthetase activity.

  10. Down-regulation of MiR-127 facilitates hepatocyte proliferation during rat liver regeneration.

    Directory of Open Access Journals (Sweden)

    Chuanyong Pan

    Full Text Available Liver regeneration (LR after partial hepatectomy (PH involves the proliferation and apoptosis of hepatocytes, and microRNAs have been shown to post-transcriptionally regulate genes involved in the regulation of these processes. To explore the role of miR-127 during LR, the expression patterns of miR-127 and its related proteins were investigated. MiR-127 was introduced into a rat liver cell line to examine its effects on the potential target genes Bcl6 and Setd8, and functional studies were undertaken. We discovered that miR-127 was down-regulated and inversely correlated with the expression of Bcl6 and Setd8 at 24 hours after PH, a time at which hypermethylation of the promoter region of the miR-127 gene was detected. Furthermore, in BRL-3A rat liver cells, we observed that overexpression of miR-127 significantly suppressed cell growth and directly inhibited the expression of Bcl6 and Setd8. The results suggest that down-regulation of miR-127 may be due to the rapid methylation of its promoter during the first 24 h after PH, and this event facilitates hepatocyte proliferation by releasing Bcl6 and Setd8. These findings support a miRNA-mediated negative regulation pattern in LR and implicate an anti-proliferative role for miR-127 in liver cells.

  11. Protective effect of liver ischemic preconditioning on rat hepatocytes

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Ischemic preconditioning (IPC) protects liver graft function following ischemia in liver transplantation and liver resection. The aim of this study was to assess the advantages and any potential disadvantages of liver IPC prior to isolation for rat hepatocytes during isolation and cryopreservation. After isolating and thawing the cryopreserved hepatocytes after 14 and 28 d,cell viability,efficiency,and lactate dehydrogenase (LDH) levels in preserve solution were examined for every group. Groups treated with IPC had better cell viability determination,assessment of plating efficiency and lactate dehydrogenase (LDH) assay than Group without IPC,suggesting that IPC prior to isolation may have a significant protective effect on hepatocytes subjected to isolation and short period cryopreservation.

  12. Acetaminophen metabolism, cytotoxicity, and genotoxicity in rat primary hepatocyte cultures

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    Milam, K.M.; Byard, J.L.

    1985-06-30

    Acetaminophen (APAP) metabolism, cytotoxicity, and genotoxicity were measured in primary cultures of rat hepatocytes. Although 3 mM APAP caused a slight increase in cellular release of lactate dehydrogenase into the culture medium, cellular glutathione concentration (an index of APAP metabolism) was reduced by 50%. APAP at 7 mM was significantly more toxic to these hepatocytes and had a similar but more marked effect on glutathione concentrations. In spite of its cytotoxicity, neither dose of APAP stimulated DNA repair synthesis when monitored by the rate of incorporation of (/sup 3/H)thymidine into DNA following exposure to APAP. Thus, although APAP has been shown to be both hepato- and nephrotoxic in several in vivo and in vitro systems, the reactive toxic metabolite of APAP is not genotoxic in rat primary hepatocyte cultures.

  13. Determination of metabolic stability using cryopreserved hepatocytes from rainbow trout (Oncorhynchus mykiss)

    Science.gov (United States)

    Standard protocols for isolating, cryopreserving, and thawing rainbow trout hepatocytes are described, along with procedures for using fresh or cryopreserved hepatocytes to assess chemical metabolic stability in fish by means of a substrate depletion approach. Variations on thes...

  14. Biological effects of extract from newborn porcine liver on hepatocytes, hepatic stellate cells, and hepatoma cell line

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Objective: Porcine liver extract has been shown to be effective in the clinical treatment of severe hepatitis. The aim of the present study was to study its antifibrotic as well as immune regulatory effect in vitro. Methods: Hepatocytes, hepatic stellate cells (HSCs), hepatoma cell line (HepG2) and human peripheral blood mononuclear cells (PMNCs) were studied with respect to proliferation, extracellular matrix production and apoptotic activities by proliferation assay, radioimmunoassay, gene transfection, reporter gene analysis and flow cytometry, respectively. Results: A strong stimulatory proliferation effect was observed in hepatocytes, and an inhibitory effect was found in HSCs. Hyaluronic acid (HA) production and reporter gene activities driven by various α1(Ⅰ) procollagen gene promoters in HSC-T6 were significantly decreased after treatment with the extract. Fluo-Anexin V binding apoptotic HepG2 cells were more prominent in the presence of 60 μg/ml extract. More CD4+/CD69+ positive T lymphocytes existed in the presence of the extract. Conclusion: Porcine liver extract is effective for antifibrogenesis via hepatocyte regeneration, HSC and hepatoma cell inhibition in vitro. The elevation of active T lymphocytes is helpful for immune surveillance. Fine mapping of the extract is necessary in order to get definite molecules which are essential in all described functions.

  15. Zinc Supplementation with Polaprezinc Protects Mouse Hepatocytes against Acetaminophen-Induced Toxicity via Induction of Heat Shock Protein 70.

    Science.gov (United States)

    Nishida, Tadashi; Ohata, Shuzo; Kusumoto, Chiaki; Mochida, Shinsuke; Nakada, Junya; Inagaki, Yoshimi; Ohta, Yoshiji; Matsura, Tatsuya

    2010-01-01

    Polaprezinc, a chelate compound consisting of zinc and l-carnosine, is clinically used as a medicine for gastric ulcers. It has been shown that induction of heat shock protein (HSP) is involved in protective effects of polaprezinc against gastric mucosal injury. In the present study, we investigated whether polaprezinc and its components could induce HSP70 and prevent acetaminophen (APAP) toxicity in mouse primary cultured hepatocytes. Hepatocytes were treated with polaprezinc, zinc sulfate or l-carnosine at the concentration of 100 microM for 9 h, and then exposed to 10 mM APAP. Polaprezinc or zinc sulfate increased cellular HSP70 expression. However, l-carnosine had no influence on it. Pretreatment of the cells with polaprezinc or zinc sulfate significantly suppressed cell death as well as cellular lipid peroxidation after APAP treatment. In contrast, pretreatment with polaprezinc did not affect decrease in intracellular glutathione after APAP. Furthermore, treatment with KNK437, an HSP inhibitor, attenuated increase in HSP70 expression induced by polaprezinc, and abolished protective effect of polaprezinc on cell death after APAP. These results suggested that polaprezinc, in particular its zinc component, induces HSP70 expression in mouse primary cultured hepatocytes, and inhibits lipid peroxidation after APAP treatment, resulting in protection against APAP toxicity. PMID:20104264

  16. Expression of p53, Bax and Bcl-2 proteins in hepatocytes in non-alcoholic fatty liver disease

    Institute of Scientific and Technical Information of China (English)

    Anatol Panasiuk; Janusz Dzieciol; Bozena Panasiuk; Danuta Prokopowicz

    2006-01-01

    AIM: To analyze the protein expression essential for apoptosis in liver steatosis.METHODS: The expression of proapoptotic proteinsp53, Bax, and antiapoptotic Bcl-2 in hepatocytes with steatosis (SH) and without steatosis (NSH) was evaluated in 84 patients at various stages of non-alcoholic fatty liver disease (NAFLD).RESULTS: Immunohistochemical staining of liver tissue showed the activation of p53 protein in SH and NSH with increased liver steatosis, diminished Bcl-2 and slightly decreased Bax protein. Positive correlation was found between the stage of liver steatosis with p53 expression in SH (r = 0.54, P < 0.01) and NSH (r = 0.49,P < 0.01).The antiapoptotic protein Bcl-2 was diminished together with the advancement of liver steatosis, especially in non-steatosed hepatocytes (r =0.43, P < 001).CONCLUSION: Apoptosis is one of the most important mechanisms leading to hepatocyte elimination in NAFLD. The intensification of inflammation in NAFLD induces proapoptotic protein p53 with the inhibition of antiapoptotic Bcl-2.

  17. Property of hepatitis B virus replication in Tupaia belangeri hepatocytes.

    Science.gov (United States)

    Sanada, Takahiro; Tsukiyama-Kohara, Kyoko; Yamamoto, Naoki; Ezzikouri, Sayeh; Benjelloun, Soumaya; Murakami, Shuko; Tanaka, Yasuhito; Tateno, Chise; Kohara, Michinori

    2016-01-01

    The northern treeshrew (Tupaia belangeri) has been reported to be an effective candidate for animal infection model with hepatitis B virus (HBV). The objective of our study was to analyze the growth characteristics of HBV in tupaia hepatocytes and the host response to HBV infection. We established primary tupaia hepatocytes (3-6-week old tupaia) and infected them with HBV genotypes A, B and C, and all the genotypes proliferated as well as those in human primary hepatocytes (>10(5) copies/ml in culture supernatant). We next generated a chimeric mouse with tupaia liver by transplantation of tupaia primary hepatocytes to urokinase-type plasminogen activator cDNA (cDNA-uPA)/severe combined immunodeficient (SCID) mice and the replacement ratio with tupaia hepatocytes was found to be more than 95%. Infection of chimeric mice with HBV (genotypes B, C, and D) resulted in HBV-DNA level of 10(4)-10(6) copies/ml after 8 weeks of infection, which were almost similar to that in humanized chimeric mouse. In contrast, serum HBV level in adult tupaia (1-year-old tupaia) was quite low (<10(3) copies/ml). Understanding the differences in the response to HBV infection in primary tupaia hepatocytes, chimeric mouse, and adult tupaia will contribute to elucidating the mechanism of persistent HBV infection and viral eradication. Thus, T. belangeri was found to be efficient for studying the host response to HBV infection, thereby providing novel insight into the pathogenesis of HBV. PMID:26654952

  18. Cytotoxic effects of 4-octylphenol on fish hepatocytes.

    Science.gov (United States)

    Kaptaner, Burak

    2016-08-01

    The present study was conducted to determine cytotoxic effects of 4-octylphenol (4-OP) on primary cultured hepatocytes of pearl mullet (Alburnus tarichi). Lactate dehydrogenase (LDH) release, malondialdehyde (MDA) level, antioxidant enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione-S-transferase (GST)] and glutathione (GSH) content were measured after 24-h exposure to 4-OP. 4-OP caused dose- and time-dependent increases in LDH release. Significant induction of MDA level and decrease in GSH content were found. SOD and GPx activities were decreased while GST activity was increased. These findings suggest that 4-OP leads to cytotoxicity by depressing antioxidant defenses in fish hepatocytes. PMID:26415925

  19. No evidence for protective erythropoietin alpha signalling in rat hepatocytes

    Directory of Open Access Journals (Sweden)

    Frede Stilla

    2009-04-01

    Full Text Available Abstract Background Recombinant human erythropoietin alpha (rHu-EPO has been reported to protect the liver of rats and mice from ischemia-reperfusion injury. However, direct protective effects of rHu-EPO on hepatocytes and the responsible signalling pathways have not yet been described. The aim of the present work was to study the protective effect of rHu-EPO on warm hypoxia-reoxygenation and cold-induced injury to hepatocytes and the rHu-EPO-dependent signalling involved. Methods Loss of viability of isolated rat hepatocytes subjected to hypoxia/reoxygenation or incubated at 4°C followed by rewarming was determined from released lactate dehydrogenase activity in the absence and presence of rHu-EPO (0.2–100 U/ml. Apoptotic nuclear morphology was assessed by fluorescence microscopy using the nuclear fluorophores H33342 and propidium iodide. Erythropoietin receptor (EPOR, EPO and Bcl-2 mRNAs were quantified by real time PCR. Activation of JAK-2, STAT-3 and STAT-5 in hepatocytes and rat livers perfused in situ was assessed by Western blotting. Results In contrast to previous in vivo studies on ischemia-reperfusion injury to the liver, rHu-EPO was without any protective effect on hypoxic injury, hypoxia-reoxygenation injury and cold-induced apoptosis to isolated cultured rat hepatocytes. EPOR mRNA was identified in these cells but specific detection of the EPO receptor protein was not possible due to the lack of antibody specificity. Both, in the cultured rat hepatocytes (10 U/ml for 15 minutes and in the rat liver perfused in situ with rHu-EPO (8.9 U/ml for 15 minutes no evidence for EPO-dependent signalling was found as indicated by missing effects of rHu-EPO on phosphorylation of JAK-2, STAT-3 and STAT-5 and on the induction of Bcl-2 mRNA. Conclusion Together, these results indicate the absence of any protective EPO signalling in rat hepatocytes. This implies that the protection provided by rHu-EPO in vivo against ischemia-reperfusion and

  20. Effect of trifluoperazine on toxicity, HIF-1α induction and hepatocyte regeneration in acetaminophen toxicity in mice

    Energy Technology Data Exchange (ETDEWEB)

    Chaudhuri, Shubhra, E-mail: SCHAUDHURI@uams.edu [Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas (United States); Arkansas Children' s Hospital Research Institute, Little Rock, AR (United States); McCullough, Sandra S., E-mail: mcculloughsandras@uams.edu [Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas (United States); Arkansas Children' s Hospital Research Institute, Little Rock, AR (United States); Hennings, Leah, E-mail: lhennings@uams.edu [Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, Arkansas (United States); Arkansas Children' s Hospital Research Institute, Little Rock, AR (United States); Brown, Aliza T., E-mail: brownalizat@uams.edu [Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas (United States); Arkansas Children' s Hospital Research Institute, Little Rock, AR (United States); Li, Shun-Hwa [Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI (United States); Simpson, Pippa M., E-mail: psimpson@mcw.edu [Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI (United States); Hinson, Jack A., E-mail: hinsonjacka@uams.edu [Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, Arkansas Children' s Hospital Research Institute, Little Rock, AR (United States); James, Laura P., E-mail: jameslaurap@uams.edu [Department of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas (United States); Arkansas Children' s Hospital Research Institute, Little Rock, AR (United States); Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, Arkansas Children' s Hospital Research Institute, Little Rock, AR (United States)

    2012-10-15

    Oxidative stress and mitochondrial permeability transition (MPT) are important mechanisms in acetaminophen (APAP) toxicity. The MPT inhibitor trifluoperazine (TFP) reduced MPT, oxidative stress, and toxicity in freshly isolated hepatocytes treated with APAP. Since hypoxia inducible factor-one alpha (HIF-1α) is induced very early in APAP toxicity, a role for oxidative stress in the induction has been postulated. In the present study, the effect of TFP on toxicity and HIF-1α induction in B6C3F1 male mice treated with APAP was examined. Mice received TFP (10 mg/kg, oral gavage) prior to APAP (200 mg/kg IP) and at 7 and 36 h after APAP. Measures of metabolism (hepatic glutathione and APAP protein adducts) were comparable in the two groups of mice. Toxicity was decreased in the APAP/TFP mice at 2, 4, and 8 h, compared to the APAP mice. At 24 and 48 h, there were no significant differences in toxicity between the two groups. TFP lowered HIF-1α induction but also reduced the expression of proliferating cell nuclear antigen, a marker of hepatocyte regeneration. TFP can also inhibit phospholipase A{sub 2}, and cytosolic and secretory PLA{sub 2} activity levels were reduced in the APAP/TFP mice compared to the APAP mice. TFP also lowered prostaglandin E{sub 2} expression, a known mechanism of cytoprotection. In summary, the MPT inhibitor TFP delayed the onset of toxicity and lowered HIF-1α induction in APAP treated mice. TFP also reduced PGE{sub 2} expression and hepatocyte regeneration, likely through a mechanism involving PLA{sub 2}. -- Highlights: ► Trifluoperazine reduced acetaminophen toxicity and lowered HIF-1α induction. ► Trifluoperazine had no effect on the metabolism of acetaminophen. ► Trifluoperazine reduced hepatocyte regeneration. ► Trifluoperazine reduced phospholipase A{sub 2} activity and prostaglandin E{sub 2} levels.

  1. Triglyceride accumulation in long-term cultures of adult rat hepatocytes by chronic exposure to Aroclor 1254

    International Nuclear Information System (INIS)

    The effect of chronic exposure to micromolar concentrations of Aroclor 1254 (Aro) on the hepatic lipid metabolism was studied in long-term cultures of adult rat hepatocytes. Hepatocytes were cocultivated with mytomicin C-treated 3T3 cells and exposed for 2 wk to Aroclor 1254 concentrations ranging from 0.01 to 20 μg/ml. The Aro-exposed cultures showed intracytoplasmic lipid droplets and a maximum increase of 55% in the triglyceride (TG) content and of 4.4-fold in the cytochrome P-450 content. Labeling studies with [14C]acetic and [14C]oleic acid showed no changes in the uptake of fatty acid and TG precursors by the Aro-treated cultures; the synthesis of cellular lipids from [14C]acetic acid was slightly inhibited by Aroclor 1254, but that from [14C]oleic acid was increased, specially for TF (37%). The secretion of total lipids and TG was 2.1- and 2.7-fold lower, respectively, in the cultures treated with 20 μg/ml of Aroclor 1254, resulting in an increase of 1.9-fold in the intracellular content of TG. The synthesis of cellular proteins labeled with [3H]leucine was unchanged in the Aro-treated cultures, but the secretion of exportable proteins was 1.7-fold lower in the cultures treated with 20 μg/ml of Aroclor 1254. Our results showed that long-term exposure to in vivo relevant concentrations of Aroclor 1254 produced morphological and biochemical changes in cultured hepatocytes, like those described in vivo, and intracellular TG accumulation due mostly to impaired secretion of TG by the hepatocytes. Our results also suggest that this culture system could be useful for the screening of toxic agents producing fatty liver and the study of the involved mechanism(s)

  2. 111Indium labeling of hepatocytes for analysis of short-term biodistribution of transplanted cells.

    Science.gov (United States)

    Gupta, S; Lee, C D; Vemuru, R P; Bhargava, K K

    1994-03-01

    Hepatocyte transplantation is useful for ex vivo gene therapy and liver repopulation. Methods for hepatic reconstitution have recently been developed but optimization of hepatocyte transplantation systems is necessary. To develop systems for noninvasive assessment of the biodistribution of transplanted cells, we labeled hepatocytes with 111indium-oxine. Our initial studies showed that hepatocytes incorporated 111indium-oxine with an efficiency of approximately 20%. After labeling, cell viability was unchanged and 111indium was present in hepatocytes after overnight culture, as well as after intrasplenic transplantation. Transplanted cells were successfully localized by means of scintigraphic imaging. The scintigraphic patterns of cell distribution were different when hepatocytes were transplanted by means of either spleen or internal jugular vein, which deposit cells into separate vascular beds. Quantitative analysis of the biodistribution of 111indium-labeled hepatocytes indicated that within 2 hr of intrasplenic transplantation, cells were predominantly localized in liver and spleen, and occasionally in lungs. To determine whether the rate of intrasplenic cell injection influenced translocation of hepatocytes, we transplanted cells in normal rats. Despite intrasplenic cell injection at a variety of rates, organ-specific distribution of 111indium-labeled hepatocytes remained unchanged. Labeling with 111indium did not affect long-term survival of transplanted hepatocytes. These results indicate that 111indium-labeling of hepatocytes should greatly assist noninvasive analysis in the short-term of the biodistribution of transplanted hepatocytes. PMID:8119703

  3. TGFbeta induces apoptosis and EMT in primary mouse hepatocytes independently of p53, p21Cip1 or Rb status

    Directory of Open Access Journals (Sweden)

    Harrison David J

    2008-07-01

    Full Text Available Abstract Background TGFβ has pleiotropic effects that range from regulation of proliferation and apoptosis to morphological changes and epithelial-mesenchymal transition (EMT. Some evidence suggests that these effects may be interconnected. We have recently reported that P53, P21Cip1 and pRB, three critical regulators of the G1/S transition are variably involved in TGFβ-induced cell cycle arrest in hepatocytes. As these proteins are also involved in the regulation of apoptosis in many circumstances, we investigated their contribution to other relevant TGFβ-induced effects, namely apoptosis and EMT, and examined how the various processes were interrelated. Methods Primary mouse hepatocytes deficient in p53, p21 and/or Rb, singly or in combination were treated with TGFβ for 24 to 96 hours. Apoptosis was quantified according to morphology and by immunostaining for cleaved-capsase 3. Epithelial and mesenchymal marker expression was studied using immunocytochemistry and real time PCR. Results We found that TGFβ similarly induced morphological changes regardless of genotype and independently of proliferation index or sensitivity to inhibition of proliferation by TGFβ. Morphological changes were accompanied by decrease in E-cadherin and increased Snail expression but the mesenchymal markers (N-cadherin, SMAα and Vimentin studied remained unchanged. TGFβ induced high levels of apoptosis in p53-/-, Rb-/-, p21cip1-/- and control hepatocytes although with slight differences in kinetics. This was unrelated to proliferation or changes in morphology and loss of cell-cell adhesion. However, hepatocytes deficient in both p53 and p21cip1were less sensitive to TGFβ-induced apoptosis. Conclusion Although p53, p21Cip1 and pRb are well known regulators of both proliferation and apoptosis in response to a multitude of stresses, we conclude that they are critical for TGFβ-driven inhibition of hepatocytes proliferation, but only slightly modulate TGF

  4. A comparative toxicogenomic investigation of oil sand water and processed water in rainbow trout hepatocytes.

    Science.gov (United States)

    Gagné, F; André, C; Turcotte, P; Gagnon, C; Sherry, J; Talbot, A

    2013-08-01

    The purpose of this study was to compare the expression of gene transcripts involved in toxic stress in rainbow trout hepatocytes exposed to oil sand water (OSW), lixiviate (OSLW), and processed water (OSPW). We pose the hypothesis that the changes in gene expression responses in cells exposed to a simulated oil sand extraction procedure (OSPW) differ from the gene expression responses of OSLW and OS. Rainbow trout hepatocytes were exposed to increasing concentrations of OSW, OSLW, and OSPW for 48 h at 15 °C. Cell viability was assessed by measuring membrane permeability, total RNA levels, and gene expression using an array of 16 genes involved in xenobiotic biotransformation (GST, CYP1A1, CYP3A4, MDR), metal homeostasis and oxidative stress (MT, SOD, and CAT), estrogenicity (VTG, ERβ), DNA repair (LIG, APEX, UNG, and OGG), cell growth (GADD45 and PCNA), and glycolysis (GAPDH). The results showed that the toxicogenomic properties of OSPW differed from those of OSLW and OSW. Gene transcripts that were influenced by OSW and OSLW, and strongly expressed in OSPW, were MT, CAT, GST (induction), CYP1A1, VTG, UNG/OGG, and PCNA. These genes are therefore considered not entirely specific to OSPW but to water in contact with OS. We also found gene transcripts that responded only with OSPW: SOD, GST (inhibition), MDR (inhibition), CYP3A4, GAPDH, GADD45, and APEX. Of these gene transcripts, the ones strongly associated with toxicity (loss of cell viability and RNA levels) were CYP3A4, GST, and GAPDH. Genes involved in DNA repair were also strongly related to the loss of cell viability but responded to both OSLW and OSPW. The observed changes in cell toxicity and gene expression therefore support the hypothesis that OSPW has a distinct toxic fingerprint from OSLW and OSW.

  5. Estrogenic activity of procymidone in rainbow trout (Oncorhynchus mykiss) hepatocytes: a possible mechanism of action.

    Science.gov (United States)

    Radice, Sonia; Fumagalli, Roberta; Chiesara, Enzo; Ferraris, Michela; Frigerio, Silvia; Marabini, Laura

    2004-03-15

    It is known that procymidone modifies sexual differentiation in vivo and in vitro, and that it induces vitellogenin (Vtg) synthesis in primary cultured rainbow trout hepatocytes. The aim of this study was to evaluate the mechanism underlying this latter in vitro estrogenic action. The cells were treated for 24 h with procymidone 150 microM (with 17beta-estradiol [E2] 20 microM as a positive control) combined with an estrogen receptor (ER) antagonist (tamoxifen 20 microM or ICI 182,780 1 microM) or, given the drug toxic action on the production of reactive oxygen species (ROS), a free radical scavenger (alpha-tocopherol 30 microM). The results from ELISA experiments provided evidence that procymidone Vtg-induction is inhibited by ER antagonists and by alpha-tocopherol suggesting that both ER and ROS are involved in this effect. The ROS detection revealed that the treatment with alpha-tocopherol and tamoxifen completely prevented ROS induction by procymidone, that was not inhibited by ICI 182,780. In exploring the mechanism mediating these events and its timing, we found that procymidone induced mitogen-activated protein kinase (MAPK) at 30 and 60 min, and that this effect was blocked by co-treatment with alpha-tocopherol. In summary, the results of the study clearly support the idea that the estrogenic activity of procymidone in primary cultured trout hepatocytes is mediated by ROS production, and that this activity is similar to that of the ligand-independent ER activation involving MAPK. PMID:15013820

  6. The use of pig hepatocytes for biotransformation and toxicity studies.

    NARCIS (Netherlands)

    Hoogenboom, L.A.P.

    1991-01-01

    The three main objectives of this study were, (1) to investigate the possibility to isolate viable hepatocytes from liver samples of pigs, (2) to study their use for biotransformation and toxicity studies, and (3) to demonstrate the value of this model, in particular in the field of residue toxicolo

  7. Inhibitory effect of picroside Ⅱ on hepatocyte apoptosis

    Institute of Scientific and Technical Information of China (English)

    Hua GAO; Ya-wei ZHOU

    2005-01-01

    Aim: To investigate the influence of picroside Ⅱ on hepatocyte apoptosis and its mechanism. Methods: Morphological changes and quantification of apoptotic cells were determined under transmission electron microscopy and flow cytometry respectively. DNA fragmentation was visualized by agarose gel electrophoresis.Semi-quantitative reverse transcription-PCR (RT-PCR) was used to analyze the expression of bcl-2 and bax genes. The content of manganese-superoxide dismutase (SOD) in liver mitochondria was detected by the Marland method. The content of malonic aldehyde (MDA) and the protein level in liver tissue were determined by thiobarbituric acid colorimetry and Lowry method. Results:Picroside Ⅱ decreased the levels of alanine aminotransferase and aspartate aminotransferase in the serum resulting from acute-liver injured mice induced with D-GalN and LPS; it also reduced the content of MDA, and thus, enhanced the activity of SOD. Picroside Ⅱ 10 mg/kg was found to protect hepatocytes against apoptosis in a dose-dependent manner; it up-regulated the expression of bcl-2 genes,thus increased the bcl-2/bax ratio. Conclusion: Picroside Ⅱ can protect hepatocytes against injury and prevent hepatocytes from apoptosis. It might by upregulating the bcl-2 gene expression and antioxidation.

  8. 3D Cultivation Techniques for Primary Human Hepatocytes

    Directory of Open Access Journals (Sweden)

    Anastasia Bachmann

    2015-02-01

    Full Text Available One of the main challenges in drug development is the prediction of in vivo toxicity based on in vitro data. The standard cultivation system for primary human hepatocytes is based on monolayer cultures, even if it is known that these conditions result in a loss of hepatocyte morphology and of liver-specific functions, such as drug-metabolizing enzymes and transporters. As it has been demonstrated that hepatocytes embedded between two sheets of collagen maintain their function, various hydrogels and scaffolds for the 3D cultivation of hepatocytes have been developed. To further improve or maintain hepatic functions, 3D cultivation has been combined with perfusion. In this manuscript, we discuss the benefits and drawbacks of different 3D microfluidic devices. For most systems that are currently available, the main issues are the requirement of large cell numbers, the low throughput, and expensive equipment, which render these devices unattractive for research and the drug-developing industry. A higher acceptance of these devices could be achieved by their simplification and their compatibility with high-throughput, as both aspects are of major importance for a user-friendly device.

  9. Asialoglycoprotein receptor mediated hepatocyte targeting - strategies and applications.

    Science.gov (United States)

    D'Souza, Anisha A; Devarajan, Padma V

    2015-04-10

    Hepatocyte resident afflictions continue to affect the human population unabated. The asialoglycoprotein receptor (ASGPR) is primarily expressed on hepatocytes and minimally on extra-hepatic cells. This makes it specifically attractive for receptor-mediated drug delivery with minimum concerns of toxicity. ASGPR facilitates internalization by clathrin-mediated endocytosis and exhibits high affinity for carbohydrates specifically galactose, N-acetylgalactosamine and glucose. Isomeric forms of sugar, galactose density and branching, spatial geometry and galactose linkages are key factors influencing ligand-receptor binding. Popular ligands for ASGPR mediated targeting are carbohydrate polymers, arabinogalactan and pullulan. Other ligands include galactose-bearing glycoproteins, glycopeptides and galactose modified polymers and lipids. Drug-ligand conjugates provide a viable strategy; nevertheless ligand-anchored nanocarriers provide an attractive option for ASGPR targeted delivery and are widely explored. The present review details various ligands and nanocarriers exploited for ASGPR mediated delivery of drugs to hepatocytes. Nanocarrier properties affecting ASGPR mediated uptake are discussed at length. The review also highlights the clinical relevance of ASGPR mediated targeting and applications in diagnostics. ASGPR mediated hepatocyte targeting provides great promise for improved therapy of hepatic afflictions.

  10. Insulin infusion reduces hepatocyte growth factor in lean humans

    DEFF Research Database (Denmark)

    de Courten, Barbora; de Courten, Maximilian; Dougherty, Sonia;

    2013-01-01

    OBJECTIVE: Plasma Hepatocyte Growth Factor (HGF) is significantly elevated in obesity and may contribute to vascular disease, metabolic syndrome or cancer in obese individuals. The current studies were done to determine if hyperinsulinemia increases plasma HGF. MATERIALS/METHODS: Twenty-two parti...

  11. Retinoic Acid-mediated Nuclear Receptor Activation and Hepatocyte Proliferation

    Science.gov (United States)

    Bushue, Nathan; Wan, Yu-Jui Yvonne

    2016-01-01

    Due to their well-known differentiation and apoptosis-inducing abilities, retinoic acid (RA) and its analogs have strong anti-cancer efficacy in human cancers. However, in vivo RA is a liver mitogen. While speculation has persisted that RA-mediated signaling is likely involved in hepatocyte proliferation during liver regeneration, direct evidence is still required. Findings in support of this proposition include observations that a release of retinyl palmitate (the precursor of RA) occurs in liver stellate cells following liver injury. Nevertheless, the biological action of this released vitamin A is virtually unknown. More likely is that the released vitamin A is converted to RA, the biological form, and then bound to a specific receptor (retinoid x receptor; RXRα), which is most abundantly expressed in the liver. Considering the mitogenic effects of RA, the RA-activated RXRα would likely then influence hepatocyte proliferation and liver tissue repair. At present, the mechanism by which RA stimulates hepatocyte proliferation is largely unknown. This review summarizes the activation of nuclear receptors (peroxisome proliferator activated receptor-α, pregnane x receptor, constitutive androstane receptor, and farnesoid x receptor) in an RXRα dependent manner to induce hepatocyte proliferation, providing a link between RA and its proliferative role.

  12. Functional activity of human hepatocytes under traumatic disease.

    Science.gov (United States)

    Kudryavtseva, M V; Stein, G I; Shashkov, B V; Kudryavtsev, B N

    1998-03-01

    Absorption and fluorescent cytophotometry techniques were applied to studies of RNA as well as of total glycogen and its fractions as the parameters of functional activity of the hepatocytes in patients with severe mechanical trauma, both with and without autointoxication (AI). Slides were stained with gallocyanine-chromalums to determine the RNA content and were processed by the fluorescent PAS-reaction for the glycogen content. To trace the dynamics of RNA and glycogen contents in the liver punction biopsies were done in the same patients. A quick increase in the RNA content took place in both groups of patients at the first period (within the first 3 days) of traumatic disease. At the second period of disease the hepatocyte RNA content in patients without AI was found to decrease up to the initial level whereas that in patients with AI increased on the average by 36% of the initial values. The total glycogen content in hepatocytes of all the patients changed insignificantly in the course of disease but its labile fraction in patients with AI decreased to 70% of the total. The increase of hepatocyte synthetic activity and the maintenance of the high glycogen level are indicative of the large compensatory potential of the liver that enables it to carry an intensive functional load under AI conditions. PMID:9570502

  13. Effect of matrine on primary human hepatocytes in vitro.

    Science.gov (United States)

    Gong, Xiaobing; Gao, Yuan; Guo, Guoqing; Vondran, Florian W R; Schwartlander, Ruth; Efimova, Ekaterina; Pless, Gesine; Sauera, Igor M; Neuhaus, Peter

    2015-03-01

    Matrine is a bioactive component of the traditional Chinese medical herb Sophora flavescens that has been used in China to treat various kinds of diseases including virus hepatitis. However, the molecular mechanisms underlying its hepatoprotective effects remains elusive. In the present study, primary human hepatocytes were employed to elucidate the protective effects and molecular mechanisms of matrine. We observed that low concentrations of matrine had no significant impact on albumin secretion, but high concentrations (>140 mg/L) of matrine decreased the albumin secretion in hepatocytes. Western blot data indicated that matrine at 140 mg/L at 72 h induced protein expression of CYP2A6, CYP2B6 and CYP3A4. Furthermore, high concentrations of matrine reduced LDH and AST levels and were cytotoxic to hepatocytes, leading to a decreased cell viability and total protein amount. Moreover, low concentrations of matrine, enhanced the ECOD activity and decreased the level of NO2 (-) induced by cytokines in human hepatocytes. Taken together, the present study sheds novel light on the molecular mechanisms of matrine and potential application of matrine in hepatic diseases.

  14. Transplantation of human hepatocytes into tolerized genetically immunocompetent rats

    Institute of Scientific and Technical Information of China (English)

    Edwin C. Ouyang; Catherine H. Wu; Cherie Walton; Kittichai Promrat; George Y. Wu

    2001-01-01

    AIM To determine whether normal geneticallyirnmunocornpetent rodent hosts could bemanipulated to accept human hepatocytetransplants with long term survival withoutirnrnunosuppression.METHODS Tolerance towards humanhepatocytes was established by injection ofprimary human hepatocytes or Huh7 humanhepatoma cells into the peritoneal cavities offetal rats. Corresponding cells weresubsequently transplanted into newborn rats viaintrasplenic injection within 24 h after birth.RESULTS Mixed lymphocyte assays showedthat spleen cells from non-tolerized rats werestimulated to proliferate when exposed to humanhepatocytes, while cells from tolerized ratswere not. Injections made between 15 d and 17 dof gestation produced optimal tolerizaton.Transplanted human hepatocytes in rat liverswere visualized by immunohistochemicalstaining of human albumin. By dot blotting ofgenomic DNA in livers of tolerized rats 16 weeksafter hepatocyte transplantation, it was foundthat approximately 2.5 × 105 human hepatocytessurvived per rat liver. Human albumin mRNA wasdetected in rat livers by RT-PCR for 15 wk, andhuman albumin protein was also detectable in ratserum.CONCLUSION Tolerization of an immuno-competent rat can permit transplantation, andsurvival of functional human hepatocytes.

  15. A Microfabricated Platform for Generating Physiologically-Relevant Hepatocyte Zonation

    Science.gov (United States)

    McCarty, William J.; Usta, O. Berk; Yarmush, Martin L.

    2016-05-01

    In vitro liver models have been important tools for more than 40 years for academic research and preclinical toxicity screening by the pharmaceutical industry. Hepatocytes, the highly metabolic parenchymal cells of the liver, are efficient at different metabolic chemistries depending on their relative spatial location along the sinusoid from the portal triad to the central vein. Although replicating hepatocyte metabolic zonation is vitally important for physiologically-relevant in vitro liver tissue and organ models, it is most often completely overlooked. Here, we demonstrate the creation of spatially-controlled zonation across multiple hepatocyte metabolism levels through the application of precise concentration gradients of exogenous hormone (insulin and glucagon) and chemical (3-methylcholanthrene) induction agents in a microfluidic device. Observed gradients in glycogen storage via periodic acid-Schiff staining, urea production via carbamoyl phosphatase synthetase I staining, and cell viability after exposure to allyl alcohol and acetaminophen demonstrated the in vitro creation of hepatocyte carbohydrate, nitrogen, alcohol degradation, and drug conjugation metabolic zonation. This type of advanced control system will be crucial for studies evaluating drug metabolism and toxicology using in vitro constructs.

  16. Comparative Metabolism of Furan in Rodent and Human Cryopreserved Hepatocytes

    Science.gov (United States)

    Gates, Leah A.; Phillips, Martin B.; Matter, Brock A.

    2014-01-01

    Furan is a liver toxicant and carcinogen in rodents. Although humans are most likely exposed to furan through a variety of sources, the effect of furan exposure on human health is still unknown. In rodents, furan requires metabolism to exert its toxic effects. The initial product of the cytochrome P450 2E1-catalyzed oxidation is a reactive α,β-unsaturated dialdehyde, cis-2-butene-1,4-dial (BDA). BDA is toxic and mutagenic and consequently is considered responsible for the toxic effects of furan. The urinary metabolites of furan in rats are derived from the reaction of BDA with cellular nucleophiles, and precursors to these metabolites are detected in furan-exposed hepatocytes. Many of these precursors are 2-(S-glutathionyl)butanedial-amine cross-links in which the amines are amino acids and polyamines. Because these metabolites are derived from the reaction of BDA with cellular nucleophiles, their levels are a measure of the internal dose of this reactive metabolite. To compare the ability of human hepatocytes to convert furan to the same metabolites as rodent hepatocytes, furan was incubated with cryopreserved human and rodent hepatocytes. A semiquantitative liquid chromatography with tandem mass spectrometry assay was developed for a number of the previously characterized furan metabolites. Qualitative and semiquantitative analysis of the metabolites demonstrated that furan is metabolized in a similar manner in all three species. These results indicate that humans may be susceptible to the toxic effects of furan. PMID:24751574

  17. Effects of 2-APB on Store-operated Ca2+ Channel Currents of Hepatocytes after Hepatic Ischemia/Reperfusion Injury in Rats

    Institute of Scientific and Technical Information of China (English)

    HUANG Changzhou; ZHANG Zongming; QIU Fazu

    2005-01-01

    The effects of hepatic ischemia/reperfusion (I/R) injuries on hepatocellular viability and store-operated calcium current (Isoc) in isolated rat hepatocytes and the effects of 2-APB on storeoperated calcium current (Isoc) in isolated rat hepatocytes after hepaticischemia/reperfusion injuries were studied. Hepatic ischemia and reperfusion injury model was established and whole cell patch-clamp techniques were used to investigate the effects of 2-APB on Isoc. The results showed that ischemia/reperfusion injuries could significantly reduce hepatocellular viability and further increase Isoc in hepatocytes and 2-APB (20, 40, 60, 80, 100 μmol/L) produced a concentration-dependent decrease of Isoc with IC50 value of 64.63±10.56 μmol/L (n= 8). It was concluded that ischemia/reperfusion injuries could reduce hepatocellular viability, probably through increased Isoc in hepatocytes and 2-APB had a protective effect on ischemia/reperfusion-induced liver injury, probably though inhibiting Isoc.

  18. Hepatoprotective and antioxidative effects of total phenolics from Laggera pterodonta on chemical-induced injury in primary cultured neonatal rat hepatocytes.

    Science.gov (United States)

    Wu, Yihang; Yang, Leixiang; Wang, Fang; Wu, Xiumei; Zhou, Changxin; Shi, Shuyun; Mo, Jianxia; Zhao, Yu

    2007-08-01

    Although Laggera pterodonta as a folk medicine has been widely used for several centuries to ameliorate some inflammatory ailments as hepatitis in China, there have been no studies of the hepatoprotective and antioxidative effects of this plant. In this paper, the hepatoprotective effect of total phenolics from L. pterodonta (TPLP) against CCI4-, D-GalN-, TAA-, and t-BHP-induced injury was examined in primary cultured neonatal rat hepatocytes. TPLP inhibited the cellular leakage of two enzymes, hepatocyte ASAT and ALAT, caused by these chemicals and improved cell viability. Moreover, TPLP afforded much stronger protection than the reference drug silibinin. Meanwhile, DPPH and superoxide radicals scavenging activities of TPLP were also determined. The present investigation is the first to report chemical-induced injury model in primary cultured neonatal rat hepatocytes and provide evidence for the hepatoprotective and antioxidative effects of L. pterodonta. Neutralizing reactive oxygen species by nonenzymatic mechanisms may be one of main mechanisms of TPLP against chemical-induced hepatocyte injury. Furthermore, The total phenolic content of L. pterodonta and its main component type were quantified, and its principle components isochlorogenic acids were isolated and authenticated. These data support the folkloric uses of L. pterodonta in the treatment of hepatitis. PMID:17329003

  19. Augmenter of liver regeneration (ALR) protects human hepatocytes against apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Ilowski, Maren [Liver Regeneration Group, Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Kleespies, Axel [Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Toni, Enrico N. de [Department of Medicine II, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Donabauer, Barbara [Liver Regeneration Group, Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Jauch, Karl-Walter [Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Hengstler, Jan G. [Leibniz Research Centre for Working Environment and Human Factors, Technical University, Dortmund (Germany); Thasler, Wolfgang E., E-mail: wolfgang.thasler@med.uni-muenchen.de [Liver Regeneration Group, Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany)

    2011-01-07

    Research highlights: {yields} ALR decreases cytochrome c release from mitochondria. {yields} ALR protects hepatocytes against apoptosis induction by ethanol, TRAIL, anti-Apo, TGF-{beta} and actinomycin D. {yields} ALR exerts a liver-specific anti-apoptotic effect. {yields} A possible medical usage of ALR regarding protection of liver cells during apoptosis inducing therapies. -- Abstract: Augmenter of liver regeneration (ALR) is known to support liver regeneration and to stimulate proliferation of hepatocytes. However, it is not known if ALR exerts anti-apoptotic effects in human hepatocytes and whether this protective effect is cell type specific. This is relevant, because compounds that protect the liver against apoptosis without undesired effects, such as protection of metastatic tumour cells, would be appreciated in several clinical settings. Primary human hepatocytes (phH) and organotypic cancer cell lines were exposed to different concentrations of apoptosis inducers (ethanol, TRAIL, anti-Apo, TGF-{beta}, actinomycin D) and cultured with or without recombinant human ALR (rhALR). Apoptosis was evaluated by the release of cytochrome c from mitochondria and by FACS with propidium iodide (PI) staining. ALR significantly decreased apoptosis induced by ethanol, TRAIL, anti-Apo, TGF-{beta} and actinomycin D. Further, the anti-apoptotic effect of ALR was observed in primary human hepatocytes and in HepG2 cells but not in bronchial (BC1), colonic (SW480), gastric (GC1) and pancreatic (L3.6PL) cell lines. Therefore, the hepatotrophic growth factor ALR acts in a liver specific manner with regards to both its mitogenic and its anti-apoptotic effect. Unlike the growth factors HGF and EGF, rhALR acts in a liver specific manner. Therefore, ALR is a promising candidate for further evaluation as a possible hepatoprotective factor in clinical settings.

  20. Investigation of the role of sigma1-receptors in inositol 1,4,5-trisphosphate dependent calcium signaling in hepatocytes.

    Science.gov (United States)

    Abou-Lovergne, A; Collado-Hilly, M; Monnet, F P; Koukoui, O; Prigent, S; Coquil, J F; Dupont, G; Combettes, L

    2011-07-01

    In hepatocytes, as in other cell types, Ca(2+) signaling is subject to complex regulations, which result largely from the intrinsic characteristics of the different inositol 1,4,5-trisphosphate receptor (InsP(3)R) isoforms and from their interactions with other proteins. Although sigma1 receptors (Sig-1Rs) are widely expressed in the liver, their involvement in hepatic Ca(2+) signaling remains unknown. We here report that in this cell type Sig-1R interact with type 1 isoforms of the InsP(3) receptors (InsP(3)R-1). These results obtained by immunoprecipitation experiments are confirmed by the observation that Sig-1R proteins and InsP(3)R-1 colocalize in hepatocytes. However, Sig-1R ligands have no effect on InsP(3)-induced Ca(2+) release in hepatocytes. This can be explained by the rather low expression level expression of InsP(3)R-1. In contrast, we find that Sig-1R ligands can inhibit agonist-induced Ca(2+) signaling via an inhibitory effect on InsP(3) synthesis. We show that this inhibition is due to the stimulation of PKC activity by Sig-1R, resulting in the well-known down-regulation of the signaling pathway responsible for the transduction of the extracellular stimulus into InsP(3) synthesis. The PKC sensitive to Sig-1R activity belongs to the family of conventional PKC, but the precise molecular mechanism of this regulation remains to be elucidated.

  1. A role for lipid rafts in the protection afforded by docosahexaenoic acid against ethanol toxicity in primary rat hepatocytes.

    Science.gov (United States)

    Aliche-Djoudi, Fatiha; Podechard, Normand; Collin, Aurore; Chevanne, Martine; Provost, Emilie; Poul, Martine; Le Hégarat, Ludovic; Catheline, Daniel; Legrand, Philippe; Dimanche-Boitrel, Marie-Thérèse; Lagadic-Gossmann, Dominique; Sergent, Odile

    2013-10-01

    Previously, we demonstrated that eicosapentaenoic acid enhanced ethanol-induced oxidative stress and cell death in primary rat hepatocytes via an increase in membrane fluidity and lipid raft clustering. In this context, another n-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA), was tested with a special emphasis on physical and chemical alteration of lipid rafts. Pretreatment of hepatocytes with DHA reduced significantly ethanol-induced oxidative stress and cell death. DHA protection could be related to an alteration of lipid rafts. Indeed, rafts exhibited a marked increase in membrane fluidity and packing defects leading to the exclusion of a raft protein marker, flotillin. Furthermore, DHA strongly inhibited disulfide bridge formation, even in control cells, thus suggesting a disruption of protein-protein interactions inside lipid rafts. This particular spatial organization of lipid rafts due to DHA subsequently prevented the ethanol-induced lipid raft clustering. Such a prevention was then responsible for the inhibition of phospholipase C-γ translocation into rafts, and consequently of both lysosome accumulation and elevation in cellular low-molecular-weight iron content, a prooxidant factor. In total, the present study suggests that DHA supplementation could represent a new preventive approach for patients with alcoholic liver disease based upon modulation of the membrane structures.

  2. Molecular mechanisms underlying the cholesterol-lowering effect of Ginkgo biloba extract in hepatocytes: a comparative study with Iovastatin

    Institute of Scientific and Technical Information of China (English)

    Zuo-quan XIE; Gai LIANG; Lu ZHANG; Qi WANG; Yi QU; Yang GAO; Li-bo LIN; Sai YE; Ji ZHANG; Hui WANG; Guo-ping ZHAO; Qing-hua ZHANG

    2009-01-01

    Aim: To explore the molecular mechanisms underlying the cholesterol-lowering effect of a Ginkgo biloba extract (GBE). Methods: Enzyme activity, cholesterol flux and changes in gene expression levels were assessed in cultured hepatocytes treated with GBE or Iovastatin. Results: GBE decreased the total cholesterol content in cultured hepatocytes and inhibited the activity of HMG-CoA reductase, as determined by an in vitro enzyme activity assay. In addition, GBE decreased cholesterol influx, whereas Iovastatin increased choles-terol influx. GBE treatment induced significant increases in the expression of cholesterogenic genes and genes involved in cholesterol metabolism, such as SREBF2, as determined by cDNA microarray and real-time RT-PCR. Furthermore, INSIG2, LDLR, LRP1, and LRP10 were differentially regulated by GBE and Iovastatin. The data imply that the two compounds modulate cholesterol metabolism through distinct mechanisms. Conclusion: By using a gene expression profiling approach, we were able to broaden the understanding of the molecular mechanisms by which GBE lowers cellular cholesterol levels. Specifically, we demonstrated that GBE exhibited dual effects on the cellular choles-terol pool by modulating both HMG-CoA reductase activity and inhibiting cholesterol influx.

  3. The cytoskeleton of digitonin-treated rat hepatocytes.

    Science.gov (United States)

    Fiskum, G; Craig, S W; Decker, G L; Lehninger, A L

    1980-06-01

    Treatment of isolated rat hepatocptes with low concentrations of digitonin increases the permeability of the plsma membrane to cytosolic proteins without causing release of organelles such as mitochondria into the surrounding medium. Electron microscopy showed that treatment of the cells with increasing concentations of digitonin results in a progressive loss in the continuity of the plasma membrane, while most other aspects of cellular morphology remain normal. Depletion of background staining material from the cytosol by digitonin treatment of the cells greatly enhances the visualization of the cytoskeleton. The use of this technique, together with immunofluorescent light microscopy, has verified the presence of an actin-containing filamentous network at the hepatocyte cortex as well as intermediate filaments distributed throughout the cell. Digitonin is thus useful both for selectively permeabilizing the plasma membrane and for intensifying the appearance of intracellular structures such as microfilaments that are normally difficult to observe in cells such as hepatocytes. PMID:6997878

  4. Effects of electroporation on primary rat hepatocytes in vitro

    Institute of Scientific and Technical Information of China (English)

    Yun-Qing Yao; Ding-Feng Zhang; Ai-Long Huang; Yun Luo; Da-Zhi Zhang; Bo Wang; Wei-Ping Zhou; Hong Ren; Shu-Hua Guo

    2002-01-01

    AIM: To investigate the effects of electroporation on primaryrat hepatocyte and to optimize the electroporation conditionsintroducing foreign genes into primary hepatocytes.METHODS: A single-pulse procedure was performed at Iowvoltage (220-400 V) but with high capacitance (500-950 μF).Hepatocytes were divided into 4 groups according to theelectroporation conditions: group Ⅰ, 220 V and 500 μF;group Ⅱ, 220 Vand 950 μF; group Ⅲ, 400 V and 950 μF,and group Ⅳ.The control group was freshly isolatedhepatocytes and directly cultured under the same conditionsas those of electroporation groups. The effects ofelectroporation on primary rat hepatocytes were detectedby trypan blue exclusion (TBE) and MTT analysis. Besides,albumin (AIb), alanine transaminase (ALT) and lactatedehydrogenase (LDH) in the supernatants of culturedhepatocytes were measured by biochemical assay.RESULTS: Between day 1 and day 15 after incubation,primary rat hepatocytes of each electroporation groupappeared normal, being the same with those of controlgroup. TBE staining showed that slight hepatocyte damageand high survival rate were found in the electroporationgroups and the control group. Cultured for 3, 7, 11 and 15days, hepatocyte viability was approximatly 92.6±2.5 %,89.5±3.3 %, 82.0±3.5 % and 74.3±1.2 %, respectively.MTT analysis indicated that the viabilities of hepatocyteshad no significant difference between each electroporationgroup, and those were similar to that of control group. Atthe 36th hour after electroporation, AIb, ALT and LDH in thesupernatants of control group were 5.3±0.1 g. L-1, 183.7±8.4 nkat. L-1 and 896.8±58.5 nkat. L-1; those of group Ⅱwere 5.7±0.1 g. L-1, 215.4±16.7 nkat. L-1 and 1063.8±51.8 nkat. L-1; and those of group Ⅲ were 5.80.2 g. L-1,217.1 ± 8.4 nkat. L-1 and 1063.8± 10.0 nkat. L-1 . Statistically,the proteins of group Ⅱ and group Ⅲ were significantlyhigher than those of control group (P<0.05), whereas theprotein production of group

  5. SREBP-1c Gene Silencing can Decrease Lipid Deposits in Bovine Hepatocytes Cultured in Vitro

    Directory of Open Access Journals (Sweden)

    Qinghua Deng

    2014-05-01

    Full Text Available Background: Fatty liver is a major metabolic disorder that occurs during early lactation in high-producing dairy cows. Sterol regulatory element-binding protein-1c (SREBP-1c is an important transcription factor that regulates lipid synthesis by regulating the expression of lipid metabolism genes. Methods: In this study, we reduced the expression of SREBP-1c by adenovirus-mediated SREBP-1c with a low expression vector (AD-GFP-SREBP-1c to study the effects of SREBP-1c on lipid deposits in bovine hepatocytes. The expression levels and enzyme activities of SERBP-1c and its target genes were determined by real-time PCR, western blot, and ELISA. Results: These results showed that Ad-GFP-SREBP-1c could inhibit SREBP-1c expression. The expression of the lipid synthesis enzyme acetyl-CoA carboxylase (ACC was down-regulated. The expression levels of the lipid oxidation enzymes long-chain fatty acyl-COA synthetase (ACSL-1, carnitine palmitoyltransferase І (CPT-І, carnitine palmitoyltransferase II (CPT- II, and β-hydroxyacyl-CoA-DH (HADH were significantly elevated. Furthermore, the expression levels of factors involved in the assembly and transport of very low-density lipoproteins (VLDLs, such as apolipoprotein B100 (ApoB, apolipoprotein E (ApoE, and microsomal triglyceride transfer protein (MTTP were decreased comparison with the negative control and the blank control groups, but the low-density lipoprotein receptor (LDLR was elevated. The concentrations of TG (triglyceride and VLDL were also reduced. Conclusion: These data suggest that low SREBP-1c expression can decrease lipid synthesis, increase lipid oxidation, and decrease the TG and VLDL content in bovine hepatocytes.

  6. Targeted in vivo delivery of siRNA and an endosome-releasing agent to hepatocytes.

    Science.gov (United States)

    Sebestyén, Magdolna G; Wong, So C; Trubetskoy, Vladimir; Lewis, David L; Wooddell, Christine I

    2015-01-01

    The discoveries of RNA interference (RNAi) and short interfering RNAs (siRNAs) have provided the opportunity to treat diseases in a fundamentally new way: by co-opting a natural process to inhibit gene expression at the mRNA level. Given that siRNAs must interact with the cells' natural RNAi machinery in order to exert their silencing effect, one of the most fundamental requirements for their use is efficient delivery to the desired cell type and, specifically, into the cytoplasm of those cells. Numerous research efforts involving the testing of a large number of delivery approaches using various carrier molecules and inventing several distinct formulation technologies during the past decade illustrate the difficulty and complexity of this task. We have developed synthetic polymer formulations for in vivo siRNA delivery named Dynamic PolyConjugates™ (DPCs) that are designed to mimic the features viruses possess for efficient delivery of their nucleic acids. These include small size, long half-life in circulation, capability of displaying distinct host cell tropism, efficient receptor binding and cell entry, disassembly in the endosome and subsequent release of the nucleic acid cargo to the cytoplasm. Here we present an example of this delivery platform composed of a hepatocyte-targeted endosome-releasing agent and a cholesterol-conjugated siRNA (chol-siRNA). This delivery platform forms the basis of ARC-520, an siRNA-based therapeutic for the treatment of chronic hepatitis B virus (HBV) infection. In this chapter, we provide a general overview of the steps in developing ARC-520 and detailed protocols for two critical stages of the discovery process: (1) verifying targeted in vivo delivery to hepatocytes and (2) evaluating in vivo drug efficacy using a mouse model of chronic HBV infection.

  7. The responses of rat hepatocytes to glucagon and adrenaline. Application of quantified elasticity analysis.

    Science.gov (United States)

    Ainscow, E K; Brand, M D

    1999-11-01

    The internal control of hepatocyte metabolism has been previously analysed using metabolic control analysis. The aim of this paper is to extend this analysis to include the responses of the cells to hormonal stimulus. Hepatocyte metabolism was divided into nine reaction blocks: glycogen breakdown, glucose release, glycolysis, lactate production, NADH oxidation, pyruvate oxidation, proton leak, mitochondrial phosphorylation and ATP consumption, linked by five intermediates: mitochondrial membrane potential, cytoplasmic NADH/NAD and total cellular ATP, glucose 6-phosphate and pyruvate. The kinetic responses of the reaction blocks to the intermediates were determined previously in the absence of added hormones. In this study, the changes in flux and intermediate levels that occurred upon addition of either glucagon or adrenaline were measured. From comparison of the fractional changes in fluxes and intermediate levels with the known kinetics of the system, it was possible to determine the primary sites of action of the hormones. The results show that the majority of processes in the cell are responsive to the hormones. The notable exception to this is the failure of adrenaline to have a direct effect on glycolysis. The activity change of each metabolic block observed in the presence of either hormone was quantified and compared to the indirect effects on each block caused by changes in metabolite levels. The second stage of the analysis was to use the calculated activity changes and the known control pattern of the system to give a semiquantitative analysis of the regulatory pathways employed by the hormones to achieve the changes in fluxes and metabolite levels. This was instructive in analysing, for example, how glucagon caused a decrease in flux through glycolysis and an increase in oxidative phosphorylation without large changes in metabolite levels (homeostasis). Conversely, it could be seen that the failure of adrenaline to maintain a constant glucose 6

  8. Metabolism of six CYP probe substrates in fetal hepatocytes

    OpenAIRE

    Shaik, Abdul Naveed; Vishwakarma, Sandeep Kumar; Khan, Aleem Ahmed

    2016-01-01

    Cytochrome P-450 (CYP) are the most common drug metabolizing enzymes and are abundantly expressed in liver apart from kidney, lungs, intestine, brain etc. Their expression levels change with physiological conditions and disease states. The expression of these CYPs is less in human foetus and neonates compared to adults, which results in lower clearance of xenobiotics in infants and neonates compared to adults. Hepatocytes are the cells which are largely used to study these CYPs. We have isola...

  9. Lessons from hepatocyte-specific cyp51 knockout mice

    OpenAIRE

    Keber, Rok; Lorbek, Gregor; Lewinska, Monika; Juvan, Peter; Perše, Martina; Bjorkhem, Ingemar; Rozman, Damjana; Horvat, Simon; Jeruc, Jera; Gutiérrez Mariscal, Francisco Miguel; Gebhardt, Rolf

    2016-01-01

    We demonstrate unequivocally that defective cholesterol synthesis is an independent determinant of liver inflammation and fibrosis. We prepared a mouse hepatocyte-specific knockout (LKO) of lanosterol 14 a -demethylase (CYP51) from the part of cholesterol synthesis that is already committed to cholesterol. LKO mice developed hepatomegaly with oval cell proliferation, fibrosis and inflammation, but without steatosis. The key trigger was reduced cholesterol esters that provoked cell cycle arres...

  10. Role of the transsulfuration pathway and of gamma-cystathionase activity in the formation of cysteine and sulfate from methionine in rat hepatocytes

    International Nuclear Information System (INIS)

    To assess the extent to which low hepatic gamma-cystathionase levels affect methionine flux to cysteine in hepatocytes, the effect of inhibition of gamma-cystathionase activity with propargylglycine on the metabolism of L-[35S]methionine was determined in studies with freshly isolated rat hepatocytes. gamma-Cystathionase activity was inhibited 25%, 42%, 63% and 76% (maximal inhibition) by treatment with 2.5 mumol/L, 0.01 mmol/L, 0.02 mmol/L and 2 mmol/l propargylglycine, respectively. Inhibition of gamma-cystathionase activity with up to 0.02 mmol/L propargylglycine had no statistically significant effect on [35S]glutathione, [35S]sulfate or [35S]cysteine formation from [35S]methionine. However, treatment of cells with 2 mmol/L propargylglycine markedly inhibited the metabolism of [35S]methionine to [35S]glutathione by 93%, to [35S]sulfate by 88% and to [35S]cysteine by 89%; [35S]cystathionine accumulation in these incubation systems was 60 times control. Hepatic gamma-cystathionase activity in premature infants has been reported to be about 23% of mature levels; this level of gamma-cystathionase activity may limit cysteine synthesis by the methionine transsulfuration pathway. No evidence for cysteine synthesis from serine and sulfide, which can be catalyzed by cystathionine beta-synthase, or for methionine metabolism by an S-adenosylmethionine-independent pathway was obtained

  11. Gene expression changes after hypoxic preconditioning in rat hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Wei Chen; Jiang-Feng Qiu; Zhi-Qi Zhang; Hai-Feng Luo; Joan Rosello-Catafau; Zhi-Yong Wu

    2006-01-01

    BACKGROUND: Hypoxic preconditioning can protect hepatocytes against hypoxic injury, but its mechanism has not been elucidated. The aim of this study was to proifle gene expression patterns involved in hypoxic preconditioning and probable mechanism at the level of gene expression. METHODS: Hepatocytes were divided into 2 groups:control group and hypoxic preconditioning group. Biotin-labeled cRNA from the control group and the hypoxic preconditioning group was hybridized by oligonucleotide microarray. Genes that were signiifcantly associated with hypoxic preconditioning were ifltered, and validated at the level of transcript expression. RESULTS: Forty-three genes with signiifcantly altered expression patterns were discovered and most of them had not been previously reported. Among these genes, genes encoding superoxide dismutase 2 (SOD2)and interleukin 10 (IL-10) in the hypoxic preconditioning group were conifrmed to be up-regulated with real-time quantitative PCR. CONCLUSIONS:Many cytokines are involved in hypoxic preconditioning and protect hepatocytes from hypoxia-reoxygenation injury, and the increase of oxygen free-radical scavengers and anti-inlfammatory factors may play a key role in this phenomenon. Diverse signal pathways are probably involved.

  12. A fast and robust hepatocyte quantification algorithm including vein processing

    Directory of Open Access Journals (Sweden)

    Homeyer André

    2010-03-01

    Full Text Available Abstract Background Quantification of different types of cells is often needed for analysis of histological images. In our project, we compute the relative number of proliferating hepatocytes for the evaluation of the regeneration process after partial hepatectomy in normal rat livers. Results Our presented automatic approach for hepatocyte (HC quantification is suitable for the analysis of an entire digitized histological section given in form of a series of images. It is the main part of an automatic hepatocyte quantification tool that allows for the computation of the ratio between the number of proliferating HC-nuclei and the total number of all HC-nuclei for a series of images in one processing run. The processing pipeline allows us to obtain desired and valuable results for a wide range of images with different properties without additional parameter adjustment. Comparing the obtained segmentation results with a manually retrieved segmentation mask which is considered to be the ground truth, we achieve results with sensitivity above 90% and false positive fraction below 15%. Conclusions The proposed automatic procedure gives results with high sensitivity and low false positive fraction and can be applied to process entire stained sections.

  13. Hepatocyte and Sertoli Cell Aquaporins, Recent Advances and Research Trends

    Directory of Open Access Journals (Sweden)

    Raquel L. Bernardino

    2016-07-01

    Full Text Available Aquaporins (AQPs are proteinaceous channels widespread in nature where they allow facilitated permeation of water and uncharged through cellular membranes. AQPs play a number of important roles in both health and disease. This review focuses on the most recent advances and research trends regarding the expression and modulation, as well as physiological and pathophysiological functions of AQPs in hepatocytes and Sertoli cells (SCs. Besides their involvement in bile formation, hepatocyte AQPs are involved in maintaining energy balance acting in hepatic gluconeogenesis and lipid metabolism, and in critical processes such as ammonia detoxification and mitochondrial output of hydrogen peroxide. Roles are played in clinical disorders including fatty liver disease, diabetes, obesity, cholestasis, hepatic cirrhosis and hepatocarcinoma. In the seminiferous tubules, particularly in SCs, AQPs are also widely expressed and seem to be implicated in the various stages of spermatogenesis. Like in hepatocytes, AQPs may be involved in maintaining energy homeostasis in these cells and have a major role in the metabolic cooperation established in the testicular tissue. Altogether, this information represents the mainstay of current and future investigation in an expanding field.

  14. Effects of 2,3,7,8-TCDD and benzo[a]pyrene on modulating vitellogenin expression in primary culture of crucian carp (Carassius auratus) hepatocytes

    Institute of Scientific and Technical Information of China (English)

    LIANG Yong; C. K. C. Wong; XU Ying; M. H. Wong

    2004-01-01

    Vitellogenin (Vtg) is the precursor of yolk protein. Its expression and secretion are estrogen-regulated and are crucial for oocyte maturation. An in vitro xenoestrogen screening model was established by measuring Vtg induction in cultured primary hepatocytes from crucian carp. Vtg production was detected by biotin-avidin sandwich ELISA method while Vtg and cytochrome P4501A1 (CYP1A1) mRNA induction were measured by semi- quantitative PCR-primer dropping technique. Vtg and Vtg mRNA were dose-dependently induced by diethylstilbestrol (DES, 0.2-200 ng/mL) in hepatocytes of crucian carp. Co-treatment of the DES-induced hepatocytes with either 2,3,7,8-TCDD (TCDD, 0.1-4 pg/mL) or benzo[a]pyrene (B[a]P, 5-1000 ng/mL) resulted in a reduction of Vtg production and an increment of CYP1A1 mRNA expression both in a dose dependent manner, indicating the anti-estro-genic effects of the compounds. However, at lower tested concentrations, TCDD (0.1, 0.2 pg/mL), B[a]P (5 ng/mL) seemed to have a potentiating effect on Vtg expression and secretion, although by their own these compounds had no observable estrogenic effect on Vtg induction. Tamoxifen (a selective estrogen receptor modulators, 1 nmol/L-1 μmol/L), and β-naphtho-flavone (β-NF, an aryl hydrocarbon receptor inducing compounds, 2.5-1000 ng/mL) also were employed to study the possible interactions in DES-induced Vtg expression. In co-treatment of the DES-induced hepatocytes with β-NF or tamoxifen, the decrease in Vtg production did parallel induction of CYP1A1 for β-NF, but tamoxifen inhibited Vtg induction did not parallel induced CYP1A1 expression in all test concentrations. On the contrary, it was found that in co-treatment of the TCDD-induced hepatocytes with DES, TCDD induced CYP1A1 mRNA production was inhibited by DES also. These results implicated a possible cross talk between estrogen receptor- and aryl hydrocarbon receptor-mediated pathways in the hepatocytes.

  15. Freshly isolated hepatocyte transplantation in acetaminophen-induced hepatotoxicity model in rats

    Directory of Open Access Journals (Sweden)

    Daniela Rodrigues

    2012-12-01

    Full Text Available CONTEXT: Hepatocyte transplantation is an attractive therapeutic modality for liver disease as an alternative for orthotopic liver transplantation. OBJECTIVE: The aim of the current study was to investigate the feasibility of freshly isolated rat hepatocyte transplantation in acetaminophen-induced hepatotoxicity model. METHODS: Hepatocytes were isolated from male Wistar rats and transplanted 24 hours after acetaminophen administration in female recipients. Female rats received either 1x10(7 hepatocytes or phosphate buffered saline through the portal vein or into the spleen and were sacrificed after 48 hours. RESULTS: Alanine aminotransferase levels measured within the experiment did not differ between groups at any time point. Molecular analysis and histology showed presence of hepatocytes in liver of transplanted animals injected either through portal vein or spleen. CONCLUSION: These data demonstrate the feasibility and efficacy of hepatocyte transplantation in the liver or spleen in a mild acetaminophen-induced hepatotoxicity model.

  16. Hepatocytes isolated from neoplastic liver-immunomagnetic purging as a new source for transplantation

    Institute of Scientific and Technical Information of China (English)

    Aravin Gunasegaram; Javed Akhter; Peng Yao; Loreena A Johnson; Stephen M Riodan; David L Morris

    2008-01-01

    AIM: To investigate whether hepatocytes isolated from macroscopically normal liver during hepatic resection for neoplasia could provide a novel source of healthy hepatocytes, including the development of reliable protocols for malignant cells removal from the hepatocyte preparation.METHODS: Hepatocytes were procured from resected liver of 18 patients with liver tumors using optimised digestion and cell-enrichment protocols. Suspensions of various known quantities of the HT-29 tumor cell line and patient hepatocytes were treated or not with Ep-CAM-antibody-coated immunomagnetic beads in order to investigate the efficacy of tumor-purging by immunomagnetic depletion, using a semi-quantitative RT-PCR method developed to detect tumor cells.Immunomagnetic bead-treated or bead-untreated tumor cell-hepatocyte suspensions were transplanted intra-peritoneally in Balb/C nude mice to assess the rates of tumor development.RESULTS: Mean viable hepatocyte yield was 9.3 x 106 cells per gram of digested liver with mean viability of 70.5%. Immunomagnetic depletion removed tumor cells to below the RT-PCR detection-threshold of 1 tumor cell in 106 hepatocytes, representing a maximum tumor purging efficacy of greater than 400000-fold.Transplanted, immunomagnetic bead-purged tumor cell-hepatocyte suspensions did not form peritoneal tumors in Balb/C nude mice. Co-transplantation of hepatocytes with tumor cells did not increase tumorigenesis of the tumor cells.CONCLUSION: Immunomagnetic depletion appears to be an effective method of purging contaminating tumor cells to below threshold for likely tumorigenesis.Along with improved techniques for isolation of large numbers of viable hepatocytes, normal liver resected for neoplasia has potential as another clinically useful source of hepatocytes for transplantation.

  17. Modeling of Hepatocytes Proliferation Isolated from Proximal and Distal Zones from Human Hepatocellular Carcinoma Lesion

    OpenAIRE

    Montalbano, Mauro; Curcurù, Giuseppe; Shirafkan, Ali; Vento, Renza; Rastellini, Cristiana; Cicalese, Luca

    2016-01-01

    Isolation of hepatocytes from cirrhotic human livers and subsequent primary culture are important new tools for laboratory research and cell-based therapeutics in the study of hepatocellular carcinoma (HCC). Using such techniques, we have previously identified different subpopulations of human hepatocytes and among them one is showing a progressive transformation of hepatocytes in HCC-like cells. We have hypothesized that increasing the distance from the neoplastic lesion might affect hepatoc...

  18. Heparanase enhances early hepatocyte inclusion in the recipient liver after transplantation in partially hepatectomized rats.

    Science.gov (United States)

    Tsiperson, Vladislav; Goldshmidt, Orit; Ilan, Neta; Shoshany, Gideon; Vlodavsky, Israel; Veitsman, Ella; Baruch, Yaacov

    2008-03-01

    Hepatocyte transplantation is an emerging approach for the treatment of liver diseases. However, broad clinical application of this method has been limited by restricted source of cells and low efficiency of cell integration within the recipient liver. Heparanase cleaves heparan sulfate proteoglycans in the extracellular matrix and basement membrane, activity that affects cellular invasion associated with cancer metastasis and inflammation. This activity has a multifunctional effect on cell-cell interaction, cell adhesion, and angiogenesis. All these factors are important for successful integration of transplanted hepatocytes. Male donor hepatocytes pretreated with heparanase or untreated were transplanted into recipient female rat spleen following partial hepatectomy. Engraftment efficacy was evaluated by PCR for Y chromosome, histology and PCNA, and heparanase immunohistochemistry. In addition, proliferative activity of hepatocytes in vitro was determined by bromodeoxyuridine immunostaining. The number of heparanase-treated cells detected in the recipient liver was significantly increased three- to fivefold within 24-48 h posttransplantation and twofold at 14 days compared with untreated cells. The transplanted hepatocytes treated with heparanase were clearly seen inside portal vein radicles as cell aggregates up to 72 h posttransplantation. The number of portal radicles filled with heparanase-treated hepatocytes was increased compared to control early after transplantation. Heparanase treatment enhanced hepatocyte and sinusoidal endothelial cell proliferation in the liver, and hepatocyte proliferation within the spleen tissue. Preliminary in vitro studies with isolated hepatocytes treated with heparanase showed increased proliferative activity within 24-48 h of cell culture. These results suggest that preincubation of hepatocytes with heparanase increases the presence of hepatocytes within the recipient liver early following cell transplantation and stimulates

  19. Species-specific toxicity of troglitazone on rats and human by gel entrapped hepatocytes

    International Nuclear Information System (INIS)

    Troglitazone, despite passing preclinical trials on animals, was shortly withdrawn from market due to its severe hepatotoxicity in clinic. As rat hepatocyte monolayer consistently showed sensitive troglitazone toxicity as human hepatocyte monolayer in contrast to the species-specific toxicity in vivo, this paper utilized both hepatocytes in three-dimensional culture of gel entrapment to reflect the species difference on hepatotoxicity. Rat hepatocytes in gel entrapment did not show obvious cellular damage even under a long-term exposure for 21 days while gel entrapped human hepatocytes significantly displayed oxidative stress, steatosis, mitochondrial damage and cell death at a short exposure for 4 days. As a result, the detected species-specific toxicity of troglitazone between gel entrapped rat and human hepatocytes consisted well with the situation in vivo but was in a sharp contrast to the performance of two hepatocytes by monolayer culture. Such contradictory toxicity of rat hepatocytes between monolayer and gel entrapment culture could be explained by the fact that troglitazone was cleared more rapidly in gel entrapment than in monolayer culture. Similarly, the differential clearance of troglitazone in rat and human might also explain its species-specific toxicity. Therefore, gel entrapment of hepatocytes might serve as a platform for evaluation of drug toxicity at early stage of drug development by reducing costs, increasing the likelihood of clinical success and limiting human exposure to unsafe drugs. -- Highlights: ► Species-specific toxicity of troglitazone reflected by rat/human hepatocytes ► 3D hepatocytes in 21 days’ long-term culture used for drug hepatotoxicity ► Oversensitive toxicity in hepatocyte monolayer by slow troglitazone clearance

  20. Basolateral and canalicular transport of xenobiotics in the hepatocyte: A review

    OpenAIRE

    Diaz, Gonzalo J.

    2000-01-01

    The molecular and functional characterization of severalproteins involved in the uptake and excretion of xenobioticsand endogenous compounds in the hepatocyte has been achievedthrough intensive research conducted in the past few years.These studies have lead to the identification of specificmembrane transporters located in the basolateral andcanalicular membrane domains of the hepatocyte. The organicanion-transporting polypeptide (OATP), present in thebasolateral membrane of the hepatocyte, i...

  1. Porcine hepatocyte isolation and reversible immortalization mediated by retroviral transfer and site-specific recombination

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM: To develop a hepatocyte cell line, we immortalized primary porcine hepatocytes with a retroviral vector SSR#69 containing the Simian Virus 40 T antigen (SV40T ag). METHODS: We first established a method of porcine hepatocyte isolation with a modified four-step retrograde perfusion technique. Then the porcine hepatocytes were immortalized with retroviral vector SSR#69 expressing SV40T and hygromycin-resistance genes flanked by paired loxP recombination targets. SV40T cDNA in the expanded cells was subse...

  2. Cholesterol Enhances the Toxic Effect of Ethanol and Acetaldehyde in Primary Mouse Hepatocytes.

    Science.gov (United States)

    López-Islas, Anayelly; Chagoya-Hazas, Victoria; Pérez-Aguilar, Benjamin; Palestino-Domínguez, Mayrel; Souza, Verónica; Miranda, Roxana U; Bucio, Leticia; Gómez-Quiroz, Luis Enrique; Gutiérrez-Ruiz, María-Concepción

    2016-01-01

    Obesity and alcohol consumption are risk factors for hepatic steatosis, and both commonly coexist. Our objective was to evaluate the effect of ethanol and acetaldehyde on primary hepatocytes obtained from mice fed for two days with a high cholesterol (HC) diet. HC hepatocytes increased lipid and cholesterol content. HC diet sensitized hepatocytes to the toxic effect of ethanol and acetaldehyde. Cyp2E1 content increased with HC diet, as well as in those treated with ethanol or acetaldehyde, while the activity of this enzyme determined in microsomes increased in the HC and in all ethanol treated hepatocytes, HC and CW. Oxidized proteins were increased in the HC cultures treated or not with the toxins. Transmission electron microscopy showed endoplasmic reticulum (ER) stress and megamitochondria in hepatocytes treated with ethanol as in HC and the ethanol HC treated hepatocytes. ER stress determined by PERK content was increased in ethanol treated hepatocytes from HC mice and CW. Nuclear translocation of ATF6 was observed in HC hepatocytes treated with ethanol, results that indicate that lipids overload and ethanol treatment favor ER stress. Oxidative stress, ER stress, and mitochondrial damage underlie potential mechanisms for increased damage in steatotic hepatocyte treated with ethanol.

  3. Lab on a chip-based hepatic sinusoidal system simulator for optimal primary hepatocyte culture.

    Science.gov (United States)

    Choi, Yoon Young; Kim, Jaehyung; Lee, Sang-Hoon; Kim, Dong-Sik

    2016-08-01

    Primary hepatocyte cultures have been used in studies on liver disease, physiology, and pharmacology. While they are an important tool for in vitro liver studies, maintaining liver-specific characteristics of hepatocytes in vitro is difficult, as these cells rapidly lose their unique characteristics and functions. Portal flow is an important condition to preserve primary hepatocyte functions and liver regeneration in vivo. We have developed a microfluidic chip that does not require bulky peripheral devices or an external power source to investigate the relationship between hepatocyte functional maintenance and flow rates. In our culture system, two types of microfluidic devices were used as scaffolds: a monolayer- and a concave chamber-based device. Under flow conditions, our chips improved albumin and urea secretion rates after 13 days compared to that of the static chips. Reverse transcription polymerase chain reaction demonstrated that hepatocyte-specific gene expression was significantly higher at 13 days under flow conditions than when using static chips. For both two-dimensional and three-dimensional culture on the chips, flow resulted in the best performance of the hepatocyte culture in vitro. We demonstrated that flow improves the viability and efficiency of long-term culture of primary hepatocytes and plays a key role in hepatocyte function. These results suggest that this flow system has the potential for long-term hepatocyte cultures as well as a technique for three-dimensional culture. PMID:27334878

  4. Hepatitis B virus infection and replication in primarily cultured human fetal hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Min Lin; Qun Chen; Li-Ye Yang; Wen-Yu Li; Xi-Biao Cao; Jiao-Ren Wu; You-Peng Peng; Mo-Rui Chen

    2007-01-01

    AIM:To investigate the infection and replication of hepatitis B virus(HBV)in primarily cultured human fetal hepatocytes(HFHs).METHODS:The human fetal hepatocytes were cultured in serum-free medium,HBV-positive serum was added into the medium to study the susceptibility of hepatocytes to HBV infection.The supernatant was collected for ELISA assay of HBsAg and HBeAg,and quantitative fluorescence PCR for HBV-DNA assay daily.Albumin and HBcAg,CK8 and CK18 expressions were detected by immunohistochemistry in cultured hepatocytes.Content of lactate dehydrogenate(LDH)was measured to find out the integrity of the cell membrane.RESULTS:A stable hepatocyte culture system was established.HBV could infect the hepatocytes and replicate,and HBcAg expression could be detected by immunohistochemistry in hepatocyte-like cells.HBV-DNA in the supernatant could be detected from d 2 to d 18 and HBsAg and HBeAg were positive on d 3-d 18 after HBV infection.HBV in medium increased from d 0 to d 6 and subsequently decreased as the cells were progressively loosing their hepatocyte phenotypes.CONCLUSION:HBV could infect human fetal hepatocytes and replicate.This in vitro model allowed a detailed Study on early events associated with human HBV entry into cells and subsequent replication.

  5. Gel entrapment culture of rat hepatocytes for investigation of tetracycline-induced toxicity

    International Nuclear Information System (INIS)

    This paper aimed to explore three-dimensionally cultured hepatocytes for testing drug-induced nonalcoholic steatohepatitis. Gel entrapped rat hepatocytes were applied for investigation of the tetracycline-induced steatohepatitis, while hepatocyte monolayer was set as a control. The toxic responses of hepatocytes were systematically evaluated by measuring cell viability, liver-specific function, lipid accumulation, oxidative stress, adenosine triphosphate content and mitochondrial membrane potential. The results suggested that gel entrapped hepatocytes showed cell death after 96 h of tetracycline treatment at 25 μM which is equivalent to toxic serum concentration in rats, while hepatocyte monolayer showed cell death at a high dose of 200 μM. The concentration-dependent accumulation of lipid as well as mitochondrial damage were regarded as two early events for tetracycline hepatotoxicity in gel entrapment culture due to their detectability ahead of subsequent increase of oxidative stress and a final cell death. Furthermore, the potent protection of fenofibrate and fructose-1,6-diphosphate were evidenced in only gel entrapment culture with higher expressions on the genes related to β-oxidation than hepatocyte monolayer, suggesting the mediation of lipid metabolism and mitochondrial damage in tetracycline toxicity. Overall, gel entrapped hepatocytes in three-dimension reflected more of the tetracycline toxicity in vivo than hepatocyte monolayer and thus was suggested as a more relevant system for evaluating steatogenic drugs.

  6. Gel entrapment culture of rat hepatocytes for investigation of tetracycline-induced toxicity.

    Science.gov (United States)

    Shen, Chong; Meng, Qin; Schmelzer, Eva; Bader, Augustinus

    2009-07-15

    This paper aimed to explore three-dimensionally cultured hepatocytes for testing drug-induced nonalcoholic steatohepatitis. Gel entrapped rat hepatocytes were applied for investigation of the tetracycline-induced steatohepatitis, while hepatocyte monolayer was set as a control. The toxic responses of hepatocytes were systematically evaluated by measuring cell viability, liver-specific function, lipid accumulation, oxidative stress, adenosine triphosphate content and mitochondrial membrane potential. The results suggested that gel entrapped hepatocytes showed cell death after 96 h of tetracycline treatment at 25 muM which is equivalent to toxic serum concentration in rats, while hepatocyte monolayer showed cell death at a high dose of 200 muM. The concentration-dependent accumulation of lipid as well as mitochondrial damage were regarded as two early events for tetracycline hepatotoxicity in gel entrapment culture due to their detectability ahead of subsequent increase of oxidative stress and a final cell death. Furthermore, the potent protection of fenofibrate and fructose-1,6-diphosphate were evidenced in only gel entrapment culture with higher expressions on the genes related to beta-oxidation than hepatocyte monolayer, suggesting the mediation of lipid metabolism and mitochondrial damage in tetracycline toxicity. Overall, gel entrapped hepatocytes in three-dimension reflected more of the tetracycline toxicity in vivo than hepatocyte monolayer and thus was suggested as a more relevant system for evaluating steatogenic drugs. PMID:19463838

  7. THE PREPARATION OF FRUCTOSE-MODIFIED CHITOSAN MICROCARRIERS AND CULTURE OF PRIMARY RAT HEPATOCYTES

    Institute of Scientific and Technical Information of China (English)

    ZHANG Liguo; PAN Jilun; LI Jieliang; YU Yaoting

    2004-01-01

    The fructose modified chitosan microcarries (CMs) were prepared by the reaction of glutaraldehyde with fructose-modified chitosan. Various factors that influence the preparation were studied and the reaction conditions were optimized. Morphology of rat hepatocytes cultured on CMs was observed using phase contrast microscope and scanning electron microscope, and the metabolic activities were measured. Rat hepatocytes cultured on CMs retained the spherical shape as they have in vivo and had high metabolic activities. Fructose can enhance the metabolic activity of hepatocytes and the modified CMs are promising scaffold for hepatocytes attachment.

  8. Cholesterol Enhances the Toxic Effect of Ethanol and Acetaldehyde in Primary Mouse Hepatocytes

    Science.gov (United States)

    López-Islas, Anayelly; Chagoya-Hazas, Victoria; Pérez-Aguilar, Benjamin; Palestino-Domínguez, Mayrel; Souza, Verónica; Miranda, Roxana U.; Bucio, Leticia; Gómez-Quiroz, Luis Enrique; Gutiérrez-Ruiz, María-Concepción

    2016-01-01

    Obesity and alcohol consumption are risk factors for hepatic steatosis, and both commonly coexist. Our objective was to evaluate the effect of ethanol and acetaldehyde on primary hepatocytes obtained from mice fed for two days with a high cholesterol (HC) diet. HC hepatocytes increased lipid and cholesterol content. HC diet sensitized hepatocytes to the toxic effect of ethanol and acetaldehyde. Cyp2E1 content increased with HC diet, as well as in those treated with ethanol or acetaldehyde, while the activity of this enzyme determined in microsomes increased in the HC and in all ethanol treated hepatocytes, HC and CW. Oxidized proteins were increased in the HC cultures treated or not with the toxins. Transmission electron microscopy showed endoplasmic reticulum (ER) stress and megamitochondria in hepatocytes treated with ethanol as in HC and the ethanol HC treated hepatocytes. ER stress determined by PERK content was increased in ethanol treated hepatocytes from HC mice and CW. Nuclear translocation of ATF6 was observed in HC hepatocytes treated with ethanol, results that indicate that lipids overload and ethanol treatment favor ER stress. Oxidative stress, ER stress, and mitochondrial damage underlie potential mechanisms for increased damage in steatotic hepatocyte treated with ethanol. PMID:26788255

  9. Cholesterol Enhances the Toxic Effect of Ethanol and Acetaldehyde in Primary Mouse Hepatocytes

    Directory of Open Access Journals (Sweden)

    Anayelly López-Islas

    2016-01-01

    Full Text Available Obesity and alcohol consumption are risk factors for hepatic steatosis, and both commonly coexist. Our objective was to evaluate the effect of ethanol and acetaldehyde on primary hepatocytes obtained from mice fed for two days with a high cholesterol (HC diet. HC hepatocytes increased lipid and cholesterol content. HC diet sensitized hepatocytes to the toxic effect of ethanol and acetaldehyde. Cyp2E1 content increased with HC diet, as well as in those treated with ethanol or acetaldehyde, while the activity of this enzyme determined in microsomes increased in the HC and in all ethanol treated hepatocytes, HC and CW. Oxidized proteins were increased in the HC cultures treated or not with the toxins. Transmission electron microscopy showed endoplasmic reticulum (ER stress and megamitochondria in hepatocytes treated with ethanol as in HC and the ethanol HC treated hepatocytes. ER stress determined by PERK content was increased in ethanol treated hepatocytes from HC mice and CW. Nuclear translocation of ATF6 was observed in HC hepatocytes treated with ethanol, results that indicate that lipids overload and ethanol treatment favor ER stress. Oxidative stress, ER stress, and mitochondrial damage underlie potential mechanisms for increased damage in steatotic hepatocyte treated with ethanol.

  10. Effect of Non-Esterified Fatty Acids on Fatty Acid Metabolism-Related Genes in Calf Hepatocytes Cultured in Vitro

    Directory of Open Access Journals (Sweden)

    Peng Li

    2013-11-01

    Full Text Available Background: NEFA plays numerous roles in the metabolism of glucose, lipids, and proteins. A number of experimental studies have shown that NEFA may have an important role in fatty acid metabolism in the liver, especially in dairy cows that experience negative energy balance (NEB during early lactation. Methods: In this study, using fluorescent quantitative RT-PCR, ELISA, and primary hepatocytes cultured in vitro, we examined the effect of NEFA (0, 0.2, 0.4, 0.8, 1.6, and 3.2 mmol/L on fatty acid metabolism by monitoring the mRNA and protein expression of the following key enzymes: long chain acyl-CoA synthetase (ACSL, carnitine palmitoyltransferase IA (CPT IA, long chain acyl-CoA dehydrogenase (ACADL, and acetyl-CoA carboxylase (ACC. Results: The mRNA and protein expression levels of ACSL and ACADL markedly increased as the concentration of NEFA in the media was increased. The mRNA and protein expression levels of CPT IA were enhanced significantly when the NEFA concentrations increased from 0 to 1.6 mmol/L and decreased significantly when the NEFA concentrations increased from 1.6 to 3.2 mmol/L. The mRNA and protein expression of ACC decreased gradually with increasing concentrations of NEFA. Conclusion: These findings indicate that increased NEFA significantly promote the activation and β-oxidation of fatty acids, but very high NEFA concentrations may inhibit the translocation of fatty acids into mitochondria of hepatocytes. This may explain the development of ketosis or liver lipidosis in dairy cows. CPT IA might be the key control enzyme of the fatty acid oxidation process in hepatocytes.

  11. Fas mRNA expression and calcium influx change in H2O2-induced apoptotic hepatocytes in vitro

    Institute of Scientific and Technical Information of China (English)

    Qi-Ping Lu; Lei Tian

    2005-01-01

    AIM: To investigate the relationship between Fas gene expression and calcium influx change in peroxide-induced apoptotic hepatocytes and the possible molecular mechanism of Rxa in protecting hepatocytes.METHODS: Single-cell Fas mRNA expression in H2O2-exposed L02 hepatocytes with or without treatment of Rxa,an extract from an anti-peroxidant, Radix Salviae Miltiorrhizae,was determined by all-cell patch clamp and single-cell reverse transcriptase polymerase chain reaction (RT-PCR). Transient calcium influx change ([Ca2+]i) in the cells was evaluated with all-cell patch clamp micro-fluorescence single-cytosolic free Ca2+ concentration technique. Fas protein expression, early apoptotic index (annexin-V+) and cell membrane change inthe cells were evaluated by immunohistochemistry, flow cytometry (FCM) and scan electron microscopy respectively.RESULTS: In cells exposed to H2O2 for 2 h, the specific lane for Fas mRNA was vivid on electrophoresis, with increased Fas protein expression, [Ca2+]i (from 143.66±34.21 to 1115.28±227.16), annexin-V+ index (from 4.00±0.79 to 16.18±0.72) and membrane vesicle formation. However, in cells exposed to H2O2 but pre-treated with Rxa, there was no increase in Fas mRNA or protein expression and [Ca2+]i (103.56±28.92). Annexin-V+ index (8.92±1.44) was lower than the controls (P<0.01), and the cell membrane was intact.CONCLUSION: H2O2 induces apoptosis of L02 cells by increasing cytosolic [Ca2+]i, and inducing Fas mRNA and protein expression. Rxa protects the L02 cells from apoptosis through anti-peroxidation, inhibition of calcium overloading and prevention of the activation of cytosolic Fas signal pathway.

  12. Effect of trifluoperazine on toxicity, HIF-1α induction and hepatocyte regeneration in acetaminophen toxicity in mice.

    Science.gov (United States)

    Chaudhuri, Shubhra; McCullough, Sandra S; Hennings, Leah; Brown, Aliza T; Li, Shun-Hwa; Simpson, Pippa M; Hinson, Jack A; James, Laura P

    2012-10-15

    Oxidative stress and mitochondrial permeability transition (MPT) are important mechanisms in acetaminophen (APAP) toxicity. The MPT inhibitor trifluoperazine (TFP) reduced MPT, oxidative stress, and toxicity in freshly isolated hepatocytes treated with APAP. Since hypoxia inducible factor-one alpha (HIF-1α) is induced very early in APAP toxicity, a role for oxidative stress in the induction has been postulated. In the present study, the effect of TFP on toxicity and HIF-1α induction in B6C3F1 male mice treated with APAP was examined. Mice received TFP (10mg/kg, oral gavage) prior to APAP (200mg/kg IP) and at 7 and 36h after APAP. Measures of metabolism (hepatic glutathione and APAP protein adducts) were comparable in the two groups of mice. Toxicity was decreased in the APAP/TFP mice at 2, 4, and 8h, compared to the APAP mice. At 24 and 48h, there were no significant differences in toxicity between the two groups. TFP lowered HIF-1α induction but also reduced the expression of proliferating cell nuclear antigen, a marker of hepatocyte regeneration. TFP can also inhibit phospholipase A(2), and cytosolic and secretory PLA(2) activity levels were reduced in the APAP/TFP mice compared to the APAP mice. TFP also lowered prostaglandin E(2) expression, a known mechanism of cytoprotection. In summary, the MPT inhibitor TFP delayed the onset of toxicity and lowered HIF-1α induction in APAP treated mice. TFP also reduced PGE(2) expression and hepatocyte regeneration, likely through a mechanism involving PLA(2).

  13. The neuronal nitric oxide synthase inhibitor NANT blocks acetaminophen toxicity and protein nitration in freshly isolated hepatocytes.

    Science.gov (United States)

    Banerjee, Sudip; Melnyk, Stepan B; Krager, Kimberly J; Aykin-Burns, Nukhet; Letzig, Lynda G; James, Laura P; Hinson, Jack A

    2015-12-01

    3-Nitrotyrosine (3NT) in liver proteins of mice treated with hepatotoxic doses of acetaminophen (APAP) has been postulated to be causative in toxicity. Nitration is by a reactive nitrogen species formed from nitric oxide (NO). The source of the NO is unclear. iNOS knockout mice were previously found to be equally susceptible to APAP toxicity as wildtype mice and iNOS inhibitors did not decrease toxicity in mice or in hepatocytes. In this work we examined the potential role of nNOS in APAP toxicity in hepatocytes using the specific nNOS inhibitor NANT (10 µM)(N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N'-nitroguanidinetris (trifluoroacetate)). Primary hepatocytes (1 million/ml) from male B6C3F1 mice were incubated with APAP (1mM). Cells were removed and assayed spectrofluorometrically for reactive nitrogen and oxygen species using diaminofluorescein (DAF) and Mitosox red, respectively. Cytotoxicity was determined by LDH release into media. Glutathione (GSH, GSSG), 3NT, GSNO, acetaminophen-cysteine adducts, NAD, and NADH were measured by HPLC. APAP significantly increased cytotoxicity at 1.5-3.0 h. The increase was blocked by NANT. NANT did not alter APAP mediated GSH depletion or acetaminophen-cysteine adducts in proteins which indicated that NANT did not inhibit metabolism. APAP significantly increased spectroflurometric evidence of reactive nitrogen and oxygen formation at 0.5 and 1.0 h, respectively, and increased 3NT and GSNO at 1.5-3.0 h. These increases were blocked by NANT. APAP dramatically increased NADH from 0.5-3.0 h and this increase was blocked by NANT. Also, APAP decreased the Oxygen Consumption Rate (OCR), decreased ATP production, and caused a loss of mitochondrial membrane potential, which were all blocked by NANT.

  14. Effects of aminoguanidine on nitric oxide production induced by inflammatory cytokines and endotoxin in cultured rat hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Guo Liang Zhang; Ye Hong Wang; Hui Ling Teng; Zhi Bin Lin

    2001-01-01

    AIM To study the effects of aminoguanidine(AG) and two L-arginine analogues Nω-nitro-L-arginine methyl ester (L-NAME) and Nω-nitro-L-arginine (L-NNA) on nitric oxide (NO) productioninduced by cytokines (TNF-α, IL-11β, and IFN-γ)and bacterial lipopolysaccharide (LPS) mixture(CM) in the cultured rat hepatocytes, andexamine their mechanisms action.METHODS Rat hepatocytes were incubatedwith AG, L-NAME, L-NNA, Actinomycin D (ActD)and dexamethasene in a medium containing CM(LPS plus TNF-α, IL-1β, and IFN-γ) for 24 h. NOproduction in the cultured supernatant wasmeasured with the Griese reaction. IntracellularcGMP level was detected with radioimmunoasey.RESULTS NO production was markedlyblocked by AG and L-NAME in a dose-dependentmanner under inflammatory stimuli conditiontriggered by CM in vitro. The rate of themaximum inhibitory effects of L-NAME (38.9%)was less potent than that obtained with AG(53.7%, P<0.05). There was no significantdifference between the inhibitory effects of AGand two L-arginine analogues on intracellularcGMP accumulation in rat cultured hepatocytes.Non-specific NOS expression inhibitordexamethasone ( DEX ) and iNOS mRNAtranscriptional inhibitor ActD also significantlyinhibited CM-induced NO production. AG(0.1mmol.L-1) and ActD (0.2ng@Lt) wereequipotent in decreasing NO production inducedby inflammatory stimuli in vitro, and botheffects were more potent than that induced bynon-selectivity NOS activity inhibitor L-NAME(0. 1 mmol@ L- 1) under similar stimuli conditions(P<O.O1).CONCLUSION AG is a potent selectiveinhibitor of inducible isoform of NOS, and themechanism of action may be not onlycompetitive inhibition in the substrate level, butalso the gene expression level in rathepatocytes .

  15. Mapping the Cell-Surface N-Glycoproteome of Human Hepatocytes Reveals Markers for Selecting a Homogeneous Population of iPSC-Derived Hepatocytes.

    Science.gov (United States)

    Mallanna, Sunil K; Cayo, Max A; Twaroski, Kirk; Gundry, Rebekah L; Duncan, Stephen A

    2016-09-13

    When comparing hepatic phenotypes between iPSC-derived hepatocyte-like cells from different liver disease patients, cell heterogeneity can confound interpretation. We proposed that homogeneous cell populations could be generated by fluorescence-activated cell sorting (FACS). Using cell-surface capture proteomics, we identified a total of 300 glycoproteins on hepatocytes. Analyses of the expression profiles during the differentiation of iPSCs revealed that SLC10A1, CLRN3, and AADAC were highly enriched during the final stages of hepatocyte differentiation. FACS purification of hepatocyte-like cells expressing SLC10A1, CLRN3, or AADAC demonstrated enrichment of cells with hepatocyte characteristics. Moreover, transcriptome analyses revealed that cells expressing the liver gene regulatory network were enriched while cells expressing a pluripotent stem cell network were depleted. In conclusion, we report an extensive catalog of cell-surface N-linked glycoproteins expressed in primary hepatocytes and identify cell-surface proteins that facilitate the purification of homogeneous populations of iPSC-derived hepatocyte-like cells.

  16. Mapping the Cell-Surface N-Glycoproteome of Human Hepatocytes Reveals Markers for Selecting a Homogeneous Population of iPSC-Derived Hepatocytes

    Directory of Open Access Journals (Sweden)

    Sunil K. Mallanna

    2016-09-01

    Full Text Available When comparing hepatic phenotypes between iPSC-derived hepatocyte-like cells from different liver disease patients, cell heterogeneity can confound interpretation. We proposed that homogeneous cell populations could be generated by fluorescence-activated cell sorting (FACS. Using cell-surface capture proteomics, we identified a total of 300 glycoproteins on hepatocytes. Analyses of the expression profiles during the differentiation of iPSCs revealed that SLC10A1, CLRN3, and AADAC were highly enriched during the final stages of hepatocyte differentiation. FACS purification of hepatocyte-like cells expressing SLC10A1, CLRN3, or AADAC demonstrated enrichment of cells with hepatocyte characteristics. Moreover, transcriptome analyses revealed that cells expressing the liver gene regulatory network were enriched while cells expressing a pluripotent stem cell network were depleted. In conclusion, we report an extensive catalog of cell-surface N-linked glycoproteins expressed in primary hepatocytes and identify cell-surface proteins that facilitate the purification of homogeneous populations of iPSC-derived hepatocyte-like cells.

  17. Mechanism of reduction of albumin expression induced by lipopolysaccharide in rat hepatocytes

    Institute of Scientific and Technical Information of China (English)

    WANG Xin-ying; LI Wei-qin; LU Jun; LI Ning; LI Jie-shou

    2005-01-01

    Background The severity of hypoalbuminemia has been shown to be related to morbidity and mortality in some critical illnesses, illustrating the need for better understanding of molecular mechanism of hypoalbuminemia. Lipopolysaccharide (LPS) is a key mediator inducing hypoalbuminemia in sepsis and septic shock. The present study was designed to identify if the reduction of albumin expression is directly induced by LPS and modulated by activated extracellular signal-regulated protein kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) in rat hepatocytes.Methods Primary rat hepatocytes were divided into five groups. In two of them, hepatocytes were treated with normal saline or 1 μg/ml LPS, then albumin mRNA expression was observed at 0, 2, 8, 12 and 24 hours after treatment. In another group, hepatocytes were pretreated with 100, 40 or 20 μmol/L of cycloheximide (CHX, an inhibitor of protein synthesis) for 30 minutes followed by 1 μg/ml LPS for 24 hours. Then the RNA was extracted from the cells for RT-PCR to detect the expression of albumin. The other two groups were administered 1 μmol/L, 10 μmol/L and 50 μmol/L of SB203580 (p38 MAPK inhibitor) or PD98059 (ERK inhibitor) 30 minutes prior to 1 μg/ml LPS treatment. After 24 hours of LPS treatment, the supernatant was collected and assayed for albumin concentrations. Data were analyzed by one-way analysis of variance, followed by the Newman-Keul test; a P<0.05 was considered significant.Results There was no marked change in albumin mRNA expression in the control group during 24-hours treatment with normal saline. The reduction did not occur until 24 hours after LPS treatment, and albumin mRNA decreased by 30% approximately compared to the control group at 24 hours (0.587 vs 0.832, P=0.007). CHX could inhibit the decline of albumin mRNA induced by LPS and the effect was correlated with the dose of CHX. The ERK inhibitor PD98059 caused a significant increase in LPS-induced albumin production at the

  18. The activation by glucose of liver membrane nitric oxide synthase in the synthesis and translocation of glucose transporter-4 in the production of insulin in the mice hepatocytes.

    Directory of Open Access Journals (Sweden)

    Suman Bhattacharya

    Full Text Available INTRODUCTION: Glucose has been reported to have an essential role in the synthesis and secretion of insulin in hepatocytes. As the efflux of glucose is facilitated from the liver cells into the circulation, the mechanism of transportation of glucose into the hepatocytes for the synthesis of insulin was investigated. METHODS: Grated liver suspension (GLS was prepared by grating intact liver from adult mice by using a grater. Nitric oxide (NO was measured by methemoglobin method. Glucose transporter-4 (Glut-4 was measured by immunoblot technique using Glut-4 antibody. RESULTS: Incubation of GLS with different amounts of glucose resulted in the uptake of glucose by the suspension with increased NO synthesis due to the stimulation of a glucose activated nitric oxide synthase that was present in the liver membrane. The inhibition of glucose induced NO synthesis resulted in the inhibition of glucose uptake. Glucose at 0.02M that maximally increased NO synthesis in the hepatocytes led to the translocation and increased synthesis of Glut-4 by 3.3 fold over the control that was inhibited by the inhibition of NO synthesis. The glucose induced NO synthesis was also found to result in the synthesis of insulin, in the presence of glucose due to the expression of both proinsulin genes I and II in the liver cells. CONCLUSION: It was concluded that glucose itself facilitated its own transportation in the liver cells both via Glut-4 and by the synthesis of NO which had an essential role for insulin synthesis in the presence of glucose in these cells.

  19. Hepatic steatosis inhibits autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression

    Energy Technology Data Exchange (ETDEWEB)

    Inami, Yoshihiro [Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Yamashina, Shunhei, E-mail: syamashi@juntendo.ac.jp [Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Izumi, Kousuke [Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Ueno, Takashi [Department of Biochemistry, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Tanida, Isei [Department of Biochemistry and Cell Biology, Laboratory of Biomembranes, National Institute of Infectious Disease, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640 (Japan); Ikejima, Kenichi; Watanabe, Sumio [Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan)

    2011-09-09

    Highlights: {yields} Acidification of autophagosome was blunted in steatotic hepatocytes. {yields} Hepatic steatosis did not disturb fusion of isolated autophagosome and lysosome. {yields} Proteinase activity of cathepsin B and L in autolysosomes was inhibited by steatosis. {yields} Hepatic expression of cathepsin B and L was suppressed by steatosis. -- Abstract: Autophagy, one of protein degradation system, contributes to maintain cellular homeostasis and cell defense. Recently, some evidences indicated that autophagy and lipid metabolism are interrelated. Here, we demonstrate that hepatic steatosis impairs autophagic proteolysis. Though accumulation of autophagosome is observed in hepatocytes from ob/ob mice, expression of p62 was augmented in liver from ob/ob mice more than control mice. Moreover, degradation of the long-lived protein leucine was significantly suppressed in hepatocytes isolated from ob/ob mice. More than 80% of autophagosomes were stained by LysoTracker Red (LTR) in hepatocytes from control mice; however, rate of LTR-stained autophagosomes in hepatocytes were suppressed in ob/ob mice. On the other hand, clearance of autolysosomes loaded with LTR was blunted in hepatocytes from ob/ob mice. Although fusion of isolated autophagosome and lysosome was not disturbed, proteinase activity of cathepsin B and L in autolysosomes and cathepsin B and L expression of liver were suppressed in ob/ob mice. These results indicate that lipid accumulation blunts autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression.

  20. Hepatic steatosis inhibits autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression

    International Nuclear Information System (INIS)

    Highlights: → Acidification of autophagosome was blunted in steatotic hepatocytes. → Hepatic steatosis did not disturb fusion of isolated autophagosome and lysosome. → Proteinase activity of cathepsin B and L in autolysosomes was inhibited by steatosis. → Hepatic expression of cathepsin B and L was suppressed by steatosis. -- Abstract: Autophagy, one of protein degradation system, contributes to maintain cellular homeostasis and cell defense. Recently, some evidences indicated that autophagy and lipid metabolism are interrelated. Here, we demonstrate that hepatic steatosis impairs autophagic proteolysis. Though accumulation of autophagosome is observed in hepatocytes from ob/ob mice, expression of p62 was augmented in liver from ob/ob mice more than control mice. Moreover, degradation of the long-lived protein leucine was significantly suppressed in hepatocytes isolated from ob/ob mice. More than 80% of autophagosomes were stained by LysoTracker Red (LTR) in hepatocytes from control mice; however, rate of LTR-stained autophagosomes in hepatocytes were suppressed in ob/ob mice. On the other hand, clearance of autolysosomes loaded with LTR was blunted in hepatocytes from ob/ob mice. Although fusion of isolated autophagosome and lysosome was not disturbed, proteinase activity of cathepsin B and L in autolysosomes and cathepsin B and L expression of liver were suppressed in ob/ob mice. These results indicate that lipid accumulation blunts autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression.

  1. Effects of eugenol on hepatic glucose production and AMPK signaling pathway in hepatocytes and C57BL/6J mice.

    Science.gov (United States)

    Jeong, Kyong Ju; Kim, Do Yeon; Quan, Hai-Yan; Jo, Hee Kyung; Kim, Go Woon; Chung, Sung Hyun

    2014-03-01

    Eugenol is a phenylpropanoid with many pharmacological activities, but its anti-hyperglycemic activity is not yet fully explored. For in vitro study, HepG2 cells and primary rat hepatocytes were used, and glucose production was induced by adding 100 nM of glucagon in the presence of gluconeogenic substrates. In animal study, hyperglycemia was induced by high fat diet (HFD) in male C57BL/6J mice, and eugenol was orally administered at 20 or 40 mg per kg (E20, E40) for 15 weeks. Eugenol significantly inhibited glucagon-induced glucose production and phosphorylated AMPK in the HepG2 and primary rat hepatocytes, and these effects were reversed in the presence of compound C (an AMPK inhibitor) or STO-609 (a CAMKK inhibitor). In addition, the protein and gene expression levels of CREB, CRTC2·CREB complex, PGC-1α, PEPCK and G6Pase were all significantly suppressed. Moreover, inhibition of AMPK by over-expression of dominant negative AMPK prevented eugenol from suppressions of gluconeogenic gene expression and hepatic glucose production. In animal study, plasma glucose and insulin levels of the E40 group were decreased by 31% and 63%, respectively, when compared to those of HFD control. In pyruvate tolerance tests, pyruvate-induced glucose excursions were decreased, indicating that the anti-hyperglycemic activity of eugenol is primarily due to the suppression of hepatic gluconeogenesis. In summary, eugenol effectively ameliorates hyperglycemia through inhibition of hepatic gluconeogenesis via modulating CAMKK-AMPK-CREB signaling pathway. Eugenol or eugenol-containing medicinal plants could represent a promising therapeutic agent to prevent type 2 diabetes.

  2. Metabolism of 1- and 2-naphthylamine in isolated rat hepatocytes.

    Science.gov (United States)

    Orzechowski, A; Schrenk, D; Bock, K W

    1992-12-01

    The liver probably plays a major role in the metabolic activation of the bladder carcinogen 2-naphthylamine (2-NA) and in the inactivation of the non-carcinogenic isomer 1-naphthylamine (1-NA). However, metabolic profiles of these compounds (including primary metabolites and directly determined conjugates) in hepatocytes are not available. Therefore metabolism of 1- and 2-NA was compared in freshly isolated hepatocytes from 3-methylcholanthrene (MC)-treated and untreated rats. At 10 microM, 2-NA was found to be mainly N-acetylated (66% of total metabolites after 1 h incubation) and N-glucuronidated (19%). Minor pathways led to C-oxidation (7%) and N-oxidation (3%; 2% present as the N-glucuronide). In hepatocytes from MC-treated rats total metabolism was slightly affected (1.5-fold increase). However, C- and N-oxidation were markedly increased (63 and 18% respectively), while N-acetylation and N-glucuronidation were diminished (5 and 2% respectively). Similar experiments were carried out with 1-NA. Its N-glucuronide was the predominant metabolite (68%) followed by the N-acetylated compound (15%) while C-oxidation was low and N-oxidized metabolites could not be detected, even after induction. The results demonstrate that MC treatment markedly shifted 2-NA metabolism from N-acetylation and N-glucuronidation to N- and C-oxidation. In the case of 1-NA metabolism extensive N-glucuronidation together with the lack of N-oxidation may prevent carcinogenesis. PMID:1473229

  3. Contextualizing Hepatocyte Functionality of Cryopreserved HepaRG Cell Cultures.

    Science.gov (United States)

    Jackson, Jonathan P; Li, Linhou; Chamberlain, Erica D; Wang, Hongbing; Ferguson, Stephen S

    2016-09-01

    Over the last decade HepaRG cells have emerged as a promising alternative to primary human hepatocytes (PHH) and have been featured in over 300 research publications. Most of these reports employed freshly differentiated HepaRG cells that require time-consuming culture (∼28 days) for full differentiation. Recently, a cryopreserved, predifferentiated format of HepaRG cells (termed here "cryo-HepaRG") has emerged as a new model that improves global availability and experimental flexibility; however, it is largely unknown whether HepaRG cells in this format fully retain their hepatic characteristics. Therefore, we systematically investigated the hepatocyte functionality of cryo-HepaRG cultures in context with the range of interindividual variation observed with PHH in both sandwich-culture and suspension formats. These evaluations uncovered a novel adaptation period for the cryo-HepaRG format and demonstrated the impact of extracellular matrix on cryo-HepaRG functionality. Pharmacologically important drug-metabolizing alleles were genotyped in HepaRG cells and poor metabolizer alleles for CYP2D6, CYP2C9, and CYP3A5 were identified and consistent with higher frequency alleles found in individuals of Caucasian decent. We observed liver enzyme inducibility with aryl hydrocarbon receptor, constitutive androstane receptor (CAR), and pregnane X receptor activators comparable to that of sandwich-cultured PHH. Finally, we show for the first time that cryo-HepaRG supports proper CAR cytosolic sequestration and translocation to hepatocyte nuclei in response to phenobarbital treatment. Taken together, these data reveal important considerations for the use of this cell model and demonstrate that cryo-HepaRG are suitable for metabolism and toxicology screening.

  4. Effects of ethanol on antioxidant capacity in isolated rat hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Sien-Sing Yang; Chi-Chang Huang; Jiun-Rong Chen; Che-Lin Chiu; Ming-Jer Shieh; Su-Jiun Lin; Suh-Ching Yang

    2005-01-01

    AIM: To investigate dose-response and time-course of the effects of ethanol on the cell viability and antioxidant capacity in isolated rat hepatocytes.METHODS: Hepatocytes were isolated from male adult Wistar rats and seeded into 100-mm dishes. Hepatocytes were treated with ethanol at concentrations between 0 (C), 10 (E10), 50 (E50), and 100 (E100) mmol/L (dose response) for 12, 24, and 36 h (time course). Then,lactate dehydrogenase (LDH) leakage, malondialdehyde (MDA) concentration, glutathione (GSH) level, and activities of glutathione peroxidase (GPX), glutathione reductase (GRD), superoxide dismutase (SOD), and catalase (CAT) were measured.RESULTS: Our data revealed that LDH leakage was significantly increased by about 30% in group E100 over those in groups C and E10 at 24 and 36 h, The MDA concentration in groups C, E10 and E50 were significantly lower than that in group E100 at 36 h. Furthermore,the concentration of MDA in group E100 at 36 h was significantly higher by 4.5- and 1.7-fold, respectively,than that at 12 and 24 h. On the other hand, the GSH level in group E100 at 24 and 36 h was significantly decreased, by 32% and 28%, respectively, compared to that at 12 h. The activities of GRD and CAT in group E100 at 36 h were significantly less than those in groups C and E10. However, The GPX and SOD activities showed no significant change in each group.CONCLUSION: These results suggest that longtime incubation with higher concentration of ethanol (100 mmol/L) decreased the cell viability by means of reducing GRD and CAT activities and increasing lipid peroxidation.

  5. Endocytosis via galactose receptors in vivo. Ligand size directs uptake by hepatocytes and/or liver macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Schlepper-Schaefer, J.; Huelsmann, D.; Djovkar, A.; Meyer, H.E.; Herbertz, L.; Kolb, H.; Kolb-Bachofen, V.

    1986-01-01

    The intrahepatic binding and uptake of variously sized ligands with terminal galactosyl residues is rat liver was followed. The ligands were administered to prefixed livers in binding studies and in vivo and in situ (serum-free perfused livers) in uptake studies. Gold sols with different particle diameters were prepared: 5 nm (Au/sub 5/), 17 nm (Au/sub 17/), 50 nm (Au/sub 50/) and coated with galactose exposing glycoproteins (asialofetuin (ASF) or lactosylated BSA (LacBSA)). Electron microscopy of mildly prefixed livers perfused with LacBSA-Au/sub 5/ in serum-free medium showed ligand binding to liver macrophages, hepatocytes and endothelial cells. Ligands bound to prefixed cell surfaces reflect the initial distribution of receptor activity: pre-aggregated clusters of ligands are found on liver macrophages, single particles statistically distributed on hepatocytes and pre-aggregated clusters of particles restricted to coated pits on endothelial cells. Ligand binding is prevented in the presence of 80 mM N-acetylgalactosamine (GalNAc), while N-acetylglucosamine (GlcNAc) is without effect. Electron microscopy of livers after ligand injection into the tail vein shows that in vivo uptake of electron-dense galactose particles by liver cells is size-dependent. In vivo uptake by liver macrophages is mediated by galactose-specific recognition as shown by inhibition with GalNAc.

  6. Effect of size and conformation of the ligand on asialoglycoprotein receptor-mediated ligand internalization and degradation in rat hepatocytes

    International Nuclear Information System (INIS)

    The rates of internalization and degradation of 125-I-labeled desialylated cyanogen bromide fragment I of orosomucoid (AS-CNBr-I) and its reduced and carboxymethylated derivative (AS-RC-CNBr-I) were compared with those of 125I-labeled asialoorosomucoid (ASOR) in rat hepatocytes. At 30 nM the rates of internalization and degradation of 125I-AS-CNBr-I were greater than those of 125I-ASOR. 125I-AS-RC-CNBr-I also had a lower rate of internalization and degradation. In contrast to 125I-ASOR, when degradation was inhibited by 5 μM colchicine there was a significant intracellular accumulation of the smaller ligands. At 40C the hepatocytes were found to bind the fragmented ligands more than 125I-ASOR. Incubation of the cells with bound ligand at 370 indicated that diacytosis of 125I-ASOR was greater than the smaller ligands. Colchincine markedly enhanced diacytosis of 125I-ASOR. On the other hand, there were marked accumulation of the smaller ligands by colchicine. These results suggest that the rates of internalization, degradation and diacytosis of the ligand are affected by the size and conformation of the ligand through different rates of receptor binding and intracellular transport

  7. Prolyl-4-Hydroxylase 2 Potentially Contributes to Hepatocellular Carcinoma-Associated Erythrocytosis by Maintaining Hepatocyte Nuclear Factor-4α Expression

    Directory of Open Access Journals (Sweden)

    Wenwen Sun

    2015-12-01

    Full Text Available Background: Increased red blood cell count (Erythrocytosis is an important paraneoplastic syndrome of hepatocellular carcinoma (HCC and is a significant risk factor for lethal lung artery thromboembolism. HCC-associated erythrocytosis is partially caused by the ability of several HCC cells to produce erythropoietin (EPO. Prolyl-4-hydroxylase 2 (PHD2 is an enzyme encoded by the gene EGLN1. The best-known function of PHD2 is to mediate the oxygen-dependent degradation of the labile α-subunit of hypoxia-inducible factor (HIF. However, there is increasing evidence that PHD2 also regulates HIF-independent pathways by interacting with other substrates. Methods: In the EPO-producing human HCC cell line HepG2, the expression of PHD2 gene was silenced with siRNA. EPO production was estimated using quantitative PCR and ELISA. Results: In HepG2 cells, PHD2 suppresses the activity of TGF-β1 pathway and consequently maintains the expression of hepatocyte nuclear factor-4α (HNF-4α, an important transcription factor promoting the EPO expression in hepatocytes. PHD2 knockdown caused a marked reduction of EPO production. HIF seemed not to be involved in this biology. Conclusion: Our findings show that PHD2 represents a potential contributing factor for HCC-associated erythrocytosis. Selective inhibition of PHD2 in HCC cells might be considered as a new way to manage erythrocytosis in HCC patients.

  8. Pharmacokinetics and effects on serum cholinesterase activities of organophosphorus pesticides acephate and chlorpyrifos in chimeric mice transplanted with human hepatocytes.

    Science.gov (United States)

    Suemizu, Hiroshi; Sota, Shigeto; Kuronuma, Miyuki; Shimizu, Makiko; Yamazaki, Hiroshi

    2014-11-01

    Organophosphorus pesticides acephate and chlorpyrifos in foods have potential to impact human health. The aim of the current study was to investigate the pharmacokinetics of acephate and chlorpyrifos orally administered at lowest-observed-adverse-effect-level doses in chimeric mice transplanted with human hepatocytes. Absorbed acephate and its metabolite methamidophos were detected in serum from wild type mice and chimeric mice orally administered 150mg/kg. Approximately 70% inhibition of cholinesterase was evident in plasma of chimeric mice with humanized liver (which have higher serum cholinesterase activities than wild type mice) 1day after oral administrations of acephate. Adjusted animal biomonitoring equivalents from chimeric mice studies were scaled to human biomonitoring equivalents using known species allometric scaling factors and in vitro metabolic clearance data with a simple physiologically based pharmacokinetic (PBPK) model. Estimated plasma concentrations of acephate and chlorpyrifos in humans were consistent with reported concentrations. Acephate cleared similarly in humans and chimeric mice but accidental/incidental overdose levels of chlorpyrifos cleared (dependent on liver metabolism) more slowly from plasma in humans than it did in mice. The data presented here illustrate how chimeric mice transplanted with human hepatocytes in combination with a simple PBPK model can assist evaluations of toxicological potential of organophosphorus pesticides.

  9. Pravastatin transport across the hepatocyte canalicular membrane requires both ATP and a transmembrane pH gradient.

    Science.gov (United States)

    Adachi, Y; Okuyama, Y; Miya, H; Matsusita, H; Kitano, M; Kamisako, T; Yamamoto, T

    1996-06-01

    Hepatic excretion of non-bile acid organic anions is reported to be ATP-dependent and a defect of this transport has been reported in congenitally jaundiced rats, animal models of human Dubin-Johnson syndrome. To investigate the effect of the transmembrane pH gradient on hepatocyte canalicular membrane transport of ATP-dependent organic anions, uptake of pravastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase-inhibiting organic anion, by hepatocyte canalicular membrane vesicles was observed in the presence or absence of transmembrane pH gradients. Uptake was assessed by a rapid filtration technique. ATP-dependent pravastatin uptake was stimulated in the presence of a transmembrane pH gradient (in > out) in Sprague-Dawley (SD) rats. Uptake was dependent on both pravastatin and ATP concentrations and showed saturation kinetics. After intravenous injection of [14C]-pravastatin (0.3 mumol), 81% of the dose was excreted in the bile within 35 min in SD rats, whereas only 20% was excreted in the bile in Eisai hyperbilirubinuria rats. ATP and the pH gradient also co-stimulated the uptake of pravastatin in Eisai hyperbilirubinuria rats, although the K(m) was much higher and Vmax was much lower than corresponding values in SD rats. This coincided well with the marked reduction in vivo biliary excretion of pravastatin in jaundiced rats.

  10. Identiifcation of Human Hepatocyte Proliferation Related Gene C2orf69

    Institute of Scientific and Technical Information of China (English)

    2014-01-01

    Objective To construct the prokaryotic expression vector pET-32a(+)-C2orf69 and induce the expression of recombinant proteins in vitro. Then the possible effects of recombinant protein on cell proliferation was observed and rabbit-anti-C2orf69 protein polyclonal antibodies was obtained. Methods Gene fragment of C2orf69 was ampliifed by PCR and then prokaryotic expression plasmid pET-32a(+)-C2orf69 was constructed. Recombinant protein C2orf69 expression was identiifed by SDS-PAGE and Western blot. The white-ear rabbits were immunized with purified recombinant protein C2orf69, and the potency and speciifcity of polyclonal antibody were evaluated by enzyme-linked immunosorbent assay (ELISA) and Western blot. Also, different liver cells were incubated with recombinant protein C2orf69 in vitro. Results C2orf69 gene fragment was successfully ampliifed, results of gene sequencing were consistent with the sequence in GenBank. Recombinant protein of C2orf69 was successfully induced and expressed. The polyclonal antibody titer was up to 1︰1 280 000 through enzyme-linked immunosorbent assay. Results of cell proliferation showed that the recombinant protein could inhibit the proliferation of different liver cells. Conclusions The recombinant protein C2orf69 could inhibit the proliferation of different liver cells, and we speculated that it may be a widely roled inhibitor of hepatocyte proliferation. Our experiment showed that the proliferation inhibition of cells may be realized by G1 phase extending and S phase shortening.

  11. Receptor binding of biosynthetic human insulin on isolated pig hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Gammeltoft, S.

    Biosynthetic human insulin (BHI) and pancreatic human insulin were compared with respect to receptor binding in a heterologous assay system: displacement of pork A14-/sup 125/I-monoiodoinsulin from receptors on pig hepatocytes. The concentrations of human insulin giving half-maximal displacement were identical for both preparations, i.e., 0.5 nM. Their relative potency was 1.01 +/- 0.14 (SD, N . 5), suggesting that biosynthetic and pancreatic human insulin exert the same biologic activity.

  12. Receptor binding of biosynthetic human insulin on isolated pig hepatocytes

    International Nuclear Information System (INIS)

    Biosynthetic human insulin (BHI) and pancreatic human insulin were compared with respect to receptor binding in a heterologous assay system: displacement of pork A14-125I-monoiodoinsulin from receptors on pig hepatocytes. The concentrations of human insulin giving half-maximal displacement were identical for both preparations, i.e., 0.5 nM. Their relative potency was 1.01 +/- 0.14 (SD, N . 5), suggesting that biosynthetic and pancreatic human insulin exert the same biologic activity

  13. Par1b induces asymmetric inheritance of plasma membrane domains via LGN-dependent mitotic spindle orientation in proliferating hepatocytes

    NARCIS (Netherlands)

    Slim, Christiaan L; Lázaro-Diéguez, Francisco; Bijlard, Marjolein; Toussaint, Mathilda J M; de Bruin, Alain; Du, Quansheng; Müsch, Anne; van Ijzendoorn, Sven C D

    2013-01-01

    The development and maintenance of polarized epithelial tissue requires a tightly controlled orientation of mitotic cell division relative to the apical polarity axis. Hepatocytes display a unique polarized architecture. We demonstrate that mitotic hepatocytes asymmetrically segregate their apical p

  14. Fagopyrum tataricum (Buckwheat Improved High-Glucose-Induced Insulin Resistance in Mouse Hepatocytes and Diabetes in Fructose-Rich Diet-Induced Mice

    Directory of Open Access Journals (Sweden)

    Chia-Chen Lee

    2012-01-01

    Full Text Available Fagopyrum tataricum (buckwheat is used for the treatment of type 2 diabetes mellitus in Taiwan. This study was to evaluate the antihyperglycemic and anti-insulin resistance effects of 75% ethanol extracts of buckwheat (EEB in FL83B hepatocytes by high-glucose (33 mM induction and in C57BL/6 mice by fructose-rich diet (FRD; 60% induction. The active compounds of EEB (100 μg/mL; 50 mg/kg bw, quercetin (6 μg/mL; 3 mg/kg bw, and rutin (23 μg/mL; 11.5 mg/kg bw were also employed to treat FL83B hepatocytes and animal. Results indicated that EEB, rutin, and quercetin + rutin significantly improved 2-NBDG uptake via promoting Akt phosphorylation and preventing PPARγ degradation caused by high-glucose induction for 48 h in FL83B hepatocytes. We also found that EEB could elevate hepatic antioxidant enzymes activities to attenuate insulin resistance as well as its antioxidation caused by rutin and quercetin. Finally, EEB also inhibited increases in blood glucose and insulin levels of C57BL/6 mice induced by FRD.

  15. Fagopyrum tataricum (buckwheat) improved high-glucose-induced insulin resistance in mouse hepatocytes and diabetes in fructose-rich diet-induced mice.

    Science.gov (United States)

    Lee, Chia-Chen; Hsu, Wei-Hsuan; Shen, Siou-Ru; Cheng, Yu-Hsiang; Wu, She-Ching

    2012-01-01

    Fagopyrum tataricum (buckwheat) is used for the treatment of type 2 diabetes mellitus in Taiwan. This study was to evaluate the antihyperglycemic and anti-insulin resistance effects of 75% ethanol extracts of buckwheat (EEB) in FL83B hepatocytes by high-glucose (33 mM) induction and in C57BL/6 mice by fructose-rich diet (FRD; 60%) induction. The active compounds of EEB (100 μg/mL; 50 mg/kg bw), quercetin (6 μg/mL; 3 mg/kg bw), and rutin (23 μg/mL; 11.5 mg/kg bw) were also employed to treat FL83B hepatocytes and animal. Results indicated that EEB, rutin, and quercetin + rutin significantly improved 2-NBDG uptake via promoting Akt phosphorylation and preventing PPARγ degradation caused by high-glucose induction for 48 h in FL83B hepatocytes. We also found that EEB could elevate hepatic antioxidant enzymes activities to attenuate insulin resistance as well as its antioxidation caused by rutin and quercetin. Finally, EEB also inhibited increases in blood glucose and insulin levels of C57BL/6 mice induced by FRD.

  16. Interactions between macrophage/Kupffer cells and hepatocytes in surgical sepsis

    International Nuclear Information System (INIS)

    Experiments were performed to investigate the role of Kupffer cell/macrophage interactions with hepatocytes in modulating liver function during infections using direct in vitro cocultivation of rat macrophages or Kupffer cells with rat hepatocytes. Protein synthesis was assayed as a sensitive indicator of integrated hepatocellular function by measuring 3H-leucine incorporation into hepatocyte protein. Septic stimuli such as lipoploysaccharide and killed bacteria were added to cocultures of hepatocytes and macrophages or Kupffer cells and the responses compared to hepatocytes alone. Information about the types of proteins synthesized by hepatocytes under various culture conditions was determined using polyacrylamide gel electrophoresis and autoradiography. These experiments showed that septic stimuli alter the amount and type of protein synthesized by hepatocytes and had no direct effect on hepatocytes in the absence of macrophages or Kupffer cells. The mediator(s) appears to be a heat labile, soluble monokine(s) which is distinct from interleukin-1 or tumor necrosis factor. The important role of Kupffer cells/macrophages in mediating alterations in hepatocellular function in sepsis may ultimately improve patient care

  17. Effects of nuclear translocation of tissue transglutaminase and the release of cytochrome C on hepatocyte apoptosis

    Institute of Scientific and Technical Information of China (English)

    宋良文; 马宪梅; 李扬; 崔雪梅; 王晓民

    2003-01-01

    Objective To assess the effects of nuclear translocation of tissue transglutaminase (TTG) and the release of cytochrome C on hepatocyte apoptosis and to reveal the mechanism of signal transduction of early apoptosis in injured hepatocytes. Methods Hepatocytes isolated from tissue transglutaminase gene knock-out rats and mice were stimulated with ethanol. Proteins from whole cell, cytoplasm and nuclei were extracted for determination of TTG activity by 14 C-putrescine incorporation. Distribution of TTG throughout the entire cell, as well as just nucleus was observed under a confocal scanning microscope. The amount of cytochrome C released from mitochondria was determined by ELISA. Cell apoptosis was observed by fluorescent cytochemistry.Results TTG activity in whole cells and nuclei was significantly increased after the hepatocytes were treated with ethanol. Cytochrome C release was remarkably increased in the cells isolated from rat and wild-type mouse after treatment with ethanol but not in TTG gene knock-out mice. Cellular apoptosis appeared in hepatocytes isolated from rats and wild-type mice but not in the hepatocytes from TTG gene knock-out mice after stimulation with ethanol.Conclusions Increased TTG in hepatocytes can be translocated into the nucleus and promote release of mitochondrial cytochrome C into the cytoplasm. Passing through a series of signal pathways, hepatocyte apoptosis is induced eventually.

  18. Differentiation of Bone Marrow: Derived Mesenchymal Stem Cells into Hepatocyte-like Cells.

    Science.gov (United States)

    Al Ghrbawy, Nesrien M; Afify, Reham Abdel Aleem Mohamed; Dyaa, Nehal; El Sayed, Asmaa A

    2016-09-01

    Cirrhosis is the end-stage liver fibrosis, whereby normal liver architecture is disrupted by fibrotic bands, parenchymal nodules and vascular distortion. Portal hypertension and hepatocyte dysfunction are the end results and give rise to major systemic complications and premature death. Mesenchymal stem cells (MSC) have the capacity of self-renew and to give rise to cells of various lineages, so MSC can be isolated from bone marrow (BM) and induced to differentiate into hepatocyte-like cells. MSC were induced to differentiate into hepatocyte-like cells by hepatotic growth factor (HGF) and fibroblast growth factor-4 (FGF-4). Differentiated cells were examined for the expression of hepatocyte-specific markers and hepatocyte functions. MSC were isolated. Flow cytometry analysis showed that they expressed the MSC-specific markers, reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated that MSC expressed the hepatocyte-specific marker cytokeratin 18 (CK-18) following hepatocyte induction. This study demonstrates that BM-derived-MSC can differentiate into functional hepatocyte-like cells following the induction of HGF and FGF-4. MSC can serve as a favorable cell source for tissue engineering in the treatment of liver disease. PMID:27429519

  19. Interactions between macrophage/Kupffer cells and hepatocytes in surgical sepsis

    Energy Technology Data Exchange (ETDEWEB)

    West, M.A.

    1988-01-01

    Experiments were performed to investigate the role of Kupffer cell/macrophage interactions with hepatocytes in modulating liver function during infections using direct in vitro cocultivation of rat macrophages or Kupffer cells with rat hepatocytes. Protein synthesis was assayed as a sensitive indicator of integrated hepatocellular function by measuring {sup 3}H-leucine incorporation into hepatocyte protein. Septic stimuli such as lipoploysaccharide and killed bacteria were added to cocultures of hepatocytes and macrophages or Kupffer cells and the responses compared to hepatocytes alone. Information about the types of proteins synthesized by hepatocytes under various culture conditions was determined using polyacrylamide gel electrophoresis and autoradiography. These experiments showed that septic stimuli alter the amount and type of protein synthesized by hepatocytes and had no direct effect on hepatocytes in the absence of macrophages or Kupffer cells. The mediator(s) appears to be a heat labile, soluble monokine(s) which is distinct from interleukin-1 or tumor necrosis factor. The important role of Kupffer cells/macrophages in mediating alterations in hepatocellular function in sepsis may ultimately improve patient care.

  20. Partial hepatectomy induces delayed hepatocyte proliferation and normal liver regeneration in ovariectomized mice

    Directory of Open Access Journals (Sweden)

    Umeda M

    2015-07-01

    Full Text Available Makoto Umeda,1 Masaki Hiramoto,1,2 Takeshi Imai1 1Department of Aging Intervention, National Center for Geriatrics and Gerontology, Obu, Aichi, Japan; 2Department of Biochemistry, Tokyo Medical University, Tokyo, Japan Abstract: Estrogens play central roles in sexual development, reproduction, and hepatocyte proliferation. The ovaries are one of the main organs for estradiol (E2 production. Ovariectomies (OVXs were performed on the female mice, and hepatocyte proliferation was analyzed. The ovariectomized mice exhibited delayed hepatocyte proliferation after partial hepatectomy (PH and also exhibited delayed and reduced E2 induction. Both E2 administration and PH induced the gene expression of estrogen receptor α (ERα. The transcripts of ERα were detected specifically in periportal hepatocytes after E2 administration and PH. Moreover, the E2 concentrations and hepatocyte proliferation rates were highest in the proestrus period of the estrous cycle. Taken together, these findings indicate that E2 accelerated ERα expression in periportal hepatocytes and hepatocyte proliferation in the female mice.Keywords: estrogen, ER, estrous cycle, hepatocyte proliferation, liver regeneration

  1. High Throughput Micro-Well Generation of Hepatocyte Micro-Aggregates for Tissue Engineering

    NARCIS (Netherlands)

    Gevaert, Elien; Dollé, Laurent; Billiet, Thomas; Dubruel, Peter; Grunsven, van Leo; Apeldoorn, van Aart; Cornelissen, Ria

    2014-01-01

    The main challenge in hepatic tissue engineering is the fast dedifferentiation of primary hepatocytes in vitro. One successful approach to maintain hepatocyte phenotype on the longer term is the cultivation of cells as aggregates. This paper demonstrates the use of an agarose micro-well chip for the

  2. High throughput micro-well generation of hepatocyte micro-aggregates for tissue engineering.

    Directory of Open Access Journals (Sweden)

    Elien Gevaert

    Full Text Available The main challenge in hepatic tissue engineering is the fast dedifferentiation of primary hepatocytes in vitro. One successful approach to maintain hepatocyte phenotype on the longer term is the cultivation of cells as aggregates. This paper demonstrates the use of an agarose micro-well chip for the high throughput generation of hepatocyte aggregates, uniform in size. In our study we observed that aggregation of hepatocytes had a beneficial effect on the expression of certain hepatocyte specific markers. Moreover we observed that the beneficial effect was dependent on the aggregate dimensions, indicating that aggregate parameters should be carefully considered. In a second part of the study, the selected aggregates were immobilized by encapsulation in methacrylamide-modified gelatin. Phenotype evaluations revealed that a stable hepatocyte phenotype could be maintained during 21 days when encapsulated in the hydrogel. In conclusion we have demonstrated the beneficial use of micro-well chips for hepatocyte aggregation and the size-dependent effects on hepatocyte phenotype. We also pointed out that methacrylamide-modified gelatin is suitable for the encapsulation of these aggregates.

  3. Primary hepatocytes : Current understanding of the regulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for the use of hepatocytes in metabolism, enzyme induction, transporter, clearance, and hepatotoxicity studies

    NARCIS (Netherlands)

    Hewitt, Nicola J.; Lechon, Maria Jose Gomez; Houston, J. Brian; Hallifax, David; Brown, Hayley S.; Maurel, Patrick; Kenna, J. Gerald; Gustavsson, Lena; Lohmann, Christina; Skonberg, Christian; Guillouzo, Andre; Tuschl, Gregor; Li, Albert P.; LeCluyse, Edward; Groothuis, Geny M. M.; Hengstler, Jan G.

    2007-01-01

    This review brings you up-to-date with the hepatocyte research on: 1) in vitro-in vivo correlations of metabolism and clearance; 2) CYP enzyme induction, regulation, and crosstalk using human hepatocytes and hepatocyte-like cell lines; 3) the function and regulation of hepatic transporters and model

  4. Hepatocyte transplantation in bile salt export pump-deficient mice: selective growth advantage of donor hepatocytes under bile acid stress

    OpenAIRE

    Chen, Huey-Ling; Chen, Hui-Ling; Yuan, Ray-Hwang; Wu, Shang-Hsin; Chen, Ya-Hui; Chien, Chin-Sung; Chou, Shi-Ping; Wang, Renxue; Ling, Victor; Chang, Mei-Hwei

    2012-01-01

    The bile salt export pump (Bsep) mediates the hepatic excretion of bile acids, and its deficiency causes progressive familial intrahepatic cholestasis. The current study aimed to induce bile acid stress in Bsep −/− mice and to test the efficacy of hepatocyte transplantation in this disease model. We fed Bsep −/− and wild-type mice cholic acid (CA) or ursodeoxycholic acid (UDCA). Both CA and UDCA caused cholestasis and apoptosis in the Bsep −/− mouse liver. Wild-type mice had minimal liver inj...

  5. Differentiation of human embryonic stem cells along a hepatocyte lineage and its application in liver regeneration

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Hepatocyte transplantation and bioartificial liver(BAL)as alternatives to liver transplantation offer the possibility of effective treatment for many inherited and acquired hepatic disorders.Unfortunately,the limited availability of donated livers and the variability of their derived hepatocytes make it difficult to obtain enough viable human hepatocytes for the hepatocyte-based therapies.Embryonic stem cells (ESCs),which could be isolated directly from the blastocyst inner cell mass,have permanent self-renewal capability and developmental pluripotency and therefore might be an ideal cell source in the treatment of hepatic discords.However,differentiation of hESCS into hepatocytes with significant numbers remains a challenge.This review updates our current understanding of differentiation of ESCs into hepatic lineage cells,their future therapeutic uses and problems in liver regeneration.

  6. Lineage tracing reveals conversion of liver sinusoidal endothelial cells into hepatocytes.

    Science.gov (United States)

    Tan, Zhaoli; Chen, Keyan; Shao, Yong; Gao, Lihua; Wang, Yan; Xu, Jianming; Jin, Yang; Hu, Xianwen; Wang, Youliang

    2016-09-01

    Although liver sinusoidal endothelial cells (LSECs) have long been known to contribute to liver regeneration following injury, the exact role of these cells in liver regeneration remains poorly understood. In this work, we performed lineage tracing of LSECs in mice carrying Tie2-Cre or VE-cadherin-Cre constructs to facilitate fate-mapping of LSECs in liver regeneration. Some YFP-positive LSECs were observed to convert into hepatocytes following a two-thirds partial hepatectomy (PH). Furthermore, human umbilical vein endothelial cells (HUVECs) could be triggered to convert into cells that closely resembled hepatocytes when cultured with serum from mice that underwent an extended PH. These findings suggest that mature non-hepatocyte LSECs play an essential role in mammalian liver regeneration by converting to hepatocytes. The conversion of LSECs to hepatocyte-like (iHep) cells may provide a new approach to tissue engineering.

  7. Hepatocytes Polyploidization and Cell Cycle Control in Liver Physiopathology

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    Géraldine Gentric

    2012-01-01

    Full Text Available Most cells in mammalian tissues usually contain a diploid complement of chromosomes. However, numerous studies have demonstrated a major role of “diploid-polyploid conversion” during physiopathological processes in several tissues. In the liver parenchyma, progressive polyploidization of hepatocytes takes place during postnatal growth. Indeed, at the suckling-weaning transition, cytokinesis failure events induce the genesis of binucleated tetraploid liver cells. Insulin signalling, through regulation of the PI3K/Akt signalling pathway, is essential in the establishment of liver tetraploidization by controlling cytoskeletal organisation and consequently mitosis progression. Liver cell polyploidy is generally considered to indicate terminal differentiation and senescence, and both lead to a progressive loss of cell pluripotency associated to a markedly decreased replication capacity. Although adult liver is a quiescent organ, it retains a capacity to proliferate and to modulate its ploidy in response to various stimuli or aggression (partial hepatectomy, metabolic overload (i.e., high copper and iron hepatic levels, oxidative stress, toxic insult, and chronic hepatitis etc.. Here we review the mechanisms and functional consequences of hepatocytes polyploidization during normal and pathological liver growth.

  8. Discovering networks of perturbed biological processes in hepatocyte cultures.

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    Christopher D Lasher

    Full Text Available The liver plays a vital role in glucose homeostasis, the synthesis of bile acids and the detoxification of foreign substances. Liver culture systems are widely used to test adverse effects of drugs and environmental toxicants. The two most prevalent liver culture systems are hepatocyte monolayers (HMs and collagen sandwiches (CS. Despite their wide use, comprehensive transcriptional programs and interaction networks in these culture systems have not been systematically investigated. We integrated an existing temporal transcriptional dataset for HM and CS cultures of rat hepatocytes with a functional interaction network of rat genes. We aimed to exploit the functional interactions to identify statistically significant linkages between perturbed biological processes. To this end, we developed a novel approach to compute Contextual Biological Process Linkage Networks (CBPLNs. CBPLNs revealed numerous meaningful connections between different biological processes and gene sets, which we were successful in interpreting within the context of liver metabolism. Multiple phenomena captured by CBPLNs at the process level such as regulation, downstream effects, and feedback loops have well described counterparts at the gene and protein level. CBPLNs reveal high-level linkages between pathways and processes, making the identification of important biological trends more tractable than through interactions between individual genes and molecules alone. Our approach may provide a new route to explore, analyze, and understand cellular responses to internal and external cues within the context of the intricate networks of molecular interactions that control cellular behavior.

  9. Extracellular phosphate requirement for insulin action on isolated rat hepatocytes

    International Nuclear Information System (INIS)

    Isolated rat hepatocytes were prepared in Krebs-Henseleit bicarbonate (KHB) buffer, pH 7.4, by a procedure described earlier. They were centrifuged and washed twice in KHB buffer containing 0, 0.05, 0.1, 0.02, 0.05, 0.89 or 1.0 mM phosphate and were incubated at 30 degrees C in the presence of tracer 2,3-14C-succinate and 0.5 mM concentration of each of the 20 natural amino acids. Hepatocytes washed and incubated in KHB buffer containing less than 0.05 mM phosphate failed to show any insulin stimulation of 2,3-14C-succinate oxidation or protein incorporation of tracer carbons. Mitochondrial oxidation of succinate carbons was significantly lower in the medium containing less than 1.0 mM phosphate. Maximal insulin stimulatory effect was observed in the presence of 1 mM phosphate in the medium. These data indicate that a diminished insulin responsiveness in hypophosphatemic patients may be due to the insensitivity of mitochondria to insulin in the hypophosphatemic state

  10. Hormonal regulation of fibrinogen synthesis in cultured hepatocytes.

    Science.gov (United States)

    Grieninger, G; Plant, P W; Liang, T J; Kalb, R G; Amrani, D; Mosesson, M W; Hertzberg, K M; Pindyck, J

    1983-06-27

    Most of what was originally known of the effects of hormones on fibrinogen synthesis was based, as noted above, on experiments involving surgical removal of endocrine glands. Some caution should be exercised when using such in vivo experiments to derive the hormonal requirements of fibrinogen synthesis, however, since multiple hormonal alterations often occur in these animals. The development of a variety of ex vivo systems has allowed investigators to more carefully control the hepatocellular environment. The work of several laboratories, including our own, has now made it clear that hormones and other agents directly stimulate hepatocellular synthesis of fibrinogen. From the studies summarized here, using chick embryo hepatocytes as a model, several generalizations emerge: Fibrinogen synthesis may be considered to be a "constitutive" liver function, since hepatocytes cultured without serum, hormones or other macromolecular supplements synthesize this protein at a basal rate for several days. Addition of certain hormones (e.g. T3, dexamethasone, insulin), individually and in physiological concentrations, elicits an increase in fibrinogen production, varying with each agent in onset, dose, minimum exposure required and accompanying effects on the synthesis of other plasma proteins. Glucocorticoids and thyroid hormones are similar in the selectivity of their stimulation (neither affects albumin or transferrin synthesis) but differ in that thyroid hormones need to be present for just a short "triggering" period. The stimulation of fibrinogen synthesis by insulin occurs only following prolonged exposure to concentrations 10-times higher than the very low doses to which albumin synthesis responds rapidly. PMID:6307104

  11. HTS compatible assay for antioxidative agents using primary cultured hepatocytes.

    Science.gov (United States)

    Gaunitz, Frank; Heise, Kerstin

    2003-06-01

    We have used primary cultured rat hepatocytes to establish a system that is compatible with HTS for screening substance libraries for biologically active compounds. The hepatocytes were treated with t-BHP to induce oxidative stress, leading to the formation ROS. The involvement of ROS in oxidative stress and pathological alterations has been of major interest in recent years, and there is great demand to identify new compounds with antioxidant potential. In most HTS programs each compound is tested in duplicate, and may only be tested once. Because of this it is important to develop assays that can identify candidate compounds accurately and with high confidence. Using newly available cell-based assay systems, we have developed a system that can detect active compounds (hits) with a high degree of confidence. As an example of an agent that can be detected from a substance library, we analyzed the effect of fisetin as an antioxidative compound using this system. All measurements were performed using the newly developed and highly versatile Multilabel-Reader Mithras LB 940 (Berthold Technologies, Bad Wildbad, Germany). The data presented show that all Z' factors determined were highly reliable. Although the protocol is primarily designed to screen for substances with antioxidative potential, it can easily be adapted to screen for other biologically active substances.

  12. Study of Valproic Acid-Enhanced Hepatocyte Steatosis

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    Renin Chang

    2016-01-01

    Full Text Available Valproic acid (VPA is one of the most widely used antiepilepsy drugs. However, several side effects, including weight gain and fatty liver, have been reported in patients following VPA treatment. In this study, we explored the molecular mechanisms of VPA-induced hepatic steatosis using FL83B cell line-based in vitro model. Using fluorescent lipid staining technique, we found that VPA enhanced oleic acid- (OLA- induced lipid accumulation in a dose-dependent manner in hepatocytes; this may be due to upregulated lipid uptake, triacylglycerol (TAG synthesis, and lipid droplet formation. Real-time PCR results showed that, following VPA treatment, the expression levels of genes encoding cluster of differentiation 36 (Cd36, low-density lipoprotein receptor-related protein 1 (Lrp1, diacylglycerol acyltransferase 2 (Dgat2, and perilipin 2 (Plin2 were increased, that of carnitine palmitoyltransferase I a (Cpt1a was not affected, and those of acetyl-Co A carboxylase α (Acca and fatty acid synthase (Fasn were decreased. Furthermore, using immunofluorescence staining and flow cytometry analyses, we found that VPA also induced peroxisome proliferator-activated receptor γ (PPARγ nuclear translocation and increased levels of cell-surface CD36. Based on these results, we propose that VPA may enhance OLA-induced hepatocyte steatosis through the upregulation of PPARγ- and CD36-dependent lipid uptake, TAG synthesis, and lipid droplet formation.

  13. Involvement of Bcl-xL degradation and mitochondrial-mediated apoptotic pathway in pyrrolizidine alkaloids-induced apoptosis in hepatocytes

    International Nuclear Information System (INIS)

    Pyrrolizidine alkaloids (PAs) are natural hepatotoxins with worldwide distribution in more than 6000 high plants including medicinal herbs or teas. The aim of this study is to investigate the signal pathway involved in PAs-induced hepatotoxicity. Our results showed that clivorine, isolated from Ligularia hodgsonii Hook, decreased cell viability and induced apoptosis in L-02 cells and mouse hepatocytes. Western-blot results showed that clivorine induced caspase-3/-9 activation, mitochondrial release of cytochrome c and decreased anti-apoptotic Bcl-xL in a time (8-48 h)- and concentration (1-100 μM)-dependent manner. Furthermore, inhibitors of pan-caspase, caspase-3 and caspase-9 significantly inhibited clivorine-induced apoptosis and rescued clivorine-decreased cell viability. Polyubiquitination of Bcl-xL was detected after incubation with 100 μM clivorine for 40 h in the presence of proteasome specific inhibitor MG132, indicating possible degradation of Bcl-xL protein. Furthermore, pretreatment with MG132 or calpain inhibitor I for 2 h significantly enhanced clivorine-decreased Bcl-xL level and cell viability. All the other tested PAs such as senecionine, isoline and monocrotaline decreased mouse hepatocytes viability in a concentration-dependent manner. Clivorine (10 μM) induced caspase-3 activation and decreased Bcl-xL was also confirmed in mouse hepatocytes. Meanwhile, another PA senecionine isolated from Senecio vulgaris L also induced apoptosis, caspase-3 activation and decreased Bcl-xL in mouse hepatocytes. In conclusion, our results suggest that PAs may share the same hepatotoxic signal pathway, which involves degradation of Bcl-xL protein and thus leading to the activation of mitochondrial-mediated apoptotic pathway

  14. Macrophage secretory products induce an inflammatory phenotype in hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Michelle Melino; Gethin P Thomas; Andrew D Clouston; Julie R Jonsson; Elizabeth E Powell; Victoria L Gadd; Gene V Walker; Richard Skoien; Helen D Barrie; Dinesh Jothimani; Leigh Horsfall; Alun Jones; Matthew J Sweet

    2012-01-01

    AIM:To investigate the influence of macrophages on hepatocyte phenotype and function.METHODS:Macrophages were differentiated from THP-1 monocytes via phorbol myristate acetate stimulation and the effects of monocyte or macrophageconditioned medium on HepG2 mRNA and protein expression determined.The in vivo relevance of these findings was confirmed using liver biopsies from 147patients with hepatitis C virus (HCV) infection.RESULTS:Conditioned media from macrophages,but not monocytes,induced a transient morphological change in hepatocytes associated with upregulation of vimentin (7.8 ± 2.5-fold,P =0.045) and transforming growth factor (TGF)-β1 (2.6 ± 0.2-fold,P < 0.001) and downregulation of epithelial cadherin (1.7 ± 0.02-fold,P =0.017) mRNA expression.Microarray analysis revealed significant upregulation of lipocalin-2 (17-fold,P < 0.001) and pathways associated with inflammation,and substantial downregulation of pathways related to hepatocyte function.In patients with chronic HCV,realtime polymerase chain reaction and immunohistochemistry confirmed an increase in lipocalin-2 mRNA (F0 1.0± 0.3,F1 2.2 ± 0.2,F2 3.0 ± 9.3,F3/4 4.0 ± 0.8,P =0.003) and protein expression (F1 1.0 ± 0.5,F2 1.3 ±0.4,F3/4 3.6 ± 0.4,P =0.014) with increasing liver injury.High performance liquid chromatography-tandem mass spectrometry analysis identified elevated levels of matrix metalloproteinase (MMP)-9 in macrophageconditioned medium,and a chemical inhibitor of MMP-9attenuated the change in morphology and mRNA expression of TGF-β1 (2.9 ± 0.2 vs 1.04 ± 0.1,P < 0.001)in macrophage-conditioned media treated HepG2 cells.In patients with chronic HCV infection,hepatic mRNA expression of CD163 (F0 1.0 ± 0.2,F1/2 2.8 ± 0.3,F3/4 5.3 ± 1.0,P =0.001) and MMP-9 (F0 1.0 ± 0.4,F1/2 2.8 ± 0.3,F3/4 4.1 ± 0.8,P =0.011) was significantly associated with increasing stage of fibrosis.CONCLUSION:Secreted macrophage products alter the phenotype and function of hepatocytes

  15. Influence of 48 hours of cold storage in University of Wisconsin organ preservation solution on metabolic capacity of rat hepatocytes

    NARCIS (Netherlands)

    Olinga, Peter; Slooff, M.JH; Meijer, D.K F; Groothuis, Geny; Merema, M.T.

    1997-01-01

    Background/Aims: Suspensions of isolated hepatocytes are a valuable tool to study liver functions, For optimal use of the isolated hepatocytes, methods are needed to preserve the hepatocytes while maintaining their viability, metabolic and transport functions, Until now little has been known about t

  16. Hepatocyte growth factor is crucial for development of the carapace in turtles.

    Science.gov (United States)

    Kawashima-Ohya, Yoshie; Narita, Yuichi; Nagashima, Hiroshi; Usuda, Ryo; Kuratani, Shigeru

    2011-01-01

    Turtles are characterized by their shell, composed of a dorsal carapace and a ventral plastron. The carapace first appears as the turtle-specific carapacial ridge (CR) on the lateral aspect of the embryonic flank. Accompanying the acquisition of the shell, unlike in other amniotes, hypaxial muscles in turtle embryos appear as thin threads of fibrous tissue. To understand carapacial evolution from the perspective of muscle development, we compared the development of the muscle plate, the anlage of hypaxial muscles, between the Chinese soft-shelled turtle, Pelodiscus sinensis, and chicken embryos. We found that the ventrolateral lip (VLL) of the thoracic dermomyotome of P. sinensis delaminates early and produces sparse muscle plate in the lateral body wall. Expression patterns of the regulatory genes for myotome differentiation, such as Myf5, myogenin, Pax3, and Pax7 have been conserved among amniotes, including turtles. However, in P. sinensis embryos, the gene hepatocyte growth factor (HGF), encoding a regulatory factor for delamination of the dermomyotomal VLL, was uniquely expressed in sclerotome and the lateral body wall at the interlimb level. Implantation of COS-7 cells expressing a HGF antagonist into the turtle embryo inhibited CR formation. We conclude that the de novo expression of HGF in the turtle mesoderm would have played an innovative role resulting in the acquisition of the turtle-specific body plan. PMID:21535464

  17. Hepatocyte growth factor, a determinant of airspace homeostasis in the murine lung.

    Directory of Open Access Journals (Sweden)

    Carla Calvi

    Full Text Available The alveolar compartment, the fundamental gas exchange unit in the lung, is critical for tissue oxygenation and viability. We explored hepatocyte growth factor (HGF, a pleiotrophic cytokine that promotes epithelial proliferation, morphogenesis, migration, and resistance to apoptosis, as a candidate mediator of alveolar formation and regeneration. Mice deficient in the expression of the HGF receptor Met in lung epithelial cells demonstrated impaired airspace formation marked by a reduction in alveolar epithelial cell abundance and survival, truncation of the pulmonary vascular bed, and enhanced oxidative stress. Administration of recombinant HGF to tight-skin mice, an established genetic emphysema model, attenuated airspace enlargement and reduced oxidative stress. Repair in the TSK/+ mouse was punctuated by enhanced akt and stat3 activation. HGF treatment of an alveolar epithelial cell line not only induced proliferation and scattering of the cells but also conferred protection against staurosporine-induced apoptosis, properties critical for alveolar septation. HGF promoted cell survival was attenuated by akt inhibition. Primary alveolar epithelial cells treated with HGF showed improved survival and enhanced antioxidant production. In conclusion, using both loss-of-function and gain-of-function maneuvers, we show that HGF signaling is necessary for alveolar homeostasis in the developing lung and that augmentation of HGF signaling can improve airspace morphology in murine emphysema. Our studies converge on prosurvival signaling and antioxidant protection as critical pathways in HGF-mediated airspace maintenance or repair. These findings support the exploration of HGF signaling enhancement for diseases of the airspace.

  18. Taurolithocholate impairs bile canalicular motility and canalicular bile secretion in isolated rat hepatocyte couplets

    Institute of Scientific and Technical Information of China (English)

    Norihito Watanabe; Tatehiro Kagawa; Sei-ichiro Kojima; Shinji Takashimizu; Naruhiko Nagata; Yasuhiro Nishizaki; Tetsuya Mine

    2006-01-01

    AIM: To investigate the effects of taurolithocholate (TLC)on the canalicular motility in isolated rat hepatocyte couplets (IRHC).METHODS: TLC was added to IRHC at concentrations of 10 and 50 μmol/L, respectively. In each group, five time-lapse movies containing 3 representative bile canaliculi were taken under phase-contrast microscopy for 12 h. The number of bile canalicular contractions and the intervals between consecutive canalicular contractions were calculated. Furthermore, the effects of TLC on IRHC were examined by transmission electron microscopy.RESULTS: The bile canalicular contractions were spontaneous and forceful in the controls. Active vesicular movement was observed in the pericanalicular region. Immediately after the addition of TLC, the bile canaliculi were deformed, and canalicular bile was incorporated into the vacuoles. The canaliculi were gradually dilated, and canalicular contractions were markedly inhibited by TLC. The vesicular movements became extremely slow in the pericanalicular region. The number of canalicular contractions significantly decreased in the TLC-treated groups, as compared with that in the controls. The time intervals were prolonged, as the TLC dosage increased,indicating that bile secretion into the canaliculi was impaired with TLC. Transmission electron microscopy revealed the lamellar transformation of the canalicular membranes in IRHC treated with TLC.CONCLUSION: TLC impairs both the bile canalicular contractions and the canalicular bile secretion, possibly by acting directly on the canalicular membranes in TLCinduced cholestasis.

  19. ADAR1 Prevents Liver Injury from Inflammation and Suppresses Interferon Production in Hepatocytes.

    Science.gov (United States)

    Wang, Guoliang; Wang, Hui; Singh, Sucha; Zhou, Pei; Yang, Shengyong; Wang, Yujuan; Zhu, Zhaowei; Zhang, Jinxiang; Chen, Alex; Billiar, Timothy; Monga, Satdarshan P; Wang, Qingde

    2015-12-01

    Adenosine deaminase acting on RNA 1 (ADAR1) is an essential protein for embryonic liver development. ADAR1 loss is embryonically lethal because of severe liver damage. Although ADAR1 is required in adult livers to prevent liver cell death, as demonstrated by liver-specific conditional knockout (Alb-ADAR1(KO)) mice, the mechanism remains elusive. We systematically analyzed Alb-ADAR1(KO) mice for liver damage. Differentiation genes and inflammatory pathways were examined in hepatic tissues from Alb-ADAR1(KO) and littermate controls. Inducible ADAR1 KO mice were used to validate regulatory effects of ADAR1 on inflammatory cytokines. We found that Alb-ADAR1(KO) mice showed dramatic growth retardation and high mortality because of severe structural and functional damage to the liver, which showed overwhelming inflammation, cell death, fibrosis, fatty change, and compensatory regeneration. Simultaneously, Alb-ADAR1(KO) showed altered expression of key differentiation genes and significantly higher levels of hepatic inflammatory cytokines, especially type I interferons, which was also verified by inducible ADAR1 knockdown in primary hepatocyte cultures. We conclude that ADAR1 is an essential molecule for maintaining adult liver homeostasis and, in turn, morphological and functional integrity. It inhibits the production of type I interferons and other inflammatory cytokines. Our findings may provide novel insight in the pathogenesis of liver diseases caused by excessive inflammatory responses, including autoimmune hepatitis.

  20. Recombinant kringle 5 from plasminogen antagonises hepatocyte growth factor-mediated signalling.

    Science.gov (United States)

    Ansell, Peter J; Zhang, Haiying; Davidson, Don J; Harlan, John E; Xue, John; Brodjian, Sevan; Lesniewski, Rick; McKeegan, Evelyn

    2010-03-01

    The blood protein plasminogen is proteolytically cleaved to produce angiostatin and kringle 5 (K5), both of which are known angiogenesis inhibitors. A common structural element between K5, angiostatin and other endogenous angiogenesis inhibitors is the presence of the kringle protein-interacting domain. Another kringle domain-containing protein, hepatocyte growth factor (HGF), promotes angiogenesis by binding to and stimulating the tyrosine kinase receptor Met. HGF binding to Met is dependent on the kringle domains of HGF. Because both K5 and HGF contain kringle motifs and because these proteins have opposite effects on angiogenesis, we hypothesised that K5 can antagonise HGF-mediated signalling in a Met-dependent manner. We determined that K5 binding to H1299 cells is competed by HGF suggesting that these two proteins bind to the same protein. Purified K5 immunoprecipitates with Met and this interaction is abolished by increasing doses of HGF. Using proliferation, phosphorylation of Met and Akt as markers of HGF activity, we determined that K5 inhibits HGF-mediated signalling. Taken together, these data support a model by which K5 binds to Met and functions as a competitive antagonist of HGF signalling and presents a novel mechanism of action of K5. PMID:20061137

  1. Dexamethasone blocks arachidonate biosynthesis in isolated hepatocytes and cultured hepatoma cells

    International Nuclear Information System (INIS)

    The effect of dexamethasone on the incorporation and conversion of [1-14C]eicosa-8,11,14-trienoic acid to arachidonic acid in isolated hepatocytes and in hepatoma tissue culture (HTC) cells was studied. In both kinds of cells, no changes in the exogenous acid incorporation were found when the hormone was added to the incubation media at 0.1 or 0.2 mM concentration, while the biosynthesis of arachidonic acid was significantly depressed. The effect on the biosynthesis was faster in isolated normal liver cells (60 min) than in tumoral cells (120 min) and reached an inhibition of ca. 50% after 3 hr of treatment. The addition of cycloheximide (10(-6) M) also caused a marked decrease in the biosynthesis of this polyunsaturated fatty acid, but when dexamethasone was added to the media simultaneously with cycloheximide, a synergistic action was not observed. The results obtained show that protein synthesis would be involved in the modulation of the biosynthesis of arachidonic acid by glucocorticoids. The changes in the delta 5 desaturation of labeled 20:3 omega 6 to arachidonic acid correlated with changes in the fatty acid composition in isolated cells

  2. Effects of aronia melanocarpa fruit juice on isolated rat hepatocytes

    Directory of Open Access Journals (Sweden)

    Magdalena Kondeva-Burdina

    2015-01-01

    Full Text Available Background: Aronia melanocarpa (Michx. Elliot fruits are very rich in polyphenols - procyanidins, flavonoids, and phenolic acids. Objective: On rat hepatocytes, isolated by two-stepped collagenase perfusion, we investigated the effect of A. melanocarpa fruit juice (AMFJ in two models of liver toxicity caused by (i metabolic bioactivation of carbon tetrachloride (CCl 4 , and (ii tert-butyl hydroperoxide (t-BuOOH-induced oxidative stress. Materials and Methods: Isolated rat hepatocytes are a suitable model for hepatotoxicity studies. We determined the main parameters of the functional and metabolic status of rat hepatocytes: Cell viability (measured by trypan blue exclusion and the levels of lactate dehydrogenase (LDH, reduced glutathione (GSH, and malondialdehyde (MDA. These parameters were used to investigate the protective effects of AMFJ in the two toxicity models. The effects of AMFJ were compared with those of silymarin. The cells were treated either with AMFJ or silymarin at increasing concentrations of 5 mg/ml, 10 mg/ml, 30 mg/ml, 50 mg/ml, and 100 mg/ml which were used for measuring of IC 50 . Results: In both toxicity models - CCl 4 and t-BuOOH, AMFJ showed statistically significant cytoprotective and antioxidant activities. AMFJ prevented the loss of cell viability and GSH depletion, decreased LDH leakage and MDA production. The effects of AMFJ at the concentrations of 5, 10, 30, and 50 mg/ml were similar to those of the same concentrations of silymarin, while the effect of the highest AMFJ concentration of 100 mg/ml was higher than that of the same silymarin concentration. The effects were concentration-dependent and more prominent in the t-BuOOH model, compared to those in the CCl 4 model. Conclusion: The cytoprotective and antioxidant effects of AMFJ established in this study might be due to its polyphenolic ingredients, which could influence the cytochrome P450-mediated metabolism of the experimental hepatotoxic substances (CCl 4

  3. Involvement of pregnane X receptor in the impaired glucose utilization induced by atorvastatin in hepatocytes.

    Science.gov (United States)

    Ling, Zhaoli; Shu, Nan; Xu, Ping; Wang, Fan; Zhong, Zeyu; Sun, Binbin; Li, Feng; Zhang, Mian; Zhao, Kaijing; Tang, Xiange; Wang, Zhongjian; Zhu, Liang; Liu, Li; Liu, Xiaodong

    2016-01-15

    Accumulating evidences demonstrated that statins impaired glucose utilization. This study was aimed to investigate whether PXR was involved in the atorvastatin-impaired glucose utilization. Rifampicin/PCN served as PXR activator control. Glucose utilization, glucose uptake, protein levels of GLUT2, GCK, PDK2, PEPCK1 and G6Pase in HepG2 cells were measured. PXR inhibitors, PXR overexpression and PXR siRNA were applied to verify the role of PXR in atorvastatin-impaired glucose utilization in cells. Hypercholesterolemia rats induced by high fat diet feeding, orally received atorvastatin (5 and 10 mg/kg), pravastatin (10 mg/kg) for 14 days, or intraperitoneally received PCN (35 mg/kg) for 4 days. Results showed that glucose utilization was markedly inhibited by atorvastatin, simvastatin, pitavastatin, lovastatin and rifampicin. Neither rosuvastatin nor pravastatin showed the similar effect. Atorvastatin and pravastatin were selected for the following study. Atorvastatin and rifampicin significantly inhibited glucose uptake and down-regulated GLUT2 and GCK expressions. Similarly, overexpressed PXR significantly down-regulated GLUT2 and GCK expressions and impaired glucose utilization. Ketoconazole and resveratrol attenuated the impaired glucose utilization by atorvastatin and rifampicin in both parental and overexpressed PXR cells. PXR knockdown significantly up-regulated GLUT2 and GCK proteins and abolished the decreased glucose consumption and uptake by atorvastatin and rifampicin. Animal experiments showed that atorvastatin and PCN significantly elicited postprandial hyperglycemia, leading to increase in glucose AUC. Expressions of GLUT2 and GCK in rat livers were markedly down-regulated by atorvastatin and PCN. In conclusion, atorvastatin impaired glucose utilization in hepatocytes via repressing GLUT2 and GCK expressions, which may be partly due to PXR activation. PMID:26616219

  4. Asialoglycoprotein Receptor-Mediated Gene Delivery to Hepatocytes Using Galactosylated Polymers.

    Science.gov (United States)

    Thapa, Bindu; Kumar, Piyush; Zeng, Hongbo; Narain, Ravin

    2015-09-14

    Highly efficient, specific, and nontoxic gene delivery vector is required for gene therapy to the liver. Hepatocytes exclusively express asialoglycoprotein receptor (ASGPR), which can recognize and bind to galactose or N-acetylgalactosamine. Galactosylated polymers are therefore explored for targeted gene delivery to the liver. A library of safe and stable galactose-based glycopolymers that can specifically deliver genes to hepatocytes were synthesized having different architectures, compositions, and molecular weights via the reversible addition-fragmentation chain transfer process. The physical and chemical properties of these polymers have a great impact on gene delivery efficacy into hepatocytes, as such block copolymers are found to form more stable complexes with plasmid and have high gene delivery efficiency into ASGPR expressing hepatocytes. Transfection efficiency and uptake of polyplexes with these polymers decreased significantly by preincubation of hepatocytes with free asialofetuin or by adding free asialofetuin together with polyplexes into hepatocytes. The results confirmed that polyplexes with these polymers were taken up specifically by hepatocytes via ASGPR-mediated endocytosis. The results from transfection efficiency and uptake of these polymers in cells without ASGPR, such as SK Hep1 and HeLa cells, further support this mechanism. Since in vitro cytotoxicity assays prove these glycopolymers to be nontoxic, they may be useful for delivery of clinically important genes specifically to the liver.

  5. Circadian rhythms of Per2::Luc in individual primary mouse hepatocytes and cultures.

    Directory of Open Access Journals (Sweden)

    Casey J Guenthner

    Full Text Available BACKGROUND: Hepatocytes, the parenchymal cells of the liver, express core clock genes, such as Period2 and Cryptochrome2, which are involved in the transcriptional/translational feedback loop of the circadian clock. Whether or not the liver is capable of sustaining rhythms independent of a central pacemaker is controversial. Whether and how circadian information may be shared among cells in the liver in order to sustain oscillations is currently unknown. RESULTS: In this study we isolated primary hepatocytes from transgenic Per2(Luc mice and used bioluminescence as a read-out of the state of the circadian clock. Hepatocytes cultured in a collagen gel sandwich configuration exhibited persistent circadian rhythms for several weeks. The amplitude of the rhythms damped, but medium changes consistently reset the phase and amplitude of the cultures. Cry2(-/- Per2(Luc cells oscillated robustly and expressed a longer period. Co-culturing with wildtype cells did not significantly shorten the period, indicating that coupling among hepatocytes is insufficient to synchronize cells with significantly differing periods. However, spatial patterns revealed by cellular imaging of wildtype cultures provided evidence of weak local coupling among the hepatocytes. CONCLUSIONS: Our results with primary hepatocyte cultures demonstrate that cultured hepatocytes are weakly coupled. While this coupling is not sufficient to sustain global synchrony, it does increase local synchrony, which may stabilize the circadian rhythms of peripheral oscillators, such as the liver, against noise in the entraining signals.

  6. In vitro induction of human embryonic stem cells into hepatocyte-like cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Human embryonic stem cells (hES cells) are pluripotent and provide a unique, unlimited resource for human hepatocytes, which can serve as a novel cell source for cell transplantation and bioartificial liver (BAL). Here, we have developed a procedure by which hES cells can differentiate into hepatocyte-like cells. After being cultured in suspension in bacteriological petri dishes for 7 d, hES cells developed into cystic embryoid bodies (EBs). The EBs were then cultured in conditional medium containing dexamethasone and insulin in collagen type I-coated tissue culture dishes for two weeks. The hES cell-derived hepatocyte-like cells (HLCs) displayed some morphologic characteristics of hepatocytes. Reverse-transcription polymerase chain reaction (RT-PCR) and immunofluorescence cell staining proved that the induced HLCs expressed several hepatocyte specific genes including AFP, ALB, CYP1B1 and cytokeratins CK18 and CK19. Furthermore, the induced cells executed a range of hepatocyte functions, such as ICG uptake/excretion, glycogen deposits, albumin production and ammonium metabolism. Taken together, our results show that HLCs exhibit similar morphologic, phenotypic, and functional characteristics to hepatocytes.

  7. Restoration of cell polarity and bile excretion function of hepatocytes in sandwich-culture

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xian-jie; WANG Ying; SUN Jia-bang; SONG Mao-min; QIAO Xin

    2007-01-01

    Objective:To investigate the nature of the restoration of cell polarity and bile excretion function in Sandwich-cultured hepatocytes.Methods:Freshly isolated hepatocytes from male Sprague-Dawley rats were cultured in a double layer collagen gel Sandwich configuration.Morphological changes were observed under a inverted microscope.The domain specific membrane associated protein DPP Ⅳ was tested by immunofluorescenee,and the bile excretion function was determined by using fluorescein diacetate.Hepatocytes cultured on a single layer of collagen gel were taken as control.Results:Adult rat hepatocytes cultured in a double layer collagen gel sandwich configuration regained its morphological and functional polarity and maintained polygonal morphology for at least 4 weeks.Immunofluorescence studies USing antibodies against DPP Ⅳ showed polarity restoration as early as 48 h.After cultured in the double layer collagen gel Sandwich configuration for 96 h the hepatocytes began to excrete bile;while hepatocytes cultured on a single layer collagen gel had no bile excretion.Conclusion:Hepatocytes cultured in a double layer collagen gel Sandwich configuration are able to regain their morphological and functional polarity givan certain conditions.Hepaotcyte culture is a useful tool for the study of polarity restoration.

  8. Nrf2 is involved in maintaining hepatocyte identity during liver regeneration.

    Directory of Open Access Journals (Sweden)

    Yuhong Zou

    Full Text Available Nrf2, a central regulator of the cellular defense against oxidative stress and inflammation, participates in modulating hepatocyte proliferation during liver regeneration. It is not clear, however, whether Nrf2 regulates hepatocyte growth, an important cellular mechanism to regain the lost liver mass after partial hepatectomy (PH. To determine this, various analyses were performed in wild-type and Nrf2-null mice following PH. We found that, at 60 h post-PH, the vast majority of hepatocytes lacking Nrf2 reduced their sizes, activated hepatic progenitor markers (CD133, TWEAK receptor, and trefoil factor family 3, depleted HNF4α protein, and downregulated the expression of a group of genes critical for their functions. Thus, the identity of hepatocytes deficient in Nrf2 was transiently but massively impaired in response to liver mass loss. This event was associated with the coupling of protein depletion of hepatic HNF4α, a master regulator of hepatocyte differentiation, and concomitant inactivation of hepatic Akt1 and p70S6K, critical hepatocyte growth signaling molecules. We conclude that Nrf2 participates in maintaining newly regenerated hepatocytes in a fully differentiated state by ensuring proper regulation of HNF4α, Akt1, and p70S6K during liver regeneration.

  9. Auxiliary liver organ formation by implantation of spleen-encapsulated hepatocytes.

    Science.gov (United States)

    Nishio, Reiji; Nakayama, Miyuki; Ikekita, Masahiko; Watanabe, Yoshifumi

    2006-09-01

    Hepatocyte transplantation is an attractive alternative to orthotopic liver transplantation. However, its application has been limited because of its short-term success only. Here we report a new approach to hepatocyte transplantation resulting in the generation of an auxiliary liver in vivo. Isolated primary hepatocytes were encapsulated in isolated spleens and then transplanted by attaching the spleens to the livers of recipient animals (mice or rats) using biodegradable adhesive. A vascular network was rapidly established, and protein molecules circulated freely between the transplanted spleen and the liver, to which they adhered. In contrast, the spleen, which did not adhere to the liver or adhered elsewhere (adipose tissue or peritoneum), did not become vascularized but shrank and died. Encapsulation of hepatocytes in an isolated spleen enhanced their survival significantly, and co-encapsulation of Engelbreth- Holm-Swarm gel together with the hepatocytes further enhanced it. The encapsulated hepatocytes expressed liver-specific differentiation genes for more than 3 weeks. Plasma albumin concentrations in Nagase analbuminemic rats began to increase 3 days after transplantation. The transplanted hepatic cells migrated into the liver parenchyma, whereas the spleen was absorbed. Thus, we have developed a novel, simple approach for the rapid and efficient formation of functional auxiliary liver using a modified hepatocyte transplantation method.

  10. 3D hepatic cultures simultaneously maintain primary hepatocyte and liver sinusoidal endothelial cell phenotypes.

    Directory of Open Access Journals (Sweden)

    Yeonhee Kim

    Full Text Available Developing in vitro engineered hepatic tissues that exhibit stable phenotype is a major challenge in the field of hepatic tissue engineering. However, the rapid dedifferentiation of hepatic parenchymal (hepatocytes and non-parenchymal (liver sinusoidal endothelial, LSEC cell types when removed from their natural environment in vivo remains a major obstacle. The primary goal of this study was to demonstrate that hepatic cells cultured in layered architectures could preserve or potentially enhance liver-specific behavior of both cell types. Primary rat hepatocytes and rat LSECs (rLSECs were cultured in a layered three-dimensional (3D configuration. The cell layers were separated by a chitosan-hyaluronic acid polyelectrolyte multilayer (PEM, which served to mimic the Space of Disse. Hepatocytes and rLSECs exhibited several key phenotypic characteristics over a twelve day culture period. Immunostaining for the sinusoidal endothelial 1 antibody (SE-1 demonstrated that rLSECs cultured in the 3D hepatic model maintained this unique feature over twelve days. In contrast, rLSECs cultured in monolayers lost their phenotype within three days. The unique stratified structure of the 3D culture resulted in enhanced heterotypic cell-cell interactions, which led to improvements in hepatocyte functions. Albumin production increased three to six fold in the rLSEC-PEM-Hepatocyte cultures. Only rLSEC-PEM-Hepatocyte cultures exhibited increasing CYP1A1/2 and CYP3A activity. Well-defined bile canaliculi were observed only in the rLSEC-PEM-Hepatocyte cultures. Together, these data suggest that rLSEC-PEM-Hepatocyte cultures are highly suitable models to monitor the transformation of toxins in the liver and their transport out of this organ. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte model, which recapitulates key features of hepatic sinusoids, is a potentially powerful medium for obtaining comprehensive knowledge on liver metabolism

  11. Proliferation of L02 human hepatocytes in tolerized geneticall yimmunocompetent rats

    Institute of Scientific and Technical Information of China (English)

    Hu Lin; Qing Mao; Yu-Ming Wang; Li Jiang

    2008-01-01

    AIM: To investigate whether human hepatocytes could proliferate after transplantation to normal immunocompetent rats treated with 2-acetaminofluorene or Retrorsine and partial hepatectomy.METHODS: L02 hepatocyte-tolerant Sprague-Dawley rats were injected with Retrorsine, 2-acetaminofluorene or normal saline. L02 hepatocytes were then transplanted via the spleen. Human albumin and its mRNA, specific proliferating cell nuclear antigen (PCNA), L02 hepatocyte dynamic distribution, number density and area density of PCNA-positive cells in the liver were determined.RESULTS: All the examined indicators were not significantly different between the rats treated with 2-acetaminofluorene and normal saline, which was not the case with rats treated with Retrorsine. A dynamic distribution of L02 hepatocytes in the rat liver was detected from wk 1 to mo 6 after transplantation in the Retrorsine group and from wk 1 to 10 in the 2-acetaminofluorene group. Human albumin and its mRNA were detected from wk 2 to mo 6 in the Retrorsine group and from wk 1 to 8 in the 2-acetaminofiuorene group.Specific human PCNA was detected in the rat liver from wk 2 to mo 6 in the Retrorsine group and from wk 2 to 6 in the 2-acetaminofluorene group. Human albumin and its mRNA contents as well as the number of PCNA positive cells reached a peak at wk 4.CONCLUSION: L02 human hepatocytes could not proliferate significiantly after transplantation to the normal,immunocompetent rats treated with 2-acetaminofluorene.L02 human hepatocytes can survive for 10 wk after transplantation and express human albumin for 8 wk. L02human hepatocytes can proliferate and express human albumin for 6 mo after transplantation to the rats treated with Retrorsine. The chimeric L02 human hepatocytes,which then underwent transplantation into tolerant rats,were normal in morphogenesis, biochemistry and function.

  12. Phosphorylation dynamics of radixin in hypoxia-induced hepatocyte injury.

    Science.gov (United States)

    Suda, Jo; Rockey, Don C; Karvar, Serhan

    2015-02-15

    The most prominent ezrin-radixin-moesin protein in hepatocytes is radixin, which is localized primarily at the canalicular microvilli and appears to be important in regulation of cell polarity and in localizing the multidrug resistance-associated protein 2 (Mrp-2) function. Our aim was to investigate how hypoxia affects radixin distribution and Mrp-2 function. We created wild-type and mutant constructs (in adenoviral vectors), which were expressed in WIF-B cells. The cellular distribution of Mrp-2 and radixin was visualized by fluorescence microscopy, and a 5-chloromethylfluorescein diacetate (CMFDA) assay was used to measure Mrp-2 function. Under usual conditions, cells infected with wild-type radixin, nonphosphorylatable radixin-T564A, and radixin-T564D (active phospho-mimicking mutant) were found to be heavily expressed in canalicular membrane compartment vacuoles, typically colocalizing with Mrp-2. In contrast, after hypoxia for 24 h, both endogenous and overexpressed wild-type radixin and the radixin-T564A mutant were found to be translocated to the cytoplasmic space. However, distribution of the radixin-T564D mutant, which mimics constant phosphorylation, was remarkably different, being associated with canalicular membranes even in hypoxic conditions. This dominant-active construct also prevented dissociation of radixin from the plasma membrane. Hypoxia also led to Mrp-2 mislocalization and caused Mrp-2 to be dissociated from radixin; the radixin phospho-mimicking mutant (T564D) abrogated this effect of hypoxia. Finally, hypoxia diminished the secretory response (measured using the CMFDA assay) in WIF-B cells, and the dominant-active construct (radixin-T567D) rescued this phenotype. Taken collectively, these findings suggest that radixin regulates Mrp-2 localization and function in hepatocytes and is important in hypoxic liver injury. PMID:25501552

  13. Uptake of palmitate by hepatocyte suspensions: facilitation by albumin?

    Science.gov (United States)

    Pond, S M; Davis, C K; Bogoyevitch, M A; Gordon, R A; Weisiger, R A; Bass, L

    1992-05-01

    Albumin-dependent uptake of unbound [3H]palmitic acid by hepatocytes isolated from female rat livers was studied and the experimental results compared with the predictions of a noncompartmental diffusion-reaction theory for the cellular uptake of protein-bound ligands. The outright theoretical predictions involve values for the parameters of the system, some newly measured (hepatocyte radii and the rate constant for the dissociation of palmitate-albumin complex) and some taken from the literature (diffusion coefficients and the equilibrium association constant for the palmitate-albumin complex). The measured unbound clearance of [3H]palmitic acid, defined as the initial uptake velocity divided by the unbound [3H]palmitic acid concentration in the medium, was enhanced 6.6-fold as the concentration of human serum albumin was increased from approximately 5 to 480 microM. This enhancement factor was predicted by the theory, according to which the enhancement reflects codiffusion of bound ligand across the unstirred layer adjacent to the cell membrane and, therefore, an increased delivery of unbound ligand to the cell surface. In contrast, the absolute magnitude of the unbound clearance was consistent with the theory only for the lowest published value for the equilibrium association constant, 15 microM-1. For higher published values (62 and 94 microM-1), the magnitude of the unbound clearance observed experimentally was severalfold higher than that predicted by the theory. If in fact the association constant exceeds 30 microM-1, the data would imply that an albumin-dependent facilitation mechanism exists which enhances the availability of palmitate to the cell over and above the enhancement predicted by the diffusion-reaction theory. PMID:1590397

  14. Electrophysiological effects of anthopleurin-Q on rat hepatocytes

    Institute of Scientific and Technical Information of China (English)

    Hong-Yi Zhou; Fang Wang; Ke-Qiang Zhang; Lan Cheng; Ji Zhou; Li-Ying Fu; Wei-Xing Yao

    2004-01-01

    AIM: To study the effects of AP-Q on CCl4-induced acute liver injury, delayed outward potassium current (Ik), inward rectifier potassium current (Ik1) and calcium release-activated calcium current (ICRAC) in isolated rat hepatocytes.METHODS: A single dose of CCl4 (10 μ g/mL, ip) was injected to induce acute liver injury in rats. Serum aminotransferase activities were determined. Whole cell patch-clamp techniques were used to investigate the effects of AP-Q on delayed outward potassium current (Ik), inward rectifier potassium current (Ik1) and calcium release-activated calcium current (ICRAC).RESULTS: AP-Q (3.5 and 7 iμg/kg) pretreatment significantly reduced ALT and AST activities. AP-Q 0.1-100 nlMl produced a concentration-dependent increase of Ik with ECs0 value of 5.55±1.8 nM (n=6). AP-Q 30 nM shifted the Ⅰ-V curve of Ik leftward and upward. CCl4 4 mM decreased Ik current 28.6±6.5% at 140 mV. After exposure to CCl4 for 5 min, AP-Q 30 nM attenuated the decrease of Ik induced by CCl4 close to normal amplitude. AP-Q 0.01-100 nM had no significant effect on either inward or outward components of Ik1 at anymembrane potential examined. AP-Q 0.1-100 nM had nosignificant influence on the peak amplitude of IcRAc, either,and did not affect the shape of its current voltage curve.CONCLUSION: AP-Q has a protective effect on CCl4-induced liver injury, probably through selectively increased Ik in hepatocytes.

  15. Development of scaffold architectures and heterotypic cell systems for hepatocyte transplantation

    Science.gov (United States)

    Alzebdeh, Dalia Abdelrahim

    In vitro assembly of functional liver tissue is needed to enable the transplantation of tissue-engineered livers. In addition, there is an increasing demand for in vitro models that replicate complex events occurring in the liver. However, tissue engineering of sizable implantable liver systems is currently limited by the difficulty of assembling three dimensional hepatocyte cultures of a useful size, while maintaining full cell viability, an issue which is closely related to the high metabolic rate of hepatocytes. In this study, we first compared two designs of highly porous chitosan-heparin scaffolds seeded with hepatocytes in dynamic perfusion bioreactor systems. The aim was to promote cell seeding efficiency by effectively entrapping 100 million hepatocytes at high density. We found that scaffolds with radially tapering pore architecture had highly efficient cell entrapment that maximized donor hepatocyte utilization, compared to alternate pore structures. Hepatocytes showed higher seeding efficiency and metabolic function when seeded as single cell suspensions as opposed to pre-formed, 100microm aggregates. Seeding efficiency was found to increase with flow rate, with single cell and aggregate suspension exhibiting different optimal flow rates. However, metabolic performance results indicated significant shear damage to cells at high efficiency flow rates. To better maintain hepatocyte basement membrane and cell polarity, spheroid co-cultures with mesenchymal stem cells (MSC) were investigated. Hepatocytes and MSCs were seeded in three different architectures in an effort to optimize the spatial arrangement of the two cell types. MSC co-culture greatly enhanced hepatocyte metabolic function in agitated cultures. Interestingly, the effects of diffusion limitations in spheroid culture, coupled with shear damage and subsequent removal of outer hepatocyte layers produced a defined oscillation of urea production rates in certain co-culture arrangements. A

  16. Role of Nuclear Constitutive Androstane Receptor in Regulation of Hepatocyte Proliferation and Hepatocarcinogenesis.

    Science.gov (United States)

    Kazantseva, Y A; Pustylnyak, Y A; Pustylnyak, V O

    2016-04-01

    Activation of the constitutive androstane receptor (CAR) in hepatocytes occurs as a body adaptation in response to a number of external influences, and its functional activity is primarily related to induction of enzymes detoxifying xenobiotics. However, special attention was recently given to CAR due to the fact that its key role becomes unveiled in various physiological and pathophysiological processes occurring in the liver: gluconeogenesis, metabolism of fatty acids and bilirubin, hormonal regulation, proliferation of hepatocytes, and hepatocarcinogenesis. Here we review the main pathways and mechanisms that elevate hepatocyte proliferative activity related to CAR and whose disturbance may be a pivotal factor in hepatocarcinogenesis.

  17. Inhibition of Crotalidae venom hemorrhagic activities by Didelphis marsupialis liver spheroids culture supernatants.

    Science.gov (United States)

    Salgueiro, L M; Rodríguez-Acosta, A; Rivas-Vetencourt, P; Zerpa, M; Carillo, G; Aguilar, I; Girón, M E; Acevedo, C E; Gendzekhadze, K

    2001-05-01

    The main aim of this work was the development of a primary hepatocyte culture from Didelphis marsupialis, to determine the possible use of culture medium supernatants as a source of inhibitors of the Bothrops lanceolatus venom hemorrhagic activity. The cellular culture was carried out from isolated hepatocytes by the double perfusion technique, and digestion of the liver with collagenase and culturing the hepatocytes in a liquid media under continuous agitation at 37 degrees C in 5% CO2. The hemorrhagic activity inhibition assays were performed inoculating intradermically, a mixture of Bothrops lanceolatus venom plus a pool of liver spheroids culture supernatants, in mice. These liver Didelphis marsupialis spheroid cultures were adequate to obtain large supernatant volumes with inhibitors of hemorrhagic activity. PMID:11405280

  18. Role of the transsulfuration pathway and of gamma-cystathionase activity in the formation of cysteine and sulfate from methionine in rat hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Rao, A.M.; Drake, M.R.; Stipanuk, M.H. (Cornell Univ., Ithaca, NY (USA))

    1990-08-01

    To assess the extent to which low hepatic gamma-cystathionase levels affect methionine flux to cysteine in hepatocytes, the effect of inhibition of gamma-cystathionase activity with propargylglycine on the metabolism of L-({sup 35}S)methionine was determined in studies with freshly isolated rat hepatocytes. gamma-Cystathionase activity was inhibited 25%, 42%, 63% and 76% (maximal inhibition) by treatment with 2.5 mumol/L, 0.01 mmol/L, 0.02 mmol/L and 2 mmol/l propargylglycine, respectively. Inhibition of gamma-cystathionase activity with up to 0.02 mmol/L propargylglycine had no statistically significant effect on ({sup 35}S)glutathione, ({sup 35}S)sulfate or ({sup 35}S)cysteine formation from ({sup 35}S)methionine. However, treatment of cells with 2 mmol/L propargylglycine markedly inhibited the metabolism of ({sup 35}S)methionine to ({sup 35}S)glutathione by 93%, to ({sup 35}S)sulfate by 88% and to ({sup 35}S)cysteine by 89%; ({sup 35}S)cystathionine accumulation in these incubation systems was 60 times control. Hepatic gamma-cystathionase activity in premature infants has been reported to be about 23% of mature levels; this level of gamma-cystathionase activity may limit cysteine synthesis by the methionine transsulfuration pathway. No evidence for cysteine synthesis from serine and sulfide, which can be catalyzed by cystathionine beta-synthase, or for methionine metabolism by an S-adenosylmethionine-independent pathway was obtained.

  19. Met inactivation by S-allylcysteine suppresses the migration and invasion of nasopharyngeal cancer cells induced by hepatocyte growth factor

    Energy Technology Data Exchange (ETDEWEB)

    Cho, O Yeon; Hwang, Hye Sook; Lee, Bok Soon; Oh, Young Taek; Kim, Chul Ho; Chun, Mi Son [Ajou University School of Medicine, Suwon (Korea, Republic of)

    2015-12-15

    Past studies have reported that S-allylcysteine (SAC) inhibits the migration and invasion of cancer cells through the restoration of E-cadherin, the reduction of matrix metalloproteinase (MMP) and Slug protein expression, and inhibition of the production of reactive oxygen species (ROS). Furthermore, evidence is emerging that shows that ROS induced by radiation could increase Met activation. Following on these reports of SAC and Met, we investigated whether SAC could suppress Met activation. Wound healing, invasion, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT), soft agar colony forming, western blotting, and gelatin zymography assays were performed in the human nasopharyngeal cancer cell lines HNE1 and HONE1 treated with SAC (0, 10, 20, or 40 mM) and hepatocyte growth factor (HGF). This study showed that SAC could suppress the migration and invasion of HNE1 and HONE1 cell lines by inhibiting p-Met. An increase of migration and invasion induced by HGF and its decrease in a dose dependent manner by SAC in wound healing and invasion assays was observed. The reduction of p-Met by SAC was positively correlated with p-focal adhesion kinase (p-FAK) and p-extracellular related kinase (p-ERK in both cell lines). SAC reduced Slug, MMP2, and MMP9 involved in migration and invasion with the inhibition of Met-FAK signaling. These results suggest that SAC inhibited not only Met activation but also the downstream FAK, Slug, and MMP expression. Finally, SAC may be a potent anticancer compound for nasopharyngeal cancer treated with radiotherapy.

  20. 小眼畸形转录因子和c-Met及组织蛋白酶-K在黑色素病变中的表达及意义%Expressions and significances of microphthalmia transcription factor, c-Met and cathepsin-K in malignant melanoma and nevi

    Institute of Scientific and Technical Information of China (English)

    李良; 张正祥; 吴波; 缪国良; 饶秋

    2014-01-01

    目的 探讨小眼畸形转录因子(microphthalmia transcription factor,MITF)、e-Met和组织蛋白酶-K (cathepsin-K)在恶性黑色素瘤、痣和非典型性痣中的表达及意义.方法 32例恶性黑色素瘤(恶黑组)、13例普通痣患者(普通痣组)和6例非典型性痣患者(非典型痣组)的组织标本,采用免疫组织化学法检测3组组织中MITF、c-Met和cathepsin-K的表达情况.结果 MITF、c-Met表达于黑色素细胞的细胞核和细胞质,cathepsin-K表达于细胞质和细胞膜;MITF、c-Met和cathepsin-K阳性表达率恶黑组分别为93.8%、90.6%和93.8%,普通痣组分别为92.3%、92.3%和100.0%,非典型痣组分别为100.0%、100.0%和100.0%,3组间比较差异均无统计学意义(P>0.05);恶黑组MITF((卅))、c-Met((卅))及cathepsin-K((卅))阳性率高于普通痣组和非典型痣组(P<0.05);恶黑组转移者MITF、c-Met及cathepsin-K阳性率明显高于非转移者(P<0.05),结节性恶性黑色素瘤者MITF、c-Met及cathepsin-K阳性率高于表浅扩散性恶性黑色素瘤者(P<0.05);恶黑组转移者MITF、c-Met及cathepsin-K阳性率均为100%,高于HMB-45阳性率(75.0%)(P<0.05).结论 MITF、c-Met及cathepsin-K对恶性黑色素瘤的诊断及评估其进展和生物学行为有重要价值.

  1. Transfection of Primary Hepatocytes with Liver-Enriched Transcription Factors Using Adenoviral Vectors.

    Science.gov (United States)

    Benet, Marta; Jover, Ramiro; Bort, Roque

    2015-01-01

    Primary cultured hepatocytes are probably the best model to study endogenous metabolic pathways, toxicity, or drug metabolism. Many of these studies require expression of ectopic genes. It would be desirable to use a method of transfection that allows dose-response studies, high efficiency of transfection, and the possibility to express several genes at the same time. Adenoviral vectors fulfill these requirements, becoming a valuable tool for primary hepatocyte transfection. Moreover, they are easy to generate and do not require a high level of biocontainment. In the present chapter, we describe the generation, cloning, amplification, and purification of an adenoviral vector capable of infecting primary cultured hepatocytes. This recombinant adenovirus induces robust expression of the protein of interest in hepatocytes within a wide range of doses. PMID:26272145

  2. Proliferative response of putative preneoplastic hepatocytes to triiodothyronine, aminoacids, glucagon and heparine.

    Science.gov (United States)

    Estadella, M D; Pujol, M J; Domingo, J

    1988-03-01

    Proliferative advantage of putative preneoplastic hepatocytes measured as the ratio of tritiated thymidine incorporation into cells of hyperplastic nodules with respect to the incorporation in hepatocytes of extranodular liver has been found to be about 2 in rats treated according to 4 carcinogenesis protocols consisting in one or two cycles of diethylnitrosamine and phenobarbital administration. The proliferative response of hepatocytes in hyperplastic nodules to the intravenous infusion of triiodothyronine, amino-acids, glucagon, and heparine (TAGH) has been found higher or similar--except in one case--than the response of surrounding liver but the proliferative advantage of preneoplastic hepatocytes is lower after TAGH stimulation than in basal conditions in all cases. PMID:3383246

  3. Recombinant Laminins Drive the Differentiation and Self-Organization of hESC-Derived Hepatocytes.

    Science.gov (United States)

    Cameron, Kate; Tan, Rosanne; Schmidt-Heck, Wolfgang; Campos, Gisela; Lyall, Marcus J; Wang, Yu; Lucendo-Villarin, Baltasar; Szkolnicka, Dagmara; Bates, Nicola; Kimber, Susan J; Hengstler, Jan G; Godoy, Patricio; Forbes, Stuart J; Hay, David C

    2015-12-01

    Stem cell-derived somatic cells represent an unlimited resource for basic and translational science. Although promising, there are significant hurdles that must be overcome. Our focus is on the generation of the major cell type of the human liver, the hepatocyte. Current protocols produce variable populations of hepatocytes that are the product of using undefined components in the differentiation process. This serves as a significant barrier to scale-up and application. To tackle this issue, we designed a defined differentiation process using recombinant laminin substrates to provide instruction. We demonstrate efficient hepatocyte specification, cell organization, and significant improvements in cell function and phenotype. This is driven in part by the suppression of unfavorable gene regulatory networks that control cell proliferation and migration, pluripotent stem cell self-renewal, and fibroblast and colon specification. We believe that this represents a significant advance, moving stem cell-based hepatocytes closer toward biomedical application.

  4. Optimizing the use of rainbow trout hepatocytes for bioaccumulation assessments with fish

    Science.gov (United States)

    Measured rates of biotransformation by cryopreserved trout hepatocytes can be extrapolated to the whole animal as a means of predicting metabolism impacts on chemical bioaccumulation. Future use of these methods within a regulatory context requires, however, that they be standar...

  5. Asialoglycoprotein receptor and liposome synergistically mediate the gene transfer into primary rat hepatocytes

    Institute of Scientific and Technical Information of China (English)

    李崇辉; 温守明; 翟海峰; 孙曼霁

    1999-01-01

    Gene transfer into primary rat hepatocytes was performed by employing cationic liposome as DNA carrier and the specific ligand of hepatic asialoglycoprotein receptor (ASGPR), asialofetuin, as liver-targeting ligand. The resuits showed that asialofetuin, when added to the gene transfer complexes, could significantly increase the hepatocyte transfeetion efficiency, and alleviate the cellular toxicity of Lipofectin. Several synthetic ligands of ASGPR (galactosyl albumin) could also increase the transfection efficiency of hepatocyte like asialofetuin. It was proved that ASGPR and cationic liposome could synergistically mediate the gene transfer into primary rat hepatoeytes. This novel gene delivery system provided a safer, more simple and efficient gene transfer method for primary hepatocytes, and showed prospecting application in hepatic gene therapy.

  6. A fat option for the pig: Hepatocytic differentiated mesenchymal stem cells for translational research

    Energy Technology Data Exchange (ETDEWEB)

    Brückner, Sandra, E-mail: sandra.brueckner@medizin.uni-leipzig.de [University Hospital Leipzig, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, Liebigstraße 21, Leipzig D-04103 (Germany); Tautenhahn, Hans-Michael, E-mail: hans-michael.tautenhahn@medizin.uni-leipzig.de [University Hospital Leipzig, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, Liebigstraße 21, Leipzig D-04103 (Germany); TRM, Translational Centre for Regenerative Medicine, Philipp-Rosenthal-Str. 55, Leipzig D-04103 (Germany); Winkler, Sandra, E-mail: sandra.pelz@medizin.uni-leipzig.de [University Hospital Leipzig, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, Liebigstraße 21, Leipzig D-04103 (Germany); Stock, Peggy, E-mail: peggy.stock@medizin.uni-leipzig.de [University Hospital Leipzig, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, Liebigstraße 21, Leipzig D-04103 (Germany); Dollinger, Matthias, E-mail: matthias.dollinger@uniklinik-ulm.de [University Hospital Ulm, First Department of Medicine, Albert-Einstein-Allee 23, Ulm D-89081 (Germany); Christ, Bruno, E-mail: bruno.christ@medizin.uni-leipzig.de [University Hospital Leipzig, Department of Visceral, Transplantation, Thoracic and Vascular Surgery, Liebigstraße 21, Leipzig D-04103 (Germany); TRM, Translational Centre for Regenerative Medicine, Philipp-Rosenthal-Str. 55, Leipzig D-04103 (Germany)

    2014-02-15

    Study background: Extended liver resection is the only curative treatment option of liver cancer. Yet, the residual liver may not accomplish the high metabolic and regenerative capacity needed, which frequently leads to acute liver failure. Because of their anti-inflammatory and -apoptotic as well as pro-proliferative features, mesenchymal stem cells differentiated into hepatocyte-like cells might provide functional and regenerative compensation. Clinical translation of basic research requires pre-clinical approval in large animals. Therefore, we characterized porcine mesenchymal stem cells (MSC) from adipose tissue and bone marrow and their hepatocyte differentiation potential for future assessment of functional liver support after surgical intervention in the pig model. Methods: Mesenchymal surface antigens and multi-lineage differentiation potential of porcine MSC isolated by collagenase digestion either from bone marrow or adipose tissue (subcutaneous/visceral) were assessed by flow cytometry. Morphology and functional properties (urea-, glycogen synthesis and cytochrome P450 activity) were determined during culture under differentiation conditions and compared with primary porcine hepatocytes. Results: MSC from porcine adipose tissue and from bone marrow express the typical mesenchymal markers CD44, CD29, CD90 and CD105 but not haematopoietic markers. MSC from both sources displayed differentiation into the osteogenic as well as adipogenic lineage. After hepatocyte differentiation, expression of CD105 decreased significantly and cells adopted the typical polygonal morphology of hepatocytes. Glycogen storage was comparable in adipose tissue- and bone marrow-derived cells. Urea synthesis was about 35% lower in visceral than in subcutaneous adipose tissue-derived MSC. Cytochrome P450 activity increased significantly during differentiation and was twice as high in hepatocyte-like cells generated from bone marrow as from adipose tissue. Conclusion: The hepatocyte

  7. A fat option for the pig: Hepatocytic differentiated mesenchymal stem cells for translational research

    International Nuclear Information System (INIS)

    Study background: Extended liver resection is the only curative treatment option of liver cancer. Yet, the residual liver may not accomplish the high metabolic and regenerative capacity needed, which frequently leads to acute liver failure. Because of their anti-inflammatory and -apoptotic as well as pro-proliferative features, mesenchymal stem cells differentiated into hepatocyte-like cells might provide functional and regenerative compensation. Clinical translation of basic research requires pre-clinical approval in large animals. Therefore, we characterized porcine mesenchymal stem cells (MSC) from adipose tissue and bone marrow and their hepatocyte differentiation potential for future assessment of functional liver support after surgical intervention in the pig model. Methods: Mesenchymal surface antigens and multi-lineage differentiation potential of porcine MSC isolated by collagenase digestion either from bone marrow or adipose tissue (subcutaneous/visceral) were assessed by flow cytometry. Morphology and functional properties (urea-, glycogen synthesis and cytochrome P450 activity) were determined during culture under differentiation conditions and compared with primary porcine hepatocytes. Results: MSC from porcine adipose tissue and from bone marrow express the typical mesenchymal markers CD44, CD29, CD90 and CD105 but not haematopoietic markers. MSC from both sources displayed differentiation into the osteogenic as well as adipogenic lineage. After hepatocyte differentiation, expression of CD105 decreased significantly and cells adopted the typical polygonal morphology of hepatocytes. Glycogen storage was comparable in adipose tissue- and bone marrow-derived cells. Urea synthesis was about 35% lower in visceral than in subcutaneous adipose tissue-derived MSC. Cytochrome P450 activity increased significantly during differentiation and was twice as high in hepatocyte-like cells generated from bone marrow as from adipose tissue. Conclusion: The hepatocyte

  8. Bile acid-induced necrosis in primary human hepatocytes and in patients with obstructive cholestasis

    Energy Technology Data Exchange (ETDEWEB)

    Woolbright, Benjamin L.; Dorko, Kenneth [Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Antoine, Daniel J.; Clarke, Joanna I. [MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, Liverpool (United Kingdom); Gholami, Parviz [Department of Internal Medicine, University of Kansas Medical Center, Kansas City, KS (United States); Li, Feng [Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Kumer, Sean C.; Schmitt, Timothy M.; Forster, Jameson [Department of Surgery, University of Kansas Medical Center, Kansas City, KS (United States); Fan, Fang [Department of Pathology, University of Kansas Medical Center, Kansas City, KS (United States); Jenkins, Rosalind E.; Park, B. Kevin [MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, Liverpool (United Kingdom); Hagenbuch, Bruno [Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Olyaee, Mojtaba [Department of Internal Medicine, University of Kansas Medical Center, Kansas City, KS (United States); Jaeschke, Hartmut, E-mail: hjaeschke@kumc.edu [Department of Pharmacology, Toxicology & Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States)

    2015-03-15

    Accumulation of bile acids is a major mediator of cholestatic liver injury. Recent studies indicate bile acid composition between humans and rodents is dramatically different, as humans have a higher percent of glycine conjugated bile acids and increased chenodeoxycholate content, which increases the hydrophobicity index of bile acids. This increase may lead to direct toxicity that kills hepatocytes, and promotes inflammation. To address this issue, this study assessed how pathophysiological concentrations of bile acids measured in cholestatic patients affected primary human hepatocytes. Individual bile acid levels were determined in serum and bile by UPLC/QTOFMS in patients with extrahepatic cholestasis with, or without, concurrent increases in serum transaminases. Bile acid levels increased in serum of patients with liver injury, while biliary levels decreased, implicating infarction of the biliary tracts. To assess bile acid-induced toxicity in man, primary human hepatocytes were treated with relevant concentrations, derived from patient data, of the model bile acid glycochenodeoxycholic acid (GCDC). Treatment with GCDC resulted in necrosis with no increase in apoptotic parameters. This was recapitulated by treatment with biliary bile acid concentrations, but not serum concentrations. Marked elevations in serum full-length cytokeratin-18, high mobility group box 1 protein (HMGB1), and acetylated HMGB1 confirmed inflammatory necrosis in injured patients; only modest elevations in caspase-cleaved cytokeratin-18 were observed. These data suggest human hepatocytes are more resistant to human-relevant bile acids than rodent hepatocytes, and die through necrosis when exposed to bile acids. These mechanisms of cholestasis in humans are fundamentally different to mechanisms observed in rodent models. - Highlights: • Cholestatic liver injury is due to cytoplasmic bile acid accumulation in hepatocytes. • Primary human hepatocytes are resistant to BA-induced injury

  9. Stimulation of phosphatidylethanolamine synthesis in isolated rat hepatocytes by phorbol 12-myristate 13-acetate

    OpenAIRE

    Tijburg, L.B.M.; Houweling, M.; Geelen, M.J.H.; Golde, L.M.G. van

    1987-01-01

    Incubation of freshly isolated rat hepatocytes in the presence of phorbol 12-myristate 13-acetate stimulates the incorporation of [1,2-14C]ethanolamine into phosphatidylethanolamines. This stimulation is strongly dependent on the ethanolamine concentration in the medium and becomes apparent at ethanolamine concentrations above 25 μM. Treatment of hepatocytes with phorbol 12-myristate 13-acetate results in a decreased labelling of intracellular ethanolamine, ethanolaminephosphate and CDPethano...

  10. Hepatocyte adhesion to carbohydrate-derivatized surfaces. II. Regulation of cytoskeletal organization and cell morphology

    OpenAIRE

    1991-01-01

    Rat hepatic lectins mediate adhesion of isolated rat hepatocytes to synthetic surfaces derivatized with galactosides. Initial weak adhesion is followed by rapid adhesion strengthening. After hepatocytes contact galactose-derivatized gels, the hepatic lectins move rapidly into an inaccessible patch at the adhesive surface (Weisz, O. A., and R. L. Schnaar. 1991. J. Cell Biol. 115:485-493). Hepatic lectin patching, which occurs both at 37 degrees C and 4 degrees C, is not responsible for adhesio...

  11. Long-Term Culture and Coculture of Primary Rat and Human Hepatocytes

    OpenAIRE

    Shulman, Maria; Nahmias, Yaakov

    2013-01-01

    The liver is the largest internal organ in mammals, serving a wide spectrum of vital functions. Loss of liver function due to drug toxicity or viral infection is a major cause of death in the United States. The development of Bioartificial Liver (BAL) devices and the demand for pharmaceutical and cosmetic toxicity screening require the development of long-term hepatocyte culture techniques. However, primary hepatocytes rapidly lose their cuboidal morphology and liver-specific functions over a...

  12. Electron microscopic observation of hepatitis B virus budding from hepatocytes into bile canaliculi.

    OpenAIRE

    Ymadada, Gotaro; Sakamoto,Yuji; Mizuno, Motowo; Kobayashi, Toshinari; Nagashima,Hideo

    1980-01-01

    In electron microscopic observation of a liver biopsy obtained from a hepatitis B surface antigen-positive patient, noncoated core particles were occasionally seen budding into the hepatocytic cisterni and many Dane particles were found in the pericanalicular vesicles of hepatocytes. Noncoated core particles were also localized in clusters within the bleb of microvilli. There were some core particles being protruded from microvilli into the lumen of bile canaliculi by budding. These findings ...

  13. HIGH GLUCOSE POTENTIATES L-FABP MEDIATED FIBRATE INDUCTION OF PPARα IN MOUSE HEPATOCYTES

    OpenAIRE

    Petrescu, Anca D.; McIntosh, Avery L.; Storey, Stephen M.; Huang, Huan; Martin, Gregory G.; Landrock, Danilo; Kier, Ann B.; Schroeder, Friedhelm

    2013-01-01

    Although liver fatty acid binding protein (L-FABP) binds fibrates and PPARα in vitro and enhances fibrate induction of PPARα in transformed cells, the functional significance of these findings is unclear, especially in normal hepatocytes. Studies with cultured primary mouse hepatocytes show that: 1) At physiological (6 mM) glucose, fibrates (bezafibrate, fenofibrate) only weakly activated PPARα transcription of genes in LCFA β-oxidation; 2) High (11–20 mM) glucose, but not maltose (osmotic co...

  14. A novel mechanism of murine hepatocyte death inducible by Concanavalin A

    OpenAIRE

    Leist, Marcel; Wendel, Albrecht

    1996-01-01

    Background: Concanavalin A (Con A) is a plant lectin that polyclonally activates T-cells. When given intravenously to mice it induces a selective liver failure. Hepatotoxicity following Con A administration involves the systemic release of tumor necrosis factor.Methods: We used primary murine hepatocyte cultures to investigate mechanisms of hepatocytotoxicity related to this animal model of inflammatory liver failure.Results: Con A was directly toxic for cultured hepatocytes. This toxicity di...

  15. Wheat proteins enhance stability and function of adhesion molecules in cryopreserved hepatocytes.

    Science.gov (United States)

    Grondin, Mélanie; Hamel, Francine; Averill-Bates, Diana A; Sarhan, Fathey

    2009-01-01

    Cryopreserved hepatocytes with good hepatospecific functions upon thawing are important for clinical transplantation and for in vitro drug toxicity testing. However, cryopreservation reduces viability and certain hepatospecific functions, but the most pronounced change is diminished attachment efficiency of hepatocytes. Adhesion of cells to the extracellular matrix and cell-cell contacts are crucial for many aspects of cellular function. These processes are partly mediated and controlled by cellular adhesion molecules. The mechanisms responsible for reduced attachment efficiency of cryopreserved hepatocytes are not well understood. To address this question, we investigated the effect of a new cryopreservation procedure, using wheat proteins (WPs) or mixtures of recombinant forms of wheat freezing tolerance-associated proteins, on the stability of three important adhesion molecules (beta1-integrin, E-cadherin, and beta-catenin). Immunoblot analyses revealed that the levels of beta1-integrin, E-cadherin, and beta-catenin were much lower in cryopreserved rat hepatocytes, when compared to fresh cells. Protein expression of the adhesion molecules was generally lower in cells cryopreserved with DMSO, compared to WPs. Moreover, the stability of the adhesion molecules was not affected by cryopreservation to the same degree, with more pronounced decreases occurring for beta1-integrin (62-74%) > beta-catenin (51-58%) > E-cadherin (21-37%). However, when hepatocytes were cryopreserved with partially purified WPs (SulWPE, AcWPE) or with mixtures of recombinant wheat proteins, there was a clear protective effect against the loss of protein expression of beta1-integrin, E-cadherin, and beta-catenin. Protein expression was only 10-20% lower than that observed in fresh hepatocytes. These findings clearly demonstrate that WPs, and more particularly, partially purified WPs and recombinant wheat proteins, were more efficient for cryopreservation of rat hepatocytes by maintaining good

  16. Molecular mechanisms of tirapazamine (SR 4233, Win 59075)-induced hepatocyte toxicity under low oxygen concentrations.

    OpenAIRE

    Khan, S.; O'Brien, P. J.

    1995-01-01

    Previously we showed that tirapazamine (SR 4233, Win 59075) is cytotoxic towards hepatocytes under conditions of hypoxia but not in 10% or 95% oxygen and that bioreduction by DT-diaphorase or cytochrome P450 is not a major pathway. In the present study, we report that tirapazamine is highly cytotoxic to isolated rat hepatocytes maintained under 1% oxygen and the molecular cytotoxic mechanism has been elucidated. Cytotoxicity was prevented by the cytochrome P450 2E1 inhibitors phenyl imidazole...

  17. Docosahexaenoic Acid Ameliorates Fructose-Induced Hepatic Steatosis Involving ER Stress Response in Primary Mouse Hepatocytes

    OpenAIRE

    Jinying Zheng; Chuan Peng; Yanbiao Ai; Heng Wang; Xiaoqiu Xiao; Jibin Li

    2016-01-01

    The increase in fructose consumption is considered to be a risk factor for developing nonalcoholic fatty liver disease (NAFLD). We investigated the effects of docosahexaenoic acid (DHA) on hepatic lipid metabolism in fructose-treated primary mouse hepatocytes, and the changes of Endoplasmic reticulum (ER) stress pathways in response to DHA treatment. The hepatocytes were treated with fructose, DHA, fructose plus DHA, tunicamycin (TM) or fructose plus 4-phenylbutyric acid (PBA) for 24 h. Intra...

  18. Repopulation of adult and neonatal mice with human hepatocytes: A chimeric animal model

    OpenAIRE

    Bissig, Karl-Dimiter; Le, Tam T.; Woods, Niels-Bjarne; Verma, Inder M.

    2007-01-01

    We report the successful transplantation of human hepatocytes in immunodeficient, fumarylacetoacetate hydrolase-deficient (fah−/−) mice. Engraftment occurs over the entire liver acinus upon transplantation. A few weeks after transplantation, increasing concentrations of human proteins (e.g., human albumin and human C3a) can be measured in the blood of the recipient mouse. No fusion between mouse and human hepatocytes can be detected. Three months after transplantation, up to 20% of the mouse ...

  19. Bile acid-induced necrosis in primary human hepatocytes and in patients with obstructive cholestasis

    International Nuclear Information System (INIS)

    Accumulation of bile acids is a major mediator of cholestatic liver injury. Recent studies indicate bile acid composition between humans and rodents is dramatically different, as humans have a higher percent of glycine conjugated bile acids and increased chenodeoxycholate content, which increases the hydrophobicity index of bile acids. This increase may lead to direct toxicity that kills hepatocytes, and promotes inflammation. To address this issue, this study assessed how pathophysiological concentrations of bile acids measured in cholestatic patients affected primary human hepatocytes. Individual bile acid levels were determined in serum and bile by UPLC/QTOFMS in patients with extrahepatic cholestasis with, or without, concurrent increases in serum transaminases. Bile acid levels increased in serum of patients with liver injury, while biliary levels decreased, implicating infarction of the biliary tracts. To assess bile acid-induced toxicity in man, primary human hepatocytes were treated with relevant concentrations, derived from patient data, of the model bile acid glycochenodeoxycholic acid (GCDC). Treatment with GCDC resulted in necrosis with no increase in apoptotic parameters. This was recapitulated by treatment with biliary bile acid concentrations, but not serum concentrations. Marked elevations in serum full-length cytokeratin-18, high mobility group box 1 protein (HMGB1), and acetylated HMGB1 confirmed inflammatory necrosis in injured patients; only modest elevations in caspase-cleaved cytokeratin-18 were observed. These data suggest human hepatocytes are more resistant to human-relevant bile acids than rodent hepatocytes, and die through necrosis when exposed to bile acids. These mechanisms of cholestasis in humans are fundamentally different to mechanisms observed in rodent models. - Highlights: • Cholestatic liver injury is due to cytoplasmic bile acid accumulation in hepatocytes. • Primary human hepatocytes are resistant to BA-induced injury

  20. Alcohol-Induced Suppression of Gluconeogenesis is Greater in Ethanol Fed Female Rat Hepatocytes Than Males

    OpenAIRE

    Sumida, Ken D.; Cogger, Alma A.; Matveyenko, Aleksey V.

    2007-01-01

    The impact of alcohol-induced suppression on hepatic gluconeogenesis (HGN) after chronic ethanol consumption between males and females is unknown. To determine the effects of chronic alcohol consumption (8 weeks) on HGN, the isolated hepatocyte technique was employed on 24 hr fasted male and female Wistar rats. Livers were initially perfused with collagenase and the hepatocytes were isolated. Aliquots of the cell suspension were placed in Krebs-Henseleit buffer and incubated for 30 minutes wi...

  1. Transcriptional profiling suggests that Nevirapine and Ritonavir cause drug induced liver injury through distinct mechanisms in primary human hepatocytes.

    Science.gov (United States)

    Terelius, Ylva; Figler, Robert A; Marukian, Svetlana; Collado, Maria S; Lawson, Mark J; Mackey, Aaron J; Manka, David; Qualls, Charles W; Blackman, Brett R; Wamhoff, Brian R; Dash, Ajit

    2016-08-01

    Drug induced liver injury (DILI), a major cause of pre- and post-approval failure, is challenging to predict pre-clinically due to varied underlying direct and indirect mechanisms. Nevirapine, a non-nucleoside reverse transcriptase inhibitor (NNRTI) and Ritonavir, a protease inhibitor, are antiviral drugs that cause clinical DILI with different phenotypes via different mechanisms. Assessing DILI in vitro in hepatocyte cultures typically requires drug exposures significantly higher than clinical plasma Cmax concentrations, making clinical interpretations of mechanistic pathway changes challenging. We previously described a system that uses liver-derived hemodynamic blood flow and transport parameters to restore primary human hepatocyte biology, and drug responses at concentrations relevant to in vivo or clinical exposure levels. Using this system, primary hepatocytes from 5 human donors were exposed to concentrations approximating clinical therapeutic and supra-therapeutic levels of Nevirapine (11.3 and 175.0 μM) and Ritonavir (3.5 and 62.4 μM) for 48 h. Whole genome transcriptomics was performed by RNAseq along with functional assays for metabolic activity and function. We observed effects at both doses, but a greater number of genes were differentially expressed with higher probability at the toxic concentrations. At the toxic doses, both drugs showed direct cholestatic potential with Nevirapine increasing bile synthesis and Ritonavir inhibiting bile acid transport. Clear differences in antigen presentation were noted, with marked activation of MHC Class I by Nevirapine and suppression by Ritonavir. This suggests CD8+ T cell involvement for Nevirapine and possibly NK Killer cells for Ritonavir. Both compounds induced several drug metabolizing genes (including CYP2B6, CYP3A4 and UGT1A1), mediated by CAR activation in Nevirapine and PXR in Ritonavir. Unlike Ritonavir, Nevirapine did not increase fatty acid synthesis or activate the respiratory electron chain

  2. Hepatocyte fate upon TGF-β challenge is determined by the matrix environment.

    Science.gov (United States)

    Meyer, Christoph; Liebe, Roman; Breitkopf-Heinlein, Katja; Liu, Yan; Müller, Alexandra; Rakoczy, Pia; Thomas, Maria; Weng, Honglei; Bachmann, Anastasia; Ebert, Matthias; Dooley, Steven

    2015-06-01

    Primary hepatocytes are a versatile tool to investigate all aspects of liver function, and frequently used in drug development and testing. Upon TGF-β challenge, hepatocytes either undergo an epithelial to mesenchymal transition (EMT) or apoptosis: culture on stiff collagen (monolayer) results in EMT whereas hepatocytes in a soft collagen matrix (sandwich) undergo programmed cell death. In this study, we analyzed the transcriptional programs triggered by TGF-β under different culture conditions. Our results indicate that TGF-β initiates an identical transcription profile in hepatocytes irrespective of the cellular environment. The fact that we nevertheless observe two vastly different phenotypes indicates that the matrix environment rather than the TGF-β induced expression signature is the major determinant of the hepatocellular response. To confirm the impact of the surrounding matrix environment on the hepatocytes׳ phenotype in response to TGF-β signaling, we studied the effect of Snail overexpression and knockout in both culture conditions. Neither genetic manipulation showed an impact on hepatocytes' fate upon TGF-β treatment, confirming the crucial role of the surrounding matrix. Our findings provide novel insights into the hepatocellular basis of the fate decision between EMT and apoptotic cell death, and might explain why liver cells can react in very different manners to identical stimuli when tissue remodeling has changed the matrix environment, as occurs in a fibrotic liver. PMID:25982745

  3. Efficient amelioration of carbon tetrachloride induced toxicity in isolated rat hepatocytes by Syzygium cumini Skeels extract.

    Science.gov (United States)

    Veigas, Jyothi M; Shrivasthava, Richa; Neelwarne, Bhagyalakshmi

    2008-09-01

    Syzygium cumini, Indian black plum or Java plum, is a rich source for anthocyanins (230 mg/100g DW) showing high antioxidant activity in vitro. In the following study it is further demonstrated that S. cumini peel extract rich in anthocyanins (SCA) offers considerable protection against carbon tetrachloride (CCl(4))-induced damage in rat hepatocytes. SCA itself being non-toxic to primary rat hepatocytes at concentrations ranging from 50 to 500 ppm, was found to suppress CCl(4)-induced LDH leakage by 54% at 50ppm, thereby improving the cell viability by 39%. The SCA significantly reversed the CCl(4) induced changes in cellular glutathione (GSH) level, lipid peroxidation and activity of the antioxidant enzyme glutathione peroxidase. Exposure of hepatocytes to SCA after CCl(4) treatment was found to elevate GSH and GPx activities by 2-folds, whereas the activities of catalase and superoxide dismutase were not significantly affected. The fruit pulp extract (SPE) was less effective in offering protection to rat hepatocytes, particularly in terms of total GSH content and a consequent increase in lipid peroxidation although the higher GPx activity suggests the probable involvement of GSH as a substrate for GPx. These observations suggest that the fruit peel extract of S. cumini, is largely responsible for the reversal of CCl(4)-induced oxidative damage in rat hepatocytes. Both peel and pulp extract appear to offer protection to rat hepatocytes through GPx along with other biological pathways independent of catalase and superoxide dismutase. PMID:18538978

  4. Differential effect of fructose on fat metabolism and clock gene expression in hepatocytes vs. myotubes.

    Science.gov (United States)

    Chapnik, Nava; Rozenblit-Susan, Sigal; Genzer, Yoni; Froy, Oren

    2016-08-01

    In the liver, fructose bypasses the main rate-limiting step of glycolysis at the level of phosphofructokinase, allowing it to act as an unregulated substrate for de novo lipogenesis. It has been reported that consumption of large amounts of fructose increases de novo lipogenesis in the liver. However, the effect of fructose on ectopic deposition of muscle fat has been under dispute. Our aim was to study the effect of fructose on levels of genes and proteins involved in fatty acid oxidation and synthesis in hepatocytes vs. muscle cells. In addition, as fat accumulation leads to disruption of daily rhythms, we tested the effect of fructose treatment on clock gene expression. AML-12 hepatocytes and C2C12 myotubes were treated with fructose or glucose for 2 consecutive 24-h cycles and harvested every 6h. In contrast to glucose, fructose disrupted clock gene rhythms in hepatocytes, but in myotubes, it led to more robust rhythms. Fructose led to low levels of phosphorylated AMP-activated protein kinase (pAMPK) and high levels of LIPIN1 in hepatocytes compared with glucose. In contrast, fructose led to high pAMPK and low LIPIN1 and microsomal triacylglycerol transfer protein (MTTP) levels in myotubes compared with glucose. Analysis of fat content revealed that fructose led to less fat accumulation in myotubes compared to hepatocytes. In summary, fructose shifts metabolism towards fatty acid synthesis and clock disruption in hepatocytes, but not in myotubes. PMID:27240446

  5. Polarized location of SLC and ABC drug transporters in monolayer-cultured human hepatocytes.

    Science.gov (United States)

    Le Vee, Marc; Jouan, Elodie; Noel, Gregory; Stieger, Bruno; Fardel, Olivier

    2015-08-01

    Human hepatocytes cultured in a monolayer configuration represent a well-established in vitro model in liver toxicology, notably used in drug transporter studies. Polarized status of drug transporters, i.e., their coordinated location at sinusoidal or canalicular membranes, remains however incompletely documented in these cultured hepatocytes. The present study was therefore designed to analyze transporter expression and location in such cells. Most of drug transporters were first shown to be present at notable mRNA levels in monolayer-cultured human hepatocytes. Cultured human hepatocytes, which morphologically exhibited bile canaliculi-like structures, were next demonstrated, through immunofluorescence staining, to express the influx transporters organic anion transporting polypeptide (OATP) 1B1, OATP2B1 and organic cation transporter (OCT) 1 and the efflux transporter multidrug resistance-associated protein (MRP) 3 at their sinusoidal pole. In addition, the efflux transporters P-glycoprotein and MRP2 were detected at the canalicular pole of monolayer-cultured human hepatocytes. Moreover, canalicular secretion of reference substrates for the efflux transporters bile salt export pump, MRP2 and P-glycoprotein as well as sinusoidal drug transporter activities were observed. This polarized and functional expression of drug transporters in monolayer-cultured human hepatocytes highlights the interest of using this human in vitro cell model in xenobiotic transport studies. PMID:25862123

  6. Hepatocyte growth factor activator is a potential target proteinase for Kazal-type inhibitor in turkey (Meleagris gallopavo) seminal plasma.

    Science.gov (United States)

    Słowińska, Mariola; Bukowska, Joanna; Hejmej, Anna; Bilińska, Barbara; Kozłowski, Krzysztof; Jankowski, Jan; Ciereszko, Andrzej

    2015-08-01

    A peculiar characteristic of turkey seminal plasma is the increased activity of serine proteinases. It is of interest if the single-domain Kazal-type inhibitor controls the activity of turkey seminal plasma proteinases. Pure preparations of the Kazal-type inhibitor and anti-Kazal-type inhibitor monospecific immunoglobulin Gs were used as ligands in affinity chromatography for proteinase isolation from turkey seminal plasma. Gene expression and the immunohistochemical detection of the single-domain Kazal-type inhibitor in the reproductive tract of turkey toms are described. The hepatocyte growth factor activator (HGFA) was identified in the binding fraction in affinity chromatography. Hepatocyte growth factor activator activity was inhibited by the Kazal-type inhibitor in a dose-dependent manner. This protease was a primary physiological target for the single-domain Kazal-type inhibitor. Numerous proteoforms of HGFA were present in turkey seminal plasma, and phosphorylation was the primary posttranslational modification of HGFA. In addition to HGFA, acrosin was a target proteinase for the single-domain Kazal-type inhibitor. In seminal plasma, acrosin was present only in complexes with the Kazal-type inhibitor and was not present as a free enzyme. The single-domain Kazal-type inhibitor was specific for the reproductive tract. The germ cell-specific expression of Kazal-type inhibitors in the testis indicated an important function in spermatogenesis; secretion by the epithelial cells of the epididymis and the ductus deferens indicated that the Kazal-type inhibitor was an important factor involved in the changes in sperm membranes during maturation and in the maintenance of the microenvironment in which sperm maturation occurred and sperm was stored. The role of HGFA in these processes remains to be established.

  7. Helicobacter hepaticus induces an inflammatory response in primary human hepatocytes.

    Directory of Open Access Journals (Sweden)

    Moritz Kleine

    Full Text Available Helicobacter hepaticus can lead to chronic hepatitis and hepatocellular carcinoma in certain strains of mice. Until now the pathogenic role of Helicobacter species on human liver tissue is still not clarified though Helicobacter species identification in human liver cancer was successful in case controlled studies. Therefore we established an in vitro model to investigate the interaction of primary human hepatocytes (PHH with Helicobacter hepaticus. Successful co-culturing of PHH with Helicobacter hepaticus was confirmed by visualization of motile bacteria by two-photon-microscopy. Isolated human monocytes were stimulated with PHH conditioned media. Changes in mRNA expression of acute phase cytokines and proteins in PHH and stimulated monocytes were determined by Real-time PCR. Furthermore, cytokines and proteins were analyzed in PHH culture supernatants by ELISA. Co-cultivation with Helicobacter hepaticus induced mRNA expression of Interleukin-1 beta (IL-1β, Tumor necrosis factor-alpha, Interleukin-8 (IL-8 and Monocyte chemotactic protein-1 (MCP-1 in PHH (p<0.05 resulting in a corresponding increase of IL-8 and MCP-1 concentrations in PHH supernatants (p<0.05. IL-8 and IL-1β mRNA expression was induced in monocytes stimulated with Helicobacter hepaticus infected PHH conditioned media (p<0.05. An increase of Cyclooxygenase-2 mRNA expression was observed, with a concomitant increase of prostaglandin E2 concentration in PHH supernatants at 24 and 48 h (p<0.05. In contrast, at day 7 of co-culture, no persistent elevation of cytokine mRNA could be detected. High expression of intercellular adhesion molecule-1 on PHH cell membranes after co-culture was shown by two-photon-microscopy and confirmed by flow-cytometry. Finally, expression of Cytochrome P450 3A4 and albumin mRNA were downregulated, indicating an impairment of hepatocyte synthesis function by Helicobacter hepaticus presence. This is the first in vitro model demonstrating a pathogenic

  8. Relationship between expression of hepatocyte grow factor and apoptosis of trophoblasts in hypertensive disorder complicating pregnancy

    Institute of Scientific and Technical Information of China (English)

    OUYANG Shan; QIAO Fuyuan; ZHANG Qinghua

    2007-01-01

    The aim of this study was to investigate the expression of hepatocyte growth factor(HGF)and Fas in placentas of uncomplicated pregnant women and those with hypertensive disorder complicating pregnancy(HDCP),and elucidate the possible relationship between HGF and apoptosis of trophoblasts.Reverse transcription-polymerase chain reaction(RT-PCR)was undertaken to examine the concentration of HGF mRNA and Fas mRNA obtained from 34 cases of HDCP and 30 cases of uncomplicated pregnancy.The expression of HGF mRNA in mild preeclampsia,severe preeclampsia and eclampsia cases was significantly lower than that in the uncomplicated cases(0.43±0.12,0.38±0.09,0.19±0.17 versus 0.67±0.19,P<0.05),while the expression of Fas mRNA in mild preeclampsia,severe preeclampsia and eclampisa cases was significantly higher than that in the uncomplicated cases(1.58±0.26,2.96±0.14,5.98±1.17versus 1.01±0.36,P<0.05).For HGF mRNA and Fas mRNA,there was no difference between gestational hypertension cases and control cases.Decreased HGF mRNA or increased Fas mRNA was found along with the progress of HDCP.Negative correlation was found between the expressions of HGF and Fas.These results indicate that HGF inhibits the apoptosis mediated by Fas,and the reduced expression of HGF in HDCP may be responsible for the apoptosis of trophoblasts.

  9. Flagging Drugs That Inhibit the Bile Salt Export Pump.

    Science.gov (United States)

    Montanari, Floriane; Pinto, Marta; Khunweeraphong, Narakorn; Wlcek, Katrin; Sohail, M Imran; Noeske, Tobias; Boyer, Scott; Chiba, Peter; Stieger, Bruno; Kuchler, Karl; Ecker, Gerhard F

    2016-01-01

    The bile salt export pump (BSEP) is an ABC-transporter expressed at the canalicular membrane of hepatocytes. Its physiological role is to expel bile salts into the canaliculi from where they drain into the bile duct. Inhibition of this transporter may lead to intrahepatic cholestasis. Predictive computational models of BSEP inhibition may allow for fast identification of potentially harmful compounds in large databases. This article presents a predictive in silico model based on physicochemical descriptors that is able to flag compounds as potential BSEP inhibitors. This model was built using a training set of 670 compounds with available BSEP inhibition potencies. It successfully predicted BSEP inhibition for two independent test sets and was in a further step used for a virtual screening experiment. After in vitro testing of selected candidates, a marketed drug, bromocriptin, was identified for the first time as BSEP inhibitor. This demonstrates the usefulness of the model to identify new BSEP inhibitors and therefore potential cholestasis perpetrators. PMID:26642869

  10. Protein phosphorylation in isolated hepatocytes of septic and endotoxemic rats

    International Nuclear Information System (INIS)

    The purpose of this study was to investigate possible alterations induced by sepsis and endotoxicosis in the late phase of Ca2+-dependent signaling in rat liver. Hepatocytes isolated from septic or chronically endotoxin (ET)-treated rats were labeled with [32P]H3PO4 and stimulated with various agents. Proteins were resolved by one-dimensional polyacrylamide gel electrophoresis and autoradiographed. Vasopressin (VP)- and phenylephrine (PE)-induced responses were attenuated in both septic and ET-treated rats for cytosolic and membrane proteins compared with their respective controls. Glucagon and 12-O-myristate phorbol-13-acetate (TPA) affected only the phosphorylation of membrane proteins. Glucagon-induced changes in the phosphorylation of membrane proteins were affected by both sepsis and endotoxicosis, whereas TPA-stimulated phosphorylation was lowered only in endotoxicosis. Response to the Ca2+ ionophore A23187 was depressed in septic rats for cytosolic proteins. The phosphorylation of two cytosolic proteins, i.e., 93 and 61 kDa (previously identified as glycogen phosphorylase and pyruvate kinase, respectively), in response to VP, PE, and A23187 was severely impaired by endotoxicosis and sepsis. TPA did not affect the phosphorylation state of these two proteins. The results show that sepsis and endotoxicosis produce perturbations of the phosphorylation step in Ca2+ transmembrane signaling. Such changes can explain alterations of glycogenolysis and gluconeogenesis associated with sepsis and endotoxicosis

  11. Protein phosphorylation in isolated hepatocytes of septic and endotoxemic rats

    Energy Technology Data Exchange (ETDEWEB)

    Deaciuc, I.V.; Spitzer, J.A. (Louisiana State Univ. Medical Center, New Orleans (USA))

    1989-11-01

    The purpose of this study was to investigate possible alterations induced by sepsis and endotoxicosis in the late phase of Ca2+-dependent signaling in rat liver. Hepatocytes isolated from septic or chronically endotoxin (ET)-treated rats were labeled with (32P)H3PO4 and stimulated with various agents. Proteins were resolved by one-dimensional polyacrylamide gel electrophoresis and autoradiographed. Vasopressin (VP)- and phenylephrine (PE)-induced responses were attenuated in both septic and ET-treated rats for cytosolic and membrane proteins compared with their respective controls. Glucagon and 12-O-myristate phorbol-13-acetate (TPA) affected only the phosphorylation of membrane proteins. Glucagon-induced changes in the phosphorylation of membrane proteins were affected by both sepsis and endotoxicosis, whereas TPA-stimulated phosphorylation was lowered only in endotoxicosis. Response to the Ca2+ ionophore A23187 was depressed in septic rats for cytosolic proteins. The phosphorylation of two cytosolic proteins, i.e., 93 and 61 kDa (previously identified as glycogen phosphorylase and pyruvate kinase, respectively), in response to VP, PE, and A23187 was severely impaired by endotoxicosis and sepsis. TPA did not affect the phosphorylation state of these two proteins. The results show that sepsis and endotoxicosis produce perturbations of the phosphorylation step in Ca2+ transmembrane signaling. Such changes can explain alterations of glycogenolysis and gluconeogenesis associated with sepsis and endotoxicosis.

  12. Cyanide toxicity in hepatocytes under aerobic and anaerobic conditions.

    Science.gov (United States)

    Aw, T Y; Jones, D P

    1989-09-01

    The effect of cyanide on cell viability and mitochondrial function was studied in hepatocytes exposed to air or argon. Cells were more susceptible to cyanide toxicity under air than under argon. Analysis of the disposition of cyanide showed that the difference in susceptibility to KCN was not due to O2-dependent differences in cyanide metabolism or elimination. Studies of mitochondrial function revealed that cyanide under aerobic conditions resulted in substantial swelling of the mitochondria, which corresponded to a matrix loading of phosphate. In addition, cyanide caused a loss of the mitochondrial protonmotive force. This was in contrast to the results for cells exposed to 30 min of anoxia alone in which there was no loss of mitochondrial delta pH, no detectable change in mitochondrial volume, and little matrix loading of phosphate. These results show that at least some of the protective mechanisms elicited by anoxia (B. S. Andersson, T. Y. Aw, and D. P. Jones. Am. J. Physiol. 252 (Cell Physiol. 21): C349-C355, 1987) are not elicited by cyanide alone. Thus cyanide under aerobic conditions does not provide a completely valid model for simple anoxia. Moreover, the results suggest that the molecular sensor necessary to signal suppression of metabolic and transport functions during neahypoxia is dependent on O2 and is neither stimulated nor antagonized by KCN. PMID:2782387

  13. Amidine-bearing lipoplex targeting to hepatocyte cells

    Institute of Scientific and Technical Information of China (English)

    Yasuya Kudo; Kazunori Koiwai; Kazuhiro Shimizu; Shota Kusuki; Mina Sakuragi; Naohiko Shimada; Yoichi Takeda; Kazuo Sakurai

    2008-01-01

    A lipoplex (i.e., pDNA#1/lipid complex and transfection reagent for pDNA delivery) containing galactosylceramide (GalCer) and an amidine-bearing lipid (TRX) was examined whether the bound pDNA was specifically ingested by hepatocyte via asialoglycoprotein receptor (ASGPR) and then expressed protein. Gel electrophoresis and small-angle X-ray scattering (SAXS) confirmed that the TRX-GalCer liposome#2 complexed with pDNA and the resultant lipoplex took a hexagonally packed inverted cylinder structure when the GalCer composition was less than 20 wt.% of the total lipid. When the lipoplex carrying pGL3 (luciferase-cording pDNA) was administrated to HepG2, the luciferase activity was increased with increasing the GalCer composition until it reached 3 wt.% and then decreased upon further addition of GalCer. When we added galactose itself as a competitor, the luciferase activity was decreased, while glucose did not show such decrease, suggesting that HepG2 ingested the lipoplex via ASGPR-mediated endocytosis. This paper indicated that the hexagonally packed inverted cylinder structures of lipoplex may not always provide excellent transfection and presented a possibility that the TRX lipoplex#3 can obtain a cellular-targeting ability through the receptors for oligosaccharide.

  14. Nuclear lactate dehydrogenase modulates histone modification in human hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Castonguay, Zachary; Auger, Christopher; Thomas, Sean C.; Chahma, M’hamed; Appanna, Vasu D., E-mail: vappanna@laurentian.ca

    2014-11-07

    Highlights: • Nuclear LDH is up-regulated under oxidative stress. • SIRT1 is co-immunoprecipitated bound to nuclear LDH. • Nuclear LDH is involved in histone deacetylation and epigenetics. - Abstract: It is becoming increasingly apparent that the nucleus harbors metabolic enzymes that affect genetic transforming events. Here, we describe a nuclear isoform of lactate dehydrogenase (nLDH) and its ability to orchestrate histone deacetylation by controlling the availability of nicotinamide adenine dinucleotide (NAD{sup +}), a key ingredient of the sirtuin-1 (SIRT1) deacetylase system. There was an increase in the expression of nLDH concomitant with the presence of hydrogen peroxide (H{sub 2}O{sub 2}) in the culture medium. Under oxidative stress, the NAD{sup +} generated by nLDH resulted in the enhanced deacetylation of histones compared to the control hepatocytes despite no discernable change in the levels of SIRT1. There appeared to be an intimate association between nLDH and SIRT1 as these two enzymes co-immunoprecipitated. The ability of nLDH to regulate epigenetic modifications by manipulating NAD{sup +} reveals an intricate link between metabolism and the processing of genetic information.

  15. Mechanisms of Hepatocyte Growth Factor Activation in Cancer Tissues

    Energy Technology Data Exchange (ETDEWEB)

    Kawaguchi, Makiko; Kataoka, Hiroaki, E-mail: mejina@med.miyazaki-u.ac.jp [Section of Oncopathology and Regenerative Biology, Department of Pathology, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692 (Japan)

    2014-09-29

    Hepatocyte growth factor/scatter factor (HGF/SF) plays critical roles in cancer progression through its specific receptor, MET. HGF/SF is usually synthesized and secreted as an inactive proform (pro-HGF/SF) by stromal cells, such as fibroblasts. Several serine proteases are reported to convert pro-HGF/SF to mature HGF/SF and among these, HGF activator (HGFA) and matriptase are the most potent activators. Increased activities of both proteases have been observed in various cancers. HGFA is synthesized mainly by the liver and secreted as an inactive pro-form. In cancer tissues, pro-HGFA is likely activated by thrombin and/or human kallikrein 1-related peptidase (KLK)-4 and KLK-5. Matriptase is a type II transmembrane serine protease that is expressed by most epithelial cells and is also synthesized as an inactive zymogen. Matriptase activation is likely to be mediated by autoactivation or by other trypsin-like proteases. Recent studies revealed that matriptase autoactivation is promoted by an acidic environment. Given the mildly acidic extracellular environment of solid tumors, matriptase activation may, thus, be accelerated in the tumor microenvironment. HGFA and matriptase activities are regulated by HGFA inhibitor (HAI)-1 (HAI-1) and/or HAI-2 in the pericellular microenvironment. HAIs may have an important role in cancer cell biology by regulating HGF/SF-activating proteases.

  16. Depot-dependent effects of adipose tissue explants on co-cultured hepatocytes.

    Directory of Open Access Journals (Sweden)

    Zhen-Yu Du

    Full Text Available We have developed an in vitro hepatocyte-adipose tissue explant (ATE co-culture model enabling examination of the effect of visceral and subcutaneous adipose tissues on primary rat hepatocytes. Initial analyses of inflammatory marker genes were performed in fractionated epididymal or inguinal adipose tissues. Expressions of inflammation related genes (IL-6, TNF-α, COX-2 were higher in the inguinal than the epididymal ATE. Similarly, expressions of marker genes of macrophage and monocyte (MPEG-1, CD68, F4/80, CD64 were higher in the stromal vascular fraction (SVF isolated from inguinal ATE than that from epididymal ATE. However, expressions of lipolysis related genes (ATGL, HSL, perilipin-1 were higher in the epididymal adipocytes than inguinal adipocytes. Moreover, secretion of IL-6 and PGE(2 was higher from inguinal ATEs than from epididymal ATEs. There was a trend that the total levels of IL-6, TNF-α and PGE(2 in the media from inguinal ATEs co-cultured with primary rat hepatocytes were higher than that in the media from epididymal ATEs co-cultured with hepatocytes, although the significant difference was only seen in PGE(2. Lipolysis, measured as glycerol release, was similar in the ATEs isolated from inguinal and epididymal adipose tissues when cultured alone, but the glycerol release was higher in the ATEs isolated from epididymal than from inguinal adipose tissue when co-cultured with hepatocytes. Compared to epididymal ATEs, the ATEs from inguinal adipose tissue elicited a stronger cytotoxic response and higher level of insulin resistance in the co-cultured hepatocytes. In conclusion, our results reveal depot-dependent effects of ATEs on co-cultured primary hepatocytes, which in part may be related to a more pronounced infiltration of stromal vascular cells (SVCs, particularly macrophages, in inguinal adipose tissue resulting in stronger responses in terms of hepatotoxicity and insulin-resistance.

  17. Oxidative stress is involved in Dasatinib-induced apoptosis in rat primary hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Xue, Tao; Luo, Peihua; Zhu, Hong; Zhao, Yuqin [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Wu, Honghai; Gai, Renhua; Wu, Youping [Center for Drug Safety Evaluation and Research of Zhejiang University, Hangzhou 310058 (China); Yang, Bo [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Yang, Xiaochun, E-mail: yangxiaochun@zju.edu.cn [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Center for Drug Safety Evaluation and Research of Zhejiang University, Hangzhou 310058 (China); He, Qiaojun, E-mail: qiaojunhe@zju.edu.cn [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058 (China); Center for Drug Safety Evaluation and Research of Zhejiang University, Hangzhou 310058 (China)

    2012-06-15

    Dasatinib, a multitargeted inhibitor of BCR–ABL and SRC kinases, exhibits antitumor activity and extends the survival of patients with chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL). However, some patients suffer from hepatotoxicity, which occurs through an unknown mechanism. In the present study, we found that Dasatinib could induce hepatotoxicity both in vitro and in vivo. Dasatinib reduced the cell viability of rat primary hepatocytes, induced the release of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in vitro, and triggered the ballooning degeneration of hepatocytes in Sprague–Dawley rats in vivo. Apoptotic markers (chromatin condensation, cleaved caspase-3 and cleaved PARP) were detected to indicate that the injury induced by Dasatinib in hepatocytes in vitro was mediated by apoptosis. This result was further validated in vivo using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays. Here we found that Dasatinib dramatically increased the level of reactive oxygen species (ROS) in hepatocytes, reduced the intracellular glutathione (GSH) content, attenuated the activity of superoxide dismutase (SOD), generated malondialdehyde (MDA), a product of lipid peroxidation, decreased the mitochondrial membrane potential, and activated nuclear factor erythroid 2-related factor 2 (Nrf2) and mitogen-activated protein kinases (MAPK) related to oxidative stress and survival. These results confirm that oxidative stress plays a pivotal role in Dasatinib-mediated hepatotoxicity. N-acetylcysteine (NAC), a typical antioxidant, can scavenge free radicals, attenuate oxidative stress, and protect hepatocytes against Dasatinib-induced injury. Thus, relieving oxidative stress is a viable strategy for reducing Dasatinib-induced hepatotoxicity. -- Highlights: ►Dasatinib shows potential hepatotoxicity both in vitro and in vivo. ►Apoptosis plays a vital role in Dasatinib

  18. Glycogenotic hepatocellular carcinoma with glycogen-ground-glass hepatocytes: A heuristically highly relevant phenotype

    Institute of Scientific and Technical Information of China (English)

    Peter Bannasch

    2012-01-01

    Glycogenotic hepatocellular carcinoma (HCC) with glycogen-ground-glass hepatocytes has recently been described as an allegedly "novel variant" of HCC,but neither the historical background nor the heuristic relevance of this observation were put in perspective.In the present contribution,the most important findings in animal models and human beings related to the emergence and further evolution of excessively glycogen storing (glycogenotic) hepatocytes with and without ground glass features during neoplastic development have been summarized.Glycogenotic HCCs with glycogen-ground-glass hepatocytes represent highly differentiated neoplasms which contain subpopulations of cells phenotypically resembling those of certain types of preneoplastic hepatic foci and benign hepatocellular neoplasms.It is questionable whether the occurrence of glycogen-ground-glass hepatocytes in a glycogenotic HCC justifies its classification as a specific entity.The typical appearance of ground-glass hepatocytes is due to a hypertrophy of the smooth endoplasmic reticulum,which is usually associated with an excessive storage of glycogen and frequently also with an expression of the hepatitis B surface antigen.Sequential studies in animal models and observations in humans indicate that glycogen-ground-glass hepatocytes are a facultative,integral part of a characteristic cellular sequence commencing with focal hepatic glycogenosis potentially progressing to benign and malignant neoplasms.During this process highly differentiated glycogenotic cells including ground-glass hepatocytes are gradually transformed via various intermediate stages into poorly differentiated glycogen-poor,basophilic (ribosome-rich) cancer cells.Histochemical,microbiochemical,and molecular biochemical studies on focal hepatic glycogenosis and advanced preneoplastic and neoplastic lesions in tissue sections and laser-dissected specimens in rat and mouse models have provided compelling evidence for an early insulinomimetic

  19. Microencapsulated hepatocytes and islets as in vivo bioartificial liver support system

    Institute of Scientific and Technical Information of China (English)

    Yue Gao; Jun Xu; Bei Sun; Hong-Chi Jiang

    2004-01-01

    AIM: To confirm the xenotransplantation of microencapsulated hepatocytes and islets as a temporary bioartificial liver support system for mice with acute liver failure (ALF).METHODS: Mice were rendered ALF by a single intraperitoneal injection of D-galactosamine (D-gal) and their tail blood was sampled to examine differences in blood ALT,albumin (ALB), total bilirubin (TB) and glucose (GLU) between 4 experimental groups. Rat hepatocytes and islets were collected and microencapsulated referring to both Sun's and Fritschy's methods. Mice were grouped into control group (CG), free hepatocyte group (FHG), microencapsulated hepatocyte group (MHG) and microencapsulated hepatocyte plus islet group (HIG). Tissue samples were subjected to microscopic and electron microscopic (EM) examinations.RESULTS: The highest survival was observed in HIG,surprisingly at 100%(16/16), while the lowest was in CG at 12.5%(2/16), with inter-group statistical difference P<0.05.ALT levels revealed no statistical difference between groups but the ALB level of HIG descended by the slightest margin {q=(0.54, 0.24, 1.33), P<0.05} at the time when it reached the lowest point in all groups. TB of HIG returned to normal reference range (NRR) statistically sooner than that of others after a fierce elevation. No statistical inter-group difference was observed in GLU levels. Fusion between hepatocytes and beta cells was demonstrated giving rise to theoretical assumptions.CONCLUSION: Hepatocytes to be microencapsulated together with islets should be a preferred in vivo hepatic functional supporting system, which can dramatically prolong survival and improve living status.

  20. Oncostatin M induces upregulation of claudin-2 in rodent hepatocytes coinciding with changes in morphology and function of tight junctions

    International Nuclear Information System (INIS)

    In rodent livers, integral tight junction (TJ) proteins claudin-1, -2, -3, -5 and -14 are detected and play crucial roles in the barrier to keep bile in bile canaculi away from the blood circulation. Claudin-2 shows a lobular gradient increasing from periportal to pericentral hepatocytes, whereas claudin-1 and -3 are expressed in the whole liver lobule. Although claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells, the physiological functions and regulation of claudin-2 in hepatocytes remain unclear. Oncostatin M (OSM) is a multifunctional cytokine implicated in the differentiation of hepatocytes that induces formation of E-cadherin-based adherens junctions in fetal hepatocytes. In this study, we examined whether OSM could induce expression and function of claudin-2 in rodent hepatocytes, immortalized mouse and primary cultured proliferative rat hepatocytes. In the immortalized mouse and primary cultured proliferative rat hepatocytes, treatment with OSM markedly increased mRNA and protein of claudin-2 together with formation of developed networks of TJ strands. The increase of claudin-2 enhanced the paracellular barrier function which depended on molecular size. The increase of claudin-2 expression induced by OSM in rodent hepatocytes was regulated through distinct signaling pathways including PKC. These results suggest that expression of claudin-2 in rodent hepatocytes may play a specific role as controlling the size of paracellular permeability in the barrier to keep bile in bile canaculi

  1. Build them up and break them down: Tight junctions of cell lines expressing typical hepatocyte polarity with a varied repertoire of claudins.

    Science.gov (United States)

    Grosse, Brigitte; Degrouard, Jeril; Jaillard, Danielle; Cassio, Doris

    2013-10-01

    Tight junctions (TJs) of cells expressing simple epithelial polarity have been extensively studied, but less is known about TJs of cells expressing complex polarity. In this paper we analyzed, TJs of four different lines, that form bile canaliculi (BC) and express typical hepatocyte polarity; WIF-B9, 11-3, Can 3-1, Can 10. Striking differences were observed in claudin expression. None of the cell lines produced claudin-1. WIF-B9 and 11-3 expressed only claudin-2 while Can 3-1 and Can 10 expressed claudin-2,-3,-4,-5. TJs of these two classes of lines differed in their ultra-stucture, paracellular permeability, and robustness. Lines expressing a large claudin repertoire, especially Can 10, had complex and efficient TJs, that were maintained when cells were depleted in calcium. Inversely, TJs of WIF-B9 and 11-3 were leaky, permissive and dismantled by calcium depletion. Interestingly, we found that during the polarization process, TJ proteins expressed by all lines were sequentially settled in a specific order: first occludin, ZO-1 and cingulin, then JAM-A and ZO-2, finally claudin-2. Claudins expressed only in Can lines were also sequentially settled: claudin-3 was the first settled. Inhibition of claudin-3 expression delayed BC formation in Can10 and induced the expression of simple epithelial polarity. These results highlight the role of claudins in the settlement and the efficiency of TJs in lines expressing typical hepatocyte polarity. Can 10 seems to be the most promising of these lines because of its claudin repertoire near that of hepatocytes and its capacity to form extended tubular BC sealed by efficient TJs. PMID:24665408

  2. S-adenosyl-methionine decreases ethanol-induced apoptosis in primary hepatocyte cultures by a c-Jun N-terminal kinase activity-independent mechanism

    Institute of Scientific and Technical Information of China (English)

    María del Pilar Cabrales-Romero; Lucrecia Márquez-Rosado; Samia Fattel-Fazenda; Cristina Trejo-Solís; Evelia Arce-Popoca; Leticia Alemén-Lazarini; Saúl Villa-Trevi(n)o

    2006-01-01

    AIM: To determine the role of c-Jun N-terminal kinase (JNK) activity in ethanol-induced apoptosis and the modulation of this signaling cascade by S-Adenosylmethionine (AdoMet).METHODS: Primary hepatocyte cultures were pretreated with 100 μmol/L SP600125, a selective JNK inhibitor, 1 mL/L DMSO or 4 mmol/L AdoMet and then exposed to 100 mmo/L ethanol. Hepatocyte apoptosis was determined by the TUNEL and DNA ladder assays.JNK activity and its inhibition by SP600125 and AdoMet were determined by Western blot analysis of c-jun phosphorylation and Bid fragmentation. SP600125 and AdoMet effects on the apoptotic signaling pathway were determined by Western blot analysis of cytochrome c release and pro-caspase 3 fragmentation. The AdoMet effect on glutathione levels was measured by Ellman's method and reactive oxygen species (ROS) generation by cell cytometry.RESULTS: The exposure of hepatocytes to ethanol induced JNK activation, c-jun phosphorylation, Bid fragmentation, cytochrome c release and pro-caspase 3 cleavage; these effects were diminished by SP600125, and caused a significant decreasein ethanol-induced apoptosis (P< 0.05). AdoMet exerted an antioxidant effect maintaining glutathione levels and decreasing ROS generation, without a significant effect on JNK activity,and prevented cytochrome c release and pro-caspase 3 cleavage.CONCLUSION: The JNK signaling cascade is a key component of the proapoptotic signaling pathway induced by ethanol. JNK activation may be independent from ROS generation, since AdoMet which exerted antioxidant properties did not have a significant effect on JNK activity. JNK pathway modulator agents and AdoMet may be components of promising therapies for alcoholic liver disease (ALD) treatment.

  3. The anti-inflammatory effects of flavanol-rich lychee fruit extract in rat hepatocytes.

    Directory of Open Access Journals (Sweden)

    Ryota Yamanishi

    Full Text Available Flavanol (flavan-3-ol-rich lychee fruit extract (FRLFE is a mixture of oligomerized polyphenols primarily derived from lychee fruit and is rich in flavanol monomers, dimers, and trimers. Supplementation with this functional food has been shown to suppress inflammation and tissue damage caused by high-intensity exercise training. However, it is unclear whether FRLFE has in vitro anti-inflammatory effects, such as suppressing the production of the proinflammatory cytokine tumor necrosis factor α (TNF-α and the proinflammatory mediator nitric oxide (NO, which is synthesized by inducible nitric oxide synthase (iNOS. Here, we analyzed the effects of FRLFE and its constituents on the expression of inflammatory genes in interleukin 1β (IL-1β-treated rat hepatocytes. FRLFE decreased the mRNA and protein expression of the iNOS gene, leading to the suppression of IL-1β-induced NO production. FRLFE also decreased the levels of the iNOS antisense transcript, which stabilizes iNOS mRNA. By contrast, unprocessed lychee fruit extract, which is rich in flavanol polymers, and flavanol monomers had little effect on NO production. When a construct harboring the iNOS promoter fused to the firefly luciferase gene was used, FRLFE decreased the luciferase activity in the presence of IL-1β, suggesting that FRLFE suppresses the promoter activity of the iNOS gene at the transcriptional level. Electrophoretic mobility shift assays indicated that FRLFE reduced the nuclear transport of a key regulator, nuclear factor κB (NF-κB. Furthermore, FRLFE inhibited the phosphorylation of NF-κB inhibitor α (IκB-α. FRLFE also reduced the mRNA levels of NF-κB target genes encoding cytokines and chemokines, such as TNF-α. Therefore, FRLFE inhibited NF-κB activation and nuclear translocation to suppress the expression of these inflammatory genes. Our results suggest that flavanols may be responsible for the anti-inflammatory and hepatoprotective effects of FRLFE and may be

  4. Renal medullary AA amyloidosis, hepatocyte dissociation and multinucleated hepatocytes in a 14-year-old free-ranging lioness (Panthera leo

    Directory of Open Access Journals (Sweden)

    J.H. Williams

    2005-06-01

    Full Text Available A 14-year-old lioness, originating from Etosha in Namibia, and a member of a pride in Pilanesberg National Park since translocation in 1994, was euthanased due to fight-related vertebral fracture and spinal injury, incurred approximately 6-8 weeks previously. Blood specimens collected at the time of death showed mild anaemia and a leukogram reflecting stress and chronic infection. Necropsy conducted within 2 hours of death was on a dehydrated, emaciated animal with hindquarter wasting and chronic traumatic friction injuries from dragging her hindlegs. There was cellulitis in the region of bite-wounds adjacent to the thoraco-lumbar vertebral fracture, at which site there was spinal cord compression, and there was marked intestinal helminthiasis. The outer renal medullae appeared pale and waxy and the liver was macroscopically unremarkable. Histopathology and electron microscopy of the kidneys revealed multifocal to coalescing deposits of proximal medullary interstitial amyloid, which fluoresced strongly with thioflavine T, and was sensitive to potassium permanganate treatment prior to Congo Red staining, thus indicating inflammatory (AA origin. There was diffuse hepatocyte dissociation, as well as numerous binucleated and scattered multinucleated (up to 8 nuclei/cell hepatocytes, with swollen hepatocyte mitochondria, in liver examined light microscopically. Ultrastructurally, the mono-, bi- and multinucleated hepatocytes contained multifocal irregular membrane-bound accumulations of finely-granular, amorphous material both intra-cytoplasmically and intra-nuclearly, as well as evidence of irreversible mitochondrial injury. The incidence and relevance in cats and other species of amyloidosis, particularly with renal medullary distribution, as well as of hepatocyte dissociation and multinucleation, as reported in selected literature, is briefly overviewed and their occurrence in this lioness is discussed.

  5. 肝细胞生长因子抗肾纤维化研究进展%Advances of Hepatocyte Growth Factor as an Anti-Fibrotic Regulator in Renal Disease

    Institute of Scientific and Technical Information of China (English)

    陈杏; 张庆林; 熊锡山; 王汉斌

    2011-01-01

    The renal function of patients suffered from chronic kidney disease shows a progressive decline over time.The pathogenesis of renal fibrosis is a progressive process, which is characterized by the progressive loss of renal parenchymal cells and an excessive accumulation of extracellular matrix, that ultimately leads to end-stage renal failure.Hepatocyte growth factor(HGF) and c-Met/HGF receptor play roles in kidney development and regeneration after acute renal injury.As to chronic renal failure and renal fibrosis, HGF also acts an nutritional and anti-fibrotic factor.The mini-review attempts to highlight the recent progress in our understanding of the cellular and molecular pathways by which HGF plays a role in anti-fibrosis, indicating the challenges and opportunities in developing therapeutic strategies.%慢性肾脏疾病患者的肾功能会随时间的推移而进行性恶化,肾实质细胞进行性丧失及细胞外基质蛋白过度沉积将导致肾纤维化形成,肾纤维化进行性发展将最终走向终末期肾衰竭.肝细胞生长因子(HGF)及其受体c-Met对肾发育和急性肾损伤后的肾脏再生修复具有重要作用,在慢性肾衰竭及肾纤维化时,HGF还具有营养肾脏及抗肾纤维化的作用.简要综述了HGF抑制肾纤维化形成的细胞分子机制的研究进展,提示HGF在治疗肾纤维化方面所具有的前景.

  6. Activated human neutrophils release hepatocyte growth factor/scatter factor.

    LENUS (Irish Health Repository)

    McCourt, M

    2012-02-03

    BACKGROUND: Hepatocyte growth factor or scatter factor (HGF\\/SF) is a pleiotropic cytokine that has potent angiogenic properties. We have previously demonstrated that neutrophils (PMN) are directly angiogenic by releasing vascular endothelial growth factor (VEGF). We hypothesized that the acute inflammatory response can stimulate PMN to release HGF. AIMS: To examine the effects of inflammatory mediators on PMN HGF release and the effect of recombinant human HGF (rhHGF) on PMN adhesion receptor expression and PMN VEGF release. METHODS: In the first experiment, PMN were isolated from healthy volunteers and stimulated with tumour necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), interleukin-8 (IL-8), and formyl methionyl-leucyl-phenylalanine (fMLP). Culture supernatants were assayed for HGF using ELISA. In the second experiment, PMN were lysed to measure total HGF release and HGF expression in the PMN was detected by Western immunoblotting. Finally, PMN were stimulated with rhHGF. PMN CD 11a, CD 11b, and CD 18 receptor expression and VEGF release was measured using flow cytometry and ELISA respectively. RESULTS: TNF-alpha, LPS and fMLP stimulation resulted in significantly increased release of PMN HGF (755+\\/-216, 484+\\/-221 and 565+\\/-278 pg\\/ml, respectively) compared to controls (118+\\/-42 pg\\/ml). IL-8 had no effect. Total HGF release following cell lysis and Western blot suggests that HGF is released from intracellular stores. Recombinant human HGF did not alter PMN adhesion receptor expression and had no effect on PMN VEGF release. CONCLUSIONS: This study demonstrates that pro-inflammatory mediators can stimulate HGF release from a PMN intracellular store and that activated PMN in addition to secreting VEGF have further angiogenic potential by releasing HGF.

  7. Reversal of acetaminophen toxicity in isolated hamster hepatocytes by dithiothreitol

    Energy Technology Data Exchange (ETDEWEB)

    Tee, L.B.; Boobis, A.R.; Huggett, A.C.; Davies, D.S.

    1986-04-01

    The toxicity of acetaminophen in freshly isolated hamster hepatocytes was investigated. Cells exposed to 2.5 mM acetaminophen for 90 min, followed by washing to completely remove unbound acetaminophen, and resuspension in fresh buffer, showed a dramatic decrease in viability over the ensuing 4.5 hr by which time only 4% of the cells could still exclude trypan blue. During the initial 90-min incubation, there was a substantial depletion of glutathione, to 19% of control values, covalent binding of (/sup 14/C)acetaminophen to cellular proteins, and evidence of morphological changes consistent with some disturbance of the plasma membrane. During subsequent incubation of these cells, covalent binding did not change nor did lipid peroxidation, despite the decrease in viability that occurred. Subsequent incubation of cells exposed to acetaminophen for 90 min in buffer containing 1.5 mM dithiothreitol (DTT), a disulfide-reducing agent, largely prevented the decrease in cell viability and reversed the morphological changes that occurred during the first 90-min incubation. However, there was no change in lipid peroxidation, glutathione content, or covalent binding. It is concluded that acetaminophen interacted with some critical target in the cell, and that this left unchecked, led eventually to the death of the cell. DTT prevented and reversed this effect. The toxicity of acetaminophen, and its reversal by DTT, appear independent of either covalent binding of acetaminophen or lipid peroxidation. In addition, the effect of DTT was independent of the concentration of glutathione, most probably acting by directly reducing oxidized SH-groups in critical enzymes, possibly membrane-bound ATP-dependent Ca2+ translocases.

  8. Safety assessment of potential food ingredients in canine hepatocytes.

    Science.gov (United States)

    Zhang, Leshuai W; Koci, Juraj; Jeffery, Brett; Riviere, Jim E; Monteiro-Riviere, Nancy A

    2015-04-01

    This research aimed to develop in vitro methods to assess hazard of canine food ingredients. Canine hepatocytes were harvested and cell viability of clove-leaf oil (CLO), eugenol (EUG), lemongrass oil (LGO), guanosine monophosphate (GMP), inosine monophosphate (IMP), sorbose, ginger-root extract (GRE), cinnamon-bark oil (CBO), cinnamaldehyde (CINA), thymol oil (TO), thymol (THYM), and citric acid were assessed with positive controls: acetaminophen (APAP), aflatoxin B1 and xylitol. Molecular Toxicology PathwayFinder array (MTPF) analyzed toxicity mechanisms for LGO. LC50 for APAP was similar among human (3.45), rat (2.35), dog (4.26 mg/ml). Aflatoxin B1 had an LC50 of 4.43 (human), 5.78 (rat) and 6.05 (dog) µg/ml; xylitol did not decrease viability. LC50 of CLO (0.185 ± 0.075(SD)), EUG (0.165 ± 0.112), LGO (0.220 ± 0.012), GRE (1.54 ± 0.31) mg/ml; GMP (166.03 ± 41.83), GMP + IMP (208.67 ± 15.27) mM; CBO (0.08 ± 0.03), CINA (0.11 ± 0.01), TO (0.21 ± 0.03), THYM (0.05 ± 0.01), citric acid (1.58 ± 0.08) mg/ml, while sorbose was non-toxic. LGO induced upregulation of 16 and down-regulation of 24 genes, which CYP and heat shock most affected. These results suggest that in vitro assays such as this may be useful for hazard assessment of food ingredients for altered hepatic function. PMID:25660481

  9. Eight paths of ERK1/2 signalling pathway regulating hepatocyte proliferation in rat liver regeneration

    Indian Academy of Sciences (India)

    J. W. Li; G. P. Wang; J. Y. Fan; C. F. Chang; C. S. Xu

    2011-12-01

    Although it is known that hormones, growth factors and integrin promote hepatocyte proliferation in liver regeneration (LR) through ERK1/2 signalling pathway, reports about regulating processes of its intracellular paths in hepatocytes of LR are limited. This study aims at exploring which paths of ERK1/2 signalling pathway participate in the regulation of rat LR, especially in hepatocyte proliferation, and how they do so. In all, 14 paths and 165 genes are known to be involved in ERK1/2 signalling pathway. Of them, 161 genes are included in Rat Genome 230 2.0 Array. This array was used to detect expression changes of genes related to ERK1/2 signalling pathway in isolated hepatocytes of rat LR, showing that 60 genes were related to hepatocytes of LR. In addition, bioinformatics and systems biology methods were used to analyse the roles of 14 above paths in regenerating hepatocytes. We found that three paths, RTK → SHC → GRB2/SOS → RAS → RAF, Integrin → FAK → RAC → PAK → RAF and G → PI3K → RAC → PAK → RAF, promoted the G1 phase progression of hepatocytes by activating ERK1/2. A further four paths, Gq → PLC → PKC → SRC/PYK2 → GRB2/SOS → RAS → RAF, RTK → PLC → PKC → SRC/PYK2 → GRB2/SOS → RAS → RAF, Integrin → FAK/SRC → GRB2/SOS → RAS → RAF and Integrin → FAK → RAC → PAK → RAF, advanced the cell progression of S phase and G2/M checkpoint by activating ERK1/2, and so did PP1/2 → Mek1/2 by decreasing the negative influence on ERK1/2. At the late phase of LR, Gs → AC → EPAC → Rap1 → Raf blocked hepatocyte proliferation by decreasing the activity of ERK1/2 and so did PP1/2 → Mek1/2. In summary, 60 genes and 8 paths of ERK1/2 signalling pathway regulated hepatocyte proliferation in rat LR.

  10. A universal system to select gene-modified hepatocytes in vivo.

    Science.gov (United States)

    Nygaard, Sean; Barzel, Adi; Haft, Annelise; Major, Angela; Finegold, Milton; Kay, Mark A; Grompe, Markus

    2016-06-01

    Many genetic and acquired liver disorders are amenable to gene and/or cell therapy. However, the efficiencies of cell engraftment and stable genetic modification are low and often subtherapeutic. In particular, targeted gene modifications from homologous recombination are rare events. These obstacles could be overcome if hepatocytes that have undergone genetic modification were to be selectively amplified or expanded. We describe a universally applicable system for in vivo selection and expansion of gene-modified hepatocytes in any genetic background. In this system, the therapeutic transgene is coexpressed with a short hairpin RNA (shRNA) that confers modified hepatocytes with resistance to drug-induced toxicity. An shRNA against the tyrosine catabolic enzyme 4-OH-phenylpyruvate dioxygenase protected hepatocytes from 4-[(2-carboxyethyl)-hydroxyphosphinyl]-3-oxobutyrate, a small-molecule inhibitor of fumarylacetoacetate hydrolase. To select for specific gene targeting events, the protective shRNA was embedded in a microRNA and inserted into a recombinant adeno-associated viral vector designed to integrate site-specifically into the highly active albumin locus. After selection of the gene-targeted cells, transgene expression increased 10- to 1000-fold, reaching supraphysiological levels of human factor 9 protein (50,000 ng/ml) in mice. This drug resistance system can be used to achieve therapeutically relevant transgene levels in hepatocytes in any setting. PMID:27280686

  11. Sensitive hepatocyte-mediated assay for the metabolism of nitrosamines to mutagens for mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Jones, C.A.; Huberman, E.

    1980-02-01

    A sensitive cell-mediated assay has been developed for testing mutagenesis in Chinese hamster V79 cells by carcinogenic nitrosamines. Mutations were characterized by resistance to ouabian and 6-thioguanine. Since V79 cells do not metabolize nitrosamines, mutagenesis in the V79 cells was tested in the presence of primary hepatocytes capable of metabolizing nitrosamines. The hepatocytes were isolated after collagenase and hyaluronidase digestion of liver slices. All seven liver carcinogens of the nine tested nitrosamines exhibited a mutagenic response in this cell-mediated assay. The potent liver carcinogens nitrosodimethylamine, nitrosodiethylamine, nitrosoethylmethylamine, and nitrosodipropylamine could be detected with doses as low as 1 ..mu..m. The noncarcinogenic nitrosodiphenylamine was not mutagenic. Nitrosomethoxymethylamine was the only nitrosamine that exhibited mutagenic activity in the absence of hepatocytes, and this activity was diminished in the presence of hepatocytes. It is suggested that the use of hepatocytes prepared by the slicing method for carcinogen metabolism and mutable V79 cells offers a highly sensitive assay for determining the mutagenic potential of carcinogenic nitrosamines and probably of other classes of hazardous chemicals occurring in the environment.

  12. Human hepatocytes derived from pluripotent stem cells: a promising cell model for drug hepatotoxicity screening.

    Science.gov (United States)

    Gómez-Lechón, María José; Tolosa, Laia

    2016-09-01

    Drug-induced liver injury (DILI) is a frequent cause of failure in both clinical and post-approval stages of drug development, and poses a key challenge to the pharmaceutical industry. Current animal models offer poor prediction of human DILI. Although several human cell-based models have been proposed for the detection of human DILI, human primary hepatocytes remain the gold standard for preclinical toxicological screening. However, their use is hindered by their limited availability, variability and phenotypic instability. In contrast, pluripotent stem cells, which include embryonic and induced pluripotent stem cells (iPSCs), proliferate extensively in vitro and can be differentiated into hepatocytes by the addition of soluble factors. This provides a stable source of hepatocytes for multiple applications, including early preclinical hepatotoxicity screening. In addition, iPSCs also have the potential to establish genotype-specific cells from different individuals, which would increase the predictivity of toxicity assays allowing more successful clinical trials. Therefore, the generation of human hepatocyte-like cells derived from pluripotent stem cells seems to be promising for overcoming limitations of hepatocyte preparations, and it is expected to have a substantial repercussion in preclinical hepatotoxicity risk assessment in early drug development stages. PMID:27325232

  13. Ultrastructure of Kupffer cells and hepatocytes in the Dubin-Johnson syndrome: A case report

    Institute of Scientific and Technical Information of China (English)

    Maria Elzbieta Sobaniec-Lotowska; Dariusz Marek Lebensztejn

    2006-01-01

    Ultrastructure of Kupffer cells and hepatocytes in liver bioptate was evaluated in a 17-year-old boy with Dubin -Johnson syndrome (DJS). The liver tissue obtained by needle biopsy was fixed in glutaraldehyde and paraformaldehyde and routinely processed for electron microscopic analysis. The ultrastructural examinations of liver bioptate revealed the accumulation of membranebound, electron-dense lysosomal granules within the cytoplasm of hepatocytes, characteristic of DJS. They were located mainly in the vicinity of the biliary pole, and preferentially in the centrilobular region that corresponded to the pigment deposits seen under light microscope. The presence of the granules was accompanied by dilated elements of the granular endoplasmic reticulum and paracrystalline mitochondrial inclusions as well as dilation of the bile canaliculi. The changes in hepatocytes coexisted with marked stimulation and enhanced phagocytic activity of Kupffer cells. This was manifested in the accumulation of pigment deposits within their cytoplasm that corresponded to those observed in hepatocytes.Hyperactive pericentral Kupffer cells which are involved in the response to pigmentary material originating from disintegrated hepatocytes may play an essential role in the development of DJS.

  14. Current Status of Human Adipose–Derived Stem Cells: Differentiation into Hepatocyte-Like Cells

    Directory of Open Access Journals (Sweden)

    Feras Al Battah

    2011-01-01

    Full Text Available The shortage of human organ donors and the low cell quality of available liver tissues represent major obstacles for the clinical application of orthotropic liver transplantation and hepatocyte transplantation, respectively. Therefore, worldwide research groups are investigating alternative extrahepatic cell sources. Recent in vitro studies have demonstrated that mesenchymal stem cells (MSCs from various sources, including human bone marrow, adipose tissue, and umbilical cord, can be differentiated into hepatocyte-like cells when appropriate conditions are used. In particular, interest exists for human adipose–derived stems cells (hASCs as an attractive cell source for generating hepatocyte-like cells. The hASCs are multipotent MSCs that reside in adipose tissue, with the ability to self-renew and differentiate into multiple cell lineages. Moreover, these cells can secrete multiple growth factors and cytokines that exert beneficial effects on organ or tissue injury. In this review, we will not only present recent data regarding hASC biology, their isolation, and differentiation capability towards hepatocytes, but also the potential application of hASC-derived hepatocytes to study drug toxicity. Additionally, this review will discuss the therapeutic potential of hASCs as undifferentiated cells in liver regeneration.

  15. Hepatocyte differentiation of human fibroblasts from cirrhotic liver in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    Yu-Ling Sun; Sheng-Yong Yin; Lin Zhou; Hai-Yang Xie; Feng Zhang; Li-Ming Wu; Shu-Sen Zheng

    2011-01-01

    BACKGROUND: Mesenchymal stem cells (MSCs) and fibro-blasts have intimate relationships, and the phenotypic homology between fibroblasts and MSCs has been recently described. The aim of this study was to investigate the hepatic differentiating potentialofhumanfibroblastsincirrhoticliver. METHODS: The phenotypes of fibroblasts in cirrhotic liver were labeled by biological methods. After that, the differentiation potential of these fibroblasts in vitro was characterized in terms of liver-specific gene and protein expression. Finally, an animal model of hepatocyte regeneration in severe combined immunodeficient (SCID) mice was created by retrorsine injection and partial hepatectomy, and the expression of human hepatocyte proteins in SCID mouse livers was checked by immunohistochemicalanalysisafterfibroblastadministration. RESULTS: Surface immunophenotyping revealed that a minority of fibroblasts expressed markers of MSCs and hepatic epithelial cytokeratins as well as alpha-smooth muscle actin, but homogeneously expressed vimentin, desmin, prolyl 4-hydroxylase and fibronectin. These fibroblasts presented the characteristics of hepatocytes in vitro and differentiated directly into functional hepatocytes in the liver of hepatecto-mized SCID mice. CONCLUSIONS: This study demonstrated that fibroblasts in cirrhotic liver have the potential to differentiate into hepatocyte-like cells in vitro and in vivo. Our findings infer that hepatic differentiation of fibroblasts may serve as a new target for reversion of liver fibrosis and a cell source for tissue engineering.

  16. Protective Effects of N-acetylcysteine Against the Statins Cytotoxicity in Freshly Isolated Rat Hepatocytes

    Directory of Open Access Journals (Sweden)

    Narges Abdoli

    2014-05-01

    Full Text Available Purpose: Hepatotoxicity is one of the most important side effects of the statins therapy as lipid-lowering agents. However, the mechanism(s of hepatotoxicity induced by these drugs is not clearly understood yet, and no hepatoprotective agent has been developed against this complication. Methods: The protective effect of N-acetylcysteine (NAC against statins-induced cytotoxicity was evaluated by using freshly isolated rat hepatocytes. Hepatocytes were prepared by the method of collagenase enzyme perfusion via portal vein. This technique is based on liver perfusion with collagenase after removal of calcium ion (Ca2+ with a chelator (ethylene glycol tetra acetic acid (EGTA 0.5 mM. The level of parameters such as cell death, ROS formation, lipid peroxidation, mitochondrial membrane potential (MMP in the statins-treated hepatocytes were determined. Additionally, the mentioned markers were assessed in the presence of NAC. Results: Incubation of hepatocytes with the statins resulted in cytotoxicity characterized by an elevation in cell death, increasing ROS generation and consequently lipid peroxidation and impairment of mitochondrial function. Administration of NAC caused a reduction in amount of ROS formation, lipid peroxidation and finally, cell viability and mitochondrial membrane potential (MMP were improved. Conclusion: This study confirms that oxidative stress and consequently mitochondrial dysfunction is one of the mechanisms underlying the statins-induced liver injury and treating hepatocytes by NAC (200 μM attenuates this cytotoxicity.

  17. Hepatic stellate cell-expressed endosialin balances fibrogenesis and hepatocyte proliferation during liver damage.

    Science.gov (United States)

    Mogler, Carolin; Wieland, Matthias; König, Courtney; Hu, Junhao; Runge, Anja; Korn, Claudia; Besemfelder, Eva; Breitkopf-Heinlein, Katja; Komljenovic, Dorde; Dooley, Steven; Schirmacher, Peter; Longerich, Thomas; Augustin, Hellmut G

    2015-03-01

    Liver fibrosis is a reversible wound-healing response to injury reflecting the critical balance between liver repair and scar formation. Chronic damage leads to progressive substitution of liver parenchyma by scar tissue and ultimately results in liver cirrhosis. Stromal cells (hepatic stellate cells [HSC] and endothelial cells) have been proposed to control the balance between liver fibrosis and regeneration. Here, we show that endosialin, a C-type lectin, expressed in the liver exclusively by HSC and portal fibroblasts, is upregulated in liver fibrosis in mouse and man. Chronic chemically induced liver damage resulted in reduced fibrosis and enhanced hepatocyte proliferation in endosialin-deficient (EN(KO)) mice. Correspondingly, acute-liver-damage-induced hepatocyte proliferation (partial hepatectomy) was increased in EN(KO) mice. A candidate-based screen of known regulators of hepatocyte proliferation identified insulin-like growth factor 2 (IGF2) as selectively endosialin-dependent hepatocyte mitogen. Collectively, the study establishes a critical role of HSC in the reciprocal regulation of fibrogenesis vs. hepatocyte proliferation and identifies endosialin as a therapeutic target in non-neoplastic settings. PMID:25680861

  18. Anti-apoptotic effects of novel phenolic antioxidant isolated from the Pacific oyster (Crassostrea gigas) on cultured human hepatocytes under oxidative stress.

    Science.gov (United States)

    Fuda, Hirotoshi; Watanabe, Mitsugu; Hui, Shu-Ping; Joko, Sae; Okabe, Hiroaki; Jin, Shigeki; Takeda, Seiji; Miki, Emiko; Watanabe, Takayuki; Chiba, Hitoshi

    2015-06-01

    The antioxidant, and hepatoprotective properties of 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), a natural phenolic antioxidant isolated from the Pacific oyster, were defined using cultured human hepatocyte-derived cells (C3A). DHMBA showed no cytotoxicity at 62.5-500μM, as well as chlorogenic acid (CGA), vitamin C, and vitamin E. However, butylated hydroxytoluene, eicosapentaenoic acid, docosahexaenoic acid and catechin reduced cell viability. In the presence of the prooxidant 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), DHMBA at 125-500μM improved cell viability, whereas CGA had no effect. DNA ladder formation and flow-cytometric studies indicated that DHMBA inhibited AAPH-induced apoptosis and necrosis. CGA was ineffective. Thus, DHMBA is a novel, potent antioxidant, effectively protecting cultured hepatocytes from apoptosis and necrosis caused by oxidative stress. Additionally, the concentration of DHMBA was determined by mass spectrometry to be 24.4μmol/kg wet oyster meat, and three polyphenols (gentisic acid, daidzein, and matairesinol) were newly identified in the oyster extracts. PMID:25624228

  19. ASGPR-Mediated Uptake of Multivalent Glycoconjugates for Drug Delivery in Hepatocytes.

    Science.gov (United States)

    Monestier, Marie; Charbonnier, Peggy; Gateau, Christelle; Cuillel, Martine; Robert, Faustine; Lebrun, Colette; Mintz, Elisabeth; Renaudet, Olivier; Delangle, Pascale

    2016-04-01

    Liver cells are an essential target for drug delivery in many diseases. The hepatocytes express the asialoglycoprotein receptor (ASGPR), which promotes specific uptake by means of N-acetylgalactosamine (GalNAc) recognition. In this work, we designed two different chemical architectures to treat Wilson's disease by intracellular copper chelation. Two glycoconjugates functionalized with three or four GalNAc units each were shown to enter hepatic cells and chelate copper. Here, we studied two series of compounds derived from these glycoconjugates to find key parameters for the targeting of human hepatocytes. Efficient cellular uptake was demonstrated by flow cytometry using HepG2 human heptic cells that express the human oligomeric ASGPR. Dissociation constants in the nanomolar range showed efficient multivalent interactions with the receptor. Both architectures were therefore concluded to be able to compete with endogeneous asialoglycoproteins and serve as good vehicles for drug delivery in hepatocytes.

  20. The use of the MTT test for determining the cytotoxicity of veterinary drugs in pig hepatocytes.

    Science.gov (United States)

    Huveneers-Oorsprong, M B; Hoogenboom, L A; Kuiper, H A

    1997-08-01

    Pig hepatocytes were used for determining the cytotoxicity of a number of veterinary drugs and known hepatotoxic compounds, using the MTT test as a marker for viability. When possible, drugs were tested in the absence and presence of dimethyl sulfoxide (DMSO), to study the possible effect of this solvent when used in the case of less hydrophilic compounds. IC(50) value