Effect of interleukin-17A on stemness of hepatoma cell lines

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LI Kexin

2017-06-01

Full Text Available ObjectiveTo investigate the effect of interleukin-17A (IL-17A on stemness of human hepatoma cell lines Hep 3B, MHCC97H, and MHCC97L and the association between IL-17A and the progression of liver cancer. MethodsHuman hepatoma cell lines Hep 3B, MHCC97H, and MHCC97L were selected, and in vitro 3D sphere formation assay was used to analyze the effect of IL-17A on sphere formation ability. The control group with common culture solution and the experimental group with 50 ng/ml IL-17A were established. Real-time cellular analysis was used to determine the effect of IL-17A on the proliferation and migration of hepatoma cells with enhanced sphere formation ability; quantitative real-time PCR was used to measure the changes in the mRNA expression of IL-17A receptors IL-17RA and IL-17RC and stemness-related genes SOX2, NANOG, OCT4, and BMI1 in hepatoma cells with enhanced sphere formation ability; Western blot was used to measure the expression of epithelial-mesenchymal transition-related proteins E-cadherin, N-cadherin, and vimentin. The t-test was used for comparison of continuous doota betwwen groups. ResultsWith the presence of 50 ng/ml IL-17A and 500 inoculated cells, Hep 3B cells had a significant increase in the number of spheres formed (113.0±10.3 vs 180.0±7.2, t=5.533, P＜0.001, while MHCC97H and MHCC97L cells showed no significant changes (t=1.087 and 0.279, P=0.325 and 0785. The analysis showed that IL-17A promoted the proliferation and migration of Hep 3B cells with an increased number of spheres formed. After the addition of 50 ng/ml IL-17A, there was an increase in the mRNA expression of IL-17A receptors IL-17RA and IL-17RC over the time of treatment; Hep 3B cells showed significant increases in the mRNA expression of stemness-related genes SOX2 (t=4.749, P=0.042, NANOG (t=19.600, P=0.003, OCT4 (t=37.310, P＜0.001, and BMI1 (t=16.810, P=0.004. Western blot showed no significant change in the expression of the epithelium

Degradation and repair of DNA from rat hepatoma cells after treatments with γ-rays and N-methyl-N-nitrosourea

International Nuclear Information System (INIS)

Zakrzhevskaya, D.G.; Kulagina, T.P.; Petrov, S.I.; Fomenko, L.A.; Gaziev, A.I.

1977-01-01

It has been shown, that DNA single-strand breaks induced in the cells of ascite hepatoma with γ-rays and metylnitrosourea (MNM) are effectively repaired. DNA two-strand breaks of hepatoma cells, treated with MNM are effectively repaired in situ as well. Only insignificant part of two-strand gamma-induced breaks in DNA of these cells is repaired during postirradiation period. Under combined effect of gamma rays and MNM on hepatoma cells a delay of DNA reparation and its further degradation as well as inhibition of nonplanned DNA synthesis and the suppression of DNA-polymerase 1 activity are observed

Human serum activates CIDEB-mediated lipid droplet enlargement in hepatoma cells

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Singaravelu, Ragunath [Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario K1N 6N5 (Canada); National Research Council of Canada, Ottawa, Ontario K1A 0R6 (Canada); Lyn, Rodney K. [Department of Chemistry, University of Ottawa, Ottawa, Ontario K1N 6N5 (Canada); National Research Council of Canada, Ottawa, Ontario K1A 0R6 (Canada); Srinivasan, Prashanth [National Research Council of Canada, Ottawa, Ontario K1A 0R6 (Canada); Delcorde, Julie [Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario K1N 6N5 (Canada); National Research Council of Canada, Ottawa, Ontario K1A 0R6 (Canada); Steenbergen, Rineke H.; Tyrrell, D. Lorne [Department of Medical Microbiology and Immunology, University of Alberta (Canada); Li Ka Shing Institute of Virology, Katz Centre for Pharmacy and Health Research, Edmonton, Alberta T6G 2S2 (Canada); Pezacki, John P., E-mail: John.Pezacki@nrc-cnrc.gc.ca [Department of Chemistry, University of Ottawa, Ottawa, Ontario K1N 6N5 (Canada); National Research Council of Canada, Ottawa, Ontario K1A 0R6 (Canada)

2013-11-15

Highlights: •Human serum induced differentiation of hepatoma cells increases cellular lipid droplet (LD) size. •The observed increase in LD size correlates with increased PGC-1α and CIDEB expression. •Induction of CIDEB expression correlates with rescue of VLDL secretion and loss of ADRP. •siRNA knockdown of CIDEB impairs the human serum mediated increase in LD size. •This system represents a cost-efficient model to study CIDEB’s role in lipid biology. -- Abstract: Human hepatocytes constitutively express the lipid droplet (LD) associated protein cell death-inducing DFFA-like effector B (CIDEB). CIDEB mediates LD fusion, as well as very-low-density lipoprotein (VLDL) maturation. However, there are limited cell culture models readily available to study CIDEB’s role in these biological processes, as hepatoma cell lines express negligible levels of CIDEB. Recent work has highlighted the ability of human serum to differentiate hepatoma cells. Herein, we demonstrate that culturing Huh7.5 cells in media supplemented with human serum activates CIDEB expression. This activation occurs through the induced expression of PGC-1α, a positive transcriptional regulator of CIDEB. Coherent anti-Stokes Raman scattering (CARS) microscopy revealed a correlation between CIDEB levels and LD size in human serum treated Huh7.5 cells. Human serum treatment also resulted in a rapid decrease in the levels of adipose differentiation-related protein (ADRP). Furthermore, individual overexpression of CIDEB was sufficient to down-regulate ADRP protein levels. siRNA knockdown of CIDEB revealed that the human serum mediated increase in LD size was CIDEB-dependent. Overall, our work highlights CIDEB’s role in LD fusion, and presents a new model system to study the PGC-1α/CIDEB pathway’s role in LD dynamics and the VLDL pathway.

Human serum activates CIDEB-mediated lipid droplet enlargement in hepatoma cells

International Nuclear Information System (INIS)

Singaravelu, Ragunath; Lyn, Rodney K.; Srinivasan, Prashanth; Delcorde, Julie; Steenbergen, Rineke H.; Tyrrell, D. Lorne; Pezacki, John P.

2013-01-01

Highlights: •Human serum induced differentiation of hepatoma cells increases cellular lipid droplet (LD) size. •The observed increase in LD size correlates with increased PGC-1α and CIDEB expression. •Induction of CIDEB expression correlates with rescue of VLDL secretion and loss of ADRP. •siRNA knockdown of CIDEB impairs the human serum mediated increase in LD size. •This system represents a cost-efficient model to study CIDEB’s role in lipid biology. -- Abstract: Human hepatocytes constitutively express the lipid droplet (LD) associated protein cell death-inducing DFFA-like effector B (CIDEB). CIDEB mediates LD fusion, as well as very-low-density lipoprotein (VLDL) maturation. However, there are limited cell culture models readily available to study CIDEB’s role in these biological processes, as hepatoma cell lines express negligible levels of CIDEB. Recent work has highlighted the ability of human serum to differentiate hepatoma cells. Herein, we demonstrate that culturing Huh7.5 cells in media supplemented with human serum activates CIDEB expression. This activation occurs through the induced expression of PGC-1α, a positive transcriptional regulator of CIDEB. Coherent anti-Stokes Raman scattering (CARS) microscopy revealed a correlation between CIDEB levels and LD size in human serum treated Huh7.5 cells. Human serum treatment also resulted in a rapid decrease in the levels of adipose differentiation-related protein (ADRP). Furthermore, individual overexpression of CIDEB was sufficient to down-regulate ADRP protein levels. siRNA knockdown of CIDEB revealed that the human serum mediated increase in LD size was CIDEB-dependent. Overall, our work highlights CIDEB’s role in LD fusion, and presents a new model system to study the PGC-1α/CIDEB pathway’s role in LD dynamics and the VLDL pathway

[Ursodeoxycholic acid induced apoptosis of human hepatoma cells HepG2 and SMMC-7721 bymitochondrial-mediated pathway].

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Wu, Duan; Zhou, Jianyin; Yin, Zhenyu; Liu, Pingguo; Zhao, Yilin; Liu, Jianming; Wang, Xiaomin

2014-12-02

To explore the effects and underlying mechanisms of ursodeoxycholic acid on human hepatoma cells. HepG2 and SMMC-7721 HCC cell lines were respectively treated with ursodeoxycholic acid. And cell proliferation, apoptosis and the expression of Bax/Bcl-2 gene were detected by methyl thiazolyl tetrazolium (MTT), inverted microscopy, fluorescent microscopy, flow cytometry and Western blot. Ursodeoxycholic acid significantly inhibited the proliferation of human hepatoma cells in a concentration- and time-dependent manner. The half maximal inhibitory concentrations (IC50) of HepG2 and SMMC-7721 were 397.3 and 387.7 µg/ml respectively after a 48-hour treatment of 400 µg /ml ursodeoxycholic acid. And it also induced the apoptosis of HepG2 and SMMC-7721 cells, up-regulated Bax gene and down-regulated Bcl-2 gene. Ursodeoxycholic acid inhibits the proliferation of hepatoma cells and induce apoptosis by mitochondrial-mediated pathway.

Complete replication of hepatitis B virus and hepatitis C virus in a newly developed hepatoma cell line.

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Yang, Darong; Zuo, Chaohui; Wang, Xiaohong; Meng, Xianghe; Xue, Binbin; Liu, Nianli; Yu, Rong; Qin, Yuwen; Gao, Yimin; Wang, Qiuping; Hu, Jun; Wang, Ling; Zhou, Zebin; Liu, Bing; Tan, Deming; Guan, Yang; Zhu, Haizhen

2014-04-01

The absence of a robust cell culture system for hepatitis B virus (HBV) and hepatitis C virus (HCV) infection has limited the analysis of the virus lifecycle and drug discovery. We have established a hepatoma cell line, HLCZ01, the first cell line, to the authors' knowledge, supporting the entire lifecycle of both HBV and HCV. HBV surface antigen (HBsAg)-positive particles can be observed in the supernatant and the lumen of the endoplasmic reticulum of the cells via electron microscopy. Interestingly, HBV and HCV clinical isolates propagate in HLCZ01 cells. Both viruses replicate in the cells without evidence of overt interference. HBV and HCV entry are blocked by antibodies against HBsAg and human CD81, respectively, and the replication of HBV and HCV is inhibited by antivirals. HLCZ01 cells mount an innate immune response to virus infection. The cell line provides a powerful tool for exploring the mechanisms of virus entry and replication and the interaction between host and virus, facilitating the development of novel antiviral agents and vaccines.

Over-expression and siRNA of a novel environmental lipopolysaccharide-responding gene on the cell cycle of the human hepatoma-derived cell line HepG2

International Nuclear Information System (INIS)

Du Kejun; Chai Yubo; Hou Lichao; Chang Wenhui; Chen Suming; Luo Wenjing; Cai Tongjian; Zhang Xiaonan; Chen Nanchun; Chen Yaoming; Chen Jingyuan

2008-01-01

Lipopolysaccharide (LPS) is the toxic determinant for Gram-negative bacterium infection. The individual response to LPS was related to its gene background. It is necessary to identify new molecules and signaling transduction pathways about LPS. The present study was undertaken to evaluate the effects of a novel environmental lipopolysaccharide-responding (Elrg) gene on the regulation of proliferation and cell cycle of the hepatoma-derived cell line, HepG2. By means of RT-PCR, the new molecule of Elrg was generated from a human dental pulp cell cDNA library. Expression level of Elrg in HepG2 cells was remarkably upgraded by the irritation of LPS. Localization of Elrg in HepG2 cells was positioned mainly in cytoplasm. HepG2 cells were markedly arrested in the G1 phase by over-expressing Elrg. The percentage of HepG2 cells in G1 phase partly decreased after Elrg-siRNA. In conclusion, Elrg is probably correlative with LPS responding. Elrg is probably a new protein in cytoplasm which plays an important role in regulating cell cycle. The results will deepen our understanding about the potential effects of Elrg on the human hepatoma-derived cell line HepG2

Downregulation of miR-210 expression inhibits proliferation, induces apoptosis and enhances radiosensitivity in hypoxic human hepatoma cells in vitro

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Yang, Wei; Sun, Ting; Cao, Jianping; Liu, Fenju; Tian, Ye; Zhu, Wei

2012-01-01

Hypoxia is a common feature of solid tumors and an important contributor to tumor radioresistance. miR-210 is the most consistently and robustly induced microRNA under hypoxia in different types of tumor cells and normal cells. In the present study, to explore the feasibility of miR-210 as an effective therapeutic target, lentiviral-mediated anti-sense miR-210 gene transfer technique was employed to downregulate miR-210 expression in hypoxic human hepatoma SMMC-7721, HepG2 and HuH7 cells, and phenotypic changes of which were analyzed. Hypoxia led to an increased hypoxia inducible factor-1α (HIF-1α) and miR-210 expression and cell arrest in the G 0 /G 1 phase in all cell lines. miR-210 downregulation significantly suppressed cell viability, induced cell arrest in the G 0 /G 1 phase, increased apoptotic rate and enhanced radiosensitivity in hypoxic human hepatoma cells. Moreover, apoptosis-inducing factor, mitochondrion-associated, 3 (AIFM3) was identified as a direct target gene of miR-210. AIFM3 downregulation by siRNA attenuated radiation induced apoptosis in miR-210 downregulated hypoxic human hepatoma cells. Taken together, these data suggest that miR-210 might be a potential therapeutic target and specific inhibition of miR-210 expression in combination with radiotherapy might be expected to exert strong anti-tumor effect on hypoxic human hepatoma cells. -- Highlights: ► miR-210 downregulation radiosensitized hypoxic hepatoma. ► AIFM3 was identified as a direct target gene of miR-210. ► miR-210 might be a therapeutic target to hypoxic hepatoma.

Effects of Uptake of Hydroxyapatite Nanoparticles into Hepatoma Cells on Cell Adhesion and Proliferation

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Meizhen Yin

2014-01-01

Full Text Available Hydroxyapatite nanoparticles (nano-HAPs were prepared by homogeneous precipitation, and size distribution and morphology of these nanoparticles were determined by laser particle analysis and transmission electron microscopy, respectively. Nano-HAPs were uniformly distributed, with rod-like shapes sizes ranging from 44.6 to 86.8 nm. Attached overnight, suspended, and proliferating Bel-7402 cells were repeatedly incubated with nano-HAPs. Inverted microscopy, transmission electron microscopy, and fluorescence microscopy were used to observe the cell adhesion and growth, the culture medium containing nano-HAPs, the cell ultrastructure, and intracellular Ca2+ labeled with a fluo-3 calcium fluorescent probe. The results showed that nano-HAPs inhibited proliferation of Bel-7402 cells and, caused an obvious increase in the concentration of intracellular Ca2+, along with significant changes in the cell ultrastructure. Moreover, nano-HAPs led suspended cells and proliferating cells after trypsinized that did not attach to the bottom of the culture bottle died. Nano-HAPs continuously entered these cells. Attached, suspended, and proliferating cells endocytosed nano-HAPs, and nanoparticle-filled vesicles were in the cytoplasm. Therefore, hepatoma cellular uptake of nano-HAPs through endocytosis was very active and occurred continuously. Nano-HAPs affected proliferation and adhesion of hepatoma cells probably because uptake of nano-HAPs blocked integrin-mediated cell adhesion, which may have potential significance in inhibiting metastatic cancer cells to their target organ.

99Tcm pertechnetate uptake by hepatoma cells induced by tissue specific hNIS gene expression

International Nuclear Information System (INIS)

Chen Libo; Luo Quanyong; Yu Yongli; Yuan Zhibin; Lu Hankui; Zhu Ruisen; Guo Lihe

2007-01-01

Objective: Human sodium/iodide symporter (hNIS) gene could be used both as an ideal reporter gene and promising therapeutic gene. Rather than radioiodine, 99 Tc m pertechnetate has been proven to be a better radiopharmaceutical for tracing and imaging purposes. Herein, the authors investigated the feasibility of monitoring hNIS gene expression in hepatoma cells using 99 Tc m pertechnetate as a tracer. Methods: Hepatoma cells MH3924A were stably transfected with recombinant retroviral vector in which hNIS cDNA was driven by murine albumin enhancer/promoter (mAlb) and coupled to hygromycin resistance gene. The uptake and efflux of 99 Tc m pertechnetate by transfected hepatoma cells were tested with 99 Tc m pertechnetate (74 kBq) solution adulterated into the culture media and counted after media suspension discharge at different intervals. In further tests, 50 μmol/L NaClO 4 and 500 μmol/L Ouabain were added into the media for 99 Tc m inhibition tests. For in vive studies, five ACI rats bearing NIS transfected hepatoma xenografts were injected with 99 Tc m pertechnetate (15.8 MBq) and followed by dynamic acquisition (0.57 1, 2 and 4 h) with small gamma camera to semi-quantitatively analyze the radioactivity distribution. Results: In vitro tests, the peak uptake of 99 Tc m pertechnetate by cultured transfected MH3924A cells was up to 254 folds higher than that by the wild type cells. 99 Tc m uptake by transfected cells were significantly inhibited by NaClO 4 down to 2.44% (P 99 Tc m pertechnetate out of cultured transfected cells became rapid immediately after renewal of culture media (half life 99 Tc m accumulations by hNIS transfected tumor xenografts were obvious in early phases of the acquisition with peak uptake at 12 min and gradually declining later on. Conclusions: hNIS transfected hepatoma cells can avidly uptake 99 Tc m pertechnetate both in vitro and in vive. It is feasible to utilize 99 Tc m pertechnetate for monitoring and even quantitatively analyzing

Recent advances in live cell imaging of hepatoma cells

Science.gov (United States)

2014-01-01

Live cell imaging enables the study of dynamic processes of living cells in real time by use of suitable reporter proteins and the staining of specific cellular structures and/or organelles. With the availability of advanced optical devices and improved cell culture protocols it has become a rapidly growing research methodology. The success of this technique relies mainly on the selection of suitable reporter proteins, construction of recombinant plasmids possessing cell type specific promoters as well as reliable methods of gene transfer. This review aims to provide an overview of the recent developments in the field of marker proteins (bioluminescence and fluorescent) and methodologies (fluorescent resonance energy transfer, fluorescent recovery after photobleaching and proximity ligation assay) employed as to achieve an improved imaging of biological processes in hepatoma cells. Moreover, different expression systems of marker proteins and the modes of gene transfer are discussed with emphasis on the study of lipid droplet formation in hepatocytes as an example. PMID:25005127

Downregulation of miR-210 expression inhibits proliferation, induces apoptosis and enhances radiosensitivity in hypoxic human hepatoma cells in vitro

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Yang, Wei, E-mail: detachedy@yahoo.com.cn [Department of Radiobiology, School of Radiological Medicine and Protection, Soochow University, Suzhou (China); Sun, Ting [Brain and Nerve Research Laboratory, The First Affiliated Hospital, Soochow University, Suzhou (China); Cao, Jianping; Liu, Fenju [Department of Radiobiology, School of Radiological Medicine and Protection, Soochow University, Suzhou (China); Tian, Ye [Department of Radiotherapy and Oncology, The Second Affiliated Hospital, Soochow University, Suzhou (China); Zhu, Wei [Department of Radiobiology, School of Radiological Medicine and Protection, Soochow University, Suzhou (China)

2012-05-01

Hypoxia is a common feature of solid tumors and an important contributor to tumor radioresistance. miR-210 is the most consistently and robustly induced microRNA under hypoxia in different types of tumor cells and normal cells. In the present study, to explore the feasibility of miR-210 as an effective therapeutic target, lentiviral-mediated anti-sense miR-210 gene transfer technique was employed to downregulate miR-210 expression in hypoxic human hepatoma SMMC-7721, HepG2 and HuH7 cells, and phenotypic changes of which were analyzed. Hypoxia led to an increased hypoxia inducible factor-1{alpha} (HIF-1{alpha}) and miR-210 expression and cell arrest in the G{sub 0}/G{sub 1} phase in all cell lines. miR-210 downregulation significantly suppressed cell viability, induced cell arrest in the G{sub 0}/G{sub 1} phase, increased apoptotic rate and enhanced radiosensitivity in hypoxic human hepatoma cells. Moreover, apoptosis-inducing factor, mitochondrion-associated, 3 (AIFM3) was identified as a direct target gene of miR-210. AIFM3 downregulation by siRNA attenuated radiation induced apoptosis in miR-210 downregulated hypoxic human hepatoma cells. Taken together, these data suggest that miR-210 might be a potential therapeutic target and specific inhibition of miR-210 expression in combination with radiotherapy might be expected to exert strong anti-tumor effect on hypoxic human hepatoma cells. -- Highlights: Black-Right-Pointing-Pointer miR-210 downregulation radiosensitized hypoxic hepatoma. Black-Right-Pointing-Pointer AIFM3 was identified as a direct target gene of miR-210. Black-Right-Pointing-Pointer miR-210 might be a therapeutic target to hypoxic hepatoma.

GEP100/Arf6 is required for epidermal growth factor-induced ERK/Rac1 signaling and cell migration in human hepatoma HepG2 cells.

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ZhenZhen Hu

Full Text Available BACKGROUND: Epidermal growth factor (EGF signaling is implicated in the invasion and metastasis of hepatoma cells. However, the signaling pathways for EGF-induced motility of hepatoma cells remain undefined. METHODOLOGY/PRINCIPAL FINDINGS: We found that EGF dose-dependently stimulated the migration of human hepatoma cells HepG2, with the maximal effect at 10 ng/mL. Additionally, EGF increased Arf6 activity, and ectopic expression of Arf6 T27N, a dominant negative Arf6 mutant, largely abolish EGF-induced cell migration. Blocking GEP100 with GEP100 siRNA or GEP100-△PH, a pleckstrin homology (PH domain deletion mutant of GEP100, blocked EGF-induced Arf6 activity and cell migration. EGF also increased ERK and Rac1 activity. Ectopic expression GEP100 siRNA, GEP100-△PH, or Arf6-T27N suppressed EGF-induced ERK and Rac1 activity. Furthermore, blocking ERK signaling with its inhibitor U0126 remarkably inhibited both EGF-induced Rac1 activation as well as cell migration, and ectopic expression of inactive mutant form of Rac1 (Rac1-T17N also largely abolished EGF-induced cell migration. CONCLUSIONS/SIGNIFICANCE: Taken together, this study highlights the function of the PH domain of GEP100 and its regulated Arf6/ERK/Rac1 signaling cascade in EGF-induced hepatoma cell migration. These findings could provide a rationale for designing new therapy based on inhibition of hepatoma metastasis.

Drug Transporter Expression and Activity in Human Hepatoma HuH-7 Cells

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Elodie Jouan

2016-12-01

Full Text Available Human hepatoma cells may represent a valuable alternative to the use of human hepatocytes for studying hepatic drug transporters, which is now a regulatory issue during drug development. In the present work, we have characterized hepatic drug transporter expression, activity and regulation in human hepatoma HuH-7 cells, in order to determine the potential relevance of these cells for drug transport assays. HuH-7 cells displayed notable multidrug resistance-associated protein (MRP activity, presumed to reflect expression of various hepatic MRPs, including MRP2. By contrast, they failed to display functional activities of the uptake transporters sodium taurocholate co-transporting polypeptide (NTCP, organic anion-transporting polypeptides (OATPs and organic cation transporter 1 (OCT1, and of the canalicular transporters P-glycoprotein and breast cancer resistance protein (BCRP. Concomitantly, mRNA expressions of various sinusoidal and canalicular hepatic drug transporters were not detected (NTCP, OATP1B1, organic anion transporter 2 (OAT2, OCT1 and bile salt export pump or were found to be lower (OATP1B3, OATP2B1, multidrug and toxin extrusion protein 1, BCRP and MRP3 in hepatoma HuH-7 cells than those found in human hepatocytes, whereas other transporters such as OAT7, MRP4 and MRP5 were up-regulated. HuH-7 cells additionally exhibited farnesoid X receptor (FXR- and nuclear factor erythroid 2-related factor 2 (Nrf2-related up-regulation of some transporters. Such data indicate that HuH-7 cells, although expressing rather poorly some main hepatic drug transporters, may be useful for investigating interactions of drugs with MRPs, notably MRP2, and for studying FXR- or Nrf2-mediated gene regulation.

Glucocorticoid-regulated and constitutive trafficking of proteolytically processed cell surface-associated glycoproteins in wild type and variant rat hepatoma cells

International Nuclear Information System (INIS)

Amacher, S.L.; Goodman, L.J.; Bravo, D.A.; Wong, K.Y.; Goldfine, I.D.; Hawley, D.M.; Firestone, G.L.

1989-01-01

Glucocorticoids regulate the trafficking of mouse mammary tumor virus (MMTV) glycoproteins to the cell surface in the rat hepatoma cell line M1.54, but not in the immunoselected sorting variant CR4. To compare the localization of MMTV glycoproteins to another proteolytically processed glycoprotein, both wild type M1.54 cells and variant CR4 cells were transfected with a human insulin receptor (hIR) expression vector, pRSVhIR. The production of cell surface hIR was monitored in dexamethasone-treated and -untreated wild type M1.54 and variant CR4 cells by indirect immunofluorescence, direct plasma membrane immunoprecipitation, and by [125I] insulin binding. In both wild type and variant rat hepatoma cells, hIR were localized at the cell surface in the presence or in the absence of 1 microM dexamethasone. In contrast, the glucocorticoid-regulated trafficking of cell surface MMTV glycoproteins occurred only in wild type M1.54 cells. We conclude that the hIR, which undergoes posttranslational processing reactions similar to MMTV glycoproteins, does not require glucocorticoids to be transported to the plasma membrane and is representative of a subset of cell surface glycoproteins whose trafficking is constitutive in rat hepatoma cells. Thus, MMTV glycoproteins and hIR provide specific cell surface markers to characterize the glucocorticoid-regulated and constitutive sorting pathways

Hydrogen sulfide (H2S)/cystathionine γ-lyase (CSE) pathway contributes to the proliferation of hepatoma cells

International Nuclear Information System (INIS)

Pan, Yan; Ye, Shuang; Yuan, Dexiao; Zhang, Jianghong; Bai, Yang; Shao, Chunlin

2014-01-01

Highlights: • Inhibition of H 2 S/CSE pathway strongly stimulates cellular apoptosis. • Inhibition of H 2 S/CSE pathway suppresses cell growth by blocking EGFR pathway. • H 2 S/CSE pathway is critical for maintaining the proliferation of hepatoma cells. - Abstract: Hydrogen sulfide (H 2 S)/cystathionine γ-lyase (CSE) pathway has been demonstrated to play vital roles in physiology and pathophysiology. However, its role in tumor cell proliferation remains largely unclear. Here we found that CSE over-expressed in hepatoma HepG2 and PLC/PRF/5 cells. Inhibition of endogenous H 2 S/CSE pathway drastically decreased the proliferation of HepG2 and PLC/PRF/5 cells, and it also enhanced ROS production and mitochondrial disruption, pronounced DNA damage and increased apoptosis. Moreover, this increase of apoptosis was associated with the activation of p53 and p21 accompanied by a decreased ratio of Bcl-2/Bax and up-regulation of phosphorylated c-Jun N-terminal kinase (JNK) and caspase-3 activity. In addition, the negative regulation of cell proliferation by inhibition of H 2 S/CSE system correlated with the blockage of cell mitogenic and survival signal transduction of epidermal growth factor receptor (EGFR) via down-regulating the extracellular-signal-regulated kinase 1/2 (ERK1/2) activation. These results demonstrate that H 2 S/CSE and its downstream pathway contribute to the proliferation of hepatoma cells, and inhibition of this pathway strongly suppress the excessive growth of hepatoma cells by stimulating mitochondrial apoptosis and suppressing cell growth signal transduction

Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines

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Chen Lei

2011-06-01

Full Text Available Abstract Background Cancer stem cells (CSCs are regarded as the cause of tumor formation and recurrence. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs. Methods Human hepatoma cell lines were plated in stem cell conditioned culture system allowed for sphere forming. To evaluate the stemness characteristics of spheres, the self-renewal, proliferation, chemoresistance, tumorigenicity of the PLC/PRF/5 sphere-forming cells, and the expression levels of stem cell related proteins in the PLC/PRF/5 sphere-forming cells were assessed, comparing with the parental cells. The stem cell RT-PCR array was performed to further explore the biological properties of liver CSCs. Results The PLC/PRF/5, MHCC97H and HepG2 cells could form clonal nonadherent 3-D spheres and be serially passaged. The PLC/PRF/5 sphere-forming cells possessed a key criteria that define CSCs: persistent self-renewal, extensive proliferation, drug resistance, overexpression of liver CSCs related proteins (Oct3/4, OV6, EpCAM, CD133 and CD44. Even 500 sphere-forming cells were able to form tumors in NOD/SCID mice, and the tumor initiating capability was not decreased when spheres were passaged. Besides, downstream proteins DTX1 and Ep300 of the CSL (CBF1 in humans, Suppressor of hairless in Drosophila and LAG1 in C. elegans -independent Notch signaling pathway were highly expressed in the spheres, and a gamma-secretase inhibitor MRK003 could significantly inhibit the sphere formation ability. Conclusions Nonadherent tumor spheres from hepatoma cell lines cultured in stem cell conditioned medium possess liver CSC properties, and the CSL-independent Notch signaling pathway may play a role in liver CSCs.

Radiation induced bystander effect on hepatoma HepG2 cells under hypoxia condition

International Nuclear Information System (INIS)

Zhang Jianghong; Jin Yizun; Shao Chunlin; Prise KM

2009-01-01

Objective: To investigate radiation induced bystander effect and its mechanism on hepatoma HepG2 cells under hypoxia condition. Methods: Non-irradiated bystander hepatoma cells were co-cultured with irradiated cells or treated with the conditioned medium (CM) from irradiated cells, then micronuclei (MN) were measured for both irradiated cells and bystander cells. Results: The MN yield of irradiated HepG2 cells under hypoxic condition was significantly lower than that under normoxia, the oxygen enhancement ratio of HepG2 cells of MN was 1.6. For both hypoxic and normoxic condition, the MN yield of bystander cells were obviously enhanced to a similar high level after co-culturing with irradiated cells or with CM treatment, and it also correlated with the irradiation dose. When the hypoxic HepG2 cells were treated with either DMSO, a scavenger of reactive oxygen species (ROS), or aminoguanidine, an iNOS inhibitor, the yield of bystander MN was partly diminished, and the reducing rate of DMSO was 42.2%-46.7%, the reducing rate of aminoguanidine was 42% . Conclusion: ROS, NO and their downstream signal factors are involved in the radiation induced bystander effect of hypoxic HepG2 cells. (authors)

Expression of rat class I major histocompatibility complex (MHC) alloantigens and hepatocytes and hepatoma cells

International Nuclear Information System (INIS)

Hunt, J.M.; Desai, P.A.; Chakraborty, S.

1986-01-01

Altered expression of Class I MHC alloantigens has been reported for murine tumors, and may be associated with the tumorigenic phenotype of tumor cells. To characterize MHC Class I alloantigen expression on a chemically-induced transplantable rat hepatoma cell line, 17X, derived from a (WF x F344) F 1 rat, polyvalent anti-F344 and anti-WF rat alloantisera were first used to immunoprecipitate the rat RT1.A Class I MHC alloantigens expressed on primary (WF x F344) F 1 hepatocyptes in short-term monolayer cultures. Two-dimensional isoelectric focusing and SDS-PAGE of immunoprecipitates from 35 S-methionine-labeled (WF x F344) F 1 hepatocytes clearly resolved the RT1.A/sup u/ (WF) and RT1.A/sup LvI/ (F344) parental alloantigens. Identical radiolabeling and immunoprecipitation failed to detect either parental alloantigen on the 17X hepatoma cells. However, indirect immunofluorescence and immunoblot analyses demonstrated the presence of parental alloantigens on the 17X cells. Immunization of F344 rats but not of WF rats with 17X cells resulted in antibodies cytotoxic for normal (WF X F344) F 1 spleen cells in the presence of complement. These findings indicate that a combination of detection techniques will be necessary to characterize altered alloantigen expression on rat hepatoma cells

Cell damage of hepatoma-22 cells exposed to continuous wave ultrasound.

Science.gov (United States)

Wang, Pan; Wang, Xiaobing; Liu, Quanhong

2012-01-01

The cellular response of hepatoma-22 cells to ultrasonic irradiation and the potential cause for the action were evaluated. Hepatoma-22 cells were subjected to ultrasound irradiation at a frequency of 2.17 MHz and a spatial average intensity of 1.6 W/cm2 for variable periods of time, and several biological parameters were analyzed. The terephthalic acid (TA) dosimetry method was used to evaluate the efficacies of irradiation parameters on the acoustic cavitation activity by monitoring hydroxyl radical (OH) production. Lactate dehydrogenase (LDH) leakage was assayed to investigate cell membrane integrity. The polarization value of fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) was measured to monitor plasma membrane fluidity. The malonaldehyde content in cells was determined to reflect lipid peroxidation. Trypan blue exclusion was used to detect cell viability. Additionally, electron microscopy was used to observe morphological changes. The generation of intracellular reactive oxygen species, mitochondria swelling and the loss of mitochondria membrane potential were also investigated. The results showed that 1) the concentration of ·OH production by ultrasonic irradiation in air-saturated cell suspensions increased as ultrasound exposure time increased; 2) compared with control, lactate dehydrogenase leakage, the polarization value of 1,6-diphenyl-1,3,5-hexatriene, malonaldehyde content and cell lysis were significantly elevated when cells were treated by ultrasound for 60 s; 3) cytotoxicity by ultrasound irradiation was also accompanied by an increase in production of intracellular reactive oxygen species and dissipation of mitochondria membrane potential as well as by mitochondria swelling. Presently available information indicates that the plasma membrane and mitochondria are the main targets for ultrasound treatment, and free radicals formation such as ·OH due to ultrasound cavitation may play an important role in mediating these cellular response

Analysis of the cytotoxicity of carbon-based nanoparticles, diamond and graphite, in human glioblastoma and hepatoma cell lines

DEFF Research Database (Denmark)

Zakrzewska, Karolina Ewa; Samluk, Anna; Wierzbicki, Mateusz

2015-01-01

carbon based nanoparticles, diamond and graphite, on glioblastoma and hepatoma cells were compared. First, we confirmed previous results that diamond nanoparticles are practically nontoxic. Second, graphite nanoparticles exhibited a negative impact on glioblastoma, but not on hepatoma cells. The studied...... carbon nanoparticles could be a potentially useful tool for therapeutics delivery to the brain tissue with minimal side effects on the hepatocytes. Furthermore, we showed the influence of the nanoparticles on the stable, fluorescently labeled tumor cell lines and concluded that the labeled cells...

Stimulation of Hepatoma Cell Invasiveness and Metastatic Potential by Proteins Secreted From Irradiated Nonparenchymal Cells

Energy Technology Data Exchange (ETDEWEB)

Zhou Leyuan [Department of Radiation Oncology, Zhongshan Hospital, Fudan University, Shanghai (China); Wang Zhiming [Department of Medical Oncology, Zhongshan Hospital, Fudan University, Shanghai (China); Gao Yabo [Department of Radiation Oncology, Zhongshan Hospital, Fudan University, Shanghai (China); Wang Lingyan [Experimental Research Center, Zhongshan Hospital, Fudan University, Shanghai (China); Zeng Zhaochong, E-mail: zeng.zhaochong@zs-hospital.sh.cn [Department of Radiation Oncology, Zhongshan Hospital, Fudan University, Shanghai (China)

2012-11-01

Purpose: To determine whether factors secreted by irradiated liver nonparenchymal cells (NPCs) may influence invasiveness and/or metastatic potential of hepatocellular carcinoma (HCC) cells and to elucidate a possible mechanism for such effect. Methods and Materials: Primary rat NPCs were cultured and divided into irradiated (10-Gy X-ray) and nonirradiated groups. Forty-eight hours after irradiation, conditioned medium from irradiated (SR) or nonirradiated (SnonR) cultures were collected and added to sublethally irradiated cultures of the hepatoma McA-RH7777 cell line. Then, hepatoma cells were continuously passaged for eight generations (RH10Gy-SR and RH10Gy-SnonR). The invasiveness and metastatic potential of McA-RH7777, RH10Gy-SnonR, and RH10Gy-SR cells were evaluated using an in vitro gelatinous protein (Matrigel) invasion and an in vivo metastasis assay. In addition, SR and SnonR were tested using rat cytokine antibody arrays and enzyme-linked immunosorbent assay (ELISA). Results: In vitro gelatinous protein invasion assay indicated that the numbers of invading cells was significantly higher in RH10Gy-SR (40 {+-} 4.74) than in RH10Gy-SnonR (30.6 {+-} 3.85) cells, and lowest in McA-RH7777 (11.4 {+-} 3.56) cells. The same pattern was observed in vivo in a lung metastasis assay, as evaluated by number of metastatic lung nodules seen with RH10Gy-SR (28.83 {+-} 5.38), RH10Gy-SnonR (22.17 {+-} 4.26), and McA-RH7777 (8.3 {+-} 3.8) cells. Rat cytokine antibody arrays and ELISA demonstrated that metastasis-promoting cytokines (tumor necrosis factor-{alpha} and interleukin-6), circulating growth factors (vascular endothelial growth factor and epidermal growth factor), and metalloproteinases (MMP-2 and MMP-9) were upregulated in SR compared with SnonR. Conclusions: Radiation can increase invasiveness and metastatic potential of sublethally irradiated hepatoma cells, and soluble mediators released from irradiated NPCs promote this potential. Increased secretion of

Hepatitis B virus X protein mutant HBxΔ127 promotes proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT

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Liu, Fabao [Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China); Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China); You, Xiaona [Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China); Chi, Xiumei [Department of Hepatology, The First Hospital, Jilin University, Changchun 130021 (China); Wang, Tao [Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China); Ye, Lihong [Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China); Niu, Junqi, E-mail: junqiniu@yahoo.com.cn [Department of Hepatology, The First Hospital, Jilin University, Changchun 130021 (China); Zhang, Xiaodong, E-mail: zhangxd@nankai.edu.cn [Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China)

2014-02-07

Highlights: • Relative to wild type HBx, HBX mutant HBxΔ127 strongly enhances cell proliferation. • Relative to wild type HBx, HBxΔ127 remarkably up-regulates miR-215 in hepatoma cells. • HBxΔ127-elevated miR-215 promotes cell proliferation via targeting PTPRT mRNA. - Abstract: The mutant of virus is a frequent event. Hepatitis B virus X protein (HBx) plays a vital role in the development of hepatocellular carcinoma (HCC). Therefore, the identification of potent mutant of HBx in hepatocarcinogenesis is significant. Previously, we identified a natural mutant of the HBx gene (termed HBxΔ127). Relative to wild type HBx, HBxΔ127 strongly enhanced cell proliferation and migration in HCC. In this study, we aim to explore the mechanism of HBxΔ127 in promotion of proliferation of hepatoma cells. Our data showed that both wild type HBx and HBxΔ127 could increase the expression of miR-215 in hepatoma HepG2 and H7402 cells. However, HBxΔ127 was able to significantly increase miR-215 expression relative to wild type HBx in the cells. We identified that protein tyrosine phosphatase, receptor type T (PTPRT) was one of the target genes of miR-215 through targeting 3′UTR of PTPRT mRNA. In function, miR-215 was able to promote the proliferation of hepatoma cells. Meanwhile anti-miR-215 could partially abolish the enhancement of cell proliferation mediated by HBxΔ127 in vitro. Knockdown of PTPRT by siRNA could distinctly suppress the decrease of cell proliferation mediated by anti-miR-215 in HepG2-XΔ127/H7402-XΔ127 cells. Moreover, we found that anti-miR-215 remarkably inhibited the tumor growth of hepatoma cells in nude mice. Collectively, relative to wild type HBx, HBxΔ127 strongly enhances proliferation of hepatoma cells through up-regulating miR-215 targeting PTPRT. Our finding provides new insights into the mechanism of HBx mutant HBxΔ127 in promotion of proliferation of hepatoma cells.

Magnetic targeting of iron-oxide-labeled fluorescent hepatoma cells to the liver

International Nuclear Information System (INIS)

Luciani, Alain; Wilhelm, Claire; Gazeau, Florence; Bruneval, Patrick; Cunin, Patrick; Autret, Gwennhael; Clement, Olivier; Rahmouni, Alain

2009-01-01

The purpose of this study was to determine whether an external magnet field can induce preferential trafficking of magnetically labeled Huh7 hepatoma cells to the liver following liver cell transplantation. Huh7 hepatoma cells were labeled with anionic magnetic nanoparticles (AMNP) and tagged with a fluorescent membrane marker (PKH67). Iron-uptake was measured by magnetophoresis. Twenty C57Bl6 mice received an intrasplenic injection of 2 x 10 6 labeled cells. An external magnet (0.29 T; 25 T/m) was placed over the liver of 13 randomly selected animals (magnet group), while the remaining 7 animals served as controls. MRI (1.5 T) and confocal fluorescence microscopy (CFM) were performed 10 days post-transplantation. The presence and location of labeled cells within the livers were compared in the magnet group and controls, and confronted with histological analysis representing the standard of reference. Mean iron content per cell was 6 pg. Based on histology, labeled cells were more frequently present within recipient livers in the magnet group (p < 0.01) where their distribution was preferentially peri-vascular (p<0.05). MRI and CFM gave similar results for the overall detection of transplanted cells (kappa=0.828) and for the identification of peri-vascular cells (kappa=0.78). Application of an external magnet can modify the trafficking of transplanted cells, especially by promoting the formation of perivascular aggregates. (orig.)

miR-150-5p inhibits hepatoma cell migration and invasion by targeting MMP14.

Directory of Open Access Journals (Sweden)

Tao Li

Full Text Available Hepatocellular carcinoma (HCC is one of the leading causes of cancer-related mortality worldwide. Despite progress in diagnostics and treatment of HCC, its prognosis remains poor because the molecular mechanisms underlying hepatocarcinogenesis are not well understood. In the study, we focused on identifying the role of miRNAs in HCC progression. miRNA microarray was used to analyze the differentially expressed miRNAs, and the results were validated by qPCR. We found that the miR-150-5p expression is down-regulated in HCC tissues compared with pair non-tumor tissues. miR-150-5p expression is also decreased in metastatic cancer tissues compared with pair primary tissues, indicating that miR-150-5p may be involved in HCC metastasis. Functionally, miR-150-5p inhibition significantly promotes hepatoma cell migration and invasion, whereas miR-150-5p overexpression suppresses cancer cell migration and invasion in vitro. The matrix metalloproteinase 14 (MMP14 is identified as a new target gene of miR-150-5p. miR-150-5p markedly inhibits MMP14 expression in hepatoma cells, and miR-150-5p expression is negative correlation with MMP14 expression in vivo. More important, re-expression of MMP14 in hepatoma cells partially reverses the effect of miR-150-5p in inhibiting cell invasion.

Hydrogen sulfide (H{sub 2}S)/cystathionine γ-lyase (CSE) pathway contributes to the proliferation of hepatoma cells

Energy Technology Data Exchange (ETDEWEB)

Pan, Yan; Ye, Shuang; Yuan, Dexiao; Zhang, Jianghong; Bai, Yang; Shao, Chunlin, E-mail: clshao@shmu.edu.cn

2014-05-15

Highlights: • Inhibition of H{sub 2}S/CSE pathway strongly stimulates cellular apoptosis. • Inhibition of H{sub 2}S/CSE pathway suppresses cell growth by blocking EGFR pathway. • H{sub 2}S/CSE pathway is critical for maintaining the proliferation of hepatoma cells. - Abstract: Hydrogen sulfide (H{sub 2}S)/cystathionine γ-lyase (CSE) pathway has been demonstrated to play vital roles in physiology and pathophysiology. However, its role in tumor cell proliferation remains largely unclear. Here we found that CSE over-expressed in hepatoma HepG2 and PLC/PRF/5 cells. Inhibition of endogenous H{sub 2}S/CSE pathway drastically decreased the proliferation of HepG2 and PLC/PRF/5 cells, and it also enhanced ROS production and mitochondrial disruption, pronounced DNA damage and increased apoptosis. Moreover, this increase of apoptosis was associated with the activation of p53 and p21 accompanied by a decreased ratio of Bcl-2/Bax and up-regulation of phosphorylated c-Jun N-terminal kinase (JNK) and caspase-3 activity. In addition, the negative regulation of cell proliferation by inhibition of H{sub 2}S/CSE system correlated with the blockage of cell mitogenic and survival signal transduction of epidermal growth factor receptor (EGFR) via down-regulating the extracellular-signal-regulated kinase 1/2 (ERK1/2) activation. These results demonstrate that H{sub 2}S/CSE and its downstream pathway contribute to the proliferation of hepatoma cells, and inhibition of this pathway strongly suppress the excessive growth of hepatoma cells by stimulating mitochondrial apoptosis and suppressing cell growth signal transduction.

Elimination of Cancer Stem-Like “Side Population” Cells in Hepatoma Cell Lines by Chinese Herbal Mixture “Tien-Hsien Liquid”

Directory of Open Access Journals (Sweden)

Chih-Jung Yao

2012-01-01

Full Text Available There are increasing pieces of evidence suggesting that the recurrence of cancer may result from a small subpopulation of cancer stem cells, which are resistant to the conventional chemotherapy and radiotherapy. We investigated the effects of Chinese herbal mixture Tien-Hsien Liquid (THL on the cancer stem-like side population (SP cells isolated from human hepatoma cells. After sorting and subsequent culture, the SP cells from Huh7 hepatoma cells appear to have higher clonogenicity and mRNA expressions of stemness genes such as SMO, ABCG2, CD133, β-catenin, and Oct-4 than those of non-SP cells. At dose of 2 mg/mL, THL reduced the proportion of SP cells in HepG2, Hep3B, and Huh7 cells from 1.33% to 0.49%, 1.55% to 0.43%, and 1.69% to 0.27%, respectively. The viability and colony formation of Huh7 SP cells were effectively suppressed by THL dose-dependently, accompanied with the inhibition of stemness genes, e.g., ABCG2, CD133, and SMO. The tumorigenicity of THL-treated Huh7 SP cells in NOD/SCID mice was also diminished. Moreover, combination with THL could synergize the effect of doxorubicin against Huh7 SP cells. Our data indicate that THL may act as a cancer stem cell targeting therapeutics and be regarded as complementary and integrative medicine in the treatment of hepatoma.

Magnetic targeting of iron-oxide-labeled fluorescent hepatoma cells to the liver

Energy Technology Data Exchange (ETDEWEB)

Luciani, Alain [Universite Rene Descartes, Hopital Europeen Georges Pompidou, Laboratoire de Recherche en Imagerie, EA 4062, Paris (France); Imagerie Medicale, Faculte de Medecine Paris XII, CHU Henri Mondor, Creteil cedex (France); Wilhelm, Claire; Gazeau, Florence [Universite Paris Diderot, Batiment Condorcet, Laboratoire Matiere et Systemes Complexes, CNRS-UMR 7057, Paris Cedex (France); Bruneval, Patrick [Anatomopathologie, Hopital Europeen Georges Pompidou, Paris (France); Cunin, Patrick [Unite de Recherche Clinique, Faculte de Medecine Paris XII, CHU Henri Mondor, Creteil cedex (France); Autret, Gwennhael; Clement, Olivier [Universite Rene Descartes, Hopital Europeen Georges Pompidou, Laboratoire de Recherche en Imagerie, EA 4062, Paris (France); Rahmouni, Alain [Imagerie Medicale, Faculte de Medecine Paris XII, CHU Henri Mondor, Creteil cedex (France)

2009-05-15

The purpose of this study was to determine whether an external magnet field can induce preferential trafficking of magnetically labeled Huh7 hepatoma cells to the liver following liver cell transplantation. Huh7 hepatoma cells were labeled with anionic magnetic nanoparticles (AMNP) and tagged with a fluorescent membrane marker (PKH67). Iron-uptake was measured by magnetophoresis. Twenty C57Bl6 mice received an intrasplenic injection of 2 x 10{sup 6} labeled cells. An external magnet (0.29 T; 25 T/m) was placed over the liver of 13 randomly selected animals (magnet group), while the remaining 7 animals served as controls. MRI (1.5 T) and confocal fluorescence microscopy (CFM) were performed 10 days post-transplantation. The presence and location of labeled cells within the livers were compared in the magnet group and controls, and confronted with histological analysis representing the standard of reference. Mean iron content per cell was 6 pg. Based on histology, labeled cells were more frequently present within recipient livers in the magnet group (p < 0.01) where their distribution was preferentially peri-vascular (p<0.05). MRI and CFM gave similar results for the overall detection of transplanted cells (kappa=0.828) and for the identification of peri-vascular cells (kappa=0.78). Application of an external magnet can modify the trafficking of transplanted cells, especially by promoting the formation of perivascular aggregates. (orig.)

Inhibitory effects of α-pinene on hepatoma carcinoma cell proliferation.

Science.gov (United States)

Chen, Wei-Qiang; Xu, Bin; Mao, Jian-Wen; Wei, Feng-Xiang; Li, Ming; Liu, Tao; Jin, Xiao-Bao; Zhang, Li-Rong

2014-01-01

Pine needle oil from crude extract of pine needles has anti-tumor effects, but the effective component is not known. In the present study, compounds from a steam distillation extract of pine needles were isolated and characterized. Alpha-pinene was identified as an active anti-proliferative compound on hepatoma carcinoma BEL-7402 cells using the MTT assay. Further experiments showed that α-pinene inhibited BEL-7402 cells by arresting cell growth in the G2/M phase of the cell cycle, downregulating Cdc25C mRNA and protein expression, and reducing cycle dependence on kinase 1(CDK1) activity. Taken together, these findings indicate that α-pinene may be useful as a potential anti-tumor drug.

Characterization of genetically engineered mouse hepatoma cells with inducible liver functions by overexpression of liver-enriched transcription factors.

Science.gov (United States)

Yamamoto, Hideaki; Tonello, Jane Marie; Sambuichi, Takanori; Kawabe, Yoshinori; Ito, Akira; Kamihira, Masamichi

2018-01-01

New cell sources for the research and therapy of organ failure could significantly alleviate the shortage of donor livers that are available to patients who suffer from liver disease. Liver carcinoma derived cells, or hepatoma cells, are the ideal cells for developing bioartificial liver systems. Such cancerous liver cells are easy to prepare in large quantities and can be maintained over long periods under standard culture conditions, unlike primary hepatocytes. However, hepatoma cells possess only a fraction of the functions of primary hepatocytes. In a previous study, by transducing cells with liver-enriched transcription factors that could be inducibly overexpressed-hepatocyte nuclear factor (HNF)1α, HNF1β, HNF3β [FOXA2], HNF4α, HNF6, CCAAT/enhancer binding protein (C/EBP)α, C/EBPβ and C/EBPγ-we created mouse hepatoma cells with high liver-specific gene expression called the Hepa/8F5 cell line. In the present study, we performed functional and genetic analyses to characterize the Hepa/8F5 cell line. Further, in three-dimensional cultures, the function of these cells improved significantly compared to parental cells. Ultimately, these cells might become a new resource that can be used in basic and applied hepatic research. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

HYPOLIPIDEMIC EFFECT OF ARGLABIN IN HEPATOMA TISSUE CULTURE

Directory of Open Access Journals (Sweden)

A. V. Ratkin

2015-01-01

Full Text Available Objective. Investigation of hypolipidemic effect of sesquiterpene γ-lactone Arglabin in hepatoma tissue culture (HTC.Materials and methods. In this study we’ve evaluated the effect of sesquiterpene γ-lactone Arglabin and gemfibrozil (reference drug on the lipid content in the hepatoma tissue culture (HTC which were incubated with a fat emulsion “Lipofundin” by fluorescent method with vital dye Nile Red. The cell viability was investigated using the MTT-test and staining by Trypan blue.Results. Cultivation of cell cultures of rat’s hepatoma cell line HTC with Arglabin and gemfibrozil in concentrations from 10 to 50 μmol and from 0.25 to 0.5 mmol, respectively, had no cytotoxic effect. HTC cell viability did not change compared with the corresponding rate in the control culture. Experimental hyperlipidemia in hepatoma culture was induced by the addition in the incubation medium of fat emulsion “Lipofundin” in a final concentration of 0.05 %. The fluorescence intensity of Nile Red in the cells was increased 4-fold (p < 0.05, which indicates a significant accumulation of lipids in the cytosol of cells. In these steady-state Arglabin and gemfibrozil at concentrations 75–100 μM and 0.25–1.0 mM, respectively, reduced the content of lipid in cells. Conclusion. In the model of hyperlipidemia induced by lipofundin, sesquiterpene γ-lactone Arglabin prevents the accumulation of lipids in the HTC cell line, as evidenced by a decrease in Nile Red fluorescence. However hypolipidemic effect of Arglabin is associated with cytotoxic effects, which is typical for anticancer drugs.

Pokemon Silencing Leads to Bim-Mediated Anoikis of Human Hepatoma Cell QGY7703

OpenAIRE

Liu, Kun; Liu, Feng; Zhang, Nannan; Liu, Shiying; Jiang, Yuyang

2012-01-01

Pokemon is an important proto-oncogene that plays a critical role in cellular oncogenic transformation and tumorigenesis. Anoikis, which is regulated by Bim-mediated apoptosis, is critical to cancer cell invasion and metastasis. We investigated the role of Pokemon in anoikis, and our results show that Pokemon renders liver cells resistant to anoikis via suppression of Bim transcription. We knocked-down Pokemon in human hepatoma cells QGY7703 with small interfering RNAs (siRNA). Knockdown of P...

Synergistic effect of intervention of glypican-3 gene transcription combined with antitumor drugs in inhibiting hepatoma cell proliferation

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YANG Jie

2016-12-01

Full Text Available ObjectiveTo investigate the inhibitory effect of intervention of glypican-3 (GPC3 gene transcription combined with antitumor drugs on hepatoma cell proliferation. MethodsFour types of GPC3-shRNA plasmids were established and transfected into HepG2 hepatoma cells. Quantitative real-time PCR and Western blot were used to measure the mRNA and protein expression of GPC3 to analyze its association with hepatoma cell proliferation and apoptosis. The independent samples t-test was used for comparison of continuous data between any two groups, and a one-way analysis of variance was used for comparison between multiple groups. ResultsAmong these four plasmids, shRNA1 had a transfection efficiency of ＞85% in the transfection of HepG2 cells and a silence efficiency of 89.3% at the mRNA level, and the protein expression of GPC3 was significantly inhibited(P＜0.01）. At 72 hours, the GPC3-shRNA1 co-intervention group had an HepG2 cell inhibition rate of 71.1%, significantly different from that in the negative group (t=18.092, P＜0.001, an inhibition rate of migration of 89.1%, significantly lower than that in the negative group (t=8.326, P＜0.001, and inhibition rates of HepG2 cell movement and invasion of 53.6% and 60.1%, which were significantly different from those in the negative group (t=52.400 and 48.245, both P＜0.001. The GPC3-shRNA1 co-intervention group had a β-catenin mRNA inhibition rate of 46.9% and a Gli1 mRNA upregulation rate of 7.4%, significantly different from those in the negative group (t=30.108 and -3.551, P＜0.001 and P=0.009. At 24 hours, 10 μmol/L sorafenib combined with shRNA1 had an inhibition rate of tumor cells of 52.6% and 100 μmol/L sorafenib combined with shRNA1 had an inhibition rate of tumor cells of 79.5%, which were significantly different from that in the control group (t=23.314 and 50.352, both P＜0.001. The half-maximal inhibitory concentrations of sorafenib, rapamycin, and erlotinib for HepG2 were 4.67±1

Comparison of the effect of interferon on two human hepatoma cell lines

Energy Technology Data Exchange (ETDEWEB)

Crespi, M; Schoub, B D; Lyons, S F; Chiu, M N [University of the Witwatersrand, Johannesburg (South Africa). Dept. of Virology

1985-06-01

Two human hepatoma cell lines, the PLC/PRF/5 and the Mahlavu cells, which differ in their production of the hepatitis B surface antigen (HBsAg), responded differently to interferon (IFN). After IFN treatment both cell lines were able to inhibit Sindbis virus replication. Oligo A synthetase (E enzyme) could be activated in the PLC/PRF/5 cells although they were not sensitive to exogenous 2 - 5 oligoadenylic acid (2 - 5 A). In contrast, the Mahlavu cells were sensitive to exogenous 2 - 5 A, but unable to activate the E enzyme. Both cell lines were unable to stimulate phosphorylation of the exogenous initiator factor eIF-2.

INVESTIGATION OF HYPOLIPIDEMIC EFFECT OF SESQUITERPENE Γ-LACTONE AHILLIN IN HEPATOMA TISSUE CULTURE (HTC CELLS

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V. V. Ivanov

2014-01-01

Full Text Available Objective. Investigation of hypolipidemic effect of sesquiterpene γ-lactone ahillin in hepatoma tissue culture (HTC cells.Material and methods. In this study we’ve evaluated the effect of γ-lactone sesquiterpene aсhillin and gemfibrozil (comparator drug on the lipid content in the hepatoma tissue culture (HTC cell which were incubated with a fat emulsion lipofundin by fluorescent method with vital dye Nile Redand staining the cells with the dye Oil Red O. The cell viability was investigated using the MTT-test and staining with Trypan blue.Results. Cultivation cells HTC with aсhillin and gemfibrozilat concentrations ranging from 0.5 to1.5 mM and from0.25 mM to0.5 mM, respectively, resulted in dose-dependent decrease of the fluorescence’s intensity Nile Red. It reflects a decrease in lipid content in the cells. At these concentrations the drugs didn’t have cytotoxic effect and the cell viability didn’t change compared to the control culture.An experimental hyperlipidemia in the hepatoma culture cells was induced by adding to the incubation medium a fat emulsion lipofundin at a final concentration 0.05%. The intensity of fluorescence Nile Red in the cells was increased 4 fold (p < 0.05. This result suggests the significant accumulation of lipids in the cell’s cytosol and confirmed by microscopy after staining neutral lipids with the dye Oil Red O. Under these conditions aсhillin and gemfibrozil reduced lipid content in cells and hadthe effect at concentrations of0.5 mM and0.25 mM respectively.Conclusion. In the lipofundin-mediated model of hyperlipidemia the sesquiterpene lactone aсhillin prevents the lipid accumulation in cells. It confirms by decrease of fluorescence Nile Red and reduction lipid drops which were stained with Oil Red O in cytosol. To establish the molecular targets of aсhillin’saction on lipid metabolism in cell culture HTC we need to investigate a gene expression of key enzymes of lipid metabolism.

Role of ROS-mediated autophagy in radiation-induced bystander effect of hepatoma cells.

Science.gov (United States)

Wang, Xiangdong; Zhang, Jianghong; Fu, Jiamei; Wang, Juan; Ye, Shuang; Liu, Weili; Shao, Chunlin

2015-05-01

Autophagy plays a crucial role in cellular response to ionizing radiation, but it is unclear whether autophagy can modulate radiation-induced bystander effect (RIBE). Here, we investigated the relationship between bystander damage and autophagy in human hepatoma cells of HepG2. HepG2 cells were treated with conditioned medium (CM) collected from 3 Gy γ-rays irradiated hepatoma HepG2 cells for 4, 12, or 24 h, followed by the measurement of micronuclei (MN), intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and protein expressions of microtubule-associated protein 1 light chain 3 (LC3) and Beclin-1 in the bystander HepG2 cells. In some experiments, the bystander HepG2 cells were respectively transfected with LC3 small interfering RNA (siRNA), Beclin-1 siRNA or treated with 1% dimethyl sulfoxide (DMSO). Additional MN and mitochondrial dysfunction coupled with ROS were induced in the bystander cells. The expressions of protein markers of autophagy, LC3-II/LC3-I and Beclin-1, increased in the bystander cells. The inductions of bystander MN and overexpressions of LC3 and Beclin-1 were significantly diminished by DMSO. However, when the bystander cells were transfected with LC3 siRNA or Beclin-1 siRNA, the yield of bystander MN was significantly enhanced. The elevated ROS have bi-functions in balancing the bystander effects. One is to cause MN and the other is to induce protective autophagy.

Radiosensitization by inhibiting survivin in human hepatoma HepG2 cells to high-LET radiation

International Nuclear Information System (INIS)

Jin Xiaodong; Li Qiang; Wu Qingfeng; Li Ping; Gong Li; Hao Jifang; Dai Zhongying; Matsumoto, Yoshitaka; Furusawa, Yoshiya

2011-01-01

In this study, whether survivin plays a direct role in mediating high-linear energy transfer (LET) radiation resistance in human hepatoma cells was investigated. Small interfering RNA (siRNA) targeting survivin mRNA was designed and transfected into human hepatoma HepG2 cells. Real-time polymerase chain reaction (PCR) and western blotting analyses revealed that survivin expression in HepG2 cells decreased at both transcriptional and post-transcriptional levels after treatment with survivin-specific siRNA. Caspase-3 activity was determined with a microplate reader assay as well. Following exposure to high-LET carbon ions, a reduced clonogenic survival effect, increased apoptotic rates and caspase-3 activity were observed in the cells treated with the siRNA compared to those untreated with the siRNA. The cells with transfection of the survivin-specific siRNA also increased the level of G 2 /M arrest. These results suggest that survivin definitely plays a role in mediating the resistance of HepG2 cells to high-LET radiation and depressing survivin expression might be useful to improve the therapeutic efficacy of heavy ions for radioresistant solid tumors. (author)

Uptake of 153Sm-DTPA-bis-biotin and 99mTc-DTPA-bis-biotin in rat as-30D-hepatoma cells

International Nuclear Information System (INIS)

Correa-Gonzalez, Luis; Arteaga de Murphy, Consuelo; Ferro-Flores, Guillermina; Pedraza-Lopez, Martha; Murphy-Stack, Eduardo; Mino-Leon, Dolores; Perez-Villasenor, Graciela; Diaz-Torres, Yaneth; Munoz-Olvera, Rodrigo

2003-01-01

Labeled biotin has been used mainly for pretargeted therapy, an approach for increasing the amount of radioactivity delivered to a cancer cell. The aim of this investigation was to prepare 153 Sm-DTPA-bis-biotin and 99m Tc-DTPA-bis-biotin in order to study their in vitro and in vivo uptake in rat AS-30D hepatoma cells found in ascites and in implanted tumor. DTPA-bis-biotin (pH 8) was 153 Sm labeled with 153 SmCl 3 and 99m Tc-DTPA-bis-biotin was prepared via SnCl 2 reduction. Radiochemical purity was >98% in both cases. AS-30D hepatoma cells were obtained from ascites of a rat with hepatoma and were propagated in the peritoneum cavity of normal rats. In vitro ascites cell 153 Sm-DTPA-bis-biotin uptake was compared with 153 SmCl 3 cell uptake. The ratio cell 153 Sm-DTPA-bis-biotin/ 153 SmCl 3 was 39.6 and when avidin was added it increased to 50. The ratio 99m Tc-DTPA-bis-biotin/TcO 4 Na was 8.7. Concentration of 153 Sm-DTPA-bis-biotin in tumor 2, 3 and 24 h after administration, was 5, 15 and 3 times higher than in normal muscle (T/nT). Biodistribution in a 0.083-24 h time period showed that 153 Sm-DTPA-bis-biotin was taken up only by ascites tumor cells and hepatoma cells. Two and 3 h ratio ascites/liver (As/Lv) was 6.4 and 6.0. For 99m Tc-DTPA-bis-biotin 2 and 3 h T/nT was 15.7 and 4.7 and 2 h As/Lv was 1.4. In conclusion, both radiopharmaceuticals show high uptake in rat AS-30D hepatoma cells in ascites and in implanted tumor. Since lung, thyroid, kidney, liver or pancreas carcinomas are ascites producing cancers 153 Sm-DTPA-bis-biotin would be an adequate therapeutic radiopharmaceutical for these patients whose life quality would be enhanced with control of ascites, and a reduction of the primary tumor and its metastases

Release of overexpressed CypB activates ERK signaling through CD147 binding for hepatoma cell resistance to oxidative stress.

Science.gov (United States)

Kim, Kiyoon; Kim, Hunsung; Jeong, Kwon; Jung, Min Hyung; Hahn, Bum-Soo; Yoon, Kyung-Sik; Jin, Byung Kwan; Jahng, Geon-Ho; Kang, Insug; Ha, Joohun; Choe, Wonchae

2012-08-01

Cyclophilin, a cytosolic receptor for the immunosuppressive drug cyclosporin A, plays a role in diverse pathophysiologies along with its receptor, CD147. Although the interaction between cyclophilin A and CD147 is well established in inflammatory disease, that of cyclophilin B (CypB) with CD147 has not been fully explored, especially in cancer cell biology, and the exact molecular mechanism underlying such an association is poorly understood. In this study, we first identified high expression levels of CypB in 54 % of hepatocellular carcinoma patient tissues but in only 12.5 % of normal liver tissues. Then, we demonstrated that CypB overexpression protects human hepatoma cells against oxidative stress through its binding to CD147; this protective effect depends on the peptidyl prolyl isomerase activity of CypB. siRNA-mediated knockdown of CypB expression rendered hepatoma cells more vulnerable to ROS-mediated apoptosis. Furthermore, we also determined that a direct interaction between secreted CypB and CD147 regulates the extracellular signal-regulated kinase intracellular signaling pathway and is indispensible for the protective functions of CypB. For the first time, we demonstrated that CypB has an essential function in protecting hepatoma cells against oxidative stress through binding to CD147 and regulating the ERK pathway.

I-123-insulin: A new marker for hepatoma

International Nuclear Information System (INIS)

Sodoyez, J.C.; Goffaux, F.S.; Fallais, C.; Bourgeois, P.

1984-01-01

Previous studies have demonstrated that carrier-free I-123-Tyr Al4 insulin was taken up by the liver (by a saturable mechanism) and by the kidneys (by a non saturable mechanism). Autoradiographs of rat liver after injection of I-125-insulin showed that binding specifically occurred at the plasma membrane of the hepatocytes. I-123-Insulin binding to the hepatocyte plasma membrane appeared mediated by specific receptors. Indeed it was blocked by antibodies to the insulin receptors and by an excess of native insulin. Futhermore insulin derivatives with low biological potency (proinsulin and desoctapeptide insulin) did not inhibit I-123-insulin binding to the hepatocytes. I-123-Insulin (1.3 mCi) was I.V. injected into a patient in whom the right liver lobe was normal (normal uptake of Tc-99m-colloid sulfur) but the left liver lobe was occupied by a voluminous hepatoma (no uptake of Tc-99m-colloid sulfur). Liver blood supply was also studied by Tc-99m-pyrophosphate-labeled red cells. Computer analysis of the data revealed that compared to the normal liver lobe, binding of I-123-insulin to the hepatoma was more precocious (vascularization through the hepatic artery and not the portal vein), more intense and more prolonged (half-lives were 6 min in the normal liver and 14 min in the hepatoma). These results are consistent with characteristics of hepatoma cells in culture in which high insulin binding capacity contrasts with a markedly decreased insulin degrading activity. It is concluded that I-123-insulin may be used as a specific marker of hepatoma in man

Bioactive chemical constituents of Curcuma longa L. rhizomes extract inhibit the growth of human hepatoma cell line (HepG2).

Science.gov (United States)

Abdel-Lateef, Ezzat; Mahmoud, Faten; Hammam, Olfat; El-Ahwany, Eman; El-Wakil, Eman; Kandil, Sherihan; Abu Taleb, Hoda; El-Sayed, Mortada; Hassenein, Hanaa

2016-09-01

The present study was designed to identify the chemical constituents of the methanolic extract of Curcuma longa L. rhizomes and their inhibitory effect on a hepatoma cell line. The methanolic extract was subjected to GC-MS analysis to identify the volatile constituents and the other part of the same extract was subjected to liquid column chromatographic separation to isolate curcumin. The inhibition of cell growth in the hepatoma cell line and the cytopathological changes were studied. GC-MS analysis showed the presence of fifty compounds in the methanolic extract of C. longa. The major compounds were ar-turmerone (20.50 %), β-sesquiphellandrene (5.20 %) and curcumenol (5.11 %). Curcumin was identified using IR, 1H and 13C NMR. The inhibition of cell growth by curcumin (IC50 = 41.69 ± 2.87 μg mL-1) was much more effective than that of methanolic extract (IC50 = 196.12 ± 5.25 μg mL-1). Degenerative and apoptotic changes were more evident in curcumin- treated hepatoma cells than in those treated with the methanol extract. Antitumor potential of the methanolic extract may be attributed to the presence of sesquiterpenes and phenolic constituents including curcumin (0.051 %, 511.39 μg g-1 dried methanol extract) in C. longa rhizomes.

Alterations in microRNA expression profile in HCV-infected hepatoma cells: Involvement of miR-491 in regulation of HCV replication via the PI3 kinase/Akt pathway

Energy Technology Data Exchange (ETDEWEB)

Ishida, Hisashi; Tatsumi, Tomohide; Hosui, Atsushi; Nawa, Takatoshi; Kodama, Takahiro; Shimizu, Satoshi; Hikita, Hayato; Hiramatsu, Naoki; Kanto, Tatsuya [Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, 2-2, Yamadaoka, Suita 565-0871 (Japan); Hayashi, Norio [Kansai Rosai Hospital, 3-1-69, Inabaso, Amagasaki 660-8511 (Japan); Takehara, Tetsuo, E-mail: takehara@gh.med.osaka-u.ac.jp [Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, 2-2, Yamadaoka, Suita 565-0871 (Japan)

2011-08-19

Highlights: {yields} HCV infection upregulated miR-192, -194, -215, downregulated miR-320, -491. {yields} Transfection of miR-192, -215, and -491 enhanced HCV replication. {yields} Transfection of miR-491 inhibited Akt phosphorylation. {yields} Akt inhibition could be responsible for augmentation of HCV replication by miR-491. -- Abstract: The aim of this study was to investigate the role of microRNA (miRNA) on hepatitis C virus (HCV) replication in hepatoma cells. Using miRNA array analysis, miR-192/miR-215, miR-194, miR-320, and miR-491 were identified as miRNAs whose expression levels were altered by HCV infection. Among them, miR-192/miR-215 and miR-491 were capable of enhancing replication of the HCV replicon as well as HCV itself. HCV IRES activity or cell proliferation was not increased by forced expression of miR-192/miR-215 or miR-491. Investigation of signaling pathways revealed that miR-491 specifically suppressed the phosphoinositol-3 (PI3) kinase/Akt pathway. Under inhibition of PI3 kinase by LY294002, the suppressive effect of miR-491 on HCV replication was abolished, indicating that suppression of HCV replication by miR-491 was dependent on the PI3 kinase/Akt pathway. miRNAs altered by HCV infection would then affect HCV replication, which implies a complicated mechanism for regulating HCV replication. HCV-induced miRNA may be involved in changes in cellular properties including hepatocarcinogenesis.

Radiation-induced cell disintegrations in cultured rat hepatoma cells JTC 2

International Nuclear Information System (INIS)

Sakka, Masatoshi

1979-01-01

Disintegration of hepatoma cells of rat were recorded by time lapse cinemicrography for more than 5 days and about 1000 pedigrees were analyzed. Five generations were followed up in control and 2 or 3 generations in irradiated cells. Cells were attached on vessel wall spreading themselves in intermitotic phase while they stood up from the wall in mitotic phase taking a roun form. When a cell disintegrates in interphase the disintegration is called D sub( s) and one in mitotic period D sub( r). The frequency of D sub( s)S' is about 3 times as much as D sub( r)S'. An age of a disintegrated cell in generation 1 and 2 was measured as the previous mitosis was age 0. Generation times of the comparable generations of surviving sister branches of the same pedigrees were used as controls. Most disintegration took place at the same age with surviving sisters indicating a determined, not at random, age of cell death. A cell in an initial state flowed to any one of the following states with or without irradiation; surviving, disintegrated, end cell or escaping out of observation field. A single exposure of 400 to 900 R induced a typical reproductive death but effective extinction of clones was observed only in small pedigrees. Temporary hypothermia and hyperthermia immediately after exposure had no remarkable lethal effects on several early generations. (author)

Kinetic imaging of NPC1L1 and sterol trafficking between plasma membrane and recycling endosomes in hepatoma cells

DEFF Research Database (Denmark)

Hartwig Petersen, Nicole; Færgeman, Nils J; Yu, Liqing

2008-01-01

fluorescent protein (NPC1L1-EGFP) and cholesterol analogues in hepatoma cells. At steady state about 42% of NPC1L1 resided in the transferrin (Tf) positive, sterol enriched endocytic recycling compartment (ERC), while time-lapse microscopy demonstrated NPC1L1 traffic between plasma membrane and ERC...... the ERC to the plasma membrane. NPC1L1-EGFP facilitated transport of fluorescent sterols from the plasma membrane to the ERC. Insulin induced translocation of vesicles containing NPC1L1 and fluorescent sterol from the ERC to the cell membrane. Upon polarization of hepatoma cells NPC1L1 resided almost...... exclusively in the canalicular membrane, where the protein is highly mobile. Our study demonstrates dynamic trafficking of NPC1L1 between cell surface and intracellular compartments and suggests that this transport is involved in NPC1L1 mediated cellular sterol uptake....

Hepatitis B virus X protein promotes interleukin-7 receptor expression via NF-κB and Notch1 pathway to facilitate proliferation and migration of hepatitis B virus-related hepatoma cells

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Fanyun Kong

2016-11-01

Full Text Available Abstract Background Interleukin-7 receptor (IL-7R is involved in the abnormal function of solid tumors, but the role and regulatory mechanisms of IL-7R in HBV-related hepatocellular carcinoma (HCC are still unclear. Methods Gene and protein expression levels of IL-7R were examined in hepatoma cells transfected with hepatitis B virus (HBV plasmids and in hepatoma cells transfected with the multifunctional nonstructural protein X (HBX. The expression of HBX and IL-7R was measured by immunohistochemical analysis in HBV-related HCC tissues. The role of NF-κB and Notch1 pathways in HBX-mediated expression of IL-7R in hepatoma cells was examined. Activation of IL-7R downstream of intracellular signaling proteins AKT, JNK, STAT5, and the associated molecules CyclinD1 and matrix metalloproteinase-9 (MMP-9, was assessed in HBX-positive cells with or without treatment with IL-7R short hairpin RNA (shRNA. Additionally, the role of IL-7R in HBX-mediated proliferation and migration of hepatoma cells was investigated. Results The expression of IL-7R was increased in hepatoma cells transfected with HBV plasmids; HBX was responsible for the HBV-mediated upregulation of IL-7R. Compared to adjacent tissues, the expression of HBX and IL-7R was increased in HBV-related HCC tissues. Additionally, the relative expression levels of HBX were associated with IL-7R in HBV-related HCC tissues. The activation of NF-κB pathways and expression of Notch1 were increased in hepatoma cells transfected with HBX, and inhibition of NF-κB and Notch1 pathways significantly decreased HBX-mediated expression of IL-7R. The activation of AKT and JNK and the expression of CyclinD1 and MMP-9 were increased in HBX-positive cells. When cells were treated with IL-7R shRNA, the activation of AKT and JNK, as well as the expression of CyclinD1 and MMP-9, were significantly inhibited. Additionally, IL-7R was responsible for HBX-induced proliferation and migration ability of hepatoma cells

Inhibitory effect of chitosan oligosaccharide on human hepatoma ...

African Journals Online (AJOL)

Background: Chitosan oligosaccharide, the degradation products of chitin, was reported to have a wide range of physiological functions and biological activities. In this study, we explored the inhibitory effect of Chitosan oligosaccharide on human hepatoma cells. Materials and Methods: MTT assay was applied to detect cell ...

Isolation and establishment of radiotolerant hepatoma cell subline

International Nuclear Information System (INIS)

Jin Wensen; Kong Zhaolu; Zhang Jianghong; Shen Zhifen; Tong Shungao; Ji Huajun; Jin Yizun

2009-01-01

Objective: To induce and isolate the monoclonal cell subline, in order to establish the experimental model for further investigating biologic characteristics in radiotolerant hepatoma cells. Methods: HepG2 cells were irradiated by γ-rays at the dose of 2 Gy each time with the total absorbed dose of 60 Gy. After monoclonal cell being selected and extensively cultured, the cell subline was named as HepG2/R60. Furthermore, HepG2/R60 cells were identified by observing the changes of morphology, ultrastructure, growth characteristics and radiosensitivity. The levels of radioresistant correlative gene mRNA in HepG2/R60 cells after exposure to 2 Gy irradiation, were also detected by RT-PCR, and then compared with parental HepG2 cells. Results: HepG2/R60 cell subline was successfully established by fractionated irradiation at 2 Gy. HepG2/R60 cells displayed higher irregularity, the clearer appearance and dissociation of cell junctions compared with parental HepG2 cells. Ultrastnictural investigations through transmission electron microscopy (TEM) showed that there was an increase of microvillus on the surfaces of HepG2/R60 cells with plenty of rough endo-plasmic reticulum, abundance of mitochondria and viable Golgi complex. Further observation found that the growth of HepG2/R60 cells was slower and its population doubling time (PDT) prolonged arrived at 34.9 h. Moreover, the radiosensitivity of HepG2/R60 cells was lower than that of parental HepG2 cells. Additionally, the levels of radioresistance correlative genes were increased in HepG2/R60 cells by 2 Gy irradiaiton Conclusions: Radiotolerant cell subline - HepG2/R60 was successfully isolated and established by fractionated irradiation. (authors)

Liver regeneration in mice bearing a transplanted hepatoma.

Science.gov (United States)

Badran, A F; Moreno, F R; Echave Llanos, J M

1984-01-01

The hepatocyte mitotic index curve in hepatectomized hepatoma-bearing mice, rises earlier, has a greater amplitude and is less synchronized than that of normal hepatectomized mice. This indicates a stimulation (more mitosis in a shorter time period) produced by the presence of the tumors. The sinusoid litoral cells mitotic index curve in hepatectomized hepatoma-bearing mice appears earlier and is much less synchronized than that of normal hepatectomized mice. Nevertheless both curves have the same amplitude for the whole sampling period and the early stimulation is quickly compensated by lower values (apparent inhibition) appearing in the resting (light) period.

Effects of interferon on cultured cells persistently infected with viruses

Energy Technology Data Exchange (ETDEWEB)

Crespi, M

1986-01-01

The role of interferon (IFN) in viral persistence at the cellular level was investigated. Two types of persistent infections were chosen. The first type was cell lines which contained hepatitis B virus (HBV) DNA (PLC/PRF/5 and Hep 3B cells) uninfected control hepatoma cells, (Mahlavu, HA22T and Hep G2 cells) or simian virus 40 (SV40) DNA (C2, C6, C11 cells) and control uninfected (CV-1 cells). In the second type of infection Vero cells persistently infected with SSPE or Sendai virus were used. The aim of this work was to determine what effect IFN had in these infections in terms of its antiviral and antiproliferative effects; which of the two major IFN-induced pathways, E enzyme or protein kinase were induced; whether there were any differences in sensitivity to IFN between the DNA and RNA virus persistent infections. The anti-viral effect of IFN was examined by its ability to inhibit Sindbis virus replication using a radioimmunoassay system. The antiproliferative effect of IFN was determined by cell counting and /sup 3/H-thymidine incorporation. The activation of the ribonuclease F, determined by the inhibition of /sup 3/H-leucine incorporation after introduction of 2-5 actin into the cells, was variable, being activated in all cell lines with the exception of the PLC/PRF/5, Hep 3B and Hep G2 cells. Major differences between the two DNA persistent infections and the two RNA persistent infections were found. No correlation was found between the presence of HBV or SV40 persistent infections and the sensitivity of the cell lines to IFN. Both the SSPE and Sendai virus persistent infections were resistant to the antiviral and antiproliferative effect of IFN.

Dual RNAseq shows the human mucosal immunity protein, MUC13, is a hallmark of Plasmodium exoerythrocytic infection

KAUST Repository

LaMonte, Gregory; Orjuela-Sanchez, Pamela; Wang, Lawrence; Li, Shangzhong; Swann, Justine; Cowell, Annie; Zou, Bing Yu; Abdel- Haleem Mohamed, Alyaa; Villa-Galarce, Zaira; Moreno, Marta; Tong-Rios, Carlos; Vinetz, Joseph; Lewis, Nathan; Winzeler, Elizabeth A

2017-01-01

The exoerythrocytic stage of Plasmodium malaria infection is a critical window for prophylactic intervention. Using a genome-wide dual RNA sequencing of flow-sorted infected and uninfected hepatoma cells we identify the human mucosal immunity gene, Mucin13 (MUC13), as strongly upregulated during Plasmodium exoerythrocytic hepatic-stage infection. We confirm that MUC13 expression is upregulated in hepatoma cell lines and primary hepatocytes. In immunofluorescence assays, host MUC13 protein expression distinguishes infected cells from adjacent uninfected cells and shows similar colocalization with parasite biomarkers such as UIS4 and HSP70. We further show that localization patterns are species independent, distinguishing both P. berghei and P. vivax infected cells, and that MUC13 can be used to identify compounds that inhibit parasite replication in hepatocytes across all Human-infecting Plasmodium species. This data presents a novel interface of host-parasite interactions in Plasmodium, in that a component of host mucosal immunity is reprogrammed to assist the progression of infection.

Dual RNAseq shows the human mucosal immunity protein, MUC13, is a hallmark of Plasmodium exoerythrocytic infection

KAUST Repository

LaMonte, Gregory

2017-10-03

The exoerythrocytic stage of Plasmodium malaria infection is a critical window for prophylactic intervention. Using a genome-wide dual RNA sequencing of flow-sorted infected and uninfected hepatoma cells we identify the human mucosal immunity gene, Mucin13 (MUC13), as strongly upregulated during Plasmodium exoerythrocytic hepatic-stage infection. We confirm that MUC13 expression is upregulated in hepatoma cell lines and primary hepatocytes. In immunofluorescence assays, host MUC13 protein expression distinguishes infected cells from adjacent uninfected cells and shows similar colocalization with parasite biomarkers such as UIS4 and HSP70. We further show that localization patterns are species independent, distinguishing both P. berghei and P. vivax infected cells, and that MUC13 can be used to identify compounds that inhibit parasite replication in hepatocytes across all Human-infecting Plasmodium species. This data presents a novel interface of host-parasite interactions in Plasmodium, in that a component of host mucosal immunity is reprogrammed to assist the progression of infection.

The interaction of HAb18G/CD147 with integrin α6β1 and its implications for the invasion potential of human hepatoma cells

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Tang Juan

2009-09-01

Full Text Available Abstract Background HAb18G/CD147 plays pivotal roles in invasion by hepatoma cells, but the underlying mechanism remains unclear. Our previous study demonstrated that overexpression of HAb18G/CD147 promotes invasion by interacting with integrin α3β1. However, it has never been investigated whether α3β1 is solely responsible for this process or if other integrin family members also interact with HAb18G/CD147 in human hepatoma cells. Methods Human SMMC-7721 and FHCC98 cells were cultured and transfected with siRNA fragments against HAb18G/CD147. The expression levels of HAb18G/CD147 and integrin α6β1 were determined by immunofluorescent double-staining and confocal imaging analysis. Co-immunoprecipitation and Western blot analyses were performed to examine the native conformations of HAb18G/CD147 and integrin α6β1. Invasion potential was evaluated with an invasion assay and gelatin zymography. Results We found that integrin α6β1 co-localizes and interacts with HAb18G/CD147 in human hepatoma cells. The enhancing effects of HAb18G/CD147 on invasion capacity and secretion of matrix metalloproteinases (MMPs were partially blocked by integrin α6β1 antibodies (P 2+ mobilization, significantly reduced cell invasion potential and secretion of MMPs in human hepatoma cells (P Conclusion These results suggest that α6β1 interacts with HAb18G/CD147 to mediate tumor invasion and metastatic processes through the PI3K pathway.

A Role for CD81 and Hepatitis C Virus in Hepatoma Mobility

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Claire L. Brimacombe

2014-03-01

Full Text Available Tetraspanins are a family of small proteins that interact with themselves, host transmembrane and cytosolic proteins to form tetraspanin enriched microdomains (TEMs that regulate important cellular functions. Several tetraspanin family members are linked to tumorigenesis. Hepatocellular carcinoma (HCC is an increasing global health burden, in part due to the increasing prevalence of hepatitis C virus (HCV associated HCC. The tetraspanin CD81 is an essential receptor for HCV, however, its role in hepatoma biology is uncertain. We demonstrate that antibody engagement of CD81 promotes hepatoma spread, which is limited by HCV infection, in an actin-dependent manner and identify an essential role for the C-terminal interaction with Ezrin-Radixin-Moesin (ERM proteins in this process. We show enhanced hepatoma migration and invasion following expression of CD81 and a reduction in invasive potential upon CD81 silencing. In addition, we reveal poorly differentiated HCC express significantly higher levels of CD81 compared to adjacent non-tumor tissue. In summary, these data support a role for CD81 in regulating hepatoma mobility and propose CD81 as a tumour promoter.

Evidence for ligand and/or receptor-specific mechanisms of internalization and processing in cultured H35 hepatoma cells

International Nuclear Information System (INIS)

Goldberg, R.I.; Smith, R.M.; Jarett, L.

1987-01-01

Total cell associated (TC) and intracellularly accumulated (IC) 125 I-labeled insulin (INS) or α-2-macroglobulin (α2M) were assessed in cultured H35 hepatoma cells which were preincubated with various agents. Cytochalasin D or sodium azide, which affect microfilament- or energy-dependent receptor internalization, had no significant effects on INS TC or IC but each decreased α2M TC and IC to 50-75% of control. Monensin and chloroquine, acidotrophic agents, each increased INS TC and IC to 150-300% of control yet decreased TC and IC of α2M to 20-50% of control. Only leupeptin, a lysosomal protease inhibitor, caused an increase in both INS and α2M TC and IC. These data suggest significant differences exist in the biochemical regulation or structural routes of INS and α2M receptors and/or receptor-ligand complexes in their (1) internalization, (2) processing in acidic organelles, (3) recycling to the cell surface or in combinations of the above. Biochemical and ultrastructural studies are being performed on the H35 hepatoma cell which will characterize the processing of INS and α2M receptors and provide an explanation for the differences observed

Hepatitis C virus E2 protein promotes human hepatoma cell proliferation through the MAPK/ERK signaling pathway via cellular receptors

International Nuclear Information System (INIS)

Zhao Lanjuan; Wang Lu; Ren Hao; Cao Jie; Li Li; Ke Jinshan; Qi Zhongtian

2005-01-01

Dysregulation of mitogen-activated protein kinase (MAPK) signaling pathways by various viruses has been shown to be responsible for viral pathogenicity. The molecular mechanism by which hepatitis C virus (HCV) infection caused human liver diseases has been investigated on the basis of abnormal intracellular signal events. Current data are very limited involved in transmembrane signal transduction triggered by HCV E2 protein. Here we explored regulation of the MAPK/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway by E2 expressed in Chinese hamster oval cells. In human hepatoma Huh-7 cells, E2 specifically activated the MAPK/ERK pathway including downstream transcription factor ATF-2 and greatly promoted cell proliferation. CD81 and low density lipoprotein receptor (LDLR) on the cell surface mediated binding of E2 to Huh-7 cells. The MAPK/ERK activation and cell proliferation driven by E2 were suppressed by blockage of CD81 as well as LDLR. Furthermore, pretreatment with an upstream kinase MEK1/2 inhibitor U0126 also impaired the MAPK/ERK activation and cell proliferation induced by E2. Our results suggest that the MAPK/ERK signaling pathway triggered by HCV E2 via its receptors maintains survival and growth of target cells

microRNA-mediated resistance to hypoglycemia in the HepG2 human hepatoma cell line

International Nuclear Information System (INIS)

Ueki, Satomi; Murakami, Yuko; Yamada, Shoji; Kimura, Masaki; Saito, Yoshimasa; Saito, Hidetsugu

2016-01-01

It is generally accepted that the energy resources of cancer cells rely on anaerobic metabolism or the glycolytic system, even if they have sufficient oxygen. This is known as the Warburg effect. The cells skillfully survive under hypoglycemic conditions when their circumstances change, which probably at least partly involves microRNA (miRNA)-mediated regulation. To determine how cancer cells exploit miRNA-mediated epigenetic mechanisms to survive in hypoglycemic conditions, we used DNA microarray analysis to comprehensively and simultaneously compare the expression of miRNAs and mRNAs in the HepG2 human hepatoma cell line and in cultured normal human hepatocytes. The hypoglycemic condition decreased the expression of miRNA-17-5p and -20a-5p in hepatoma cells and consequently upregulated the expression of their target gene p21. These regulations were also confirmed by using antisense inhibitors of these miRNAs. In addition to this change, the hypoglycemic condition led to upregulated expression of heat shock proteins and increased resistance to caspase-3-induced apoptosis. However, we could not identify miRNA-mediated regulations, despite using comprehensive detection. Several interesting genes were also found to be upregulated in the hypoglycemic condition by the microarray analysis, probably because of responding to this cellular stress. These results suggest that cancer cells skillfully survive in hypoglycemic conditions, which frequently occur in malignancies, and that some of the gene regulation of this process is manipulated by miRNAs. The online version of this article (doi:10.1186/s12885-016-2762-7) contains supplementary material, which is available to authorized users

Effects of JS-K, a novel anti-cancer nitric oxide prodrug, on gene expression in human hepatoma Hep3B cells.

Science.gov (United States)

Dong, Ray; Wang, Xueqian; Wang, Huan; Liu, Zhengyun; Liu, Jie; Saavedra, Joseph E

2017-04-01

JS-K is a novel anticancer nitric oxide (NO) prodrug effective against a variety of cancer cells, including the inhibition of AM-1 hepatoma cell growth in rats. To further evaluate anticancer effects of JS-K, human hepatoma Hep3B cells were treated with JS-K and the compound control JS-43-126 at various concentrations (0-100μM) for 24h, and cytotoxicity was determined by the MTS assay. The compound control JS-43-126 was not cytotoxic to Hep3B cells at concentrations up to 100μM, while the LC 50 for JS-K was about 10μM. To examine the molecular mechanisms of antitumor effects of JS-K, Hep3B cells were treated with 1-10μM of JS-K for 24h, and then subjected to gene expression analysis via real time RT-PCR and protein immunostain via confocal images. JS-K is a GST-α targeting NO prodrug, and decreased immunostaining for GST-α was associated with JS-K treatment. JS-K activated apoptosis pathways in Hep3B cells, including induction of caspase-3, caspase-9, Bax, TNF-α, and IL-1β, and immunostaining for caspase-3 was intensified. The expressions of thrombospondin-1 (TSP-1) and the tissue inhibitors of metalloproteinase-1 (TIMP-1) were increased by JS-K at both transcript and protein levels. JS-K treatment also increased the expression of differentiation-related genes CD14 and CD11b, and depressed the expression of c-myc in Hep3B cells. Thus, multiple molecular events appear to be associated with anticancer effects of JS-K in human hepatoma Hep3B cells, including activation of genes related to apoptosis and induction of genes involved in antiangiogenesis and tumor cell migration. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

CD81 Receptor Regions outside the Large Extracellular Loop Determine Hepatitis C Virus Entry into Hepatoma Cells

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Pia Banse

2018-04-01

Full Text Available Hepatitis C virus (HCV enters human hepatocytes using four essential entry factors, one of which is human CD81 (hCD81. The tetraspanin hCD81 contains a large extracellular loop (LEL, which interacts with the E2 glycoprotein of HCV. The role of the non-LEL regions of hCD81 (intracellular tails, four transmembrane domains, small extracellular loop and intracellular loop is poorly understood. Here, we studied the contribution of these domains to HCV susceptibility of hepatoma cells by generating chimeras of related tetraspanins with the hCD81 LEL. Our results show that non-LEL regions in addition to the LEL determine susceptibility of cells to HCV. While closely related tetraspanins (X. tropicalis CD81 and D. rerio CD81 functionally complement hCD81 non-LEL regions, distantly related tetraspanins (C. elegans TSP9 amd D. melanogaster TSP96F do not and tetraspanins with intermediate homology (hCD9 show an intermediate phenotype. Tetraspanin homology and susceptibility to HCV correlate positively. For some chimeras, infectivity correlates with surface expression. In contrast, the hCD9 chimera is fully surface expressed, binds HCV E2 glycoprotein but is impaired in HCV receptor function. We demonstrate that a cholesterol-coordinating glutamate residue in CD81, which hCD9 lacks, promotes HCV infection. This work highlights the hCD81 non-LEL regions as additional HCV susceptibility-determining factors.

Anti-hepatoma activity of a novel compound glaucocalyxin H in vivo and in vitro.

Science.gov (United States)

Hai, Guangfan; Zhang, Chong; Jia, Yanlong; Bai, Suping; Han, Jinfen; Guo, Lanqing; Cui, Taizhen; Niu, Bingxuan; Huang, Feng; Song, Yu

2015-06-01

Glaucocalyxin H (GLH) is a new compound isolated from a traditional Chinese medical herb Isodon japonica var. glaucocalyx which has been used for folk medicine. This study was carried out for the first time to investigate the potential role of GLH in anti-hepatoma activity and underlying mechanisms in it. GLH could inhibit the growth of tumor in mice and induce HepG2 cells to death as assessed by the tumor reduction assay, toxic assay, morphological change, and survival rate assay. Many antitumor drugs originated from plants could inhibit the growth of tumor by inducing cells to apoptosis. The morphological changes of HepG2 cells treated with different concentrations of GLH under fluorescence and electron microscope and apoptotic rates were detected to verify its effect on apoptosis. As shown in the study, GLH could induce HepG2 cells to apoptosis in a dose-dependent manner. Bcl2 and Bax proteins played important roles in apoptosis and the disequilibrium between Bcl2 and Bax might result in apoptosis. The expression of Bax protein was upregulated and Bcl2 protein was downregulated in HepG2 cells treated with GLH assessed by Western blotting, and they were in a dose-dependent manner. Taken together, GLH can inhibit the growth of hepatoma cells in vivo and in vitro by inducing cell apoptosis due to the decreased Bcl2 and increased Bax proteins suggesting that GLH could be a potential candidate as an anti-hepatoma agent for the therapeutic treatment of hepatoma.

The oncoprotein HBXIP suppresses gluconeogenesis through modulating PCK1 to enhance the growth of hepatoma cells.

Science.gov (United States)

Shi, Hui; Fang, Runping; Li, Yinghui; Li, Leilei; Zhang, Weiying; Wang, Huawei; Chen, Fuquan; Zhang, Shuqin; Zhang, Xiaodong; Ye, Lihong

2016-11-28

Hepatitis B X-interacting protein (HBXIP) as an oncoprotein plays crucial roles in the development of cancer, involving glucose metabolism reprogramming. In this study, we are interested in whether the oncoprotein HBXIP is involved in the modulation of gluconeogenesis in liver cancer. Here, we showed that the expression level of phosphoenolpyruvate carboxykinase (PCK1), a key enzyme of gluconeogenesis, was lower in clinical hepatocellular carcinoma (HCC) tissues than that in normal tissues. Mechanistically, HBXIP inhibited the expression of PCK1 through down-regulating transcription factor FOXO1 in hepatoma cells, and up-regulated miR-135a targeting the 3'UTR of FOXO1 mRNA in the cells. In addition, HBXIP increased the phosphorylation levels of FOXO1 protein by activating PI3K/Akt pathway, leading to the export of FOXO1 from nucleus to cytoplasm. Strikingly, over-expression of PCK1 could abolish the HBXIP-promoted growth of hepatoma cells in vitro and in vivo. Thus, we conclude that the oncoprotein HBXIP is able to depress the gluconeogenesis through suppressing PCK1 to promote hepatocarcinogenesis, involving miR-135a/FOXO1 axis and PI3K/Akt/p-FOXO1 pathway. Our finding provides new insights into the mechanism by which oncoprotein HBXIP modulates glucose metabolism reprogramming in HCC. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Regulation of low density lipoprotein receptor function in a human hepatoma cell line

International Nuclear Information System (INIS)

Leichtner, A.M.; Krieger, M.; Schwartz, A.L.

1984-01-01

Low density lipoprotein (LDL) processing was investigated in a human hepatoma-derived cell line, Hep G2. Hep G2 cells bound, internalized and degraded LDL via a saturable, high affinity pathway similar to that present in other mammalian cells. Although 80% of the uptake and degradation of 125 I-LDL was inhibited by 40-fold excess native LDL, the same concentration of methylated LDL, which cannot bind to LDL receptors, had virtually no effect on processing. When added at low concentrations, the lysosomotropic agent, chloroquine, inhibited degradation without affecting the rate of lipoprotein internalization. Receptor activity was decreased 60% by preincubation of the cells in medium containing a source of cholesterol (LDL or unesterified cholesterol) and increased 1.7-fold by preincubation with compactin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase. The Hep G2 cell line may prove a useful system both for the further study of hepatic lipoprotein metabolism and for the evaluation of new antihypercholesterolemic agents

A case report of hepatoma with cystic calcification

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Jeon, Byung Hee; Choi, Sung Wook; Kim, Byung So [Busan National University College of Medicine, Busan (Korea, Republic of)

1974-10-15

A case of hepatoma with cystic calcification radiographically which confirmed by pathological examination, was reported. The patients was 19 years old boy who had abdominal mass and pain in left upper quadrant for 1 month. His family history was not contributary. The upper G-I series revealed slight posterior displacement of the fundus with a cyst like calcification, about 4.5 X 5 cm, in diameter at the left upper quadrant. Liver scanning showed normal concentration of 198{sup A}u on the right lobe but nonvisualization of the left lobe area. Biopsy specimen showed hepatoma cells invading the portal vein and intrahepatic blood vessels, and the cystic structure which was a blood vessel invaded by the tumor consisting of the organized thrombi.

Hepatoma targeting peptide conjugated bio-reducible polymer complexed with oncolytic adenovirus for cancer gene therapy.

Science.gov (United States)

Choi, Joung-Woo; Kim, Hyun Ah; Nam, Kihoon; Na, Youjin; Yun, Chae-Ok; Kim, SungWan

2015-12-28

Despite adenovirus (Ad) vector's numerous advantages for cancer gene therapy, such as high ability of endosomal escape, efficient nuclear entry mechanism, and high transduction, and therapeutic efficacy, tumor specific targeting and antiviral immune response still remain as a critical challenge in clinical setting. To overcome these obstacles and achieve cancer-specific targeting, we constructed tumor targeting bioreducible polymer, an arginine grafted bio-reducible polymer (ABP)-PEG-HCBP1, by conjugating PEGylated ABP with HCBP1 peptides which has high affinity and selectivity towards hepatoma. The ABP-PEG-HCBP1-conjugated replication incompetent GFP-expressing ad, (Ad/GFP)-ABP-PEG-HCBP1, showed a hepatoma cancer specific uptake and transduction compared to either naked Ad/GFP or Ad/GFP-ABP. Competition assays demonstrated that Ad/GFP-ABP-PEG-HCBP1-mediated transduction was specifically inhibited by HCBP1 peptide rather than coxsackie and adenovirus receptor specific antibody. In addition, ABP-PEG-HCBP1 can protect biological activity of Ad against serum, and considerably reduced both innate and adaptive immune response against Ad. shMet-expressing oncolytic Ad (oAd; RdB/shMet) complexed with ABP-PEG-HCBP1 delivered oAd efficiently into hepatoma cancer cells. The oAd/ABP-PEG-HCBP1 demonstrated enhanced cancer cell killing efficacy in comparison to oAd/ABP complex. Furthermore, Huh7 and HT1080 cancer cells treated with oAd/shMet-ABP-PEG-HCBP1 complex had significantly decreased Met and VEGF expression in hepatoma cancer, but not in non-hepatoma cancer. In sum, these results suggest that HCBP1-conjugated bioreducible polymer could be used to deliver oncolytic Ad safely and efficiently to treat hepatoma. Copyright © 2015 Elsevier B.V. All rights reserved.

Percutaneous hepatic arterial catheterization for infusion chemotherapy in treatment of primary hepatoma

International Nuclear Information System (INIS)

Juhn, Jae Ryang; Chang, Jae Yong; Cha, Seong Sook; Han, Sang Suk; Bae, Cheol; Kim, Sung Rok; Chae, Yoo Soon

1984-01-01

Chemotherapy offers palliative treatment to patient with advanced nonresectable hepatoma. The usefulness of systemic chemotherapy is limited because of serious side reaction and low concentration of drug at tumor. But this problem may be overcome by intraarterial infusion. Nonsurgical percutaneous hepatic arterial catheterization was done in 21 patients with primary hepatoma, and infusion chemotherapy was done in 19 patients who were successful in catheterization. The results were as follows: 1. Selective catheterization of hepatic artery proper, common hepatic artery, and celiac artery were successful in 4, 9 and 4 patients respectively. The success rate of selective catheterization is 80.9% including celiac artery among 21 patients with hepatoma. 2. Simple catheterization method was applied in 14 patients, and catheter exchange and Loop methods were applied in 2 and 1 patient respectively. 3. Complication related to catheterization, such as infection and bleeding on punctured site, intimal injury and dislodgement of catheter were not serious. 4. Drugs were well tolerated without serious toxicity or complication. 5. 3 patients showed objective response and median survival time of treated patients is 2.5 months.

Fluoro-sorafenib (Regorafenib) effects on hepatoma cells: growth inhibition, quiescence and recovery

Science.gov (United States)

Carr, Brian I.; Cavallini, Aldo; Lippolis, Catia; D’Alessandro, Rosalba; Messa, Caterina; Refolo, Maria Grazia; Tafaro, Angela

2015-01-01

To evaluate the growth-inhibitory properties of the potent multi-kinase antagonist Regorafenib (Fluoro-Sorafenib), which was synthesized as a more potent Sorafenib, a Raf inhibitor and to determine whether similar mechanisms were involved, human hepatoma cell lines were grown in the presence or absence of Regorafanib and examined for growth inhibition. Western blots were performed for Raf targets, for apoptosis and autophagy. Regorafenib inhibited growth of human Hep3B, PLC/PRF/5 and HepG2 cells in a concentration- and time-dependent manner. Multiple signaling pathways were altered, including MAP kinases phospho-ERK and phospho-JNK and its target phospho-c-Jun. There was evidence for apoptosis by FACS, cleavage of caspases and increased Bax levels; as well as induction of autophagy, as judged by increased Beclin-1 and LC3 (II) levels. Prolonged drug exposure resulted in cell quiescence. Full growth recovery occurred after drug removal, unlike with doxorubicin chemotherapy. Regorafenib is a potent inhibitor of cell growth. Cells surviving Regorafenib treatment remain viable, but quiescent and capable of regrowth following drug removal. The reversibility of tumor cell growth suppression after drug removal may have clinical implications. PMID:22777740

SirT1 confers hypoxia-induced radioresistance via the modulation of c-Myc stabilization on hepatoma cells

International Nuclear Information System (INIS)

Xie Yuexia; Zhang Jianghong; Shao Chunlin; Xu Yanwu

2012-01-01

Intratumoral hypoxia is an important contributory factor to tumor cell resistance to radiotherapy. SirT1, a nicotinamide adenine dinucleotide (NAD + )-dependent histone/protein deacetylase, has been linked to the decrease of radiation-induced DNA damage and seems to be critical for cancer therapy. The purpose of this study was to investigate the role of SirT1 in hypoxia-induced radiation response on hepatoma cells. It was found that the administration with resveratrol, a putative SirT1 activator, enhanced the resistance of HepG2 cells against radiation-induced DNA damage of MN formation under hypoxia condition; while nicotinamide, a well-known SirT1 inhibitor, sensitized this radiation damage. Nevertheless, pretreatment of cells with 10058-F4, a specific inhibitor of c-Myc, almost eliminated the nicotinamide-induced radiosensitive effect. Further studies revealed that resveratrol inhibited c-Myc protein accumulation via up-regulation of SirT1 expression and deacetylase activity, and this loss of c-Myc protein was abolished by inhibiting its degradation in the presence of MG132, a potent inhibitor of proteasome. In contrast, nicotinamide attenuated c-Myc protein degradation induced by radiation under hypoxia through inhibition of SirT1 deacetylase activity. Our findings suggest that SirT1 could serve as a novel potent target of radiation-induced DNA damage and thus as a potential strategy to advance the efficiency of radiation therapy in hepatoma entities. (author)

Rapid internalization of the insulin receptor in rat hepatoma cells

International Nuclear Information System (INIS)

Backer, J.M.; White, M.F.; Kahn, C.R.

1987-01-01

The authors have studied the internalization of the insulin receptor (IR) in rat hepatoma cells (Fao). The cells were surface-iodinated at 4 0 C, stimulated with insulin at 37 0 C, and then cooled rapidly, trypsinized at 4 0 C and solubilized. The IR was immunoprecipitated with a specific antibody, and internalization of the IR was assessed by the appearance of trypsin-resistant bands on SDS-PAGE. Insulin induced the internalization of surface receptors with a t 1/2 of 9-10 mins; cells not exposed to insulin internalized less than 20% of the IR during 1 h at 37 0 C. Further experiments demonstrated that the accumulation of trypsin-resistant IR paralleled a loss of receptor from the cell surface. Insulin-stimulated cells were chilled and iodinated at 4 0 C, followed by solubilization, immunoprecipitation and SDS-PAGE; alternatively, insulin-stimulated cells were chilled, surface-bound ligand removed by washing the cells at pH 4.2, and specific [ 125 I]insulin binding measured at 4 0 C. Both techniques confirmed the disappearance of IR from the cell surface at rates comparable to the insulin-stimulated internalization described above. The total amount of phosphotyrosine-containing IR, as assessed by immunoprecipitation with an anti-phosphotyrosine antibody, remained constant during this time interval, suggesting that active kinase is translocated into the cell. In summary, the authors data indicate that insulin binding increases the rate of IR internalization of Fao cells. This relocation may facilitate the interaction of the activated tyrosine kinase in the IR with intracellular substrates, thus transmitting the insulin signal to metabolic pathways

CD147 stimulates hepatoma cells escaping from immune surveillance of T cells by interaction with Cyclophilin A.

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Ren, Yi-Xin; Wang, Shu-Jing; Fan, Jian-Hui; Sun, Shi-Jie; Li, Xia; Padhiar, Arshad Ahmed; Zhang, Jia-Ning

2016-05-01

T cells play an important role in tumor immune surveillance. CD147 is a member of immunoglobulin superfamily present on the surface of many tumor cells and mediates malignant cell behaviors. Cyclophilin A (CypA) is an intracellular protein promoting inflammation when released from cells. CypA is a natural ligand for CD147. In this study, CD147 specific short hairpin RNAs (shRNA) were transfected into murine hepatocellular carcinoma Hepa1-6 cells to assess the effects of CD147 on hepatoma cells escaping from immune surveillance of T cells. We found extracellular CypA stimulated cell proliferation through CD147 by activating ERK1/2 signaling pathway. Downregulation of CD147 expression on Hepa1-6 cells significantly suppressed tumor progression in vivo, and decreased cell viability when co-cultured with T cells in vitro. Importantly, knockdown of CD147 on Hepa1-6 cells resulted in significantly increased T cells chemotaxis induced by CypA both in vivo and in vitro. These findings provide novel mechanisms how tumor cells escaping from immune surveillance of T cells. We provide a potential therapy for hepatocellular carcinoma by targeting CD147 or CD147-CypA interactions. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Differential expression of candidate virus receptors in human T lymphocytes prone or resistant to infection with patient-derived hepatitis C virus.

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Mohammed A Sarhan

Full Text Available Accumulated evidence implies that hepatitis C virus (HCV infects not only the liver but also the immune system. A lymphocyte-specific CD5 molecule was recently identified as essential for infection of T cells with native, patient-derived HCV. To assess whether the proposed hepatocyte receptors may also contribute to HCV lymphotropism, expression of scavenger receptor-class B type 1 (SR-B1, claudin-1 (CLDN-1, claudin-6 (CLDN-6, occludin (OCLN, CD5 and CD81 was examined by real-time RT-PCR and the respective proteins quantified by immunoblotting in HCV-prone and resistant T cell lines, peripheral blood mononuclear cells (PBMC, primary T cells and their subsets, and compared to hepatoma Huh7.5 and HepG2 cells. SR-B1 protein was found in T and hepatoma cell lines but not in PBMC or primary T lymphocytes, CLDN-1 in HCV-resistant PM1 T cell line and hepatoma cells only, while CLDN-6 equally in the cells investigated. OCLN protein occurred in HCV-susceptible Molt4 and Jurkat T cells and its traces in primary T cells, but not in PBMC. CD5 was displayed by HCV-prone T cell lines, primary T cells and PBMC, but not by non-susceptible T and hepatoma cell lines, while CD81 in all cell types except HepG2. Knocking-down OCLN in virus-prone T cell line inhibited HCV infection, while de novo infection downregulated OCLN and CD81, and upregulated CD5 without modifying SR-B1 expression. Overall, while no association between SR-B1, CLDN-1 or CLDN-6 and the susceptibility to HCV was found, CD5 and CD81 expression coincided with virus lymphotropism and that of OCLN with permissiveness of T cell lines but unlikely primary T cells. This study narrowed the range of factors potentially utilized by HCV to infect T lymphocytes amongst those uncovered using laboratory HCV and Huh7.5 cells. Together with the demonstrated role for CD5 in HCV lymphotropism, the findings indicate that virus utilizes different molecules to enter hepatocytes and lymphocytes.

Pokemon Silencing Leads to Bim-Mediated Anoikis of Human Hepatoma Cell QGY7703

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Kun Liu

2012-05-01

Full Text Available Pokemon is an important proto-oncogene that plays a critical role in cellular oncogenic transformation and tumorigenesis. Anoikis, which is regulated by Bim-mediated apoptosis, is critical to cancer cell invasion and metastasis. We investigated the role of Pokemon in anoikis, and our results show that Pokemon renders liver cells resistant to anoikis via suppression of Bim transcription. We knocked-down Pokemon in human hepatoma cells QGY7703 with small interfering RNAs (siRNA. Knockdown of Pokemon alone did not significantly affect the growth and survival of QGY7703 cells but notably enhanced their sensitivity to apoptotic stress due to the presence of chemical agents or cell detachment, thereby inducing anoikis, as evidenced by flow cytometry and caspase-3 activity assays. In contrast, ectopic expression of Pokemon in HL7702 cells led to resistance to anoikis. Dual-luciferase reporter and ChIP assays illustrated that Pokemon suppressed Bim transcription via direct binding to its promoter. Our results suggest that Pokemon prevents anoikis through the suppression of Bim expression, which facilitates tumor cell invasion and metastasis. This Pokemon-Bim pathway may be an effective target for therapeutic intervention for cancer.

Pokemon silencing leads to Bim-mediated anoikis of human hepatoma cell QGY7703.

Science.gov (United States)

Liu, Kun; Liu, Feng; Zhang, Nannan; Liu, Shiying; Jiang, Yuyang

2012-01-01

Pokemon is an important proto-oncogene that plays a critical role in cellular oncogenic transformation and tumorigenesis. Anoikis, which is regulated by Bim-mediated apoptosis, is critical to cancer cell invasion and metastasis. We investigated the role of Pokemon in anoikis, and our results show that Pokemon renders liver cells resistant to anoikis via suppression of Bim transcription. We knocked-down Pokemon in human hepatoma cells QGY7703 with small interfering RNAs (siRNA). Knockdown of Pokemon alone did not significantly affect the growth and survival of QGY7703 cells but notably enhanced their sensitivity to apoptotic stress due to the presence of chemical agents or cell detachment, thereby inducing anoikis, as evidenced by flow cytometry and caspase-3 activity assays. In contrast, ectopic expression of Pokemon in HL7702 cells led to resistance to anoikis. Dual-luciferase reporter and ChIP assays illustrated that Pokemon suppressed Bim transcription via direct binding to its promoter. Our results suggest that Pokemon prevents anoikis through the suppression of Bim expression, which facilitates tumor cell invasion and metastasis. This Pokemon-Bim pathway may be an effective target for therapeutic intervention for cancer.

Study on the damage effect of 131I-iodinated oil internal radiation in SMMC-7721 hepatoma model in rat

International Nuclear Information System (INIS)

Wu Shuyan; Zhang Xuguang; Wang Xiangying; Li Su'an; Mao Dihua

2004-01-01

Objective: To investigate the damage effect of 131 I-iodinated oil internal radiation in hepatoma. Methods: SMMC-7721 rat hepatoma model was used to evaluate the damage of 131 I-iodinated oil internal radiation in carcinoma. 131 I-iodinated oil was injected sector-shapely into tumor model of SMMC-7721 hepatoma with arc-needle, matched with routine straight-needle injection. Tumor damage induced by 131 I-iodinated oil intralesion radiation in the carcinoma models are recorded through survival time, weight of rat, local carcinoma, pathology, electron microscopy. Results: Arc-needle injection 131 I-iodinated oil in SMMC-7721 hepatoma at subcutis could increase rat's survival time, the body weight kept less descent, the lumps necrosed wholly. Pathology and ultrastructure detection revealed cell necrosis and collapse, sever nuclear damage was observed in the death cells. The early characteristics of necrosis such as margination of heterochromatin was also found in some tumor cells. Besides, well differentiated tumor cells, degenerative tumor cells and some lymphocytes were seen. Conclusion: Arc-needle injection 131 I-iodinated oil step-by step sector-shapely into tumor is a better method and necrosis is the major effect of 131 I-iodinated oil internal radiation in carcinoma at the level of treated dosage

Actions of exogenous histones and other proteins on [3H]-thymidine incorporation into DNA of Novikoff hepatoma cells

International Nuclear Information System (INIS)

Barra, R.; Beres, B.; Koch, M.R.; Lea, M.A.

1976-01-01

The effects of exogenous proteins on the incorporation of [ 3 H]-thymidine into DNA was studied in Novikoff hepatoma ascites cells incubated in Eagle's minimal essential medium. A liver cytosol fraction (8 mg protein/ml) caused approximately 80% inhibition of isotope incorporation. The inhibitory activity of cytosol fractions from Morris hepatomas 9618A 2 , 5123C and 20 were inversely related to their growth rate. Under conditions in which there appeared to be a density dependent inhibition of growth, a mean 10 to 20% stimulation of isotope incorporation was observed after addition of total calf thymus histones and individual fractions in the concentration range of 100 to 400μg/ml. In experiments with lower cell concentrations, a 60% or greater increase in [ 3 H]-thymidine incorporation could be obtained with total calf thymus histone and with Fl and arginine-rich histones from rat liver. At concentrations of 1 to 2 mg/ml, histones inhibited DNA synthesis. Bovine serum albumin had little effect on DNA synthesis. Polylysine caused an 80 to 90% inhibition at a concentration of 1 mg/ml, but stimulatory effects were detected under certain conditions at 10μg/ml. The results suggest critical dependence on the ratio of cell and exogenous protein concentration in the action of proteins on DNA synthesis. (author)

Elemental trace analysis of hepatomas and normal tissues by proton induced x-ray emission spectroscopy

International Nuclear Information System (INIS)

Matsuzawa, Taiju; Shishido, Fumio; Sera, Koichiro; Sato, Tachio; Morita, Tasuku.

1977-01-01

Specimens taken from liver, brain, serum and ascites hepatoma 130 in rats, were bombarded with 3.5 MeV protons accelerated by a Van de graaff generator, and the induced x-ray fluorescence was analysed with a Si(Li) detector. Absolute concentrations were determined with reference to a known concentration of uranium in the specimen. Small amounts of Ga, Yb and Tl which are known as metals having tumor affinity were injected into rats implanted with ascites hepatoma and several of its derivatives. Twenty-four hours after injection, liver, brain, serum and hepatoma were removed from the rats and these specimens were analysed by the same method. Relative concentrations of Fe, Cu, Zn and Br in liver, brain, serum and hepatoma specimens showed characteristic patterns. Patterns of liver and ascites hepatoma were quite similar, but the total amount of metals in liver was greater. The serum contained a large quantity of Br. Each AH 130 tumor cell line and its derivatives showed a different accumulation rate for Ga, Yb and Tl. Tl accumulated peculiarly in the brain. There was excellent co-relation between the concentrations of the elements and the biological characteristics of the tumor. (Evans, J.)

Preparation of a radioactive boron compound (B-I-131-lipiodol) for neutron capture therapy of hepatoma

International Nuclear Information System (INIS)

Chou, F.I.; Chung, H.P.; Chung, R.J.; Wen, H.W.; Wei, Y.Y.; Kai, J.J.; Lui, W.Y.; Chi, C.W.

2000-01-01

In our research, a radioactive boron compound, B-I-131-lipiodol, that can be selectively retained in hepatoma cells was prepared. Combining the effect of α particles produced by boron neutron capture reaction with the β particles released by radionuclides in the radioactive boron compounds will produce a synergistic killing effect on cancer cells. Human hepatoma HepG2 cell cultures were used to examine the stability and the intracellular distribution of the radioactive boron drug. Microscopes were used to examine the interaction and retention of B-I-131-lipiodol globules in the individual hepatoma cell. Moreover, ICP-AES and NaI scintillation counter were performed to determine boron concentrations and I-131 radioactivity, respectively. Results showed that B-I-131-lipiodol with a boron concentration and a specific radioactivity ranged from 500-2000 ppm and 0.05-10 mCi/mL respectively was stably retained in serum. The radiochemical purity of B-I-131-lipiodol was 98%. After supplement with a medium containing B-I-131-lipiodol, the HepG2 cells had intracellular B-I-131-lipiodol globules in the cytoplasm as seen by inverted light microscope, the I-131 and boron can be stably retained in HepG2 cells. (author)

The chicken c-erbA alpha-product induces expression of thyroid hormone-responsive genes in 3,5,3'-triiodothyronine receptor-deficient rat hepatoma cells

DEFF Research Database (Denmark)

Muñoz, A; Höppner, W; Sap, J

1990-01-01

To determine the capacity of the chicken c-erbA (cTR-alpha) gene product in regulating expression of known thyroid hormone-responsive genes, both the cTR-alpha and the viral v-erbA genes were expressed in FAO cells, a rat hepatoma cell line defective for functional thyroid hormone receptors. Upon...

Contribution of ketone bodies to cholesterogenesis in Morris hepatoma 7777 cells

International Nuclear Information System (INIS)

Hilderbrandt, L.; Elson, C.; Shrago, E.

1990-01-01

Cholesterol synthesis in neoplastic tissues is typically measured in incubations of minced tissue or tissue slices with 10 mM concentrations of individual substrates. Carbon incorporation into cholesterol from [ 14 C] labelled substrates by freshly isolated hepatoma cells was measured after one hour incubation with 10 mm single substrates. These observations were extended by measuring cholesterol synthesis supported by [ 14 C] substrates in a media containing a mixture of substrates at physiological concentrations: 5.0 mM glucose, 1.3 mM D(-)-3-hydroxybutyrate, 0.5 mM acetoacetate, 0.3 mM acetate, 0.3 mM oleate, 0.3 mM palmitate, 0.65 mM glutamine, 1.4 mM lactate and 0.1 mM pyruvate in Eagle's modified essential medium. Under single substrate conditions, the ketone bodies contribute substantially to cholesterogenesis. Estimates of the quantitative contribution of each substrate to total cholesterol synthesis are reported

CXCL10 Decreases GP73 Expression in Hepatoma Cells at the Early Stage of Hepatitis C Virus (HCV Infection

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Yuan Liu

2013-12-01

Full Text Available Golgi protein 73 (GP73, which is up-regulated in hepatocellular carcinoma (HCC, has recently been identified as a novel serum marker for HCC diagnosis. Several reports also noted the increased levels of GP73 expression in chronic liver disease in patients with acute hepatitis of various etiologies, chronic Hepatitis C virus (HCV infection and alcoholic liver disease. The molecular mechanisms of GP73 expression in HCV related liver disease still need to be determined. In this study, we aimed to evaluate the effect of HCV infection on GP73 expression. GP73 was highly expressed in Huh7, Hep3B, 293T and HUVEC cells, and was low-expressed in HepG2 cells. HCV infection led to down-regulation of GP73 in Huh7 and HepG2/CD81 cells at the early stage of infection. CXCL10 decreased GP73 expression in Huh7 and HepG2 cells. Up-regulation of GP73 was noted in hepatocytes with cytopathic effect at advanced stage of HCV infection, and further research is needed to determine the unknown factors affecting GP73 expression. In conclusion, our study provided additional evidence for the roles of GP73 in liver disease.

Studies on Anti-Hepatoma Effect of Gan-Ai-Xiao Decoction

African Journals Online (AJOL)

Nowadays, the people's lifestyles such as obesity [1], smoking [2] and alcohol drinking [3] enhance the incidence of hepatoma. Hepatoma is the second leading cause of cancer mortality worldwide [4] and a number of therapies have been used to decrease the mortality of patients with hepatoma, such as surgical treatment ...

Protection of betulin against cadmium-induced apoptosis in hepatoma cells

International Nuclear Information System (INIS)

Oh, Seon-Hee; Choi, Jeong-Eun; Lim, Sung-Chul

2006-01-01

The protective effects of betulin (BT) against cadmium (Cd)-induced cytotoxicity have been previously reported. However, the mechanisms responsible for these protective effects are unclear. Therefore, this study investigated the mechanisms responsible for the protection of BT against Cd-induced cytotoxicity in human hepatoma cell lines. The protection of BT against Cd cytotoxicity was more effective in the HepG2 than in the Hep3B cells. The protection of BT on Cd-induced cytotoxicity in the HepG2 cells appeared to be related to the inhibition of apoptosis, as determined by PI staining and DNA fragmentation analysis. The anti-apoptosis exerted by BT involved the blocking of Cd-induced reactive oxygen species (ROS) generation, the abrogation of the Cd-induced Fas upregulation, the blocking of caspase-8-dependent Bid activation, and subsequent inhibition of mitochondrial pathway. The BT pretreatment did not affect the p21 and p53 expression levels, when compared with those of the treated cells with Cd alone. BT induced the transient S phase arrest at an early stage and the G /G 1 arrest at a relatively late stage, but it did not observe the sub-G1 apoptotic peak. In the Hep3B cells, Cd did not induce ROS generation. The BT pretreatment partially inhibited the Cd-induced apoptosis, which was related with the incomplete blockage in caspase-9 or -3 activation, as well as in Bax activation. Taken together, it was found that Cd can induce apoptosis via the Fas-dependent and -independent apoptosis pathways. However, the observed protective effects of BT were clearly more sensitive to Fas-expressing HepG2 cells than to Fas-deficient Hep3B cells

Camel Milk Triggers Apoptotic Signaling Pathways in Human Hepatoma HepG2 and Breast Cancer MCF7 Cell Lines through Transcriptional Mechanism

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Hesham M. Korashy

2012-01-01

Full Text Available Few published studies have reported the use of crude camel milk in the treatment of stomach infections, tuberculosis and cancer. Yet, little research was conducted on the effect of camel milk on the apoptosis and oxidative stress associated with human cancer. The present study investigated the effect and the underlying mechanisms of camel milk on the proliferation of human cancer cells using an in vitro model of human hepatoma (HepG2 and human breast (MCF7 cancer cells. Our results showed that camel milk, but not bovine milk, significantly inhibited HepG2 and MCF7 cells proliferation through the activation of caspase-3 mRNA and activity levels, and the induction of death receptors in both cell lines. In addition, Camel milk enhanced the expression of oxidative stress markers, heme oxygenase-1 and reactive oxygen species production in both cells. Mechanistically, the increase in caspase-3 mRNA levels by camel milk was completely blocked by the transcriptional inhibitor, actinomycin D; implying that camel milk increased de novo RNA synthesis. Furthermore, Inhibition of the mitogen activated protein kinases differentially modulated the camel milk-induced caspase-3 mRNA levels. Taken together, camel milk inhibited HepG2 and MCF7 cells survival and proliferation through the activation of both the extrinsic and intrinsic apoptotic pathways.

Immunological response induced by cryoablation against murine H22 hepatoma cell line in vivo.

Science.gov (United States)

Yang, Xueling; Li, Xiaoli; Guo, Zhi; Si, Tongguo; Yu, Haipeng; Xing, Wenge

2018-02-01

To describe immunological consequences induced by cryoablation against H22 cells in vivo. Adult BALB/c mice underwent subcutaneous implantation of H22 cells. All of them were assigned into three groups randomly: group A (false surgery), group B (cryoablation) and group C (cryoablation plus Freund's adjuvant). Animals were sacrificed 1, 2 and 3 weeks after treatment. Serum IFN-γ and IL-4, Th1/Th2 in spleens and cytotoxicity were detected. Compared with that of group A, (1) INF-γ of group B was higher, but IL-4 was lower; cryoablation plus Freund's adjuvant enhanced these effects. (2) Th1/Th2 rose significantly in both group B and group C. (3) Strong cytolytic activity against H22 cells of group B and group C was found on day 7, 14 and 21. Our study showed a marked shift toward Th1 and IFN-γ expression after cryoablation, with an immuno-stimulatory effect against murine H22 hepatoma Cell. Copyright © 2017. Published by Elsevier Inc.

RhoC is essential for TGF-β1-induced invasive capacity of rat ascites hepatoma cells

International Nuclear Information System (INIS)

Mukai, M.; Endo, H.; Iwasaki, T.; Tatsuta, M.; Togawa, A.; Nakamura, H.; Inoue, M.

2006-01-01

Transforming growth factor-β1 (TGF-β1) is a multifunctional growth factor that plays a role in cell proliferation, differentiation, extracellular matrix production, apoptosis, and cell motility. We show here that TGF-β1 increased the invasiveness of MM1 cells, which are a highly invasive clone of rat ascites hepatoma cells. Both mRNA and protein levels of RhoC but not RhoA in TGF-β1-treated MM1 cells increased. In parallel with this increase in expression, RhoC activity was induced by TGF-β1 treatment. When RhoC was overexpressed in MM1 cells, the invasive capacity increased. The RhoC-overexpressing cells formed more nodules than did mock cells when injected into rat peritoneum. Furthermore, when RhoC expression was reduced by transfection with shRNA/RhoC, the invasiveness of MM1 cells decreased with concomitant suppression of RhoC expression. Thus, the induced expression of RhoC by TGF-β1 in MM1 cells plays a critical role in TGF-β1-induced cell migration

In vitro uptakes of radiolabeled IVDU and IVFRU in herpes simplex virus type-1 thymidine kinase (HSV1-tk) gene transduced morris hepatoma cell line

Energy Technology Data Exchange (ETDEWEB)

Lee, Tae Sup; Choi, Tae Hyun; Ahn, Soon Hyuk; Woo, Kwang Sun; Jeong, Wee Sup; Kwon, Hee Chung; Choi, Chang Woon; Lim, Sang Moo [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Awh, Ok Doo [College of Health Sciences, Yonsei Univ., Wonju (Korea, Republic of)

2004-02-01

The herpes simplex virus type 1 thymidine kinase gene(HSV1-tk) is an attractive candidate as a reporter gene in noninvasive reporter gene monitoring system. The HSV1-tk gene was chosen as a reporter gene, because it has been extensively studied, and there are appropriate reporter probes, substrates of HSV1-tk gene product, to apply for HSV1-tk gene imaging. We used radiolabeled 5-iodovinyl-2'-deoxyuridine (IVDU) and 5-lodovinyl-2'-fluoro-2'-deoxyuridine (IVFRU) as reporter probes for HSV1-tk gene monitoring system. We prepared HSV1-tk gene transduced Morris hepatoma cell line using retroviral vector, MOLTEN containing HSV1-tk gene. And we confirmed the HSV1-tk gene expression by Northern blotting and Western blotting. We compared in vitro uptakes of radioiodinated IVDU and IVFRU to monitor HSV1-tk gene expression in Morris hepatoma cell line (MCA) and HSV1-tk gene tranduced MCA (MAC-tk) cells until 480 minutes. We also performed correlation analysis between percentage of HSV1-tk gene tranduced MCA cell % (MCA-tk%) and uptakes of radiolabeled IVDU or IVFRU. MCA-tk cell expressed HSV1-tk mRNA and HSV1-TK protein. Two compounds showed minimal uptake in MCA, but increased uptake was observed in MCA-tk. IVDU showed 4-fold higher accumulation than IVFRU at 480 min in MCA-tk (p<0.01). Both IVDU and IVFRU uptake were linearly correlated (R{sup 2}>0.96) with increasing MCA-tk%. The rediolabeld IVDU and IVFRU showed higher specific accumulation in retrovirally HSV1-tk gene transfected Morris hepatoma cell line. Both IVDU and IVFRU could be used as good substrates for evaluation of HSV1-tk gene expression.

Studies on the propagation in cell culture and the infectivity for baboons of human hepatitis A virus

International Nuclear Information System (INIS)

Taylor, M.B.

1985-05-01

Current aspects of hepatitis A and hepatitis A virus (HAV) research and the techniques used for the propagation and monitoring of HAV and HAV antigen (HA Ag) production in vitro and HAV infection in vivo, and its sequelae are reviewed. Radioimmunoassay, immunofluorescence and electron microscopic techniques for the demonstration of HA Ag were adapted for this investigation. The cell-adapted strain of HAV(MBB) was successfully propagated in the human hepatoma cell line PLC/PRF/5 at 32 degrees Celsius. A crystalline structure was demonstrated in the cytoplasm of HAV-infected cells by thin-section electron microscopy. The origin and significance of this structure is uncertain. A possible temperature variant of HAV (strain MBB) or an HAV-related baboon virus was detected in PLC/PRF/5 cells maintained at 37 degrees Celsius after infection with a faecal extract prepared from baboons which had been infected with the cell-cultured HAV. Baboons, both free-ranging and in captivity, were found to have antibodies to HAV, which suggests susceptibility to human HAV or another cross-reacting virus. The experimental infection of the Cape baboon orally, intravenously or by both routes with HAV were investigated. The results of the study suggest reasons for the presence of anti-HAV antibodies in certain baboon populations and show that the baboon is not an ideal model for hepatitis A investigations

Studies on the propagation in cell culture and the infectivity for baboons of human hepatitis A virus

Energy Technology Data Exchange (ETDEWEB)

Taylor, M B

1985-01-01

Current aspects of hepatitis A and hepatitis A virus (HAV) research and the techniques used for the propagation and monitoring of HAV and HAV antigen (HA Ag) production in vitro and HAV infection in vivo, and its sequelae are reviewed. Radioimmunoassay, immunofluorescence and electron microscopic techniques for the demonstration of HA Ag were adapted for this investigation. The cell-adapted strain of HAV(MBB) was successfully propagated in the human hepatoma cell line PLC/PRF/5 at 32 degrees Celsius. A crystalline structure was demonstrated in the cytoplasm of HAV-infected cells by thin-section electron microscopy. The origin and significance of this structure is uncertain. A possible temperature variant of HAV (strain MBB) or an HAV-related baboon virus was detected in PLC/PRF/5 cells maintained at 37 degrees Celsius after infection with a faecal extract prepared from baboons which had been infected with the cell-cultured HAV. Baboons, both free-ranging and in captivity, were found to have antibodies to HAV, which suggests susceptibility to human HAV or another cross-reacting virus. The experimental infection of the Cape baboon orally, intravenously or by both routes with HAV were investigated. The results of the study suggest reasons for the presence of anti-HAV antibodies in certain baboon populations and show that the baboon is not an ideal model for hepatitis A investigations.

Studies on Anti-Hepatoma Effect of Gan-Ai-Xiao Decoction | Yuan ...

African Journals Online (AJOL)

Purpose: To explore the anti-hepatoma effect of Gan-Ai-Xiao Decoction (GAXD), a folk remedy. Methods: High performance liquid chromatography (HPLC) was used to identify the major chemical components of GAXD ethanol extract (EE). The cytotoxic effect of GAXD EE against HepG2 cells was measured by methyl ...

[Knockdown of STAT3 inhibits proliferation and migration of HepG2 hepatoma cells induced by IFN1].

Science.gov (United States)

Li, Xiaofang; Wang, Yuqi; Yan, Ben; Fang, Peipei; Ma, Chao; Xu, Ning; Fu, Xiaoyan; Liang, Shujuan

2018-02-01

Objective To prepare lentiviruses expressing shRNA sequences targeting human signal transducer and activator of transcription 3 (STAT3) and detect the effect of STAT3 knockdown on type I interferon (IFN1)-induced proliferation and migration in HepG2 cells. Methods Four STAT3-targeting shRNA sequences (shRNA1-shRNA4) and one control sequence (Ctrl shRNA) were selected and cloned respectively into pLKO.1-sp6-pgk-GFP to construct shRNA-expressing vectors. Along with backbone psPAX2 and pMD2.G vectors, they were separately transfected into HEK293T cells to prepare lentiviruses. HepG2 cells were infected with the lentiviruses. Cytoplastic STAT3 level was detected by Western blotting to screen effective shRNA sequence(s) targeting STAT3. Proliferation and migration of HepG2 cells were analyzed by CCK-8 assay and Transwell TM migration and scratching assay, respectively. To detect the effect of IFN1 on cell proliferation and migration of HepG2 cells, the cells were treated with 2000 U/mL IFNα2b for indicated time and the activation of IFN-triggered STAT1 signal transduction was assayed by Western blotting. Results Two most effective STAT3-targeting shRNA sequences shRNA1 and shRNA2 were selected, and the expression of both STAT3 shRNA significantly decreased proliferation and migration of HepG2 cells. When treated with IFNα2b, 2000 U/mL of IFN1 showed more competent in attenuating growth and migration of HepG2 cells. Our data further proved that knockdown of STAT3 increased the phosphorylation of STAT1, and IFNα2b further enhanced the activation of STAT1 signaling in HepG2 cells. Conclusion Knockdown of STAT3 inhibits cell migration and growth, and rescues IFN response through up-regulating STAT1 signal transduction in HepG2 hepatoma cells.

Chemopreventive Activities of Sulforaphane and Its Metabolites in Human Hepatoma HepG2 Cells

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Peng Liu

2018-05-01

Full Text Available Sulforaphane (SFN exhibits chemopreventive effects through various mechanisms. However, few studies have focused on the bioactivities of its metabolites. Here, three metabolites derived from SFN were studied, known as sulforaphane glutathione, sulforaphane cysteine and sulforaphane-N-acetylcysteine. Their effects on cell viability, DNA damage, tumorigenicity, cell migration and adhesion were measured in human hepatoma HepG2 cells, and their anti-angiogenetic effects were determined in a 3D co-culture model of human umbilical vein endothelial cells (HUVECs and pericytes. Results indicated that these metabolites at high doses decreased cancer cell viability, induced DNA damage and inhibited motility, and impaired endothelial cell migration and tube formation. Additionally, pre-treatment with low doses of SFN metabolites protected against H2O2 challenge. The activation of the nuclear factor E2-related factor 2 (Nrf2-antioxidant response element (ARE pathway and the induction of intracellular glutathione (GSH played an important role in the cytoprotective effects of SFN metabolites. In conclusion, SFN metabolites exhibited similar cytotoxic and cytoprotective effects to SFN, which proves the necessity to study the mechanisms of action of not only SFN but also of its metabolites. Based on the different tissue distribution profiles of these metabolites, the most relevant chemical forms can be selected for targeted chemoprevention.

Hepatoma SK Hep-1 cells exhibit characteristics of oncogenic mesenchymal stem cells with highly metastatic capacity.

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Jong Ryeol Eun

Full Text Available SK Hep-1 cells (SK cells derived from a patient with liver adenocarcinoma have been considered a human hepatoma cell line with mesenchymal origin characteristics, however, SK cells do not express liver genes and exhibit liver function, thus, we hypothesized whether mesenchymal cells might contribute to human liver primary cancers. Here, we characterized SK cells and its tumourigenicity.We found that classical mesenchymal stem cell (MSC markers were presented on SK cells, but endothelial marker CD31, hematopoietic markers CD34 and CD45 were negative. SK cells are capable of differentiate into adipocytes and osteoblasts as adipose-derived MSC (Ad-MSC and bone marrow-derived MSC (BM-MSC do. Importantly, a single SK cell exhibited a substantial tumourigenicity and metastatic capacity in immunodefficient mice. Metastasis not only occurred in circulating organs such as lung, liver, and kidneys, but also in muscle, outer abdomen, and skin. SK cells presented greater in vitro invasive capacity than those of Ad-MSC and BM-MSC. The xenograft cells from subcutaneous and metastatic tumors exhibited a similar tumourigenicity and metastatic capacity, and showed the same relatively homogenous population with MSC characteristics when compared to parental SK cells. SK cells could unlimitedly expand in vitro without losing MSC characteristics, its tumuorigenicity and metastatic capacity, indicating that SK cells are oncogenic MSC with enhanced self-renewal capacity. We believe that this is the first report that human MSC appear to be transformed into cancer stem cells (CSC, and that their derivatives also function as CSCs.Our findings demonstrate that SK cells represent a transformation mechanism of normal MSC into an enhanced self-renewal CSC with metastasis capacity, SK cells and their xenografts represent a same relative homogeneity of CSC with substantial metastatic capacity. Thus, it represents a novel mechanism of tumor initiation, development and

Excision of foreign gene product with cathepsin D in chicken hepatoma cell line

International Nuclear Information System (INIS)

Sato, Masaharu; Kawashima, Tsuyoshi; Aosasa, Masayoshi; Horiuchi, Hiroyuki; Furusawa, Shuichi; Matsuda, Haruo

2005-01-01

To easily and rapidly recover exogenous gene products from chicken egg yolk, we constructed pVTG-catD (VTG, vitellogenin; catD, cathepsin D), a vector cassette carrying two catD-recognition signal peptides (catD-RSPs) in addition to the cloning site. An enhanced green fluorescence protein (EGFP)-encoding DNA fragment was ligated into the pVTG-catD. When the resultant construct pVTG-EGFP-catD containing histidine- and myc-tags was transfected into the chicken hepatoma cell line LMH, EGFP-expression at 24 h post-cultivation was confirmed by fluorescence microscopy. Because a signal peptide (NTVLAEF) encoded in pVTG-EGFP-catD is recognized by catD, the VTG-EGFP fusion protein digested with catD was detectable by Western blotting. Digested exogenous gene product was recovered with nickel resin. These results indicate that catD-recognition sites bearing pVTG-catD and His-tags are functional in chicken LMH cells. Therefore, the system described here may be of use in making excision exogenous gene products in the chicken and in creating homozygous knock-in chickens

Inhibition effects of 125I-triplex forming oligonucleotide to hepatoma cells

International Nuclear Information System (INIS)

Lv Zhongwei; Hou Min; Cai Haidong; Yuan Xueyu; Yang Yuehua; Yuan Shidong; He Junmin

2007-01-01

Objective: Triplex forming oligonucleotide (TFO) has been reported as a new antigene strategy. The purpose of this study was to observe the inhibition effects of 125 I-TFO on hepatoma cells and to investigate the possibility of using 125 I-TFO as an antigene radiotherapy technique for hepatocellular carcinoma (HCC) related to HBV. Methods: TFO complementary to the initiator of S gene of HBV was synthesized and labeled with 125 I. HepG2.2.15 cells, in which HBV genome was integrated, were incubated with 125 I-TFO, TFO and 125 I respectively. After incubation, hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) of each group were assayed with ELISA and the survival rate of cells in each group was determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTT) reduction assay. Results: 125 I-TFO showed a high stability with a radiolabeling rate of >93%. The radiochemical purity of labeled compound was 90.8%, 81.1% and 73.2% respectively after 12, 48 and 72 h at 37 degree C. The peak inhibition effect of 125 I-TFO on synthesizing HBsAg and HBeAg by HepG2.2.15 cells were found at 48 h after transfection, with significantly the highest inhibition rate of 45.2% for HBsAg and 74.5% for HBeAg expression among the three groups(P 125 I-TFO may inhibit the antigen expression of HBV and the growth of hepatocarcinoma cells, thus it may provide a new approach to develop gene-based radiotherapeutic pharmaceuticals for anti-HBV and HCC. (authors)

Size-dependent cytotoxicity of Fe3O4 nanoparticles induced by biphasic regulation of oxidative stress in different human hepatoma cells

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Xie Y

2016-07-01

Full Text Available Yuexia Xie,1,2,* Dejun Liu,3,* Chenlei Cai,1,* Xiaojing Chen,1 Yan Zhou,1 Liangliang Wu,1 Yongwei Sun,3 Huili Dai,1,2 Xianming Kong,1,2 Peifeng Liu1,2 1Central Laboratory, 2State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, 3Department of Biliary-Pancreatic Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, People’s Republic of China *These authors contributed equally to this work Abstract: The application of Fe3O4 nanoparticles (NPs has made great progress in the diagnosis of disease and in the drug delivery system for cancer therapy, but the relative mecha­nisms of potential toxicity induced by Fe3O4 have not kept pace with its development in the application, which has hampered its further clinical application. In this article, we used two kinds of human hepatoma cell lines, SK-Hep-1 and Hep3B, to investigate the cytotoxic effects and the involved mechanisms of small Fe3O4 NPs with different diameters (6 nm, 9 nm, and 14 nm. Results showed that the size of NPs effectively influences the cytotoxicity of hepatoma cells: 6 nm Fe3O4 NPs exhibited negligible cytotoxicity and 9 nm Fe3O4 NPs affected cytotoxicity via cellular mitochondrial dysfunction and by inducing necrosis mediated through the mitochondria-dependent intracellular reactive oxygen species generation. Meanwhile, 14 nm Fe3O4 NPs induced cytotoxicity by impairing the integrity of plasma membrane and promoting massive lactate dehydrogenase leakage. These results explain the detailed mechanism of different diameters of small Fe3O4 NPs-induced cytotoxicity. We anticipate that this study will provide different insights into the cytotoxicity mechanism of Fe3O4 NPs, so as to make them safer to use in clinical application. Keywords: hepatoma cells, nanoparticles, cytotoxicity, mechanism, oxidative stress

Arecoline inhibits the 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced cytochrome P450 1A1 activation in human hepatoma cells

International Nuclear Information System (INIS)

Chang, Eddy Essen; Miao Zhifeng; Lee, W.-J.; Chao, H.-R.; Li, Lih-Ann; Wang, Y.-F.; Ko, Y.-C.; Tsai, F.-Y.; Yeh, S.C.; Tsou, T.-C.

2007-01-01

In the present study, we investigated the effect of arecoline, a major areca nut alkaloid, on the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced activation of cytochrome P4501A1 (CYP1A1) in a human hepatoma cell line Huh-7. We treated Huh-7 cells with 10 nM TCDD in the presence of different concentrations of arecoline (50-300 μM). Our results indicated that arecoline attenuated the TCDD-induced CYP1A1 enzyme activation with an inhibitory effect on cell proliferation. By using real-time RT-PCR, we demonstrated that arecoline inhibited the TCDD-induced activations of CYP1A1 and AhR repressor (AhRR) mRNA expression in a similar pattern. Our results revealed that arecoline inhibited AhR mRNA expression with no direct effect on CYP1A1 enzyme activity. Therefore, in our present study, the observed inhibitory effect of arecoline on CYP1A1 activation was not due to the up-regulation of AhRR or direct inhibitory effect on CYP1A1. Taken together, here we have demonstrated that arecoline attenuates the TCDD-induced CYP1A1 activation mainly via down-regulation of AhR expression in human hepatoma cells, suggesting the possible involvement of arecoline in the AhR-mediated metabolism of environmental toxicants in liver

Stable Human Hepatoma Cell Lines for Efficient Regulated Expression of Nucleoside/Nucleotide Analog Resistant and Vaccine Escape Hepatitis B Virus Variants and Woolly Monkey Hepatitis B Virus.

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Xin Cheng

Full Text Available Hepatitis B virus (HBV causes acute and chronic hepatitis B (CHB. Due to its error-prone replication via reverse transcription, HBV can rapidly evolve variants that escape vaccination and/or become resistant to CHB treatment with nucleoside/nucleotide analogs (NAs. This is particularly problematic for the first generation NAs lamivudine and adefovir. Though now superseded by more potent NAs, both are still widely used. Furthermore, resistance against the older NAs can contribute to cross-resistance against more advanced NAs. For lack of feasible HBV infection systems, the biology of such variants is not well understood. From the recent discovery of Na+-taurocholate cotransporting polypeptide (NTCP as an HBV receptor new in vitro infection systems are emerging, yet access to the required large amounts of virions, in particular variants, remains a limiting factor. Stably HBV producing cell lines address both issues by allowing to study intracellular viral replication and as a permanent source of defined virions. Accordingly, we generated a panel of new tetracycline regulated TetOFF HepG2 hepatoma cell lines which produce six lamivudine and adefovir resistance-associated and two vaccine escape variants of HBV as well as the model virus woolly monkey HBV (WMHBV. The cell line-borne viruses reproduced the expected NA resistance profiles and all were equally sensitive against a non-NA drug. The new cell lines should be valuable to investigate under standardized conditions HBV resistance and cross-resistance. With titers of secreted virions reaching >3 x 10(7 viral genome equivalents per ml they should also facilitate exploitation of the new in vitro infection systems.

Effects of the peroxisome proliferator clofibric acid on superoxide dismutase expression in the human HepG2 hepatoma cell line.

Science.gov (United States)

Bécuwe, P; Bianchi, A; Keller, J M; Dauça, M

1999-09-15

We examined the effects of clofibric acid, a peroxisome proliferator, on the production of superoxide radicals, on the levels of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), and on the expression of superoxide dismutases (SODs) in the human HepG2 hepatoma cell line. To this end, HepG2 cells were treated for 1 or 5 days with 0.25, 0.50, or 0.75 mM clofibric acid. The production of superoxide radicals was only enhanced in HepG2 cells exposed for 5 days to the different clofibric acid concentrations. However, this overproduction of superoxide radicals was not accompanied by increased rates of lipid peroxidation, as the MDA and 4-HNE levels did not change significantly. Manganese (Mn) SOD activity was increased when HepG2 cells were treated for 1 day with 0.50 or 0.75 mM clofibric acid. For this duration of treatment, no change was observed in total SOD and copper/zinc (Cu/Zn) SOD activities. For a 5-day treatment, total SOD and MnSOD activities as well as the enzyme apoprotein and MnSOD mRNA levels increased whatever the clofibric acid concentration used. This transcriptional induction of the MnSOD gene was correlated with an activation of the activator protein-1 transcription factor for 1 and 5 days of treatment, but was independent of nuclear factor-kappa B and of peroxisome proliferator-activated receptor. On the other hand, the PP exerted very little effect if any on Cu,ZnSOD expression. In contrast to rodent data, PP treatment of human hepatoma cells induces MnSOD expression.

Synthesis of PBAD-lipiodol nanoparticles for combination treatment with boric acid in boron neutron capture therapy for hepatoma in-vitro

International Nuclear Information System (INIS)

Chou, F.I.; Chung, H.P.; Liu, H.M.; Wen, H.W.; Chi, C.W.; Lin, Shanyang; Lui, W.Y.; Kai, J.J.

2006-01-01

This study attempted to increase BNCT efficiency for hepatoma by a combined treatment of phenylboric acid derivative entrapped lipiodol nanoparticles (PBAD-L nanoparticles) with boric acid. The size of PBAD-L nanoparticles were 400-750 nm at the boron concentrations of 0.3-2.7 mg/ml. After 24 hours the boron concentration in PBAD-L nanoparticles treated human hepatoma HepG2 cells was 112 ppm, while that in rat liver Clone 9 cells was 52 ppm. With the use of 25 μg B/ml boric acid, after 6 hours the boron concentration in HepG2 and Clone 9 cells were 75 ppm and 40 ppm, respectively. In a combined treatment, boron concentration in HepG2 cells which were treated with PBAD-L nanoparticles for 18 hours and then combined with boric acid for 6 hours was 158 ppm. After neutron irradiation, the surviving fraction of HepG2 cells treated with PBAD-L nanoparticles was 12.6%, while that in the ones with a combined treatment was 1.3%. In conclusion, the combined treatment provided a higher boron concentration in HepG2 cells than treatments with either PBAD-L nanoparticles or boric acid, resulting in a higher therapeutic efficacy of BNCT in hepatoma cells. (author)

Molecular switch of Cre/loxP for radiation modulated gene therapy on hepatoma

Energy Technology Data Exchange (ETDEWEB)

Hsieh, Y.-J. [Institute of Radiological Sciences, National Yang-Ming University, Taiwan (China); Chen, Fu-Du [Institute of Radiological Sciences, National Yang-Ming University, Taiwan (China); Institute of Radiological Sciences, Central Taiwan University of Science and Technology, Taiwan (China); Wang, F.H. [National Yang-Ming University Medical School, Taiwan (China); Ke, C.C. [National PET/Cyclotron Center, Taipei Veterans General Hospital, Taiwan (China); Wang, H.-E. [Institute of Radiological Sciences, National Yang-Ming University, Taiwan (China); Liu, R.-S. [Institute of Radiological Sciences, National Yang-Ming University, Taiwan (China) and National Yang-Ming University Medical School, Taiwan (China) and National PET/Cyclotron Center, Taipei Veterans General Hospital, Taiwan (China)]. E-mail: maimai5010@yahoo.com.tw

2007-02-01

For the purpose of enhancement of AFP promoter for the use of radiation modulated gene therapy for hepatocellular carcinoma (HCC), we combined hepatitis B virus (HBV) enhancer II with AFP promoter which shows the selectivity to the target cells to control the Cre/loxP system. Different gene constructs, pE4luc, pE4Tk, EIIAPA-Cre, E4CMV-STOP-Tk and chimeric promoters combined with HBV enhancer were constructed and transfected into HepG2, HeLa and NIH-3T3 cell lines. Cell experiments revealed that E4 enhancer responses to radiation best after 60 h irradiation at a dose range of 5-7 Gy in HepG2 stable clone. The EIIAPA promoter provided high specificity to hepatoma and activated the Cre downstream and removed the stop cassette only in hepatoma cells. After removal of the stop cassette, the E4 response to radiation could encode more Tk protein and kill more tumor cells. In summary, the chimeric EIIAPA promoter can stringently control the expression of Cre recombinase only in HCC. The radiation effect of the EIIAPA-Cre and E4CMV-STOP-Tk system shows promising results in terms of cell survival of HCC.

Molecular switch of Cre/loxP for radiation modulated gene therapy on hepatoma

International Nuclear Information System (INIS)

Hsieh, Y.-J.; Chen, Fu-Du; Wang, F.H.; Ke, C.C.; Wang, H.-E.; Liu, R.-S.

2007-01-01

For the purpose of enhancement of AFP promoter for the use of radiation modulated gene therapy for hepatocellular carcinoma (HCC), we combined hepatitis B virus (HBV) enhancer II with AFP promoter which shows the selectivity to the target cells to control the Cre/loxP system. Different gene constructs, pE4luc, pE4Tk, EIIAPA-Cre, E4CMV-STOP-Tk and chimeric promoters combined with HBV enhancer were constructed and transfected into HepG2, HeLa and NIH-3T3 cell lines. Cell experiments revealed that E4 enhancer responses to radiation best after 60 h irradiation at a dose range of 5-7 Gy in HepG2 stable clone. The EIIAPA promoter provided high specificity to hepatoma and activated the Cre downstream and removed the stop cassette only in hepatoma cells. After removal of the stop cassette, the E4 response to radiation could encode more Tk protein and kill more tumor cells. In summary, the chimeric EIIAPA promoter can stringently control the expression of Cre recombinase only in HCC. The radiation effect of the EIIAPA-Cre and E4CMV-STOP-Tk system shows promising results in terms of cell survival of HCC

Up-regulation of ALG-2 in hepatomas and lung cancer tissue

DEFF Research Database (Denmark)

la Cour, Jonas Marstrand; Mollerup, Jens; Winding, Pernille

2003-01-01

, a result confirmed by immunohistochemical analysis. Staining of four different lung cancer tissue microarrays including specimens of 263 patients showed that ALG-2 is mainly localized to epithelial cells and significantly up-regulated in small-cell lung cancers and in non-small-cell lung cancers. Our...... using Western blot analysis and immunohistochemistry. Western blot analysis of 15 different adult mouse tissues demonstrated that ALG-2 is ubiquitously expressed. We found that ALG-2 was more than threefold overexpressed in rat liver hepatoma compared to normal rat liver using Western blot analysis...

MiR-520b suppresses proliferation of hepatoma cells through targeting ten-eleven translocation 1 (TET1) mRNA

Energy Technology Data Exchange (ETDEWEB)

Zhang, Weiying; Lu, Zhanping; Gao, Yuen [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin (China); Ye, Lihong [State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin (China); Song, Tianqiang, E-mail: tjchi@hotmai.com [Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin (China); Zhang, Xiaodong, E-mail: zhangxd@nankai.edu.cn [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin (China)

2015-05-08

Accumulating evidence indicates that microRNAs are able to act as oncogenes or tumor suppressor genes in human cancer. We previously reported that miR-520b was down-regulated in hepatocellular carcinoma (HCC) and its deregulation was involved in hepatocarcinogenesis. In the present study, we report that miR-520b suppresses cell proliferation in HCC through targeting the ten-eleven translocation 1 (TET1) mRNA. Notably, we identified that miR-520b was able to target 3′-untranslated region (3′UTR) of TET1 mRNA by luciferase reporter gene assays. Then, we revealed that miR-520b was able to reduce the expression of TET1 at the levels of mRNA and protein using reverse transcription-polymerase chain reaction and Western blotting analysis. In terms of function, 5-ethynyl-2-deoxyuridine (EdU) incorporation and colony formation assays demonstrated that the forced miR-520b expression remarkably inhibited proliferation of hepatoma cells, but TET1 overexpression could rescue the inhibition of cell proliferation mediated by miR-520b. Furthermore, anti-miR-520b enhanced proliferation of hepatoma cells, whereas silencing of TET1 abolished anti-miR-520b-induced acceleration of cell proliferation. Then, we validated that the expression levels of miR-520b were negatively related to those of TET1 mRNA in clinical HCC tissues. Thus, we conclude that miR-520b depresses proliferation of liver cancer cells through targeting 3′UTR of TET1 mRNA. Our finding provides new insights into the mechanism of hepatocarcinogenesis. - Highlights: • TET1 is a novel target gene of miR-520b. • TET1 is upregulated in clinical HCC tissues. • MiR-520b is negatively correlated with TET1 in clinical HCC tissues. • MiR-520b depresses the proliferation of HCC cells through targeting TET1 mRNA.

CdTe quantum dots with daunorubicin induce apoptosis of multidrug-resistant human hepatoma HepG2/ADM cells: in vitro and in vivo evaluation

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Shi Lixin

2011-01-01

Full Text Available Abstract Cadmium telluride quantum dots (Cdte QDs have received significant attention in biomedical research because of their potential in disease diagnosis and drug delivery. In this study, we have investigated the interaction mechanism and synergistic effect of 3-mercaptopropionic acid-capped Cdte QDs with the anti-cancer drug daunorubicin (DNR on the induction of apoptosis using drug-resistant human hepatoma HepG2/ADM cells. Electrochemical assay revealed that Cdte QDs readily facilitated the uptake of the DNR into HepG2/ADM cells. Apoptotic staining, DNA fragmentation, and flow cytometry analysis further demonstrated that compared with Cdte QDs or DNR treatment alone, the apoptosis rate increased after the treatment of Cdte QDs together with DNR in HepG2/ADM cells. We observed that Cdte QDs treatment could reduce the effect of P-glycoprotein while the treatment of Cdte QDs together with DNR can clearly activate apoptosis-related caspases protein expression in HepG2/ADM cells. Moreover, our in vivo study indicated that the treatment of Cdte QDs together with DNR effectively inhibited the human hepatoma HepG2/ADM nude mice tumor growth. The increased cell apoptosis rate was closely correlated with the enhanced inhibition of tumor growth in the studied animals. Thus, Cdte QDs combined with DNR may serve as a possible alternative for targeted therapeutic approaches for some cancer treatments.

Analytical study of cell liver proliferation and serum AFP in various liver diseases other than hepatomas

Energy Technology Data Exchange (ETDEWEB)

Takino, T; Okuda, K; Kitamura, O; Takahashi, T; Ashihara, T [Kyoto Prefectural Univ. of Medicine (Japan)

1974-12-01

Cell proliferative activity in the liver tissue obtained in 50 cases by liver biopsy, was analyzed using in vitro labeling of /sup 3/H-thymidine autoradiography. The proliferating cells were found to be located mainly in the periportal areas of the lobules. The mean labeling indices of the liver cells were 0.06 % in chronic hepatitis in its active form, 0.05 % in pre-cirrhosis of the liver, 0.03 % in liver cirrhosis, 0.02 % in chronic hepatitis in an inactive form and 0.018 % in acute hepatitis at the restoractive stage. The labeling indices of the liver parenchymal cells of each specimen studied were very low being at most 0.2 %. On the other hand, when the serum AFP was analyzed by radioimmunoassay technique in 185 patients with various liver diseases, level of the mean serum AFP in each group of the liver diseases was found to correspond to that of the proliferative activity of the liver cells in its respective group. From these data it was suggested that the proliferative activity of the liver cells in various liver diseases, with the exception of hepatomas, was closely related to release of AFP into the serum.

Cytotoxicity of the dicarboximide fungicides, vinclozolin and iprodione, in rat hepatoma-derived Fa32 cells.

Science.gov (United States)

Dierickx, Paul J

2004-10-01

Dicarboximide fungicides are widely used to control various fungal species. Their primary action is not known, due to a lack of knowledge concerning the mechanism of action of the dicarboximide group. The cytotoxicities of vinclozolin and iprodione in rat hepatoma-derived Fa32 cells were investigated. Cytotoxicity was measured by neutral red uptake inhibition after treatment for 24 hours. Iprodione was more toxic than vinclozolin. Vinclozolin was less toxic in glutathione-depleted cells than in control cells. This was also true for iprodione at lower concentrations, but iprodione became more toxic at higher concentrations. Both the fungicides increased the endogenous glutathione content by 20% after 1 hour. After 24 hours, the glutathione content was doubled by vinclozolin, but was not affected by iprodione. No effect on glutathione S-transferase activity or reactive oxygen species formation could be observed. Cytochrome P450-dependent ethoxyresorufin-O-deethylase and pentoxyresorufin-O-depentylase activities were moderately activated by iprodione and strongly activated by vinclozolin. A glutathione-related cytochrome P450-dependent metabolic attack of vinclozolin and iprodione could be responsible for their cytotoxicity in Fa32 cells. Further research is needed to fully elucidate these (or other) mechanisms.

Metabolic Flux Distribution during Defatting of Steatotic Human Hepatoma (HepG2 Cells

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Gabriel Yarmush

2016-01-01

Full Text Available Methods that rapidly decrease fat in steatotic hepatocytes may be helpful to recover severely fatty livers for transplantation. Defatting kinetics are highly dependent upon the extracellular medium composition; however, the pathways involved are poorly understood. Steatosis was induced in human hepatoma cells (HepG2 by exposure to high levels of free fatty acids, followed by defatting using plain medium containing no fatty acids, or medium supplemented with a cocktail of defatting agents previously described before. We measured the levels of 28 extracellular metabolites and intracellular triglyceride, and fed the data into a steady-state mass balance model to estimate strictly intracellular fluxes. We found that during defatting, triglyceride content decreased, while beta-oxidation, the tricarboxylic acid cycle, and the urea cycle increased. These fluxes were augmented by defatting agents, and even more so by hyperoxic conditions. In all defatting conditions, the rate of extracellular glucose uptake/release was very small compared to the internal supply from glycogenolysis, and glycolysis remained highly active. Thus, in steatotic HepG2 cells, glycolysis and fatty acid oxidation may co-exist. Together, these pathways generate reducing equivalents that are supplied to mitochondrial oxidative phosphorylation.

Overexpression of thyroid hormone beta1 nuclear receptor is associated with an increased proliferation of human hepatoma cells

International Nuclear Information System (INIS)

Lin, K.; Lin, Y.; McPhie, P.; Cheng S.

1994-01-01

It is evaluated the expression of thyroid hormone nuclear receptors (TRs) and their possible roles in the carcinogenesis of human hepatocarcinoma. The expression of TRβ and TRα genes was evaluated at both the mRNA and protein levels. The expression of TRβ1 and TRα1 mRNAs is similar to those found in normal liver. However, the expression of TR isoform proteins depends on the cell-type. The expression of TRα1 protein is low in all cell lines examined. However, TRβ1 protein is overexpressed in Mahlavu, SK-Hep-1, and HA22T, moderately expressed in J5, J7, and J328 and is very low in HepG2, Hep3B, and PLC/PRF/5 cells. The proliferation of cells in which TRβ1 is overexpressed is stimulated by the thyroid hormone, 3,3',5-triiodo-L-thyronine. These results suggest that TRβ1 not TRα1, is probably involved in the proliferation of hepatoma cells

The spleen can influence the metastasis of AH130 hepatoma cells in rats.

Science.gov (United States)

Toyonaga, M; Hiraoka, T; Tanaka, H; Miyauchi, Y

1993-06-01

The effect of pathophysiological conditions due to disturbance of the spleen is still unclear. We studied the effects of splenectomy in normal and methylcellulose-induced hypersplenic rats on the development of pulmonary metastases created by intravenous injection of ascites containing AH130 hepatoma cells from male Hos-Donryu rats. Growth of metastatic lesions in the lung was not affected by splenectomy in normal rats, but was increased by splenectomy in hypersplenic rats. Overall, there were fewer pulmonary metastases in rats with hypersplenism, but after splenectomy rats with hypersplenism had a significantly greater number of metastases than did normal rats. The metastases rate correlated somewhat with changes in the blood coagulation and T lymphocyte profile. There is a relationship between the spleen and formation of metastases in cancer. Formation of metastases in the lung was affected most by splenectomy in hypersplenism. To elucidate the mechanism by which metastases are formed in the lung under these pathologic conditions, further studies on the exact role of the spleen are required.

Skeletal metastases from hepatoma: frequency, distribution, and radiographic features

International Nuclear Information System (INIS)

Kuhlman, J.E.; Fishman, E.K.; Leichner, P.K.; Magid, D.; Order, S.E.; Siegelman, S.S.

1986-01-01

Over the past 6 years, the authors evaluated 300 patients with hepatoma as part of phase 1 and 2 treatment protocol trials. Analysis of the available clinical data and radiographic studies revealed 22 patients (7.3%) with skeletal metastases demonstrated by radiography, computed tomography (CT), and/or nuclear scintigraphy. The plain film appearance of skeletal metastases from hepatoma was osteolytic in all cases. CT scanning best demonstrated the expansile, destructive nature of these metastases, which were often associated with large, bulky soft-tissue masses. Skeletal metastases from hepatomas demonstrated increased radiotracer uptake on standard bone scans and were gallium avid, similar to the hepatoma itself. In addition, they could be targeted therapeutically with I-131 antiferritin immunoglobulin. The most frequent sites of skeletal metastases were the ribs, spine, femur, pelvis, and humerus. An initial symptom in ten patients was skeletal pain corresponding to the osseous metastases. In five patients, pathologic fractures of the proximal femur or humerus developed and required total hip replacement or open-reduction internal fixation. Patients with long-standing cirrhosis or known hepatocellular carcinoma who also have skeletal symptoms should be evaluated for possible osseous metastases

Insulin regulation of Na/K pump activity in rat hepatoma cells

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Gelehrter, T.D.; Shreve, P.D.; Dilworth, V.M.

1984-01-01

Insulin rapidly increases Na/K pump activity in HTC rat hepatoma cells in tissue culture, as measured by the ouabain-sensitive influx of the potassium analogue 86Rb+. Increased influx is observed within minutes and is maximal (70% above control) within 1-2 h. The effect appears to be mediated by the insulin receptors, as: the concentration dependence on insulin is identical to that for insulin induction of tyrosine aminotransferase and stimulation of 2-aminoisobutyric acid transport, proinsulin is 6% as potent as insulin, and the effect is blocked by anti-receptor antibodies. The early stimulation of potassium influx is not blocked by cycloheximide and is not associated with an increased number of pump sites as measured by 3 H-ouabain binding. The insulin effect is blocked by amiloride, which blocks sodium influx, and is mimicked by the sodium ionophore monensin, which increases sodium influx and intracellular accumulation. Insulin also rapidly increases the initial rate of 22 Na+ influx, suggesting that insulin may enhance Na/K pump activity, in part, by increasing intracellular sodium concentration. Incubation of HTC cells with insulin for 24 h causes complete unresponsiveness to the insulin induction of transaminase and stimulation of amino acid transport, a phenomenon mediated by postbinding mechanisms. In contrast, similar incubation with insulin does not cause unresponsiveness to the insulin stimulation of Na/K pump activity. Therefore, the site of regulation of responsiveness to insulin must be distal to, or separate from, those events causing stimulation of ion fluxes

Iso-suillin isolated from Suillus luteus, induces G1 phase arrest and apoptosis in human hepatoma SMMC-7721 cells.

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Jia, Zhi-Qiang; Chen, Ying; Yan, Yong-Xin; Zhao, Jun-Xia

2014-01-01

Iso-suillin, a natural product isolated from Suillus luteus, has been shown to inhibit the growth of some cancer cell lines. However, the molecular mechanisms of action of this compound are poorly understood. The purpose of this study was to investigate how iso-suillin inhibits proliferation and induces apoptosis in a human hepatoma cell line (SMMC-7721). We demonstrated the effects of iso-suillin on cell proliferation and apoptosis in SMMC-7721 cells, with no apparent toxicity in normal human lymphocytes, using colony formation assays and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis. Western blotting was used to examine the expression of G1 phase-regulated and apoptosis-associated protein levels in iso-suillin treated SMMC-7721 cells. The results indicated that iso-suillin significantly decreased viability, induced G1 phase arrest and triggered apoptosis in SMMC-7721cells. Taken together, these results suggest the potential of iso-suillin as a candidate for liver cancer treatment.

Species-specific differences in peroxisome proliferation, catalase, and SOD2 upregulation as well as toxicity in human, mouse, and rat hepatoma cells induced by the explosive and environmental pollutant 2,4,6-trinitrotoluene.

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Naumenko, Ekaterina Anatolevna; Ahlemeyer, Barbara; Baumgart-Vogt, Eveline

2017-03-01

2,4,6-Trinitrotoluene (TNT) has been widely used as an explosive substance and its toxicity is still of interest as it persisted in polluted areas. TNT is metabolized in hepatocytes which are prone to its toxicity. Since analysis of the human liver or hepatocytes is restricted due to ethical reasons, we investigated the effects of TNT on cell viability, reactive oxygen species (ROS) production, peroxisome proliferation, and antioxidative enzymes in human (HepG2), mouse (Hepa 1-6), and rat (H4IIEC3) hepatoma cell lines. Under control conditions, hepatoma cells of all three species were highly comparable exhibiting identical proliferation rates and distribution of their cell cycle phases. However, we found strong differences in TNT toxicity with the lowest IC 50 values (highest cell death rate) for rat cells, whereas human and mouse cells were three to sevenfold less sensitive. Moreover, a strong decrease in cellular dehydrogenase activity (MTT assay) and increased ROS levels were noted. TNT caused peroxisome proliferation with rat hepatoma cells being most responsive followed by those from mouse and human. Under control conditions, rat cells contained fivefold higher peroxisomal catalase and mitochondrial SOD2 activities and a twofold higher capacity to reduce MTT than human and mouse cells. TNT treatment caused an increase in catalase and SOD2 mRNA and protein levels in human and mouse, but not in rat cells. Similarly, human and mouse cells upregulated SOD2 activity, whereas rat cells failed therein. We conclude that TNT induced oxidative stress, peroxisome proliferation and mitochondrial damage which are highest in rat cells rendering them most susceptible toward TNT. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 989-1006, 2017. © 2016 Wiley Periodicals, Inc.

Caspase-8 acts as a key upstream executor of mitochondria during justicidin A-induced apoptosis in human hepatoma cells.

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Su, Chun-Li; Huang, Lynn L H; Huang, Li-Min; Lee, Jenq-Chang; Lin, Chun-Nan; Won, Shen-Jeu

2006-05-29

Justicia procumbens is a traditional Taiwanese herbal remedy used to treat fever, pain, and cancer. Justicidin A, isolated from Justicia procumbens, has been reported to suppress in vitro growth of several tumor cell lines as well as hepatoma cells. In this study, justicidin A activated caspase-8 to increase tBid, disrupted mitochondrial membrane potential (Delta psi(m)), and caused the release of cytochrome c and Smac/DIABLO in Hep 3B and Hep G2 cells. Justicidin A also reduced Bcl-x(L) and increased Bax and Bak in mitochondria. Caspase-8 inhibitor (Z-IETD) attenuated the justicidin A-induced disruption of Delta psi(m). Growth of Hep 3B implanted in NOD-SCID mice was suppressed significantly by oral justicidin A (20 mg/kg/day). These results indicate that justicidin A-induced apoptosis in these cells proceeds via caspase-8 and is followed by mitochondrial disruption.

Hepatitis B virus X protein accelerates the development of hepatoma

International Nuclear Information System (INIS)

Zhang, Xiao-Dong; Wang, Yuan; Ye, Li-Hong

2014-01-01

The chronic infection of hepatitis B virus (HBV) is closely related to the occurrence and development of hepatocellular carcinoma (HCC). Accumulated evidence has shown that HBV X protein (HBx protein) is a multifunctional regulator with a crucial role in hepatocarcinogenesis. However, information on the mechanism by which HBV induces HCC is lacking. This review focuses on the pathological functions of HBx in HBV-induced hepatocarcinogenesis. As a transactivator, HBx can modulate nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and transcription factor AP-2. Moreover, HBx can affect regulatory non-coding RNAs (ncRNAs) including microRNAs and long ncRNAs (lncRNAs), such as miRNA-205 and highly upregulated in liver cancer (HULC), respectively. HBx is also involved in epigenetic modification, including methylation and acetylation. HBx interacts with various signal-transduction pathways, such as protein kinase B/Akt, Wnt/β-catenin, signal transducer and activator of transcription, and NF-κB pathways. Moreover, HBx affects cellular fate by shifting the balance toward cell survival. HBx may lead to the loss of apoptotic functions or directly contributes to oncogenesis by achieving transforming functions, which induce hepatocarcinogenesis. Additionally, HBx can modulate apoptosis and immune response by direct or indirect interaction with host factors. We conclude that HBx hastens the development of hepatoma

Immunoprecipitation assay of alpha-fetoprotein synthesis by cultured mouse hepatoma cells treated with estrogens and glucocorticoids.

Science.gov (United States)

Rosebrock, J A; Parker, C L; Kute, T E

1981-01-01

This investigation was to study the biosynthesis of 3H-labeled alpha-fetoprotein (AFP) by cultured mouse hepatoma (HEPA-2) cells. Both the function and regulation of this oncodevelopmental gene are unknown. However, evidence indicates that mechanisms controlling the expression of AFP involve aspects of both normal embryonic development and neoplastic transformation. the secretion of AFP was analyzed during different phases of the growth cycle to provide information on AFP production using standard culture conditions. The highest rate of secretion occurred during the stationary phase, followed by the late logarithmic and early logarithmic phases of growth, respectively. The production of AFP was then determined following the addition of glucocorticoids and estrogens in an attempt to understand hormonal factors that may be involved. Studies utilizing estradiol-17 beta indicated that the secretion of AFP did not appear to be sensitive to this steroid even though sucrose density gradient analysis of HEPA-2 cytosol, for estrogenic receptors, revealed competitive binding moieties on the 8S and 4S regions of the gradient. In contrast, the secretion of the total complement of proteins, including AFP, was significantly stimulated by the glucocorticoids, dexamethasone and corticosterone. Analysis of HEPA-2 cytosol for glucocorticoid receptors revealed binding components in the 7S and 3-4S regions of the gradient. The 3H-dexamethasone binding appeared to be stereospecific since nonlabeled dexamethasone, but not nonlabeled estradiol-17 beta, effectively displaced the bound radioactivity. The glucocorticoid-binding component in HEPA-2 therefore displayed characteristics reported for glucocorticoid receptors in normal liver and other hepatomas.

Effects of PARP-1 inhibitors AG-014699 and AZD2281 on proliferation and apoptosis of human hepatoma cell line HepG2

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DU Senrong

2015-06-01

Full Text Available ObjectiveTo observe the inhibitory and pro-apoptotic effects of two poly(ADP-ribose polymerase (PARP-1 inhibitors, AG-014699 and AZD2281, on human hepatoma HepG2 cells and preliminarily explore the mechanism by which AG-014699 induces HepG2 cell apoptosis, and to provide a new therapeutic target for hepatoma. MethodsThe effects of different concentrations of AG-014699 and AZD2281 on HepG2 cell proliferation were determined by MTT assay. The cell apoptosis rate was measured by flow cytometry. The expression levels of caspase-3 and caspase-8 were measured by Western Blot. Inter-group comparison was made by t test. ResultsBoth AG-014699 and AZD2281 suppressed HepG2 cell proliferation in a time- and dose-dependent manner. However, the sensitivity of HepG2 cells to the two PARP-1 inhibitors was different. The half-maximal inhibitory concentrations of AG-014699 and AZD2281 at 48 h determined by MTT assay were about 20 μmol/L and 400 μmol/L, respectively. Flow cytometry and Western blot were not used to evaluate the apoptosis of HepG2 cells exposed to AZD2281 to which these cells were not sensitive. HepG2 cell apoptosis could be induced by 10, 30, and 50 μmol/L AG-014699, and the highest apoptosis rate at 48 h was significantly higher than that of the control group (3100%±2.13% vs 09%±0013%, P＜0.01. Compared with those in the control group, the protein levels of caspase-3 and caspase-8 in HepG2 cells after 48-h exposure to 30, and 50 μmol/L AG-014699 increased. ConclusionThe two PARP-1 inhibitors AG-014699 and AZD2281 can inhibit the proliferation of HepG2 cells, which showed different sensitivities to the two inhibitors. AG-014699 can induce HepG2 cell apoptosis by up-regulating the protein expression of caspase-3 and caspase-8.

Chylomicron remnant-vitamin A metabolism by the human hepatoma cell line HepG2

International Nuclear Information System (INIS)

Lenich, C.M.

1985-01-01

The binding and metabolism of [ 3 H] vitamin A-containing chylomicron remnants (CMR) by the human hepatoma cell line Hep G2 was studied. Mesenteric lymph chylomicrons (CM) were collected from [ 3 H] retinol-fed rats and incubated with lipoprotein-lipase to obtain CMR. At 4 0 C, specific CMR binding was inhibited by excess unlabeled CMR. Specific binding predominated at low concentrations and approached saturation while total binding continued to increase over an extensive concentration range (0.45-32 μg triglyceride/ml). CMR uptake at 37 0 C was greater than that of CM and at least 100 times more efficient than the fluid-phase pinocytosis of sucrose. CMR binding increased as the extent of lipolysis obtained by incubation with lipoprotein-lipase increased. Addition of human apolipoprotein E enhanced both CMR and CM binding. After internalization, Hep G2 cells hydrolyzed CMR-[ 3 H]retinyl esters and radiolabeled metabolites accumulated as a function of time and temperature. As a function of the concentration of [ 3 H] VA initially cell-bound, retinol and retinyl esters accumulated as the major cell-associated metabolites. By contrast, retinol was the major metabolite in the medium only at low VA concentrations as other more polar metabolites accumulated at higher concentrations (> 110 pmol VA/mg cell protein). The accumulation of CMR-VA metabolites in the medium was reduced when cells were preincubated in retinol-supplemented media. Also, the specific activity of retinol in the medium closely resembled that in the cell indicating that CMR-VA mixed with the cellular store prior to its secretion

Inhibition effects of {sup 125}I-triplex forming oligonucleotide to hepatoma cells

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Zhongwei, Lv; Min, Hou; Haidong, Cai; Xueyu, Yuan; Yuehua, Yang; Shidong, Yuan [Department of Nuclear Medicine, 10th People' s Hospital, Tongji Univ., Shanghai (China); Junmin, He

2007-08-15

Objective: Triplex forming oligonucleotide (TFO) has been reported as a new antigene strategy. The purpose of this study was to observe the inhibition effects of {sup 125}I-TFO on hepatoma cells and to investigate the possibility of using {sup 125}I-TFO as an antigene radiotherapy technique for hepatocellular carcinoma (HCC) related to HBV. Methods: TFO complementary to the initiator of S gene of HBV was synthesized and labeled with {sup 125}I. HepG2.2.15 cells, in which HBV genome was integrated, were incubated with {sup 125}I-TFO, TFO and {sup 125}I respectively. After incubation, hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) of each group were assayed with ELISA and the survival rate of cells in each group was determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTT) reduction assay. Results: {sup 125}I-TFO showed a high stability with a radiolabeling rate of >93%. The radiochemical purity of labeled compound was 90.8%, 81.1% and 73.2% respectively after 12, 48 and 72 h at 37 degree C. The peak inhibition effect of {sup 125}I-TFO on synthesizing HBsAg and HBeAg by HepG2.2.15 cells were found at 48 h after transfection, with significantly the highest inhibition rate of 45.2% for HBsAg and 74.5% for HBeAg expression among the three groups(P<0.01 ). As the transfection time prolonged its inhibition effects were stronger. Conclusion: {sup 125}I-TFO may inhibit the antigen expression of HBV and the growth of hepatocarcinoma cells, thus it may provide a new approach to develop gene-based radiotherapeutic pharmaceuticals for anti-HBV and HCC. (authors)

Overexpression of thyroid hormone beta1 nuclear receptor is associated with an increased proliferation of human hepatoma cells

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Lin, K; Lin, Y; McPhie, P [Chang-Gung College of Medicine and Technology, Graduate Institute of Clinical Medicine, Taoyuan (Taiwan, Province of China); Cheng, S [National Cancer Inst., Bethesda, MD (United States)

1994-12-31

It is evaluated the expression of thyroid hormone nuclear receptors (TRs) and their possible roles in the carcinogenesis of human hepatocarcinoma. The expression of TR{beta}1 and TR{alpha} genes was evaluated at both the mRNA and protein levels. The expression of TR{beta}1 and TR{alpha}1 mRNAs is similar to those found in normal liver. However, the expression of TR isoform proteins depends on the cell-type. The expression of TRaplha1 protein is low in all cell lines examined. However, TR{Beta}1 protein is overexpressed in Mahlavu, SK-Hep-1, and HA22T, moderately expressed in J5, J7, and J328 and is very low HepG2, Hep3B, and PLC/PRF/5 cells. The proliferation of cells in which TR{beta}1 is overexpressed is stimulated by the thyroid hormone, 3,3`,5- triiodo-L-thyronine. These results suggest that TR{beta}1, not TR{alpha}1, is probably involved in the prolifaration of hepatoma cells.

Size-mediated cytotoxicity of nanocrystalline titanium dioxide, pure and zinc-doped hydroxyapatite nanoparticles in human hepatoma cells

International Nuclear Information System (INIS)

Devanand Venkatasubbu, G.; Ramasamy, S.; Avadhani, G. S.; Palanikumar, L.; Kumar, J.

2012-01-01

Nanoparticles are highly used in biological applications including nanomedicine. In this present study, the interaction of HepG2 hepatocellular carcinoma cells (HCC) with hydroxyapatite (HAp), zinc-doped hydroxyapatite, and titanium dioxide (TiO 2 ) nanoparticles were investigated. Hydroxyapatite, zinc-doped hydroxyapatite and titanium dioxide nanoparticles were prepared by wet precipitation method. They were subjected to isochronal annealing at different temperatures. Particle morphology and size distribution were characterized by X-ray diffraction and transmission electron microscope. The nanoparticles were co-cultured with HepG2 cells. MTT assay was employed to evaluate the proliferation of tumor cells. The DNA damaging effect of HAp, Zn-doped HAp, and TiO 2 nanoparticles in human hepatoma cells (HepG2) were evaluated using DNA fragmentation studies. The results showed that in HepG2 cells, the anti-tumor activity strongly depend on the size of nanoparticles in HCC cells. Cell cycle arrest analysis for HAp, zinc-doped HAp, and TiO 2 nanoparticles revealed the influence of HAp, zinc-doped HAp, and titanium dioxide nanoparticles on the apoptosis of HepG2 cells. The results imply that the novel nano nature effect plays an important role in the biomedicinal application of nanoparticles.

BlueBerry Isolate, Pterostilbene, Functions as a Potential Anticancer Stem Cell Agent in Suppressing Irradiation-Mediated Enrichment of Hepatoma Stem Cells

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Chi-Ming Lee

2013-01-01

Full Text Available For many malignancies, radiation therapy remains the second option only to surgery in terms of its curative potential. However, radiation-induced tumor cell death is limited by a number of factors, including the adverse response of the tumor microenvironment to the treatment and either intrinsic or acquired mechanisms of evasive resistance, and the existence of cancer stem cells (CSCs. In this study, we demonstrated that using different doses of irradiation led to the enrichment of CD133+ Mahlavu cells using flow cytometric method. Subsequently, CD133+ Mahlavu cells enriched by irradiation were characterized for their stemness gene expression, self-renewal, migration/invasion abilities, and radiation resistance. Having established irradiation-enriched CD133+ Mahlavu cells with CSC properties, we evaluated a phytochemical, pterostilbene (PT, found abundantly in blueberries, against irradiation-enriched CSCs. It was shown that PT treatment dose-dependently reduced the enrichment of CD133+ Mahlavu cells upon irradiation; PT treatment also prevented tumor sphere formation, reduced stemness gene expression, and suppressed invasion and migration abilities as well as increasing apoptosis of CD133+ Mahlavu CSCs. Based on our experimental data, pterostilbene could be used to prevent the enrichment of CD133+ hepatoma CSCs and should be considered for future clinical testing as a combined agent for HCC patients.

Preparation of [[sup 131]I]lipiodol as a hepatoma therapeutic agent

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Jiunnguang Lo; Aiyih Wang; Yuanyaw Wei (National Tsinghua Univ., Hsinchu (Taiwan). Inst. of Nuclear Science); Wingyiu Lui; Chinwen Chi (Taipei Veterans General Hospital, Taipei (Taiwan)); Wingkai Chan (Academia Sinica, Taipei (Taiwan). Inst. of Biomedical Sciences)

1992-12-01

An isotopic exchange method was used to label lipiodol with [sup 131]I. The labelling efficiency was > 92.5%, and the radiochemical purity of [[sup 131]I]lipiodol was above 98% as determined by ITLC. The influencing factors e.g. the heating temperature, reaction, pH and storage conditions were studied and the optimum conditions were determined. In a pilot study injecting [[sup 131]I]lipiodol for the treatment of hepatoma, about 70% of hepatoma patients had a response to the treatment with a reduction of [alpha]-fetoprotein and decrease of hepatoma sizes. The overall median survival was 9 months (range 2-17 months). (author).

Up-regulation of hepatoma-derived growth factor facilitates tumor progression in malignant melanoma [corrected].

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Han-En Tsai

Full Text Available Cutaneous malignant melanoma is the fastest increasing malignancy in humans. Hepatoma-derived growth factor (HDGF is a novel growth factor identified from human hepatoma cell line. HDGF overexpression is correlated with poor prognosis in various types of cancer including melanoma. However, the underlying mechanism of HDGF overexpression in developing melanoma remains unclear. In this study, human melanoma cell lines (A375, A2058, MEL-RM and MM200 showed higher levels of HDGF gene expression, whereas human epidermal melanocytes (HEMn expressed less. Exogenous application of HDGF stimulated colony formation and invasion of human melanoma cells. Moreover, HDGF overexpression stimulated the degree of invasion and colony formation of B16-F10 melanoma cells whereas HDGF knockdown exerted opposite effects in vitro. To evaluate the effects of HDGF on tumour growth and metastasis in vivo, syngeneic mouse melanoma and metastatic melanoma models were performed by manipulating the gene expression of HDGF in melanoma cells. It was found that mice injected with HDGF-overexpressing melanoma cells had greater tumour growth and higher metastatic capability. In contrast, mice implanted with HDGF-depleted melanoma cells exhibited reduced tumor burden and lung metastasis. Histological analysis of excised tumors revealed higher degree of cell proliferation and neovascularization in HDGF-overexpressing melanoma. The present study provides evidence that HDGF promotes tumor progression of melanoma and targeting HDGF may constitute a novel strategy for the treatment of melanoma.

Effects of exogenous cyclic AMP on growth characteristics and radiation response of Reuber H35 hepatoma cells

International Nuclear Information System (INIS)

van Rijn, J.; van Den Berg, J.; van Meeteren, A.; van Wijk, R.

1983-01-01

Reuber H35 rat hepatoma cells, clone KRC, were used to study the effect of cyclic AMP on radiation-induced cell death. Treatment of logarithmically growing cultures with 0.5 mM cAMP for 17 hr prior to irradiation resulted in a decreased cell survival. Similar results were obtained with cultures irradiated after treatment with Bt 2 cAMP. Treatment of H35 cells with cAMP or Bt 2 cAMP caused inhibition of their proliferation and resulted in an accumulation of cells in early S phase and depletion of G2-phase cells. In synchronized cultures cells were relatively radioresistant during their S phase. In addition to single-dose treatment with X rays, the effect of Bt 2 cAMP on radiation-induced cell death was studied during fractionated irradiation wtih 2.5 Gy per day. This fractionated irradiation resulted in a dose-reduction factor of 1.6 at the 10% survival level and a 10-fold decrease in the surviving cell population due to the cooperative effects of Bt 2 cAMP on growth rate and radiation survival. The effect of cAMP on radiation-induced mitotic delay was also studied. It appeared that where cAMP had on effect on the progression of G2 cells into mitosis, it prevented cells from recovery from the X-ray mitotic delay in G2

Incidence and significance of pleural effusion after hepatoma surgery

International Nuclear Information System (INIS)

Song, Jae Uoo; Im, Jung Gi; Ahn, Joong Mo; Kim, Seung Cheol; Kim, Sam Soo; Kim, Seung Hoon; Yeon, Kyung Mo

1994-01-01

We performed this study to evaluate the clinical significance and temporal changes of pleural effusion developed after the resection of hepatoma. We reviewed retrospectively follow-up chest radiographs of 97 patients who had undergone operation for hepatoma and had no radiologically demonstrable postoperative complications. The duration of pleural effusion was classified into five groups and the amount of pleural effusion at one week after operation was graded into four groups. Statistical significance of the relationship between the duration, amount of pleural effusion and five factors, which are location and size of tumor, age of the patients, methods of operation, and preoperative liver function, was studied respectively. Pleural effusion was developed in 63.9% (62/97) and the mean duration was 2.5 weeks. In 92% (52/56), pleural effusion disappeared spontaneously within four weeks. Patients who had hepatoma in upper portion of the right lobe developed more frequent pleural effusion which persisted longer, and was larger in amount at one week after operation(p<0.05). There were no statistically significant differences between pleural effusion and the other four factors. Pleural effusion following hepatoma surgery should not be regarded as a sign of post-operative complication, as it invariably disappears spontaneously within four weeks. Development of pleural effusion is considered to be caused by local irritation and disturbance of lymphatic flow at the diaphragm

Thyroid hormone receptor inhibits hepatoma cell migration through transcriptional activation of Dickkopf 4

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Chi, Hsiang-Cheng; Liao, Chen-Hsin [Department of Biochemistry, School of Medicine, Chang-Gung University, Taoyuan 333, Taiwan, ROC (China); Huang, Ya-Hui [Medical Research Central, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan, ROC (China); Wu, Sheng-Ming; Tsai, Chung-Ying; Liao, Chia-Jung; Tseng, Yi-Hsin; Lin, Yang-Hsiang; Chen, Cheng-Yi; Chung, I-Hsiao; Wu, Tzu-I [Department of Biochemistry, School of Medicine, Chang-Gung University, Taoyuan 333, Taiwan, ROC (China); Chen, Wei-Jan [First Cardiovascular Division, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan, ROC (China); Lin, Kwang-Huei, E-mail: khlin@mail.cgu.edu.tw [Department of Biochemistry, School of Medicine, Chang-Gung University, Taoyuan 333, Taiwan, ROC (China)

2013-09-13

Highlights: •T{sub 3} affects DKK4 mRNA and protein expression in HepG2-TR cells. •Regulation of DKK4 by T{sub 3} is at transcriptional level. •DKK4 overexpression suppresses hepatoma cell metastasis. -- Abstract: Triiodothyronine (T{sub 3}) is a potent form of thyroid hormone mediates several physiological processes including cellular growth, development, and differentiation via binding to the nuclear thyroid hormone receptor (TR). Recent studies have demonstrated critical roles of T{sub 3}/TR in tumor progression. Moreover, long-term hypothyroidism appears to be associated with the incidence of human hepatocellular carcinoma (HCC), independent of other major HCC risk factors. Dickkopf (DKK) 4, a secreted protein that antagonizes the canonical Wnt signaling pathway, is induced by T{sub 3} at both mRNA and protein levels in HCC cell lines. However, the mechanism underlying T{sub 3}-mediated regulation of DKK4 remains unknown. In the present study, the 5′ promoter region of DKK4 was serially deleted, and the reporter assay performed to localize the T{sub 3} response element (TRE). Consequently, we identified an atypical direct repeat TRE between nucleotides −1645 and −1629 conferring T{sub 3} responsiveness to the DKK4 gene. This region was further validated using chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA). Stable DKK4 overexpression in SK-Hep-1 cells suppressed cell invasion and metastatic potential, both in vivo andin vitro, via reduction of matrix metalloproteinase-2 (MMP-2) expression. Our findings collectively suggest that DKK4 upregulated by T{sub 3}/TR antagonizes the Wnt signal pathway to suppress tumor cell progression, thus providing new insights into the molecular mechanism underlying thyroid hormone activity in HCC.

DNA binding properties of dioxin receptors in wild-type and mutant mouse hepatoma cells

International Nuclear Information System (INIS)

Cuthill, S.; Poellinger, L.

1988-01-01

The current model of action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) entails stimulation of target gene transcription via the formation of dioxin-receptor complexes and subsequent accumulation of the complexes within the cell nucleus. Here, the authors have analyzed the DNA binding properties of the dioxin receptor in wild-type mouse hepatoma (Hepa 1c1c7) cells and a class of nonresponsive mutant cells which fail to accumulate dioxin-receptor complexes within the nucleus in vivo. In vitro, both the wild-type and mutant [ 3 H]dioxin-receptor complexes exhibited low affinity for DNA-cellulose (5-8% and around 4% retention, respectively) in the absence of prior biochemical manipulations. However, following chromatography on heparin-Sepharose, the wild-type but not the mutant dioxin receptor was transformed to a species with an increased affinity for DNA (40-50% retention on DNA-cellulose). The gross molecular structure of the mutant, non DNA binding dioxin receptor did not appear to be altered as compared to that of the wild-type receptor. These results imply that the primary deficiency in the mutant dioxin receptor form may reside at the DNA binding level and that, in analogy to steroid hormone receptors, DNA binding of the receptor may be an essential step in the regulation of target gene transcription by dioxin

Ginsenoside Rh2 Induces Human Hepatoma Cell Apoptosisvia Bax/Bak Triggered Cytochrome C Release and Caspase-9/Caspase-8 Activation

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Xiao-Xi Guo

2012-11-01

Full Text Available Ginsenoside Rh2 (G-Rh2 has been shown to induce apoptotic cell death in a variety of cancer cells. However, the details of the signal transduction cascade involved in G-Rh2-induced cell death is unclear. In this manuscript we elucidate the molecular mechanism of G-Rh2-induced apoptosis in human hepatoma SK-HEP-1 cells by demonstrating that G-Rh2 causes rapid and dramatic translocation of both Bak and Bax, which subsequently triggers mitochondrial cytochrome c release and consequent caspase activation. Interestingly, siRNA-based gene inactivation of caspase-8 effectively delays caspase-9 activation and apoptosis induced by G-Rh2, indicating that caspase-8 also plays an important role in the G-Rh2-induced apoptosis program. Taken together, our results indicate that G-Rh2 employs a multi pro-apoptotic pathway to execute cancer cell death, suggesting a potential role for G-Rh2 as a powerful chemotherapeutic agent.

Various imaging methods in the detection of small hepatomas

International Nuclear Information System (INIS)

Nakatsuka, Haruki; Kaminou, Toshio; Takemoto, Kazumasa; Takashima, Sumio; Kobayashi, Nobuyuki; Nakamura, Kenji; Onoyama, Yasuto; Kurioka, Naruto

1985-01-01

Fifty-one patients with small hepatomas under 5 cm in diameter were studied to compare the detectability of various imaging methods. Positive finding was obtained in 50 % of the patients by scintigraphy, in 74 % by ultrasonography and in 79 % by CT during screening tests. Rate of detection in retrospective analysis, after the site of the tumor had been known, were 73 %, 93 % and 87 % respectively. Rate of detection was 92 % by celiac arteriography and 98 % by selective hepatic arteriography. In 21 patients, who had the tumor under 3 cm, the rate was 32 % for scintigraphy, 74 % for ultrasonography and 65 % for CT during screening, whereas it was 58 %, 84 % and 75 % retrospectively. By celiac arteriography, it was 85 %, and by hepatic arteriography, 95 %. Rate of detection of small hepatomas in screening tests differed remarkably from that in retrospective analysis. No single method of imaging can disclose reliably the presense of small hepatoma, therefore more than one method should be used in screening. (author)

Comparative study on lysosomal accumulation of 67Ga and 111In in Morris hepatoma 7316A

International Nuclear Information System (INIS)

Takeda, S.; Uchida, T.; Matsuzawa, T.

1977-01-01

Intracellular localization of 67 Ga and 111 In was investigated in Morris hepatoma 7316A and in normal Buffalo rat liver cells by a cell fractionation method at 48 hr after an intraperitoneal injection of the nuclides. Lysosomal fractions of the tumor and normal liver cells had the highest relative specific radioactivities of the nuclides (p 67 Ga (p 67 Ga seemed to indicate that 67 Ga determines lysosomal functions of tumor cells more precisely than 111 In

Serum concentration of alpha-1-fetoprotein suggestive of, or pathognomonic for hepatoma

International Nuclear Information System (INIS)

Polterauer, P.; Horak, W.; Legenstein, E.; Mueller, M.

1979-01-01

A short review of alpha-1-fetoprotein (AFP), is followed by a presentation of the serum AFP concentrations obtained in healthy subjects and in patients with hepatoma, cirrhosis of the liver or metastatic liver cancer, measured by radioimmunoassay (RIA). A calculation is made from these results of the upper limit of normal (9 ng/ml), a limit which is suggestive of hepatome (215 ng/ml) and a limit which is pathognomonic for hepatoma (7500 ng/ml). It is concluded that the quantitative determination of AFP by RIA represents a sensitive method which provides valuable clinical information for the early diagnosis of hepatoma. (author)

Three-dimensional Huh7 cell culture system for the study of Hepatitis C virus infection

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Uprichard Susan L

2009-07-01

Full Text Available Abstract Background In order to elucidate how Hepatitis C Virus (HCV interacts with polarized hepatocytes in vivo and how HCV-induced alterations in cellular function contribute to HCV-associated liver disease, a more physiologically relevant hepatocyte culture model is needed. As such, NASA-engineered three-dimensional (3-D rotating wall vessel (RWV bioreactors were used in effort to promote differentiation of HCV-permissive Huh7 hepatoma cells. Results When cultured in the RWV, Huh7 cells became morphologically and transcriptionally distinct from more standard Huh7 two-dimensional (2-D monolayers. Specifically, RWV-cultured Huh7 cells formed complex, multilayered 3-D aggregates in which Phase I and Phase II xenobiotic drug metabolism genes, as well as hepatocyte-specific transcripts (HNF4α, Albumin, TTR and α1AT, were upregulated compared to 2-D cultured Huh7 cells. Immunofluorescence analysis revealed that these HCV-permissive 3-D cultured Huh7 cells were more polarized than their 2D counterparts with the expression of HCV receptors, cell adhesion and tight junction markers (CD81, scavenger receptor class B member 1, claudin-1, occludin, ZO-1, β-Catenin and E-Cadherin significantly increased and exhibiting apical, lateral and/or basolateral localization. Conclusion These findings show that when cultured in 3-D, Huh7 cells acquire a more differentiated hepatocyte-like phenotype. Importantly, we show that these 3D cultures are highly permissive for HCV infection, thus providing an opportunity to study HCV entry and the effects of HCV infection on host cell function in a more physiologically relevant cell culture system.

Dioxin-like activity of brominated dioxins as individual compounds or mixtures in in vitro reporter gene assays with rat and mouse hepatoma cell lines.

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Suzuki, G; Nakamura, M; Michinaka, C; Tue, N M; Handa, H; Takigami, H

2017-10-01

In vitro reporter gene assays detecting dioxin-like compounds have been developed and validated since the middle 1990's, and applied to the determination of dioxin-like activities in various samples for their risk management. Data on characterizing the potency of individual brominated dioxins and their activity in mixture with chlorinated dioxins are still limited on the cell-based assay. This study characterized the dioxin-like activities of the 32 brominated dioxins, such as polybrominated dibenzo-p-dioxins, polybrominated dibenzofurans (PBDFs), coplanar polybrominated biphenyls, mixed halogenated dibenzo-p-dioxins and dibenzofurans (PXDFs), as a sole component or in a mixture by DR-CALUX (dioxin-responsive chemically activated luciferase expression) using the rat hepatoma H4IIE cell line and XDS-CALUX (xenobiotic detection systems-chemically activated luciferase expression) assays using the mouse hepatoma H1L6.1 cell line. The 2,3,7,8-TCDD-relative potencies (REPs) of most of the brominated dioxins were within a factor of 10 of the WHO toxicity equivalency factor (WHO-TEF) for the chlorinated analogues. The REPs of a few PXDFs were an order of magnitude higher than the corresponding WHO-TEFs, indicating their toxicological importance. Results with reconstituted mixtures suggest that the activity of brominated and chlorinated dioxins in both CALUX assays was dose-additive. Thus, obtained results indicated the applicability of the CALUX assays as screening tools of brominated dioxins together with their chlorinated analogues. Copyright © 2017 Elsevier Ltd. All rights reserved.

The synergistic radiosensitizing effect of tirapazamine-conjugated gold nanoparticles on human hepatoma HepG2 cells under X-ray irradiation

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Liu X

2016-07-01

Full Text Available Xi Liu,1–4 Yan Liu,1–4 Pengcheng Zhang,1–4 Xiaodong Jin,1–3 Xiaogang Zheng,1–4 Fei Ye,1–4 Weiqiang Chen,1–3 Qiang Li1–3 1Institute of Modern Physics, 2Key Laboratory of Heavy Ion Radiation Biology and Medicine, Chinese Academy of Sciences, 3Key Laboratory of Basic Research on Heavy Ion Radiation Application in Medicine, Gansu Province, Lanzhou, 4School of Life Science, University of Chinese Academy of Sciences, Beijing, People’s Republic of China Abstract: Reductive drug-functionalized gold nanoparticles (AuNPs have been proposed to enhance the damage of X-rays to cells through improving hydroxyl radical production by secondary electrons. In this work, polyethylene glycol-capped AuNPs were conjugated with tirapazamine (TPZ moiety, and then thioctyl TPZ (TPZs-modified AuNPs (TPZs-AuNPs were synthesized. The TPZs-AuNPs were characterized by transmission electron microscopy, ultraviolet-visible spectra, dynamic light scattering, and inductively coupled plasma mass spectrometry to have a size of 16.6±2.1 nm in diameter and a TPZs/AuNPs ratio of ~700:1. In contrast with PEGylated AuNPs, the as-synthesized TPZs-AuNPs exhibited 20% increment in hydroxyl radical production in water at 2.0 Gy, and 19% increase in sensitizer enhancement ratio at 10% survival fraction for human hepatoma HepG2 cells under X-ray irradiation. The production of reactive oxygen species in HepG2 cells exposed to X-rays in vitro demonstrated a synergistic radiosensitizing effect of AuNPs and TPZ moiety. Thus, the reductive drug-conjugated TPZs-AuNPs as a kind of AuNP radiosensitizer with low gold loading provide a new strategy for enhancing the efficacy of radiation therapy. Keywords: AuNPs, radiation enhancement, synergistic effect, human hepatoma cells, hydroxyl radical production

Anti-infectivity of camel polyclonal antibodies against hepatitis C virus in Huh7.5 hepatoma

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EL-Fakharany Esmail M

2012-09-01

Full Text Available Abstract Purpose To extend the study of the camel milk proteins which have antiviral activity against HCV, camel naïve polyclonal IgGs, α-lactalbumin were purified from camel milk and their anti-HCV effect was examined using PBMCs and Huh7.5 cell-lines. They were compared with the activity of human polyclonal IgGs and camel lactoferrin and casein. Material and methods Three types of experiments were performed on PBMCs and HuH7.5 cell. HCV was directly incubated with the purified proteins and then mixed with both cell types, or the proteins were incubated with the cells and then exposed to HCV, or the HCV pre-infected cells were treated with the proteins to inhibit intracellular replication. The proteins were added to cells or virus at different concentrations and time intervals. Results Pretreated PBMCs and Huh7.5 cells with milk proteins were not protected when exposed to HCV infection. The direct interaction between HCV and camel IgGs and camel lactoferrin (cLf led to a complete inhibition of HCV entry into cells, while casein, α-lactalbumin and human IgGs failed to inhibit HCV entry at any tested concentration. Camel IgGs showed ability to recognize HCV peptides with a significant titer (12 × 103 in comparison with human IgGs which failed to do it. Camel lactoferrin was capable of inhibiting the intracellular HCV replication at concentrations of 0.25-1.25 mg/ml. Conclusion Camel milk naïve polyclonal IgGs isolated from camel milk could inhibit the HCV infectivity and demonstrated strong signal against its synthetic peptides. Lactoferrin inhibit the HCV infectivity started from 0.25 mg/ml. However, α-lactalbumin, human IgGs and casein failed to demonstrate any activity against HCV infectivity.

The hyper-radiosensitivity effect of human hepatoma SMMC-7721 cells exposed to low dose γ-rays and 12C ions

International Nuclear Information System (INIS)

Jin Xiaodong; Li Qiang; Li Wenjian; Wang Jufang; Guo Chuanling; Hao Jifang

2006-01-01

Hypersensitive response of mammalian cells in cell killing to X- and γ-rays has been reported at doses below 1 Gy. The purpose of this study was to examine the low dose sensitivity of human hepatoma SMMC-7721 cells irradiated with 6 Co γ-rays and 50 MeV/u 12 C ions. Experiments using γ-rays and charged particle irradiation were performed, particularly in the low dose range from 0 to 2 Gy. The survival effect of SMMC-7721 cells was measured by means of standard clonogenic assay in conjunction with a cell sorter. The result indicates SMMC-7721 cells showed hyper-radiosensitive response at low doses and increased radio-resistance at larger single doses for the carbon ions (LET = 45.2 keV/μm) and the γ-rays. However, the HRS/IRR effect caused by high-LET irradiation is different from that by low-LET radiation. This might possibly be due to the difference in the mode of energy deposition by particle beam and low-LET irradiation

Arbidol: a broad-spectrum antiviral that inhibits acute and chronic HCV infection

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Pécheur Eve-Isabelle

2006-07-01

Full Text Available Abstract Arbidol (ARB is an antiviral compound that was originally proven effective for treatment of influenza and several other respiratory viral infections. The broad spectrum of ARB anti-viral activity led us to evaluate its effect on hepatitis C virus (HCV infection and replication in cell culture. Long-term ARB treatment of Huh7 cells chronically replicating a genomic length genotype 1b replicon resulted in sustained reduction of viral RNA and protein expression, and eventually cured HCV infected cells. Pre-treatment of human hepatoma Huh7.5.1 cells with 15 μM ARB for 24 to 48 hours inhibited acute infection with JFH-1 virus by up to 1000-fold. The inhibitory effect of ARB on HCV was not due to generalized cytotoxicity, nor to augmentation of IFN antiviral signaling pathways, but involved impaired virus-mediated membrane fusion. ARB's affinity for membranes may inhibit several aspects of the HCV lifecycle that are membrane-dependent.

Computed tomographic evaluation of the portal vein in the hepatomas

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Lee, Kee Hyung; Lee, Seung Chul; Bae, Man Gil; Seo, Heung Suk; Kim, Soon Yong; Lee, Min Ho; Kee, Choon Suhk; Park, Kyung Nam [Hanyang University College of Medicine, Seoul (Korea, Republic of)

1986-10-15

Computed tomography and pornographic findings of 63 patients with hepatoma, undergone hepatic angiography and superior mesenteric pornography for evaluation of tumor and thrombosis of portal vein and determination of indication of transcatheter arterial embolization for palliative treatment of hepatoma from April, 85 to June, 86 in Hanyang university hospital, were reviewed. The results were as follows: 1. In 36 cases, portal vein thrombosis was detected during photography. Nineteen of 37 cases which revealed localized hepatoma in the right lobe of the liver showed portal vein thrombosis; 9 of 11 cases of the left lobe; 8 of 14 cases which were involved in entire liver revealed thrombosis. One case localized in the caudate lobe showed no evidence of invasion to portal vein. 2. Twenty-four of 34 cases with diffuse infiltrative hepatoma revealed portal vein thrombosis and the incidence of portal vein thrombosis in this type were higher than in the cases of the nodular type. 3. The portal vein thrombosis appeared as filling defects of low density in the lumen of the portal veins in CT and they did not reveal contrast enhancement. 4. CT revealed well the evidence of obstructions in the cases of portal vein thrombosis and the findings were well-corresponded to the findings of the superior mesenteric photography. 5. Five of the cases of the portal vein thrombosis were missed in the CT and the causes were considered as due to partial volume effect of enhanced portal vein with partial occlusion or arterioportal shunts. 6. Six of 13 cases with occlusion of main portal vein showed cavernous transformation and they were noted as multiple small enhanced vascularities around the porta hepatis in the CT. According to the results, we conclude that CT is a useful modality to detect the changes of the portal veins in the patients of the hepatoma.

Computed tomographic evaluation of the portal vein in the hepatomas

International Nuclear Information System (INIS)

Lee, Kee Hyung; Lee, Seung Chul; Bae, Man Gil; Seo, Heung Suk; Kim, Soon Yong; Lee, Min Ho; Kee, Choon Suhk; Park, Kyung Nam

1986-01-01

Computed tomography and pornographic findings of 63 patients with hepatoma, undergone hepatic angiography and superior mesenteric pornography for evaluation of tumor and thrombosis of portal vein and determination of indication of transcatheter arterial embolization for palliative treatment of hepatoma from April, 85 to June, 86 in Hanyang university hospital, were reviewed. The results were as follows: 1. In 36 cases, portal vein thrombosis was detected during photography. Nineteen of 37 cases which revealed localized hepatoma in the right lobe of the liver showed portal vein thrombosis; 9 of 11 cases of the left lobe; 8 of 14 cases which were involved in entire liver revealed thrombosis. One case localized in the caudate lobe showed no evidence of invasion to portal vein. 2. Twenty-four of 34 cases with diffuse infiltrative hepatoma revealed portal vein thrombosis and the incidence of portal vein thrombosis in this type were higher than in the cases of the nodular type. 3. The portal vein thrombosis appeared as filling defects of low density in the lumen of the portal veins in CT and they did not reveal contrast enhancement. 4. CT revealed well the evidence of obstructions in the cases of portal vein thrombosis and the findings were well-corresponded to the findings of the superior mesenteric photography. 5. Five of the cases of the portal vein thrombosis were missed in the CT and the causes were considered as due to partial volume effect of enhanced portal vein with partial occlusion or arterioportal shunts. 6. Six of 13 cases with occlusion of main portal vein showed cavernous transformation and they were noted as multiple small enhanced vascularities around the porta hepatis in the CT. According to the results, we conclude that CT is a useful modality to detect the changes of the portal veins in the patients of the hepatoma.

Tyramine-O-sulfate is produced and secreted by human hepatoma cells, line HepG2

International Nuclear Information System (INIS)

Liu, M.C.; Yu, S.; Suiko, M.

1987-01-01

Human hepatoma cells, line HepG2, were metabolically labeled with [ 35 S]sulfate. The spent medium separated following 24 hr labeling was subjected to ultrafiltration using an Amicon Centricon unit. The filtrate obtained was analyzed by a two-dimensional separation procedure combining high-voltage electrophoresis and thin-layer chromatography. The autoradiograph taken from the cellulose thin-layer plate following the analysis revealed the presence of tyramine-O-[ 35 ]sulfate in addition to tyrosine-O-[ 35 ]sulfate. Using adenosine, 3'-phosphate, 5'-phospho[ 35 S]sulfate as the sulfate donor, it was shown that tyramine was actively sulfated to form tyramine-O-[ 35 S]sulfate as catalyzed by the sulfotransferase(s) present in dog liver homogenate. Attempts to decarboxylate tyrosine-O-sulfate to tyramine-O-sulfate using intrinsic p-tyrosine decarboxylase present in dog liver homogenate, however, were unsuccessful. Employing purified Streptococcus faecalis tyrosine decarboxylase, it was shown that L-tyrosine was actively decarboxylated to tyramine, whereas tyrosine-O-sulfate could not serve as a substrate

Enhancement of esculetin on Taxol-induced apoptosis in human hepatoma HepG2 cells

International Nuclear Information System (INIS)

Kuo, H.-C.; Lee, H.-J.; Hu, C.-C.; Shun, H.-I; Tseng, T.-H.

2006-01-01

The potential use of low dose chemotherapy has been appealing since lower dosages are more attainable during cancer therapy and cause less toxicity in patients. Combination therapy of Taxol, a promising frontline chemotherapy agent, with natural anti-tumor agents that are considerably less toxic with a capability of activating additional apoptotic signals or inhibiting survival signals may provide a rational molecular basis for novel chemotherapeutic strategies. Esculetin, a well-known lipoxygenase inhibitor, showed an inhibitory effect on the cell cycle progression of HL-60 cells in our previous study. In this report, the effects of a concomitant administration of esculetin and Taxol were investigated in human hepatoma HepG2 cells. Firstly, esculetin alone could exert an antiproliferation effect together with an inhibitory effect on the activation of ERKs and p38 MAPK. As compared to the treatment with Taxol only, a co-administration with esculetin and Taxol could result in a further enhancement of apoptosis as revealed by DNA fragmentation assay and Annexin-V-based assay. Meanwhile, immunoblotting analysis also showed that the co-administration of esculetin and Taxol could increase the expression of Bax and the cytosolic release of cytochrome C and enhance the expression of Fas and Fas ligand while the activation of caspase-8 and caspase-3 was also increased. Finally, the ERK cascade was proven to be involved in the enhancement of esculetin on the Taxol-induced apoptosis

Synergistic cytotoxicity and mechanism of caffeine and lysozyme on hepatoma cell line HepG2

Science.gov (United States)

Yang, Hongchao; Li, Jingjuan; Cui, Lin; Ren, Yanqing; Niu, Liying; Wang, Xinguo; Huang, Yun; Cui, Lijian

2018-03-01

The influences of caffeine, lysozyme and the joint application of them on the hepatoma cell line HepG2 proliferation inhibition and cell apoptosis were observed by 3-(4, 5-dimethyl-2-thiazyl)-2, 5-diphenyl-2H-tetrazolium bromide assay and Hoechst 33342, which showed the proliferation inhibition rate of the joint application on HepG2 cells was 47.21%, significantly higher than caffeine or lysozyme, and the joint application promoted the apoptosis of HepG2 cells obviously. Van't Hoff classical thermodynamics formula, the Föster theory of non-radiation energy transfer and fluorescence phase diagram were used to manifest that the process of lysozyme binding to caffeine followed a two-state model, which was spontaneous at low temperature driven by enthalpy change, and the predominant intermolecular force was hydrogen bonding or Van der Waals force to stabilize caffeine-lysozyme complex with the distance 5.86 nm. The attenuated total reflection-Fourier transform infrared spectra indicated that caffeine decreased the relative contents of α-helix and β-turn, which inferred the structure of lysozyme tended to be "loose". Synchronous fluorescence spectra and ultraviolet spectra supported the above conclusion. The amino acid residues in the cleft of lysozyme were exposed and electropositivity was increased attributing to the loose structure, which were conducive to increasing caffeine concentration on the HepG2 cell surface by electrostatic interaction to show synergistic effect. The great quantities of microvilli on the liver cancer cell membrane surface, is beneficial for the lysozyme-caffeine compound to aggregate on cell surface to increase the concentration of caffeine to play stronger physiological role by electrostatic effect.

The inhibition effect of 2,3,7,8-tetrachlorinated dibenzo-p-dioxin-induced aryl hydrocarbon receptor activation in human hepatoma cells with the treatment of cadmium chloride

International Nuclear Information System (INIS)

Chao, How-Ran; Tsou, Tsui-Chun; Chen, Hung-Ta; Chang, Eddy Essen; Tsai, Feng-Yuan; Lin, Ding-Yan; Chen, Fu-An; Wang, Ya-Fen

2009-01-01

Polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs), considered as endocrine disruptors, tend to accumulate in fatty tissues. Dioxin-responsive element chemical activated luciferase gene expression assay (DRE-luciferase assay) has been recognized as a semi-quantitative method for screening dioxins for its fast and low-cost as compared with HRGC/HRMS. However, some problems with the bioassay, including specificity, detection variation resulted from different cleanup strategies, and uncertainty of false-negative or false-positive results, remain to be overcome. Cadmium is a prevalent environmental contaminant around the world. This study was aimed to examine the effects of cadmium on the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced activation of aryl hydrocarbon receptor (AhR)-mediated gene expression in human hepatoma cells (Huh7-DRE-Luc cells and Huh7 cells). Ethoxyresorufin-O-deethylase (EROD) and DRE-luciferase assay were employed to determine the enzyme activity of cytochrome P450 1A1 (CYP1A1) and activation of AhR, respectively. The results showed that Cd 2+ levels significantly inhibited the induction of TCDD-induced CYP1A1 and DRE luciferase activation in hepatoma cells. The 50% inhibited concentrations (IC 50 ) of CdCl 2 were 0.414 μM (95% confidence interval (C.I.): 0.230-0.602 μM) in Huh7-DRE-Luc cells and 23.2 μM (95% C.I.: 21.7-25.4 μM) in Huh7 cells. Accordingly, prevention of interference with non-dioxin-like compounds in a DRE-luciferase assay is of great importance in an extensive cleanup procedure.

A simple and rapid Hepatitis A Virus (HAV titration assay based on antibiotic resistance of infected cells: evaluation of the HAV neutralization potency of human immune globulin preparations

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Kaplan Gerardo G

2008-12-01

Full Text Available Abstract Background Hepatitis A virus (HAV, the causative agent of acute hepatitis in humans, is an atypical Picornaviridae that grows poorly in cell culture. HAV titrations are laborious and time-consuming because the virus in general does not cause cytopathic effect and is detected by immunochemical or molecular probes. Simple HAV titration assays could be developed using currently available viral construct containing selectable markers. Results We developed an antibiotic resistance titration assay (ARTA based on the infection of human hepatoma cells with a wild type HAV construct containing a blasticidin (Bsd resistance gene. Human hepatoma cells infected with the HAV-Bsd construct survived selection with 2 μg/ml of blasticidin whereas uninfected cells died within a few days. At 8 days postinfection, the color of the pH indicator phenol red in cell culture media correlated with the presence of HAV-Bsd-infected blasticidin-resistant cells: an orange-to-yellow color indicated the presence of growing cells whereas a pink-to-purple color indicated that the cells were dead. HAV-Bsd titers were determined by an endpoint dilution assay based on the color of the cell culture medium scoring orange-to-yellow wells as positive and pink-to-purple wells as negative for HAV. As a proof-of-concept, we used the ARTA to evaluate the HAV neutralization potency of two commercially available human immune globulin (IG preparations and a WHO International Standard for anti-HAV. The three IG preparations contained comparable levels of anti-HAV antibodies that neutralized approximately 1.5 log of HAV-Bsd. Similar neutralization results were obtained in the absence of blasticidin by an endpoint dilution ELISA at 2 weeks postinfection. Conclusion The ARTA is a simple and rapid method to determine HAV titers without using HAV-specific probes. We determined the HAV neutralization potency of human IG preparations in 8 days by ARTA compared to the 14 days required by the

Basil extract inhibits the sulfotransferase mediated formation of DNA adducts of the procarcinogen 1'-hydroxyestragole by rat and human liver S9 homogenates and in HepG2 human hepatoma cells

NARCIS (Netherlands)

Jeurissen, S.M.F.; Punt, A.; Delatour, T.; Rietjens, I.M.C.M.

2008-01-01

The effects of a basil extract on the sulfation and concomitant DNA adduct formation of the proximate carcinogen 1¿-hydroxyestragole were studied using rat and human liver S9 homogenates and the human hepatoma cell line HepG2. Basil was chosen since it contains the procarcinogen estragole that can

Preliminary result in patients with primary hepatoma treated by stereotactic radiotherapy

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Kang, Ki Mun; Choi, Ihl Bohng; Kim, In Ah; Choi, Byung Ock; Kang, Young Nam; Han, Sung Tae; Chung, Gyu Won [College of Medicine, Catholic Univ., Seoul (Korea, Republic of); Chai, Gyu Young [College of Medicine, Gyeongsang National Univ., Chinju (Korea, Republic of)

2001-03-01

It is not common to evaluate the response of the fractionated stereotactic radiotherapy (SRT) to primary hepatoma as compared with conventional radiotherapy. The purpose of the study was to take the preliminary result on the clinical trial of primary hepatoma by SRT. From July 1999 to March 2000, thirty three patients were hospitalized in the St. Mary's Hospital, and treated with SRT for extracranial tumors. Among them, 13 patients were diagnosed to primary hepatoma and then applied by frameless SRT using 6 MV linac accelerator. There were 12 male and 1 female patients. They had the age of 44-66 year old (median: 59) and the tumor size of 10-825 cc (median: 185 cc). SRT was given to them 3-5 fractions a week (5 Gy/fraction, 90% isodose line) for 2-3 weeks. Median dose of SRT was 50 Gy and the range was 30-50 Gy. Follow-up period ranged from 3 months to 13 months with median of 8 months. After treating SRT to thirteen patients with primary hepatoma, the response of the tumor was examined by abdominal CT: they are classified by 1 complete regression (7.7%), 7 partial regression (53.8%), 4 minimal regression (30.8%), 1 stable disease (7.7%). The positive responses more than partial remission were 8 patients (61.5%) after the treatment. The level of serum alpha-fetoprotein (AFP) after the treatment as compared with pretreatment had been 92.3% decreased. There was no severe complication except dyspepsia 84.6%, mild nausea 69.2%, transient decreased of hepatic function 15.4% and fever 7.7%. SRT to the patients with primary hepatoma was potentially suggested to become the safe and more effective tool than the conventional radiotherapy even though there were relatively short duration of follow-up and small numbers to be tested.

Basil extract inhibits the sulfotransferase mediated formation of DNA adducts of the procarcinogen 1′-hydroxyestragole by rat and human liver S9 homogenates and in HepG2 human hepatoma cells

NARCIS (Netherlands)

Jeurissen, S.M.F.; Punt, A.; Delatour, T.; Rietjens, I.M.C.M.

2008-01-01

The effects of a basil extract on the sulfation and concomitant DNA adduct formation of the proximate carcinogen 1′-hydroxyestragole were studied using rat and human liver S9 homogenates and the human hepatoma cell line HepG2. Basil was chosen since it contains the procarcinogen estragole that can

Expression of 65-kDa oncofetal protein in experimental hepatoma after antivancer therapy

International Nuclear Information System (INIS)

Mirowski, M.; Rozalski, M.; Krajewska, U.; Wierbicki, R.; Hanausek, M.

1997-01-01

We have tested the expression of 65-kDa oncofetal protein (p65) after combined treatment with menadione and methotrexate in hamsters transplanted with Kirkman-Robbins hepatoma. The treatment of tumor-bearing animals with these compounds significantly inhibited both the tumor development and the expression of p65. This inhibition in tumor tissue was calculated from densitograms of Western blots. The inhibition of p65 was also confirmed in the serum of hepatoma bearing animals by using solid-phase radioimmunoassay (RIA) to quantify the specificity of polyclonal antibodies to fetal p65 molecules. Additionally, p65 was shown to localize both in cytoplasm an in the nuclear extracts prepared from hepatoma tissue. (author)

Immunopathogenesis of Hepatitis B Virus Infection and Related Complications

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Mankgopo M. Kgatle

2016-05-01

Full Text Available Chronic hepatitis B (CHB is a serious consequence of hepatitis B virus (HBV, which infects and replicates in the liver. It is characterised by prolonged hepatitis B surface antigen seropositivity; this can lead to both cirrhosis and hepatocellular carcinoma (HCC. The infection begins when HBV binds its only known functional receptor, sodium taurocholate cotransporting polypeptide (NTCP, which was identified recently. The discovery of NTCP was a significant breakthrough in the field of HBV research, and has facilitated the establishment of a susceptible hepatoma cell line model for studying the mechanisms underlying HBV pathogenesis. Following productive HBV infection, both cellular and humoral immune cells and molecules, such as T cells and chemokines, are activated to resolve infection by destroying HBV-infected hepatocytes. However, host immunity to HBV is not always protective, most likely due to immune evasion mechanisms employed by HBV. These mechanisms may result in viral persistence, accumulation of mutations, and aberrant epigenetic alterations that lead to HCC. Here we highlight our current understanding of the HBV replication cycle, immunopathogenesis, and related mechanisms underlying the progression of CHB to advanced liver disease, along with the attendant complications.

Radiosensitizing effects of 9401 on mice bearing H22 hepatoma

International Nuclear Information System (INIS)

Liu Xiaoqiu; Wang Qin; Zhou Zewei; Han Ying; Wang Dezhi; Shen Xiu

2013-01-01

Objective: To investigate the radiosensitizing effects of 9401 on mice bearing H22 hepatoma. Methods: Mouse model bearing H22 hepatoma cells were established. Mice were randomly divided into six groups, the control group,the radiation group and four treatment groups including 9401 at high, medium and low dosages and nicotinamide combined with radiation. After irradiated, the growth of tumor was observed, the time of tumor growth was recorded, the delay time of tumor growth and enhancement factor (EF) were calculated. After 28 days, the mice were killed, the tumors were stripped and inhibition rate was calculated. Results: Groups of 9401 combined with radiation could postpone tumor growth. The difference was statistically significant between 9401 groups at high, medium dosages combined with radiation and nicotinamide combined with radiation group (t=24.7 and 7.5, both P<0.01). Compared with radiation alone group, groups of 9401 combined with radiation had significant radiosensitizing effect. The enhancement factor of 9401 combined with radiation groups at high and medium dosages were 2.13 and 1.73 respectively, they were significant higher than nicotinamide combined with radiation group (t=2.26 and 9.04, both P<0.05). The inhibition rate of 9401 groups at high, medium and low dosages combined with radiation were 64.5%, 50.9% and 42.6% respectively. The inhibition rate of nicotinamide group combined radiation was 53.2%. The inhibition rate of 9401 at high dosage combined with radiation had significant difference with nicotinamide combined radiation (t =2.8, P<0.05). Nicotinamide combined with radiation group, 9401 combined with radiation groups could significant inhibit the growth of tumors compared with radiation alone group (t=5.7, 4.0 and 2.2, all P<0.05). Conclusion: 9401 can inhibit the tumor growth and the inhibition effect increases gradually with the drug dose increasing. It also has radiosensitizing effects on mice bearing H22 hepatoma and present broadly

Plasmid Transfer of Plasminogen K1-5 Reduces Subcutaneous Hepatoma Growth by Affecting Inflammatory Factors

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Lea A. Koch

2014-01-01

Full Text Available There is evidence that plasminogen K1-5 (PlgK1-5 directly affects tumour cells and inflammation. Therefore, we analysed if PlgK1-5 has immediate effects on hepatoma cells and inflammatory factors in vitro and in vivo. In vitro, effects of plasmid encoding PlgK1-5 (pK1-5 on Hepa129, Hepa1-6, and HuH7 cell viability, apoptosis, and proliferation as well as VEGF and TNF-alpha expression and STAT3-phosphorylation were investigated. In vivo, tumour growth, proliferation, vessel density, and effects on vascular endothelial growth factor (VEGF and tumour necrosis factor alpha (TNF-alpha expression were examined following treatment with pK1-5. In vivo, pK1-5 halved cell viability; cell death was increased by up to 15% compared to the corresponding controls. Proliferation was not affected. VEGF, TNF-alpha, and STAT3-phosphorylation were affected following treatment with pK1-5. In vivo, ten days after treatment initiation, pK1-5 reduced subcutaneous tumour growth by 32% and mitosis by up to 77% compared to the controls. Vessel density was reduced by 50%. TNF-alpha levels in tumour and liver tissue were increased, whereas VEGF levels in tumours and livers were reduced after pK1-5 treatment. Taken together, plasmid gene transfer of PlgK1-5 inhibits hepatoma (cell growth not only by reducing vessel density but also by inducing apoptosis, inhibiting proliferation, and triggering inflammation.

The concentration of cadmium in hepatoma among Filipinos

International Nuclear Information System (INIS)

Alejandrino, A.L.; Goze, C.B.; Paradero, R.R.

1977-08-01

The concentration of cadmium in liver hepatoma and in normal liver in Filipinos was determined by atomic absorption spectrophotometry. Using NBS Bovine Liver (SRM1577) as reference material, a value of 0.28+-0.025 ug/g dry weight was obtained for cadmium which is close to the certified NBS value of 0.27+-0.04 ug/g. The mean percentage recovery for cadmium determination by AAS was 98.38%. A mean value of 2.14+-1.58 ug Cd/g liver hepatoma was observed for the 12 cases investigated, showing decreased cadmium levels in the cancerous liver compared to the mean value of 12.62 ug Cd/g observed for normal liver obtained from 10 cases of accidental deaths. The values are expressed on a dry weight basis

Enhancement of 1,3-bis(2-chloroethyl)-1-nitrosourea resistance by gamma-irradiation or drug pretreatment in rat hepatoma cells

International Nuclear Information System (INIS)

Habraken, Y.; Laval, F.

1991-01-01

Treatment of rat hepatoma cells (H4 cells) with various DNA-damaging agents increases the number of O6-methylguanine-DNA-methyltransferase (transferase) molecules per cell. Because the cellular resistance to chloroethylnitrosoureas depends on the number of transferase molecules, we studied the influence of pretreatment with gamma-irradiation, cis-dichlorodiammineplatinum(II), or 2-methyl-9-hydroxyellipticinium on the sensitivity of H4 cells to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). The BCNU resistance depends on the gamma-ray dose and increases with time after irradiation: it is maximum when the drug is added 48 h after irradiation, which corresponds to the maximum enhancement of the transferase activity in the cells. Pretreatment with a single dose of cis-dichlorodiammineplatinum(II) or 2-methyl-9-hydroxyellipticinium also increases the cellular resistance to BCNU. This resistance is not due to a modification of the alkylation of the cellular DNA in the pretreated cells but is related to the increased transferase activity, as it is no longer observed when this activity is depleted by incubating the pretreated cells with the free base O6-methylguanine before BCNU treatment. These results suggest that tumor cells surviving after gamma-irradiation or drug treatment may become resistant to chemotherapy with chloroethylnitrosoureas

Induction of DNA double-strand breaks in hepatoma cell SMMC-7721 by accelerated carbon ion 12C6+

International Nuclear Information System (INIS)

Lei Suwen; Su Xu; Wang Jufang; Zhao Jing; Li Wenjian

2004-01-01

DNA lesions, especially DNA double-strand breaks (dsbs), are looked upon as the dominant molecular effect of radiation action. Dsbs mark the beginning of a cascade of cellular processes that either results in complete repair of the DNA damage or lead to deleterious stages such as mutation, transformation or even cell death. Changing the radiation quality can influence the radiosensitivity of cells in culture. Accelerated particles provide an excellent means of varying the ionization density of the test radiation. With ion beams, the molecular mechanisms underlying the biological consequences of high linear energy transfer (LET) irradiation can be studied and describing radiation action with biophysical models can be tested. In this paper, radiation-induced DNA double-strand breaks (dsbs) were measured in hepatoma SMMC-7721 cells by means of an experimental approach involving pulsed-field gel electrophoresis and densitometric scanning of ethidium bromide stained gels. With this set-up, the induction of dsbs was investigated in SMMC-7721 cells after irradiation with accelerated carbon ions with specific LET 70 keV/μm. The fraction of DNA retained was taken as quantitative measure to calculate absolute yields of induced DNA dsbs. Experimental data shows that the induction of DNA dsbs increasing with the dose of irradiation. Data are compared with published results on dsbs induction in mammalian cells by radiations of comparable LET

The involvement of splenic artery in the blood supply of hepatomas: its DSA findings and interventional treatment

International Nuclear Information System (INIS)

Duan Xuhua; Liang Huiming; Feng Gansheng; Zheng Chuangsheng; Ren Jianzhuang

2009-01-01

Objective: To investigate the DSA manifestations of the involvement of splenic artery in supplying blood to hepatomas and to assess the therapeutic value of super-selective interventional embolization. Methods: During the period of March 2005-June 2008, 897 patients with hepatoma underwent angiography and the involvement of splenic artery in the blood supply of hepatoma was confirmed in 7 cases. Splenic arteriography was performed by means of super-selective catheterization with 5 F Yashiro catheter together with 3 F SP catheter. The splenic arteries which supplied blood to hepatomas were embolized with hyper-liquid iodized-oil emulsion mixed with chemotherapy drug, which was followed by the injection of sufficient gelatin sponge or ethanol. The clinical results were analyzed. Results: Splenic arteriography revealed that the splenic artery was the main supplying vessel of the hepatoma in two cases, and was not the main supplying vessel of the hepatoma in five cases. The splenic supplying vessels were completely embolized in all 7 cases. After the procedure, AFP level was decreased over 50%, and in two patients it dropped to normal. CT checkup 4-6 weeks after the surgery revealed that the diameter of tumor decreased to 2.5 - 4.6 cm. Conclusion: The involvement of splenic artery in supplying blood to hepatomas is not common. Super-selective catheterization and sufficient embolization of the splenic supplying vessels are very important for improving the interventional effectiveness. (authors)

Coating independent cytotoxicity of citrate- and PEG-coated silver nanoparticles on a human hepatoma cell line.

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Bastos, Verónica; Ferreira-de-Oliveira, José M P; Carrola, Joana; Daniel-da-Silva, Ana L; Duarte, Iola F; Santos, Conceição; Oliveira, Helena

2017-01-01

The antibacterial potential of silver nanoparticles (AgNPs) resulted in their increasing incorporation into consumer, industrial and biomedical products. Therefore, human and environmental exposure to AgNPs (either as an engineered product or a contaminant) supports the emergent research on the features conferring them different toxicity profiles. In this study, 30nm AgNPs coated with citrate or poly(ethylene glycol) (PEG) were used to assess the influence of coating on the effects produced on a human hepatoma cell line (HepG2), namely in terms of viability, apoptosis, apoptotic related genes, cell cycle and cyclins gene expression. Both types of coated AgNPs decreased cell proliferation and viability with a similar toxicity profile. At the concentrations used (11 and 5μg/mL corresponding to IC50 and ~IC10 levels, respectively) the amount of cells undergoing apoptosis was not significant and the apoptotic related genes BCL2 (anti-apoptotic gene) and BAX (pro-apoptotic gene) were both downregulated. Moreover, both AgNPs affected HepG2 cell cycle progression at the higher concentration (11μg/mL) by increasing the percentage of cells in S (synthesis phase) and G2 (Gap 2 phase) phases. Considering the cell-cycle related genes, the expression of cyclin B1 and cyclin E1 genes were decreased. Thus, this work has shown that citrate- and PEG-coated AgNPs impact on HepG2 apoptotic gene expression, cell cycle dynamics and cyclin regulation in a similar way. More research is needed to determine the properties that confer AgNPs at lower toxicity, since their use has proved helpful in several industrial and biomedical contexts. Copyright © 2016. Published by Elsevier B.V.

Increased PRPP synthetase activity in cultured rat hepatoma cells containing mutations in the hypoxanthine-guanine phosphoribosyltransferase gene.

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Graf, L H; McRoberts, J A; Harrison, T M; Martin, D W

1976-07-01

Nine independently derived clones of mutagenized rat hepatoma cells selected for resistance to 6-mercaptopurine (6-MP) or 6-thioguanine (6-ThioG) have been isolated. Each has severely reduced catalytic activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) and seven of them possess significantly increased activities of phosphoribosylpyrophosphate (PRPP) synthetase. The degrees of elevations of PRPP synthetase activities do not correlate with the degrees of deficiencies of HPRT activities. The cells from one of these clones, 1020/12, posses 40% of the normal HPRT catalytic activity and overproduce purines. We have extensively examined the cells from this clone. Immunotration studies of 1020/12 cells indicate that there is a mutation in the structural gene for HPRT. Although they possess increased specific catalytic activities of the enzyme. PRPP synthetase, the catalytic parameters, heat stability, and isoelectric pH of PRPP synthetase from 1020/12 cells are indistinguishable from those of the enzyme from wild-type cells. The cause of purine overproduction by 1020/12 cells appears to be the elevated PRPP synthetase activity, rather than a PRPP "sparing" effect stemming from reduced HPRT activity. Support for this idea is provided by the observation that the complete loss of HPRT activity in a clone derived from 1020/12 cells does not further enhance the levels of PRPP synthetase or purine overproduction. We propose that the elevated levels of PRPP synthetase activity in these HPRT deficient cells result from a mutational event in the structural gene for HPRT, and that this causes the disruption of a previously undescribed regulatory function of this gene on the expression of the PRPP synthetase gene.

Diagnosis of hepatoma using grayscale and Doppler ultrasound in patients with chronic liver disease

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Idris S

2011-10-01

Full Text Available Wasim A Memon, Zishan Haider, Mirza Amanullah Beg, Muhammad Idris, Tanveer-ul-Haq, Waseem Akhtar, Sidra IdrisRadiology Department, Aga Khan University Hospital, Karachi, Pakistan Every author contributed equally to the workObjective: To determine the diagnostic accuracy of liver ultrasound for the detection of hepatoma in chronic liver disease (CLD patients by either taking histopathology or serum α-fetoprotein levels or a biphasic computed tomography (CT scan (whichever is available as the gold standard.Study design: Cross-sectional.Place and duration of study: Radiology Department, The Aga Khan University Hospital, Karachi, Pakistan, from January 2007 to January 2010.Methods: A total of 239 patients (156 males and 83 females with clinical suspicion or surveillance of hepatoma in CLD referred to the radiology department for ultrasound evaluation followed by either liver biopsy and histopathology or serum α-fetoprotein level or biphasic CT scan.Results: The sensitivity of ultrasound for hepatoma detection in CLD was 65%, specificity was 85%, and accuracy was 70%, and positive predictive value and negative predictive value were 92% and 45%, respectively.Conclusion: Ultrasound is a relatively quick, safe, reasonably accurate, and noninvasive imaging modality for the detection of hepatoma in CLD and can be complemented with clinical assessment of screening high-risk patients.Keywords: hepatoma, ultrasound, radiology, chronic liver disease

Composition of Lycium barbarum polysaccharides and their apoptosis-inducing effect on human hepatoma SMMC-7721 cells

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Qian Zhang

2015-11-01

Full Text Available Background: Lycium barbarum polysaccharide (LBP is a natural functional component that has a variety of biological activities. The molecular structures and apoptosis-inducing activities on human hepatoma SMMC-7721 cells of two LBP fractions, LBP-d and LBP-e, were investigated. Results: The results showed that LBP-d and LBP-e both consist of protein, uronic acid, and neutral sugars in different proportions. The structure of LBP was characterized by gas chromatography, periodate oxidation, and Smith degradation. LBP-d was composed of eight kinds of monosaccharides (fucose, ribose, rhamnose, arabinose, xylose, mannose, galactose, and glucose, while LBP-e was composed of six kinds of monosaccharides (fucose, rhamnose, arabinose, mannose, galactose, and glucose. LBP-d and LBP-e blocked SMMC-7721 cells at the G0/G1 and S phases with an inhibition ratio of 26.70 and 45.13%, respectively, and enhanced the concentration of Ca2 + in the cytoplasm of SMMC-7721. Conclusion: The contents of protein, uronic acid, and galactose in LBP-e were much higher than those in LBP-d, which might responsible for their different bioactivities. The results showed that LBP can be provided as a potential chemotherapeutic agent drug to treat cancer.

The dietary flavonoid kaempferol effectively inhibits HIF-1 activity and hepatoma cancer cell viability under hypoxic conditions.

Science.gov (United States)

Mylonis, Ilias; Lakka, Achillia; Tsakalof, Andreas; Simos, George

2010-07-16

Hepatocellular carcinoma (HCC) is characterized by high mortality rates and resistance to conventional treatment. HCC tumors usually develop local hypoxia, which stimulates proliferation of cancer cells and renders them resilient to chemotherapy. Adaptation of tumor cells to the hypoxic conditions depends on the hypoxia-inducible factor 1 (HIF-1). Over-expression of its regulated HIF-1alpha subunit, an important target of anti-cancer therapy, is observed in many cancers including HCC and is associated with severity of tumor growth and poor patient prognosis. In this report we investigate the effect of the dietary flavonoid kaempferol on activity, expression levels and localization of HIF-1alpha as well as viability of human hepatoma (Huh7) cancer cells. Treatment of Huh7 cells with kaempferol under hypoxic conditions (1% oxygen) effectively inhibited HIF-1 activity in a dose-dependent manner (IC(50)=5.16microM). The mechanism of this inhibition did not involve suppression of HIF-1alpha protein levels but rather its mislocalization into the cytoplasm due to inactivation of p44/42 MAPK by kaempferol (IC(50)=4.75microM). Exposure of Huh7 cells to 10microM kaempferol caused significant reduction of their viability, which was remarkably more evident under hypoxic conditions. In conclusion, kaempferol, a non-toxic natural food component, inhibits both MAPK and HIF-1 activity at physiologically relevant concentrations (5-10microM) and suppresses hepatocarcinoma cell survival more efficiently under hypoxia. It has, therefore, potential as a therapeutic or chemopreventive anti-HCC agent. Copyright 2010 Elsevier Inc. All rights reserved.

A study on the thermochemotherapy effect of nanosized As2O3/MZF thermosensitive magnetoliposomes on experimental hepatoma in vitro and in vivo

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Wang, Li; Zhang, Jia; An, Yanli; Wang, Ziyu; Liu, Jing; Li, Yutao; Zhang, Dongsheng

2011-08-01

In this paper, we describe the synthesis and characterization of a nanosized, thermosensitive magnetoliposome encapsulating magnetic nanoparticles (MZFs) and antitumor drugs (As2O3). The nanoliposomes were spherical and mostly single volume, with an average diameter of 128.2 nm. Differential scanning calorimetry (DSC) showed a liposome phase transition temperature of 42.71 °C. After that, we studied the liposomes' anti-hepatoma effect in vitro and in vivo. The antitumor effect of the nanoliposomes on human hepatoma cells, SMMC-7721, and changes in expression of apoptosis-related proteins were examined in vitro. The results show that As2O3/MZF thermosensitive magnetoliposomes combined with hyperthermia had a great impact on the Bax/Bcl-2 ratio, which increased to 1.914 and exhibited a rapid response to induce apoptosis of tumor cells. An in situ rabbit liver tumor model was established and used to evaluate the antitumor effect of combined hyperthermia and chemotherapy following transcatheter arterial embolization with As2O3/MZF thermosensitive magnetoliposomes. The results demonstrated a strong anti-hepatoma effect, with a tumor volume inhibition rate of up to 85.22%. Thus, As2O3/MZF thermosensitive magnetoliposomes may play a great role in the treatment of hepatocarcinoma.

Magnetic nanoparticles for targeted therapeutic gene delivery and magnetic-inducing heating on hepatoma

International Nuclear Information System (INIS)

Yuan, Chenyan; Zhang, Jia; Li, Hongbo; Zhang, Hao; Wang, Ling; Zhang, Dongsheng; An, Yanli

2014-01-01

Gene therapy holds great promise for treating cancers, but their clinical applications are being hampered due to uncontrolled gene delivery and expression. To develop a targeted, safe and efficient tumor therapy system, we constructed a tissue-specific suicide gene delivery system by using magnetic nanoparticles (MNPs) as carriers for the combination of gene therapy and hyperthermia on hepatoma. The suicide gene was hepatoma-targeted and hypoxia-enhanced, and the MNPs possessed the ability to elevate temperature to the effective range for tumor hyperthermia as imposed on an alternating magnetic field (AMF). The tumoricidal effects of targeted gene therapy associated with hyperthermia were evaluated in vitro and in vivo. The experiment demonstrated that hyperthermia combined with a targeted gene therapy system proffer an effective tool for tumor therapy with high selectivity and the synergistic effect of hepatoma suppression. (paper)

Number of infection events per cell during HIV-1 cell-free infection.

Science.gov (United States)

Ito, Yusuke; Remion, Azaria; Tauzin, Alexandra; Ejima, Keisuke; Nakaoka, Shinji; Iwasa, Yoh; Iwami, Shingo; Mammano, Fabrizio

2017-07-26

HIV-1 accumulates changes in its genome through both recombination and mutation during the course of infection. For recombination to occur, a single cell must be infected by two HIV strains. These coinfection events were experimentally demonstrated to occur more frequently than would be expected for independent infection events and do not follow a random distribution. Previous mathematical modeling approaches demonstrated that differences in target cell susceptibility can explain the non-randomness, both in the context of direct cell-to-cell transmission, and in the context of free virus transmission (Q. Dang et al., Proc. Natl. Acad. Sci. USA 101:632-7, 2004: K. M. Law et al., Cell reports 15:2711-83, 2016). Here, we build on these notions and provide a more detailed and extensive quantitative framework. We developed a novel mathematical model explicitly considering the heterogeneity of target cells and analysed datasets of cell-free HIV-1 single and double infection experiments in cell culture. Particularly, in contrast to the previous studies, we took into account the different susceptibility of the target cells as a continuous distribution. Interestingly, we showed that the number of infection events per cell during cell-free HIV-1 infection follows a negative-binomial distribution, and our model reproduces these datasets.

MiR-30e suppresses proliferation of hepatoma cells via targeting prolyl 4-hydroxylase subunit alpha-1 (P4HA1) mRNA

Energy Technology Data Exchange (ETDEWEB)

Feng, Guoxing [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin (China); Shi, Hui [State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin (China); Li, Jiong; Yang, Zhe [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin (China); Fang, Runping; Ye, Lihong [State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin (China); Zhang, Weiying, E-mail: zhwybao@nankai.edu.cn [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin (China); Zhang, Xiaodong, E-mail: zhangxd@nankai.edu.cn [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin (China)

2016-04-08

Aberrant microRNA expression has been shown to be characteristic of many cancers. It has been reported that the expression levels of miR-30e are decreased in liver cancer tissues. However, the role of miR-30e in hepatocellular carcinoma remains poorly understood. In the present study, we investigated the significance of miR-30e in hepatocarcinogenesis. Bioinformatics analysis reveals a putative target site of miR-30e in the 3′-untranslated region (3′UTR) of prolyl 4-hydroxylase subunit alpha-1 (P4HA1) mRNA. Moreover, luciferase reporter gene assays verified that miR-30e directly targeted 3′UTR of P4HA1 mRNA. Then, we demonstrated that miR-30e was able to reduce the expression of P4HA1 at the levels of mRNA and protein using reverse transcription-polymerase chain reaction and Western blot analysis. Enforced expression of miR-30e suppressed proliferation of HepG2 cells by 5-ethynyl-2-deoxyuridine (EdU) assay and reduced colony formation of these cells by colony formation analysis. Conversely, anti-miR-30e enhanced the proliferation of hepatoma cells in vitro. Interestingly, the ectopic expression of P4HA1 could efficiently rescue the inhibition of cell proliferation mediated by miR-30e in HepG2 cells. Meanwhile, silencing of P4HA1 abolished the anti-miR-30e-induced proliferation of cells. Clinically, quantitative real-time PCR showed that miR-30e was down-regulated in liver tumor tissues relative to their peritumor tissues. The expression levels of miR-30e were negatively correlated to those of P4HA1 mRNA in clinical liver tumor tissues. Thus, we conclude that miR-30e suppresses proliferation of hepatoma cells through targeting P4HA1 mRNA. Our finding provides new insights into the mechanism of hepatocarcinogenesis. - Highlights: • P4HA1 is a novel target gene of miR-30e. • P4HA1 is increased in clinical HCC tissues. • MiR-30e is negatively correlated with P4HA1 in clinical HCC tissues. • MiR-30e suppresses the proliferation of HCC cells through

The dietary flavonoid kaempferol effectively inhibits HIF-1 activity and hepatoma cancer cell viability under hypoxic conditions

International Nuclear Information System (INIS)

Mylonis, Ilias; Lakka, Achillia; Tsakalof, Andreas; Simos, George

2010-01-01

Research highlights: → Kaempferol inhibits HIF-1 activity in hepatocarcinoma cells; → Kaempferol causes cytoplasmic mislocalization of HIF-1α by impairing the MAPK pathway. → Viability of hepatocarcinoma cells under hypoxia is reduced by kaempferol. -- Abstract: Hepatocellular carcinoma (HCC) is characterized by high mortality rates and resistance to conventional treatment. HCC tumors usually develop local hypoxia, which stimulates proliferation of cancer cells and renders them resilient to chemotherapy. Adaptation of tumor cells to the hypoxic conditions depends on the hypoxia-inducible factor 1 (HIF-1). Over-expression of its regulated HIF-1α subunit, an important target of anti-cancer therapy, is observed in many cancers including HCC and is associated with severity of tumor growth and poor patient prognosis. In this report we investigate the effect of the dietary flavonoid kaempferol on activity, expression levels and localization of HIF-1α as well as viability of human hepatoma (Huh7) cancer cells. Treatment of Huh7 cells with kaempferol under hypoxic conditions (1% oxygen) effectively inhibited HIF-1 activity in a dose-dependent manner (IC 50 = 5.16 μM). The mechanism of this inhibition did not involve suppression of HIF-1α protein levels but rather its mislocalization into the cytoplasm due to inactivation of p44/42 MAPK by kaempferol (IC 50 = 4.75 μM). Exposure of Huh7 cells to 10 μΜ kaempferol caused significant reduction of their viability, which was remarkably more evident under hypoxic conditions. In conclusion, kaempferol, a non-toxic natural food component, inhibits both MAPK and HIF-1 activity at physiologically relevant concentrations (5-10 μM) and suppresses hepatocarcinoma cell survival more efficiently under hypoxia. It has, therefore, potential as a therapeutic or chemopreventive anti-HCC agent.

The dietary flavonoid kaempferol effectively inhibits HIF-1 activity and hepatoma cancer cell viability under hypoxic conditions

Energy Technology Data Exchange (ETDEWEB)

Mylonis, Ilias; Lakka, Achillia; Tsakalof, Andreas [Laboratory of Biochemistry, School of Medicine, University of Thessaly, BIOPOLIS, 41110 Larissa (Greece); Institute of Biomedical Research and Technology (BIOMED), 51 Papanastasiou str., 41222 Larissa (Greece); Simos, George, E-mail: simos@med.uth.gr [Laboratory of Biochemistry, School of Medicine, University of Thessaly, BIOPOLIS, 41110 Larissa (Greece); Institute of Biomedical Research and Technology (BIOMED), 51 Papanastasiou str., 41222 Larissa (Greece)

2010-07-16

Research highlights: {yields} Kaempferol inhibits HIF-1 activity in hepatocarcinoma cells; {yields} Kaempferol causes cytoplasmic mislocalization of HIF-1{alpha} by impairing the MAPK pathway. {yields} Viability of hepatocarcinoma cells under hypoxia is reduced by kaempferol. -- Abstract: Hepatocellular carcinoma (HCC) is characterized by high mortality rates and resistance to conventional treatment. HCC tumors usually develop local hypoxia, which stimulates proliferation of cancer cells and renders them resilient to chemotherapy. Adaptation of tumor cells to the hypoxic conditions depends on the hypoxia-inducible factor 1 (HIF-1). Over-expression of its regulated HIF-1{alpha} subunit, an important target of anti-cancer therapy, is observed in many cancers including HCC and is associated with severity of tumor growth and poor patient prognosis. In this report we investigate the effect of the dietary flavonoid kaempferol on activity, expression levels and localization of HIF-1{alpha} as well as viability of human hepatoma (Huh7) cancer cells. Treatment of Huh7 cells with kaempferol under hypoxic conditions (1% oxygen) effectively inhibited HIF-1 activity in a dose-dependent manner (IC{sub 50} = 5.16 {mu}M). The mechanism of this inhibition did not involve suppression of HIF-1{alpha} protein levels but rather its mislocalization into the cytoplasm due to inactivation of p44/42 MAPK by kaempferol (IC{sub 50} = 4.75 {mu}M). Exposure of Huh7 cells to 10 {mu}{Mu} kaempferol caused significant reduction of their viability, which was remarkably more evident under hypoxic conditions. In conclusion, kaempferol, a non-toxic natural food component, inhibits both MAPK and HIF-1 activity at physiologically relevant concentrations (5-10 {mu}M) and suppresses hepatocarcinoma cell survival more efficiently under hypoxia. It has, therefore, potential as a therapeutic or chemopreventive anti-HCC agent.

[Pseudolaric acid B induces G2/M arrest and inhibits invasion and migration in HepG2 hepatoma cells].

Science.gov (United States)

Li, Shuai; Guo, Lianyi

2018-01-01

Objective To investigate the mechanisms of pseudolaric acid B (PAB) blocks cell cycle and inhibits invasion and migration in human hepatoma HepG2 cells. Methods The proliferation effect of PAB on HepG2 cells was evaluated by MTT assay. The effect of PAB on the cell cycle of HepG2 cells was analyzed by flow cytometry. Immunofluorescence cytochemical staining was applied to observe the effect of PAB on the α-tubulin polymerization and expression in HepG2 cells. Transwell TM chamber invasion assay and wound healing assay were performed to detect the influence of PAB on the migration and invasion ability of HepG2 cells. Western blotting was used to determine the expressions of α-tubulin, E-cadherin and MMP-9 in HepG2 cells after treated with PAB. Results PAB inhibited the proliferation of HepG2 cells in a dose-dependent manner and blocked the cell cycle in G2/M phase. PAB significantly changed the polymerization and decreased the expression of α-tubulin. The capacities of invasion and migration of HepG2 cells treated by PAB were significantly depressed. The protein levels of α-tubulin and MMP-9 decreased while the E-cadherin protein level increased. Conclusion PAB can inhibits the proliferation of HepG2 cells by down-regulating the expression of α-tubulin and influencing its polymerization, arresting HepG2 cells in G2/M phase. Meanwhile, PAB also can inhibit the invasion and migration of HepG2 cells by lowering cytoskeleton α-tubulin and MMP-9, and increasing E-cadherin.

Mechanism(s of Toxic Action of Zn2+ and Selenite: A Study on AS-30D Hepatoma Cells and Isolated Mitochondria

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Elena A. Belyaeva

2011-01-01

Full Text Available Mitochondria of AS-30D rat ascites hepatoma cells are found to be the main target for Zn2+ and sodium selenite (Na2SeO3. High [mu]M concentrations of Zn2+ or selenite were strongly cytotoxic, killing the AS-30D cells by both apoptotic and necrotic ways. Both Zn2+ and selenite produced strong changes in intracellular generation of reactive oxygen species (ROS and the mitochondrial dysfunction via the mitochondrial electron transport chain (mtETC disturbance, the membrane potential dissipation, and the mitochondrial permeability transition pore opening. The significant distinctions in toxic action of Zn2+ and selenite on AS-30D cells were found. Selenite induced a much higher intracellular ROS level (the early event compared to Zn2+ but a lower membrane potential loss and a lower decrease of the uncoupled respiration rate of the cells, whereas the mtETC disturbance was the early and critical event in the mechanism of Zn2+ cytotoxicity. Sequences of events manifested in the mitochondrial dysfunction produced by the metal/metalloid under test are compared with those obtained earlier for Cd2+, Hg2+, and Cu2+ on the same model system.

Cells for bioartificial liver devices: the human hepatoma-derived cell line C3A produces urea but does not detoxify ammonia.

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Mavri-Damelin, Demetra; Damelin, Leonard H; Eaton, Simon; Rees, Myrddin; Selden, Clare; Hodgson, Humphrey J F

2008-02-15

Extrahepatic bioartificial liver devices should provide an intact urea cycle to detoxify ammonia. The C3A cell line, a subclone of the hepatoma-derived HepG2 cell line, is currently used in this context as it produces urea, and this has been assumed to be reflective of ammonia detoxification via a functional urea cycle. However, based on our previous findings of perturbed urea-cycle function in the non-urea producing HepG2 cell line, we hypothesized that the urea produced by C3A cells was via a urea cycle-independent mechanism, namely, due to arginase II activity, and therefore would not detoxify ammonia. Urea was quantified using (15)N-ammonium chloride metabolic labelling with gas chromatography-mass spectrometry. Gene expression was determined by real-time reverse transcriptase-PCR, protein expression by western blotting, and functional activities with radiolabelling enzyme assays. Arginase inhibition studies used N(omega)-hydroxy-nor-L-arginine. Urea was detected in C3A conditioned medium; however, (15)N-ammonium chloride-labelling indicated that (15)N-ammonia was not incorporated into (15)N-labelled urea. Further, gene expression of two urea cycle genes, ornithine transcarbamylase and arginase I, were completely absent. In contrast, arginase II mRNA and protein was expressed at high levels in C3A cells and was inhibited by N(omega)-hydroxy-nor-L-arginine, which prevented urea production, thereby indicating a urea cycle-independent pathway. The urea cycle is non-functional in C3A cells, and their urea production is solely due to the presence of arginase II, which therefore cannot provide ammonia detoxification in a bioartificial liver system. This emphasizes the continued requirement for developing a component capable of a full repertoire of liver function. (c) 2007 Wiley Periodicals, Inc.

Co-operation of the transcription factor hepatocyte nuclear factor-4 with Sp1 or Sp3 leads to transcriptional activation of the human haem oxygenase-1 gene promoter in a hepatoma cell line.

Science.gov (United States)

Takahashi, Shigeru; Matsuura, Naomi; Kurokawa, Takako; Takahashi, Yuji; Miura, Takashi

2002-11-01

We reported previously that the 5'-flanking region (nucleotides -1976 to -1655) of the human haem oxygenase-1 ( hHO-1 ) gene enhances hHO-1 promoter activity in human hepatoma HepG2 cells, but not in HeLa cells [Takahashi, Takahashi, Ito, Nagano, Shibahara and Miura (1999) Biochim. Biophys. Acta 1447, 231-235]. To define more precisely the regulatory elements involved, in the present study we have functionally dissected this region and localized the enhancer to a 50 bp fragment (-1793 to -1744). Site-direct mutagenesis analysis revealed that two regions were responsible for this enhancer activity, i.e. a hepatocyte nuclear factor-4 (HNF-4) homologous region and a GC box motif homologous region. Mutation in either region alone moderately decreased enhancer activity. However, mutations in both regions reduced promoter activity to the basal level. Electrophoretic mobility-shift assays demonstrated that the P5-2 fragment (-1793 to -1744) interacted with at least two nuclear factors, i.e. HNF-4 and Sp1/Sp3. Co-transfection experiments using Drosophila SL2 cells revealed that HNF-4 and Sp1/Sp3 synergistically stimulated the enhancer activity of the P5-2 fragment. These results indicate that co-operation of HNF-4 with Sp1 or Sp3 leads to the activation of hHO-1 gene expression in hepatoma cells.

A Taiwanese Propolis Derivative Induces Apoptosis through Inducing Endoplasmic Reticular Stress and Activating Transcription Factor-3 in Human Hepatoma Cells

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Fat-Moon Suk

2013-01-01

Full Text Available Activating transcription factor-(ATF- 3, a stress-inducible transcription factor, is rapidly upregulated under various stress conditions and plays an important role in inducing cancer cell apoptosis. NBM-TP-007-GS-002 (GS-002 is a Taiwanese propolin G (PPG derivative. In this study, we examined the antitumor effects of GS-002 in human hepatoma Hep3B and HepG2 cells in vitro. First, we found that GS-002 significantly inhibited cell proliferation and induced cell apoptosis in dose-dependent manners. Several main apoptotic indicators were found in GS-002-treated cells, such as the cleaved forms of caspase-3, caspase-9, and poly(ADP-ribose polymerase (PARP. GS-002 also induced endoplasmic reticular (ER stress as evidenced by increases in ER stress-responsive proteins including glucose-regulated protein 78 (GRP78, growth arrest- and DNA damage-inducible gene 153 (GADD153, phosphorylated eukaryotic initiation factor 2α (eIF2α, phosphorylated protein endoplasmic-reticular-resident kinase (PERK, and ATF-3. The induction of ATF-3 expression was mediated by mitogen-activated protein kinase (MAPK signaling pathways in GS-002-treated cells. Furthermore, we found that GS-002 induced more cell apoptosis in ATF-3-overexpressing cells. These results suggest that the induction of apoptosis by the propolis derivative, GS-002, is partially mediated through ER stress and ATF-3-dependent pathways, and GS-002 has the potential for development as an antitumor drug.

HBx induced AFP receptor expressed to activate PI3K/AKT signal to promote expression of Src in liver cells and hepatoma cells

International Nuclear Information System (INIS)

Zhu, Mingyue; Guo, Junli; Li, Wei; Xia, Hua; Lu, Yan; Dong, Xu; Chen, Yi; Xie, Xieju; Fu, Shigan; Li, Mengsen

2015-01-01

Hepatitis B virus (HBV)-X protein(HBx) is a transactivator of host several cellular genes including alpha-fetoprotein(AFP) and AFP receptor(AFPR) which contributes to HBV-associated tumor development. The expression of AFP/AFPR are correlated with hepatocellular carcinoma(HCC)-initial cells. But the role of AFP and AFPR in promoting occurrence of HBV-related HCC were still unclear. A total of 71 clinical patients’ liver specimens, normal human liver cells L-02 and HCC cell lines, PLC/PRF/5 were selected for analyzing the effects of HBx on expression of AFP, AFPR and Src. The expression of goal proteins were detected by Immunohistochemical stained and Western blotting; HBx-expressed vectors were constructed and transfected into L-02 cells, laser confocal microscopy was applied to observe expression and location of AFP, AFPR and Src in the normal liver cells and HCC cells, soft agar colony formation assay was used to observe colonies formed of the cells. We confirmed HBx gives preference to promote the expression of AFP and AFPR; HBx priors to up-regulate the expression of AFPR and AFP in L-02 cells and in normal liver specimens; AFPR signal been able to stimulate Src expression. The results also indicated that phosphatidylinositol 3-kinase(PI3K) inhibitors Ly294002 and GDC0941 effectively suppress AFPR mediated up-regulation expression of Src in AFPR positive HCC lines. HBx priors to drive the expression of AFP and AFPR to promote expression of Src in normal liver cells and hepatoma cells; AFP and AFPR maybe play pivotal role in HBV-related hepatocarcinogenesis; Targeting AFPR is an available therapeutic strategy of HCC. The online version of this article (doi:10.1186/s12885-015-1384-9) contains supplementary material, which is available to authorized users

Involvement of c-Met- and phosphatidylinositol 3-kinase dependent pathways in arsenite-induced downregulation of catalase in hepatoma cells.

Science.gov (United States)

Kim, Soohee; Lee, Seung Heon; Kang, Sukmo; Lee, Lyon; Park, Jung-Duck; Ryu, Doug-Young

2011-01-01

Catalase protects cells from reactive oxygen species-induced damage by catalyzing the breakdown of hydrogen peroxide to oxygen and water. Arsenite decreases catalase activity; it activates phosphatidylinositol 3-kinase (PI3K) and its key downstream effector Akt in a variety of cells. The PI3K pathway is known to inhibit catalase expression. c-Met, an upstream regulator of PI3K and Akt, is also involved in the regulation of catalase expression. To examine the involvement of c-Met and PI3K pathways in the arsenite-induced downregulation of catalase, catalase mRNA and protein expression were analyzed in the human hepatoma cell line HepG2 treated with arsenite and either an inhibitor of c-Met (PHA665752 (PHA)) or of PI3K (LY294002 (LY)). Arsenite treatment markedly activated Akt and decreased the levels of both catalase mRNA and protein. Both PHA and LY attenuated arsenite-induced activation of Akt. PHA and LY treatment also prevented the inhibitory effect of arsenite on catalase protein expression but did not affect the level of catalase mRNA. These findings suggest that arsenite-induced inhibition of catalase expression is regulated at the mRNA and post-transcriptional levels in HepG2 cells, and that the post-transcriptional regulation is mediated via c-Met- and PI3K-dependent mechanisms.

Effects of drugs in subtoxic concentrations on the metabolic fluxes in human hepatoma cell line Hep G2

International Nuclear Information System (INIS)

Niklas, Jens; Noor, Fozia; Heinzle, Elmar

2009-01-01

Commonly used cytotoxicity assays assess the toxicity of a compound by measuring certain parameters which directly or indirectly correlate to the viability of the cells. However, the effects of a given compound at concentrations considerably below EC 50 values are usually not evaluated. These subtoxic effects are difficult to identify but may eventually cause severe and costly long term problems such as idiosyncratic hepatotoxicity. We determined the toxicity of three hepatotoxic compounds, namely amiodarone, diclofenac and tacrine on the human hepatoma cell line Hep G2 using an online kinetic respiration assay and analysed the effects of subtoxic concentrations of these drugs on the cellular metabolism by using metabolic flux analysis. Several changes in the metabolism could be detected upon exposure to subtoxic concentrations of the test compounds. Upon exposure to diclofenac and tacrine an increase in the TCA-cycle activity was observed which could be a signature of an uncoupling of the oxidative phosphorylation. The results indicate that metabolic flux analysis could serve as an invaluable novel tool for the investigation of the effects of drugs. The described methodology enables tracking the toxicity of compounds dynamically using the respiration assay in a range of concentrations and the metabolic flux analysis permits interesting insights into the changes in the central metabolism of the cell upon exposure to drugs.

Plectin deficiency in liver cancer cells promotes cell migration and sensitivity to sorafenib treatment.

Science.gov (United States)

Cheng, Chiung-Chi; Chao, Wei-Ting; Liao, Chen-Chun; Tseng, Yu-Hui; Lai, Yen-Chang Clark; Lai, Yih-Shyong; Hsu, Yung-Hsiang; Liu, Yi-Hsiang

2018-01-02

Plectin involved in activation of kinases in cell signaling pathway and plays important role in cell morphology and migration. Plectin knockdown promotes cell migration by activating focal adhesion kinase and Rac1-GTPase activity in liver cells. Sorafenib is a multi-targeting tyrosine kinase inhibitor that improves patient survival on hepatocellular carcinoma. The aim of this study is to investigate the correlation between the expression of plectin and cell migration as well as the sensitivity of hepatoma cell lines exposing to sorafenib. Hepatoma cell lines PLC/PRF/5 and HepG2 were used to examine the level of plectin expression and cell migration in comparison with Chang liver cell line. In addition, sensitivity of the 3 cell lines to sorafenib treatment was also measured. Expression of plectin was lower in PLC/PRF/5 and HepG2 hepatoma cells than that of Chang liver cells whereas HepG2 and PLC/PRF/5 cells exhibit higher rate of cell migration in trans-well migration assay. Immunohistofluorecent staining on E-cadherin revealed the highest rate of collective cell migration in HepG2 cells and the lowest was found in Chang liver cells. Likewise, HepG2 cell line was most sensitive to sorafenib treatment and Chang liver cells exhibited the least sensitivity. The drug sensitivity to sorafenib treatment showed inverse correlation with the expression of plectin. We suggest that plectin deficiency and increased E-cadherin in hepatoma cells were associated with higher rates of cell motility, collective cell migration as well as higher drug sensitivity to sorafenib treatment.

A study on the thermochemotherapy effect of nanosized As{sub 2}O{sub 3}/MZF thermosensitive magnetoliposomes on experimental hepatoma in vitro and in vivo

Energy Technology Data Exchange (ETDEWEB)

Wang Li; Zhang Jia; Wang Ziyu; Liu Jing; Li Yutao; Zhang Dongsheng [School of Medicine, Southeast University, NO. 87 Ding jia qiao, Nanjing 210009 (China); An Yanli, E-mail: wangli040418@163.com, E-mail: zdszds1222@163.com [Affiliated Zhong-Da Hospital of Southeast University, Nanjing 210009 (China)

2011-08-05

In this paper, we describe the synthesis and characterization of a nanosized, thermosensitive magnetoliposome encapsulating magnetic nanoparticles (MZFs) and antitumor drugs (As{sub 2}O{sub 3}). The nanoliposomes were spherical and mostly single volume, with an average diameter of 128.2 nm. Differential scanning calorimetry (DSC) showed a liposome phase transition temperature of 42.71 deg. C. After that, we studied the liposomes' anti-hepatoma effect in vitro and in vivo. The antitumor effect of the nanoliposomes on human hepatoma cells, SMMC-7721, and changes in expression of apoptosis-related proteins were examined in vitro. The results show that As{sub 2}O{sub 3}/MZF thermosensitive magnetoliposomes combined with hyperthermia had a great impact on the Bax/Bcl-2 ratio, which increased to 1.914 and exhibited a rapid response to induce apoptosis of tumor cells. An in situ rabbit liver tumor model was established and used to evaluate the antitumor effect of combined hyperthermia and chemotherapy following transcatheter arterial embolization with As{sub 2}O{sub 3}/MZF thermosensitive magnetoliposomes. The results demonstrated a strong anti-hepatoma effect, with a tumor volume inhibition rate of up to 85.22%. Thus, As{sub 2}O{sub 3}/MZF thermosensitive magnetoliposomes may play a great role in the treatment of hepatocarcinoma.

Acetyl-CoA carboxylase in Reuber hepatoma cells: variation in enzyme activity, insulin regulation, and cellular lipid content.

Science.gov (United States)

Bianchi, A; Evans, J L; Nordlund, A C; Watts, T D; Witters, L A

1992-01-01

Reuber hepatoma cells are useful cultured lines for the study of insulin action, lipid and lipoprotein metabolism, and the regulation of acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of fatty acid biosynthesis. During investigations in different clonal lines of these cells, we have uncovered marked intercellular variability in the activity, enzyme content, and insulin regulation of ACC paralleled by differences in cellular neutral lipid (triglyceride) content. Two contrasting clonal lines, Fao and H356A-1, have been studied in detail. Several features distinguish these two lines, including differences in ACC activity and enzyme kinetics, the content of the two major hepatic ACC isozymes (Mr 280,000 and 265,000 Da) and their heteroisozymic complex, the extent of ACC phosphorylation, and the ability of ACC to be activated on stimulation by insulin and insulinomimetic agonists. As studied by Nile Red staining and fluorescence-activated cell sorting, these two lines also display marked differences in neutral lipid content, which correlates with both basal levels of ACC activity and inhibition of ACC by the fatty acid analog, 5-(tetradecyloxy)-2-furoic acid (TOFA). These results emphasize the importance of characterization of any particular clonal line of Reuber cells for studies of enzyme regulation, substrate metabolism, and hormone action. With respect to ACC, studies in contrasting clonal lines of Reuber cells could provide valuable clues to understanding both the complex mechanisms of intracellular ACC regulation in the absence and presence of hormones and its regulatory role(s) in overall hepatic lipid metabolism.

Clinical and biological significance of hepatoma-derived growth factor in Ewing's sarcoma.

Science.gov (United States)

Yang, Yang; Li, Hui; Zhang, Fenfen; Shi, Huijuan; Zhen, Tiantian; Dai, Sujuan; Kang, Lili; Liang, Yingjie; Wang, Jin; Han, Anjia

2013-11-01

We sought to investigate the clinicopathological significance and biological function of hepatoma-derived growth factor (HDGF) in Ewing's sarcoma. Our results showed that HDGF expression is up-regulated in Ewing's sarcoma. Nuclear HDGF expression is significantly associated with tumour volume (p Ewing's sarcoma cell growth, proliferation and enhances tumourigenesis, both in vitro and in vivo. Meanwhile, HDGF knock-down causes cell cycle arrest and enhanced sensitization to serum starvation-induced apoptosis. Furthermore, recombinant HDGF promotes proliferation and colony formation of Ewing's sarcoma cells. Ninety-eight candidate HDGF downstream genes were identified in Ewing's sarcoma cells using cDNA microarray analysis. In addition, we found that HDGF knock-down inhibited FLI1 expression in Ewing's sarcoma cells at the mRNA and protein levels. Our findings suggest that HDGF exhibits oncogenic properties and may be a novel prognostic factor in Ewing's sarcoma. Targeting HDGF might be a potential therapeutic strategy for Ewing's sarcoma. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Efferocytosis of Pathogen-Infected Cells

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Niloofar Karaji

2017-12-01

Full Text Available The prompt and efficient clearance of unwanted and abnormal cells by phagocytes is termed efferocytosis and is crucial for organism development, maintenance of tissue homeostasis, and regulation of the immune system. Dying cells are recognized by phagocytes through pathways initiated via “find me” signals, recognition via “eat me” signals and down-modulation of regulatory “don’t eat me” signals. Pathogen infection may trigger cell death that drives phagocytic clearance in an immunologically silent, or pro-inflammatory manner, depending on the mode of cell death. In many cases, efferocytosis is a mechanism for eliminating pathogens and pathogen-infected cells; however, some pathogens have subverted this process and use efferocytic mechanisms to avoid innate immune detection and assist phagocyte infection. In parallel, phagocytes can integrate signals received from infected dying cells to elicit the most appropriate effector response against the infecting pathogen. This review focuses on pathogen-induced cell death signals that drive infected cell recognition and uptake by phagocytes, and the outcomes for the infected target cell, the phagocyte, the pathogen and the host.

Synthesis and preliminary evaluation of 9-(4-[{sup 18}F]fluoro-3-hydroxymethylbutyl) guanine ([{sup 18}F]FHBG) in HSV1-tk gene transduced hepatoma cell

Energy Technology Data Exchange (ETDEWEB)

Moon, Byung Seok; Lee, Tae Sup [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Lee, Myoung Keun [Yonsei University, Wonju (Korea, Republic of)] (and others)

2006-08-15

The HSV1-tk reporter gene system is the most widely used system because of its advantage that direct monitoring is possible without the introduction of a separate reporter gene in case of HSV1-tk suicide gene therapy. In this study, we investigate the usefulness of the reporter probe (substrate), 9-(4-[{sup 18}F]fluoro-3-hydroxymethylbutyl) guanine ([{sup 18}F]FHBG) for non-invasive reporter gene imaging using PET in HSV1-tk expressing hepatoma model. Radiolabeled FHBG was prepared in 8 steps from a commercially available triester. The labeling reaction was carried out by NCA nucleophilic substitution with K[{sup 18}F]/K2.2.2. in acetonitrile using N2-monomethoxytrityl-9-[4-(tosly)-3-monomethoxytritylmethylbutl] guanine as a precursor, followed by deprotection with 1 N HCI. Preliminary biological properties of the probe were evaluated with MCA cells and MCA-tk cells transduced with HSV1-tk reporter gene. In vitro uptake and release-out studies of [{sup 18}F]FHBG were performed, and was analyzed correlation between [{sup 18}F]FHBG uptake ratio according to increasing numeric count of MCA-tk cells and degree of gene expression. MicroPET scan image was obtained with MCA and MCA-tk tumor beating Balb/c-nude mouse model. [{sup 18}F]FHBG was purified by reverse phase semi-HPLC system and collected at around 16-18 min. Radiochemical yield was about 20-25% (corrected for decay), radiochemical purity was > 95% and specific activity was around > 55.5 GBq/ {mu} mol. Specific accumulation of [{sup 18}F]FHBG was observed in HSV1-tk gene transduced MCA-tk cells but not MCA cells, and consecutive 1 hour release-out results showed more than 86% of uptaked [{sup 18}F]FHBG was retained inside of cells. The uptake of [{sup 18}F]FHBG was showed a highly significant linear correlation (R{sup 2} = 0.995) with increasing percentage of MCA-tk numeric cell count. In microPET scan images, remarkable difference of accumulation was observed for the two type of tumors. [{sup 18}F]FHBG appears

Cyclic Mechanical Stretch Up-regulates Hepatoma-Derived Growth Factor Expression in Cultured Rat Aortic Smooth Muscle Cells.

Science.gov (United States)

Kao, Ying-Hsien; Chen, Po-Han; Sun, Cheuk-Kwan; Chang, Yo-Chen; Lin, Yu-Chun; Tsai, Ming-Shian; Lee, Po-Huang; Cheng, Cheng-I

2018-02-21

Hepatoma-derived growth factor (HDGF) is a potent mitogen for vascular smooth muscle cells (SMCs) during embryogenesis and injury repair of vessel walls. Whether mechanical stimuli modulate HDGF expression remains unknown. This study aimed at investigating whether cyclic mechanical stretch plays a regulatory role in HDGF expression and regenerative cytokine production in aortic SMCs. A SMC cell line was grown on a silicone-based elastomer chamber with extracellular matrix coatings (either type I collagen or fibronectin) and received cyclic and uni-axial mechanical stretches with 10% deformation at frequency 1 Hz. Morphological observation showed that fibronectin coating provided better cell adhesion and spreading and that consecutive 6 hours of cyclic mechanical stretch remarkably induced reorientation and realignment of SMCs. Western blotting detection demonstrated that continuous mechanical stimuli elicited up-regulation of HDGF and PCNA, a cell proliferative marker. Signal kinetic profiling study indicated that cyclic mechanical stretch induced signaling activity in RhoA/ROCK and PI3K/Akt cascades. Kinase inhibition study further showed that blockade of PI3K activity suppressed the stretch-induced TNF-a, whereas RhoA/ROCK inhibition significantly blunted the IL-6 production and HDGF over-expression. Moreover, siRNA-mediated HDGF gene silencing significantly suppressed constitutive expression of IL-6, but not TNF-α, in SMCs. These findings support the role of HDGF in maintaining vascular expression of IL-6, which has been regarded a crucial regenerative factor for acute vascular injury. In conclusion, cyclic mechanical stretch may maintain constitutive expression of HDGF in vascular walls and be regarded an important biophysical regulator in vascular regeneration. ©2018 The Author(s).

Dosimetric considerations in radioimmunotherapy of patients with hepatoma

International Nuclear Information System (INIS)

Leichner, P.K.; Klein, J.L.; Order, S.E.

1986-01-01

Dosimetric studies of I-131 labeled antiferritin have provided the foundation for preparative and administrative aspects of radiolabeled antibody treatment of patients with hepatoma. Tumor response to I-131 labeled antiferritin IgG was encouraging and radioimmunotherapy with Y-90 labeled antiferritin IgG was recently initiated. For these patients, In-111 labeled antiferritin IgG was used as the imaging agent, with administered activities ranging from 0.8 - 7 mCi. Serial gamma camera imaging from 30 minutes to 6 days post injection demonstrated that 5-30% of the administered activity localized in hepatomas (8/12 patients). The mean value of the effective half-life in the tumor and liver was 2.8 d. Disappearance curves for the blood circulation, spleen, and other normal tissues were biphasic such that 50% of the activity disappeared within 24 hours post injection. The eight patients who demonstrated sufficient tumor localization where subsequently treated with Y-90 labeled antiferritin IgG. Administered activities were dependent on tumor volume and uptake of radiolabeled IgG and ranged from 8 - 20 mCi. The remaining patients were treated under other existing protocols. 10 references

Sphingoid bases from sea cucumber induce apoptosis in human hepatoma HepG2 cells through p-AKT and DR5.

Science.gov (United States)

Hossain, Zakir; Sugawara, Tatsuya; Hirata, Takashi

2013-03-01

Biofunctional marine compounds have recently received substantial attention for their nutraceutical characteristics. In this study, we investigated the apoptosis-inducing effects of sphingoid bases prepared from sea cucumber using human hepatoma HepG2 cells. Apoptotic effects were determined by cell viability assay, DNA fragmentation assay, caspase-3 and caspase-8 activities. The expression levels of apoptosis-inducing death receptor-5 (DR5) and p-AKT were assayed by western blot analysis, and mRNA expression of bax, GADD45 and PPARγ was assayed by quantitative RT-PCR analysis. Sphingoid bases from sea cucumber markedly reduced the cell viability of HepG2 cells. DNA fragmentation indicative of apoptosis was observed in a dose-dependent manner. The expression levels of the apoptosis inducer protein Bax were increased by the sphingoid bases from sea cucumber. GADD45, which plays an important role in apoptosis-inducing pathways, was markedly upregulated by sphingoid bases from sea cucumber. Upregulation of PPARγ mRNA was also observed during apoptosis induced by the sphingoid bases. The expression levels of DR5 and p-AKT proteins were increased and decreased, respectively, as a result of the effects of sphingoid bases from sea cucumber. The results indicate that sphingoid bases from sea cucumber induce apoptosis in HepG2 cells through upregulation of DR5, Bax, GADD45 and PPARγ and downregulation of p-AKT. Our results show for the first time the functional properties of marine sphingoid bases as inducers of apoptosis in HepG2 cells.

Modulation of the DNA repair system and ATR-p53 mediated apoptosis is relevant for tributyltin-induced genotoxic effects in human hepatoma G2 cells.

Science.gov (United States)

Li, Bowen; Sun, Lingbin; Cai, Jiali; Wang, Chonggang; Wang, Mengmeng; Qiu, Huiling; Zuo, Zhenghong

2015-01-01

The toxic effects of tributyltin (TBT) have been extensively documented in several types of cells, but the molecular mechanisms related to the genotoxic effects of TBT have still not been fully elucidated. Our study showed that exposure of human hepatoma G2 cells to 1-4 μmol/L TBT for 3 hr caused severe DNA damage in a concentration-dependent manner. Moreover, the expression levels of key DNA damage sensor genes such as the replication factor C, proliferating cell nuclear antigen and poly (ADP-ribose) polymerase-1 were inhabited in a concentration-dependent manner. We further demonstrated that TBT induced cell apoptosis via the p53-mediated pathway, which was most likely activated by the ataxia telangiectasia mutated and rad-3 related (ATR) protein kinase. The results also showed that cytochrome c, caspase-3, caspase-8, caspase-9, and the B-cell lymphoma 2 were involved in this process. Taken together, we demonstrated for the first time that the inhibition of the DNA repair system might be more responsible for TBT-induced genotoxic effects in cells. Then the generated DNA damage induced by TBT initiated ATR-p53-mediated apoptosis. Copyright © 2014. Published by Elsevier B.V.

Genotoxicity assessment of membrane concentrates of landfill leachate treated with Fenton reagent and UV-Fenton reagent using human hepatoma cell line.

Science.gov (United States)

Wang, Guifang; Lu, Gang; Yin, Pinghe; Zhao, Ling; Yu, Qiming Jimmy

2016-04-15

Membrane concentrates of landfill leachates contain organic and inorganic contaminants that could be highly toxic and carcinogenic. In this paper, the genotoxicity of membrane concentrates before and after Fenton and UV-Fenton reagent was assessed. The cytotoxicity and genotoxicity was determined by using the methods of methyltetrazolium (MTT), cytokinesis-block micronucleus (CBMN) and comet assay in human hepatoma cells. MTT assay showed a cytotoxicity of 75% after 24h of exposure to the highest tested concentration of untreated concentrates, and no cytotoxocity for UV-Fenton and Fenton treated concentrates. Both CBMN and comet assays showed increased levels of genotoxicity in cells exposed to untreated concentrates, compared to those occurred in cells exposed to UV-Fenton and Fenton reagent treated concentrates. There was no significant difference between negative control and UV-Fenton treated concentrates for micronucleus and comet assay parameters. UV-Fenton and Fenton treatment, especially the former, were effective methods for degradation of bisphenol A and nonylphenol in concentrates. These findings showed UV-Fenton and Fenton reaction were effective methods for treatment of such complex concentrates, UV-Fenton reagent provided toxicological safety of the treated effluent, and the genotoxicity assays were found to be feasible tools for assessment of toxicity risks of complex concentrates. Copyright © 2016 Elsevier B.V. All rights reserved.

Adaptive mutations enhance assembly and cell-to-cell transmission of a high-titer hepatitis C virus genotype 5a Core-NS2 JFH1-based recombinant

DEFF Research Database (Denmark)

Mathiesen, Christian K; Prentoe, Jannick; Meredith, Luke W

2015-01-01

UNLABELLED: Recombinant hepatitis C virus (HCV) clones propagated in human hepatoma cell cultures yield relatively low infectivity titers. Here, we adapted the JFH1-based Core-NS2 recombinant SA13/JFH1C3405G,A3696G (termed SA13/JFH1orig), of the poorly characterized genotype 5a, to Huh7.5 cells......-titer production of diverse HCV strains would be advantageous. Our study offers important functional data on how cell culture-adaptive mutations identified in genotype 5a JFH1-based HCVcc permit high-titer culture by affecting HCV genesis through increasing virus assembly and HCV fitness by enhancing the virus...... specific infectivity and cell-to-cell transmission ability, without influencing the biophysical particle properties. High-titer HCVcc like the one described in this study may be pivotal in future vaccine-related studies where large quantities of infectious HCV particles are necessary....

Total Saponin from Root of Actinidia valvata Dunn Inhibits Hepatoma 22 Growth and Metastasis In Vivo by Suppression Angiogenesis

Directory of Open Access Journals (Sweden)

Guo-Yin Zheng

2012-01-01

Full Text Available The root of Actinidia valvata dunn has been widely used in the treatment of hepatocellular carcinoma (HCC, proved to be beneficial for a longer and better life in China. In present work, total saponin from root of Actinidia valvata Dunn (TSAVD was extracted, and its effects on hepatoma H22-based mouse in vivo were observed. Primarily transplanted hypodermal hepatoma H22-based mice were used to observe TSAVD effect on tumor growth. The microvessel density (MVD, vascular endothelial growth factor (VEGF, basic fibroblast growth factor (bFGF are characterized factors of angiogenesis, which were compared between TSAVD-treated and control groups. Antimetastasis effect on experimental pulmonary metastasis hepatoma mice was also observed in the study. The results demonstrated that TSAVD can effectively inhibit HCC growth and metastasis in vivo, inhibit the formation of microvessel, downregulate expressions of VEGF and bFGF, and retrain angiogenesis of hepatoma 22 which could be one of the reasons.

Antioxidative and apoptotic properties of polyphenolic extracts from edible part of artichoke (Cynara scolymus L.) on cultured rat hepatocytes and on human hepatoma cells.

Science.gov (United States)

Miccadei, Stefania; Di Venere, Donato; Cardinali, Angela; Romano, Ferdinando; Durazzo, Alessandra; Foddai, Maria Stella; Fraioli, Rocco; Mobarhan, Sohrab; Maiani, Giuseppe

2008-01-01

Cultured rat hepatocytes and human hepatoma HepG2 cells were used to evaluate the hepatoprotective properties of polyphenolic extracts from the edible part of artichoke (AE). The hepatocytes were exposed to H2O2generated in situ by glucose oxidase and were treated with either AE, or pure chlorogenic acid (ChA) or with the well known antioxidant, N, N'-diphenyl-p-phenilenediamine (DPPD). Addition of glucose oxidase to the culture medium caused depletion of intracellular glutathione (GSH) content, accumulation of malondialdehyde (MDA) in the cultures, as a lipid peroxidation indicator, and cell death. These results demonstrated that AE protected cells from the oxidative stress caused by glucose oxidase, comparable to DPPD. Furthermore, AE, as well as ChA, prevented the loss of total GSH and the accumulation of MDA. Treatment of HepG2 cells for 24 h with AE reduced cell viability in a dose-dependent manner, however, ChA had no prominent effects on the cell death rate. Similarly, AE rather than ChA induced apoptosis, measured by flow cytometric analysis of annexin and by activation of caspase-3, in HepG2 cells. Our findings indicate that AE had a marked antioxidative potential that protects hepatocytes from an oxidative stress. Furthermore, AE reduced cell viability and had an apoptotic activity on a human liver cancer cell line.

Productive homologous and non-homologous recombination of hepatitis C virus in cell culture

DEFF Research Database (Denmark)

Scheel, Troels K H; Galli, Andrea; Li, Yi-Ping

2013-01-01

. In addition, recombination is an important regulatory mechanism of cytopathogenicity for the related pestiviruses. Here we describe recombination of HCV RNA in cell culture leading to production of infectious virus. Initially, hepatoma cells were co-transfected with a replicating JFH1ΔE1E2 genome (genotype 2a......) lacking functional envelope genes and strain J6 (2a), which has functional envelope genes but does not replicate in culture. After an initial decrease in the number of HCV positive cells, infection spread after 13-36 days. Sequencing of recovered viruses revealed non-homologous recombinants with J6...

Vinclozolin, a widely used fungizide, enhanced BaP-induced micronucleus formation in human derived hepatoma cells by increasing CYP1A1 expression.

Science.gov (United States)

Wu, Xin-Jiang; Lu, Wen-qing; Roos, Peter H; Mersch-Sundermann, Volker

2005-10-15

Vinclozolin, a widely used fungicide, can be identified as a residue in numerous vegetable and fruit samples. To get insight in its genetic toxicity, we investigated the genotoxic effect of vinclozolin in the human derived hepatoma cell line HepG2 using the micronucleus (MN) assay. Additionally, to evaluate the co- or anti-mutagenic potency of vinclozolin, we treated HepG2 cells with different concentrations of vinclozolin for 24 h. Subsequently, the cells were exposed to benzo[a]pyrene (BaP) for 1h. Exposure of HepG2 cells to 50-400 microM vinclozolin alone did not cause any induction of micronuclei. However, a pronounced co-mutagenic effect was observed. MN frequencies caused by BaP increased by 30.6%, 52.8% and 65.3% after pretreatment of the cell cultures with 50, 100 and 200 microM vinclozolin, respectively. The highest concentration (400 microM) of vinclozolin tested caused cytotoxicity. Therefore, micronuclei were not considered for that concentration. To clarify the mechanism of cogenotoxicity, we assayed cytochrome P450 1A1 (CYP1A1), which plays a pivotal role in activation of BaP. Cells exposed to vinclozolin led to significant increase of CYP1A1 expression in Western blot. The result suggested that induction of CYP1A1 by vinclozolin account for its enhancing effect on genotoxicity caused by BaP.

Inhibition of glutathione synthesis eliminates the adaptive response of ascitic hepatoma 22 cells to nedaplatin that targets thioredoxin reductase

Energy Technology Data Exchange (ETDEWEB)

Wang, Yijun [School of Tea and Food Science, Anhui Agricultural University, Hefei 230036, Anhui (China); Lu, Hongjuan [Productivity Center of Jiangsu Province, Nanjing 210042, Jiangsu (China); Wang, Dongxu; Li, Shengrong; Sun, Kang; Wan, Xiaochun [School of Tea and Food Science, Anhui Agricultural University, Hefei 230036, Anhui (China); Taylor, Ethan Will [Department of Nanoscience, Joint School of Nanoscience and Nanoengineering, University of North Carolina at Greensboro, Greensboro, NC 27402 (United States); Zhang, Jinsong, E-mail: zjs@ahau.edu.cn [School of Tea and Food Science, Anhui Agricultural University, Hefei 230036, Anhui (China)

2012-12-15

Thioredoxin reductase (TrxR) is a target for cancer therapy and the anticancer mechanism of cisplatin involves TrxR inhibition. We hypothesize that the anticancer drug nedaplatin (NDP), an analogue of cisplatin and a second-generation platinum complex, also targets TrxR. Furthermore, we investigate whether the therapeutic efficacy of NDP can be enhanced by simultaneous modulation of 1) TrxR, via NDP, and 2) glutathione (GSH), via the GSH synthesis inhibitor buthionine sulfoximine (BSO). Mice bearing ascitic hepatoma 22 (H22) cells were treated with NDP alone or NDP plus BSO. TrxR activity of H22 cells was inhibited by NDP in a dose-dependent manner. A high correlation between the inhibition of TrxR activity at 6 h and the inhibition of ascitic fluid volume at 72 h was established (r = 0.978, p < 0.01). As an adaptive response, the viable ascitic cancer cells after NDP treatment displayed an enlarged cell phenotype, assembled with several-fold more antioxidant enzymes and GSH-predominant non-protein free thiols. This adaptive response was largely eliminated when BSO was co-administered with NDP, leading to the decimation of the H22 cell population without enhancing renal toxicity, since at this dose, NDP did not inhibit renal TrxR activity. In conclusion, the pharmacological effect of NDP involves TrxR inhibition, and the adaptive response of NDP-treated ascitic H22 cells can be efficiently counteracted by BSO. Simultaneous modulation of TrxR and GSH on ascitic H22 cells using NDP plus BSO greatly enhances therapeutic efficacy as compared with the single modulation of TrxR using NDP alone. -- Highlights: ► Nedaplatin at a pharmacological dose inhibits TrxR in cancer cells but not in kidney. ► The nedaplatin-treated cancer cells exhibit adaptive response. ► Buthionine sulfoximine inhibits glutathione in both cancer cells and kidney. ► Buthionine sulfoximine counteracts the adaptive response to the nedaplatin treatment. ► Buthionine sulfoximine does not

Inhibition of glutathione synthesis eliminates the adaptive response of ascitic hepatoma 22 cells to nedaplatin that targets thioredoxin reductase

International Nuclear Information System (INIS)

Wang, Yijun; Lu, Hongjuan; Wang, Dongxu; Li, Shengrong; Sun, Kang; Wan, Xiaochun; Taylor, Ethan Will; Zhang, Jinsong

2012-01-01

Thioredoxin reductase (TrxR) is a target for cancer therapy and the anticancer mechanism of cisplatin involves TrxR inhibition. We hypothesize that the anticancer drug nedaplatin (NDP), an analogue of cisplatin and a second-generation platinum complex, also targets TrxR. Furthermore, we investigate whether the therapeutic efficacy of NDP can be enhanced by simultaneous modulation of 1) TrxR, via NDP, and 2) glutathione (GSH), via the GSH synthesis inhibitor buthionine sulfoximine (BSO). Mice bearing ascitic hepatoma 22 (H22) cells were treated with NDP alone or NDP plus BSO. TrxR activity of H22 cells was inhibited by NDP in a dose-dependent manner. A high correlation between the inhibition of TrxR activity at 6 h and the inhibition of ascitic fluid volume at 72 h was established (r = 0.978, p < 0.01). As an adaptive response, the viable ascitic cancer cells after NDP treatment displayed an enlarged cell phenotype, assembled with several-fold more antioxidant enzymes and GSH-predominant non-protein free thiols. This adaptive response was largely eliminated when BSO was co-administered with NDP, leading to the decimation of the H22 cell population without enhancing renal toxicity, since at this dose, NDP did not inhibit renal TrxR activity. In conclusion, the pharmacological effect of NDP involves TrxR inhibition, and the adaptive response of NDP-treated ascitic H22 cells can be efficiently counteracted by BSO. Simultaneous modulation of TrxR and GSH on ascitic H22 cells using NDP plus BSO greatly enhances therapeutic efficacy as compared with the single modulation of TrxR using NDP alone. -- Highlights: ► Nedaplatin at a pharmacological dose inhibits TrxR in cancer cells but not in kidney. ► The nedaplatin-treated cancer cells exhibit adaptive response. ► Buthionine sulfoximine inhibits glutathione in both cancer cells and kidney. ► Buthionine sulfoximine counteracts the adaptive response to the nedaplatin treatment. ► Buthionine sulfoximine does not

Neuraminidase treatment of respiratory syncytial virus-infected cells or virions, but not target cells, enhances cell-cell fusion and infection

International Nuclear Information System (INIS)

Barretto, Naina; Hallak, Louay K.; Peeples, Mark E.

2003-01-01

Respiratory syncytial virus (RSV) infection of HeLa cells induces fusion, but transient expression of the three viral glycoproteins induces fusion poorly, if at all. We found that neuraminidase treatment of RSV-infected cells to remove sialic acid (SA) increases fusion dramatically and that the same treatment of transiently transfected cells expressing the three viral glycoproteins, or even cells expressing the fusion (F) protein alone, results in easily detectable fusion. Neuraminidase treatment of the effector cells, expressing the viral glycoproteins, enhanced fusion while treatment of the target cells did not. Likewise, infectivity was increased by treating virions with neuraminidase, but not by treating target cells. Reduction of charge repulsion by removal of the negatively charged SA is unlikely to explain this effect, since removal of negative charges from either membrane would reduce charge repulsion. Infection with neuraminidase-treated virus remained heparan-sulfate-dependent, indicating that a novel attachment mechanism is not revealed by SA removal. Interestingly, neuraminidase enhancement of RSV infectivity was less pronounced in a virus expressing both the G and the F glycoproteins, compared to virus expressing only the F glycoprotein, possibly suggesting that the G protein sterically hinders access of the neuraminidase to its fusion-enhancing target

Merkel cell polyomavirus infection and Merkel cell carcinoma.

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Liu, Wei; MacDonald, Margo; You, Jianxin

2016-10-01

Merkel cell polyomavirus is the only polyomavirus discovered to date that is associated with a human cancer. MCPyV infection is highly prevalent in the general population. Nearly all healthy adults asymptomatically shed MCPyV from their skin. However, in elderly and immunosuppressed individuals, the infection can lead to a lethal form of skin cancer, Merkel cell carcinoma. In the last few years, new findings have established links between MCPyV infection, host immune response, and Merkel cell carcinoma development. This review discusses these recent discoveries on how MCPyV interacts with host cells to achieve persistent infection and, in the immunocompromised population, contributes to MCC development. Copyright © 2016 Elsevier B.V. All rights reserved.

The role of hypoxia response element in TGFβ-induced carbonic anhydrase IX expression in Hep3B human hepatoma cells

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Yildirim Hatice

2017-01-01

Full Text Available Carbonic anhydrase IX (CAIX is a hypoxia-regulated gene. It is over expressed in a variety of cancers, including hepatocellular cancer. Transforming growth factor β (TGFβ is considered to have an impact on cancer biology due to its important roles in cell proliferation and differentiation. The effect of the TGFβ on CAIX expression under hypoxia and the mechanism underlying the role of the hypoxia response element (HRE on this expression are unknown. In this study, we demonstrate that TGFβ upregulates CAIX expression under hypoxic conditions in the Hep3B hepatoma cell line, indicating that the mitogen-activated protein kinase (MAPK- and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K-signaling pathways might be responsible for this response. Site-directed mutagenesis of the HRE region in CAIX promoter reduced the TGFβ-induced CAIX promoter activity, pointing to the significance of HRE for this response. Up regulation of TGFβ-stimulated CAIX expression was consistent with the up regulation of promoter activity of five different truncated constructs of the CAIX promoter under hypoxia. Our findings show that the HRE region is critical for TGFβ-induced CAIX expression, which is mainly controlled by MAPK and PI3K pathways.

Differential Cytotoxicity of Acetaminophen in Mouse Macrophage J774.2 and Human Hepatoma HepG2 Cells: Protection by Diallyl Sulfide.

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Haider Raza

Full Text Available Non-steroidal anti-inflammatory drugs (NSAIDs, including acetaminophen (APAP, have been reported to induce cytotoxicity in cancer and non-cancerous cells. Overdose of acetaminophen (APAP causes liver injury in humans and animals. Hepatic glutathione (GSH depletion followed by oxidative stress and mitochondrial dysfunction are believed to be the main causes of APAP toxicity. The precise molecular mechanism of APAP toxicity in different cellular systems is, however, not clearly understood. Our previous studies on mouse macrophage J774.2 cells treated with APAP strongly suggest induction of apoptosis associated with mitochondrial dysfunction and oxidative stress. In the present study, using human hepatoma HepG2 cells, we have further demonstrated that macrophages are a more sensitive target for APAP-induced toxicity than HepG2 cells. Using similar dose- and time-point studies, a marked increase in apoptosis and DNA fragmentation were seen in macrophages compared to HepG2 cells. Differential effects of APAP on mitochondrial respiratory functions and oxidative stress were observed in the two cell lines which are presumably dependent on the varying degree of drug metabolism by the different cytochrome P450s and detoxification by glutathione S-transferase enzyme systems. Our results demonstrate a marked increase in the activity and expression of glutathione transferase (GST and multidrug resistance (MDR1 proteins in APAP-treated HepG2 cells compared to macrophages. This may explain the apparent resistance of HepG2 cells to APAP toxicity. However, treatment of these cells with diallyl sulfide (DAS, 200 μM, a known chemopreventive agent from garlic extract, 24 h prior to APAP (10 μmol/ml for 18h exhibited comparable cytoprotective effects in the two cell lines. These results may help in better understanding the mechanism of cytotoxicity caused by APAP and cytoprotection by chemopreventive agents in cancer and non-cancerous cellular systems.

HCV Infection and B-Cell Lymphomagenesis

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Masahiko Ito

2011-01-01

Full Text Available Hepatitis C virus (HCV has been recognized as a major cause of chronic liver diseases worldwide. It has been suggested that HCV infects not only hepatocytes but also mononuclear lymphocytes including B cells that express the CD81 molecule, a putative HCV receptor. HCV infection of B cells is the likely cause of B-cell dysregulation disorders such as mixed cryoglobulinemia, rheumatoid factor production, and B-cell lymphoproliferative disorders that may evolve into non-Hodgkin's lymphoma (NHL. Epidemiological data indicate an association between HCV chronic infection and the occurrence of B-cell NHL, suggesting that chronic HCV infection is associated at least in part with B-cell lymphomagenesis. In this paper, we aim to provide an overview of recent literature, including our own, to elucidate a possible role of HCV chronic infection in B-cell lymphomagenesis.

Independent, parallel pathways to CXCL10 induction in HCV-infected hepatocytes.

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Brownell, Jessica; Wagoner, Jessica; Lovelace, Erica S; Thirstrup, Derek; Mohar, Isaac; Smith, Wesley; Giugliano, Silvia; Li, Kui; Crispe, I Nicholas; Rosen, Hugo R; Polyak, Stephen J

2013-10-01

The pro-inflammatory chemokine CXCL10 is induced by HCV infection in vitro and in vivo, and is associated with outcome of IFN (interferon)-based therapy. We studied how hepatocyte sensing of early HCV infection via TLR3 (Toll-like receptor 3) and RIG-I (retinoic acid inducible gene I) led to expression of CXCL10. CXCL10, type I IFN, and type III IFN mRNAs and proteins were measured in PHH (primary human hepatocytes) and hepatocyte lines harboring functional or non-functional TLR3 and RIG-I pathways following HCV infection or exposure to receptor-specific stimuli. HuH7 human hepatoma cells expressing both TLR3 and RIG-I produced maximal CXCL10 during early HCV infection. Neutralization of type I and type III IFNs had no impact on virus-induced CXCL10 expression in TLR3+/RIG-I+ HuH7 cells, but reduced CXCL10 expression in PHH. PHH cultures were positive for monocyte, macrophage, and dendritic cell mRNAs. Immunodepletion of non-parenchymal cells (NPCs) eliminated marker expression in PHH cultures, which then showed no IFN requirement for CXCL10 induction during HCV infection. Immunofluorescence studies also revealed a positive correlation between intracellular HCV Core and CXCL10 protein expression (r(2) = 0.88, p ≤ 0.001). While CXCL10 induction in hepatocytes during the initial phase of HCV infection is independent of hepatocyte-derived type I and type III IFNs, NPC-derived IFNs contribute to CXCL10 induction during HCV infection in PHH cultures. Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Saponins isolated from Asparagus induce apoptosis in human hepatoma cell line HepG2 through a mitochondrial-mediated pathway

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Ji, Y.; Ji, C.; Yue, L.; Xu, H.

2012-01-01

Objective Many scientific studies have shown that Asparagus officinalis has an antitumour effect and enhances human immunity, but the active components and the antitumour mechanisms are unclear. We investigated the effects of saponins isolated from Asparagus on proliferation and apoptosis in the human hepatoma cell line HepG2. Methods HepG2 cells were treated with varying concentrations of Asparagus saponins at various times. Using mtt and flow cytometry assays, we evaluated the effects of Asparagus saponins on the growth and apoptosis of HepG2 cells. Transmission electron microscopy was used to observe the morphology of cell apoptosis. Confocal laser scanning microscopy was used to analyze intracellular calcium ion concentration, mitochondrial permeability transition pore (mptp), and mitochondrial membrane potential (mmp). Spectrophotometry was applied to quantify the activity of caspase-9 and caspase-3. Flow cytometry was used to investigate the levels of reactive oxygen species (ros) and pH, and the expressions of Bcl2, Bax, CytC, and caspase-3, in HepG2 cells. Results Asparagus saponins inhibited the growth of HepG2 cells in a dose-dependent manner. The median inhibitory concentration (IC50) was 101.15 mg/L at 72 hours. The apoptosis morphology at 72 hours of treatment was obvious, showing cell protuberance, concentrated cytoplasm, and apoptotic bodies. The apoptotic rates at 72 hours were 30.9%, 51.7%, and 62.1% (for saponin concentrations of 50 mg/L, 100 mg/L, 200 mg/L). Treatment with Asparagus saponins for 24 hours increased the intracellular level of ros and Ca2+, lowered the pH, activated intracellular mptp, and decreased mmp in a dose-dependent manner. Treatment also increased the activity of caspase-9 and caspase-3, downregulated the expression of Bcl2, upregulated the expression of Bax, and induced release of CytC and activation of caspase-3. Conclusions Asparagus saponins induce apoptosis in HepG2 cells through a mitochondrial-mediated and caspase

Clinicopathological study on the primary hepatoma of the atomic bomb survivors

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Satoh, T; Takahara, O; Takahashi, N; Matsuo, K; Tsunoo, S [Nagasaki Atomic Bomb Hospital (Japan)

1978-09-01

Fifty-eight cases autopsied during 10 years from 1968 to 1977 were divided into (A) a non-exposed group, (B) a group exposed within 2 km of the center of explosion, and (C) a group exposed over 2 km from the center of explosion or a group who entered Nagasaki city after the explosion, and clinicopathological study on them was made in detail. These three groups were studied as to incidences according to age and sex, prognosis, histological types and hepatic lesion complicated by hepatoma in survival cases for more than one year, histological diagnosis, gross types, histological types, degree of atypia, hepatic lesion at sites not injured with hepatoma, and the detection rate of B-type virus hepatitis surface antigen (HBs-Ag). Pathohistologically, ordinary hematoxylin-eosin staining, van Gieson staining, silvering staining, iron staining were performed, and aldehyde-fuchsine staining was also performed HBs-Ag detection in a part of cases for.

Bystander effect in human hepatoma HepG2 cells caused by medium transfers at different times after high-LET carbon ion irradiation

International Nuclear Information System (INIS)

Wu Qingfeng; Li Qiang; Jin Xiaodong; Liu Xinguo; Dai Zhongying

2011-01-01

Although radiation-induced bystander effects have been well documented in a variety of biological systems, whether irradiated cells have the ability to generate bystander signaling persistently is still unclear and the clinical relevance of bystander effects in radiotherapy remains to be elucidated. This study examines tumor cellular bystander response to autologous medium from cell culture irradiated with high-linear energy transfer (LET) heavy ions at a therapeutically relevant dose in terms of clonogenic cell survival. In vitro experiments were performed using human hepatoma HepG2 cell line exposed to 100 keV/μm carbon ions at a dose of 2 Gy. Two different periods (2 and 12 h) after irradiation, irradiated cell conditioned medium (ICCM) and replenished fresh medium were harvested and then transferred to unirradiated bystander cells. Cellular bystander responses were measured with the different medium transfer protocols. Significant higher survival fractions of unirradiated cells receiving the media from the irradiated cultures at the different times post-irradiation than those of the control were observed. Even replenishing fresh medium for unirradiated cells which had been exposed to the ICCM for 12 h could not prevent the bystander cells from the increased survival fraction. These results suggest that the irradiated cells could release unidentified signal factor(s), which induced the increase in survival fraction for the unirradiated bystander cells, into the media sustainedly and the carbon ions triggered a cascade of signaling events in the irradiated cells rather than secreting the soluble signal factor(s) just at a short period after irradiation. Based on the observations in this study, the importance of bystander effect in clinical radiotherapy was discussed and incorporating the bystander effect into the current radiobiological models, which are applicable to heavy ion radiotherapy, is needed urgently.

Hepatitis C virus replication and Golgi function in brefeldin a-resistant hepatoma-derived cells.

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Rayan Farhat

Full Text Available Recent reports indicate that the replication of hepatitis C virus (HCV depends on the GBF1-Arf1-COP-I pathway. We generated Huh-7-derived cell lines resistant to brefeldin A (BFA, which is an inhibitor of this pathway. The resistant cell lines could be sorted into two phenotypes regarding BFA-induced toxicity, inhibition of albumin secretion, and inhibition of HCV infection. Two cell lines were more than 100 times more resistant to BFA than the parental Huh-7 cells in these 3 assays. This resistant phenotype was correlated with the presence of a point mutation in the Sec7 domain of GBF1, which is known to impair the binding of BFA. Surprisingly, the morphology of the cis-Golgi of these cells remained sensitive to BFA at concentrations of the drug that allowed albumin secretion, indicating a dichotomy between the phenotypes of secretion and Golgi morphology. Cells of the second group were about 10 times more resistant than parental Huh-7 cells to the BFA-induced toxicity. The EC50 for albumin secretion was only 1.5-1.8 fold higher in these cells than in Huh-7 cells. However their level of secretion in the presence of inhibitory doses of BFA was 5 to 15 times higher. Despite this partially effective secretory pathway in the presence of BFA, the HCV infection was almost as sensitive to BFA as in Huh-7 cells. This suggests that the function of GBF1 in HCV replication does not simply reflect its role of regulator of the secretory pathway of the host cell. Thus, our results confirm the involvement of GBF1 in HCV replication, and suggest that GBF1 might fulfill another function, in addition to the regulation of the secretory pathway, during HCV replication.

Degradation of surface-labeled hepatoma membrane polypeptides: effect of inhibitors

International Nuclear Information System (INIS)

Hare, J.F.; Huston, M.

1984-01-01

When their membrane proteins were labeled with 125I by lactoperoxidase, dividing hepatoma cells lost radioactivity to the medium in a biphasic manner (T1/2 . 16-26 h, greater than 40 h). Lysosomotropic weak bases, chloroquine, and NH4Cl inhibited the rapid phase by 59%. More than 50% of the radioactivity which accumulates in the media from dividing cells during the first 4 h after labeling was trichloroacetic acid-soluble, and was identified as iodotyrosine. Iodotyrosine release from labeled membrane proteins was 60-71% inhibited by lysosomotropic agents chloroquine and NH4Cl as well as the sodium-proton ionophore, monensin. The inhibitory effect of NH4Cl and monensin was reversible. Inhibitors of microtubule and microfilament function and transglutamination had no effect on release of iodotyrosine to the medium, but trypsin-like protease inhibitors, p-aminobenzamidine, tosyl-L-lysine/chloromethylketone, and phenylmethylsulfonyl fluoride, as well as the cathepsin B inhibitor, leupeptin, inhibited by 21-24%. Iodotyrosine release showed a biphasic Arrhenius plot with an activation energy of 17 kcal/mol above but 27 kcal/mol below 20 degrees C. These results indicate that cell membrane polypeptides require a temperature-limiting event as well as passage through an ion-sensitive compartment prior to their complete degradation to constituent amino acids. In contrast to other lysosomal-mediated events, however, iodinated membrane proteins of dividing cells are degraded in a manner insensitive to agents which disrupt the cytoskeleton

Oral epithelial cells are susceptible to cell-free and cell-associated HIV-1 infection in vitro

International Nuclear Information System (INIS)

Moore, Jennifer S.; Rahemtulla, Firoz; Kent, Leigh W.; Hall, Stacy D.; Ikizler, Mine R.; Wright, Peter F.; Nguyen, Huan H.; Jackson, Susan

2003-01-01

Epithelial cells lining the oral cavity are exposed to HIV-1 through breast-feeding and oral-genital contact. Genital secretions and breast milk of HIV-1-infected subjects contain both cell-free and cell-associated virus. To determine if oral epithelial cells can be infected with HIV-1 we exposed gingival keratinocytes and adenoid epithelial cells to cell-free virus and HIV-1-infected peripheral blood mononuclear cells and monocytes. Using primary isolates we determined that gingival keratinocytes are susceptible to HIV-1 infection via cell-free CD4-independent infection only. R5 but not X4 viral strains were capable of infecting the keratinocytes. Further, infected cells were able to release infectious virus. In addition, primary epithelial cells isolated from adenoids were also susceptible to infection; both cell-free and cell-associated virus infected these cells. These data have potential implications in the transmission of HIV-1 in the oral cavity

Possible reduction of hepatoma formation by Smmu 7721 cells in SCID mice and metastasis formation by B16F10 melanoma cells in C57BL/6 mice by Agaricus blazei murill extract.

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Wu, Ming-Fang; Lu, Hsu-Feng; Hsu, Yu-Ming; Tang, Ming-Chu; Chen, Hsueh-Chin; Lee, Ching-Sung; Yang, Yi-Yuan; Yeh, Ming-Yang; Chung, Hsiung-Kwang; Huang, Yi-Ping; Wu, Chih-Chung; Chung, Jing-Gung

2011-01-01

Agaricus blazei Murill extract (ABM) has been reported to possess antitumor effects. In this study, the role of ABM in tumor growth and metastasis in vivo was evaluated in experimental Smmu 7721 hepatoma cells in severe combined immunodeficiency (SCID) mice and B16F10 melanoma cells lung metastasis in C57BL/6 mice. For the tumor growth model, the size of the liver tumor mass was about 10 mm to 20 mm in the control group. In comparison with the control group, the tumor mass seem to grow slowly with ABM treatment, especially at the high dose. For the tumor metastasis model, after a six-week treatment, the survival rates of B6 mice were 0%, 30%, 10% and 50% for control group, low, median and high concentration ABM treatment groups, respectively. The survival rate showed that pretreatment of C57BL/6 (B6) mice with ABM lengthened their lifespan after tumor cell inoculation, which supports the notion that ABM successfully reduced lung metastasis formation by B16F10 melanoma cells. The treatment effect was dependent on the concentration of ABM for tumor growth and metastasis in these models.

Localization of 131I-chTNT in a nude mice model with human hepatoma

International Nuclear Information System (INIS)

Chen Shaoliang; Sun Xiaoguang; Xiu Yan; Zhong Gaoren; Qiao Weiwei; Xu Lanwen; Li Wenzheng

1998-01-01

Purpose: In order to evaluate the targeting activity in the animal model with human hepatoma, the 131 I-chTNT radioimmunoimaging was explored. Methods: Radioimmunoimages were taken on different intervals after injection of 131 I-chTNT 5.55 MBq to the nude mice, and tissue distribution was measured. The results of 131 I-chTNT monoclonal antibody group were compared with that of 131 I control group. Results: The experimental group developed tumor positive images after one day of radio-labelled monoclonal antibodies injection and held on until the end of the experiment. The radioactivity in tumor mass was stable, and the half life of 131 I-chTNT in hepatoma mass was 6.0 +- 1.6 days. there was no special radioactivity accumulation in normal liver tissue in the nude mice and the radioactivity in it disappeared rapidly. Statistics indicated the tumor/liver ratio in 1, 2, 3, 5, 7 days were 1.03, 2.43, 5.71, 7.96, 10.67, respectively. Conclusions: The results suggest that 131 I-chTNT monoclonal antibody has a considerable targeting activity, and provide an evidence for that it can be used as a new radiopharmaceutical agent for the imaging and radio therapy of hepatoma

Dynamic MR imaging of hepatoma treated by transcatheter arterial embolization therapy

International Nuclear Information System (INIS)

Yamashita, Y.; Yoshimatsu, S.; Sumi, M.; Harada, M.; Takahashi, M.

1993-01-01

The effect of transcatheter arterial chemo-embolization theory (TACE) for hepatoma was evaluated with dynamic MR imaging with Gd-DTPA in 37 patients (44 tumors). TACE was performed using Lipiodol/cis-platinum and gelatin sponge (or microspheres) as an embolic material. All patients were examined with dynamic CT and MR imaging before and after treatment. On conventional spin echo images, changes of signal intensity after treatment varied regardless of presence of Lipiodol. Dynamic MR imaging revealed changes of tumor vascularity before and after treatment. On histologic correlation, areas of persistent tumor enhancement on dynamic MR imaging corresponded to areas of viable tumor cells while areas of nonenhancement corresponded to areas of necrosis. Dynamic MR imaging was superior in contrast resolution and was not influenced by the presence of Lipiodol compared with dynamic CT, and therefore residual viable tumors were better defined by dynamic MR imaging. (orig.)

Salmonella typhimurium strain SL7207 induces apoptosis and inhibits the growth of HepG2 hepatoma cells in vitro and in vivo

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Baowei Li

2012-12-01

Full Text Available Salmonella typhimurium is probably most extensively studied tumor-targeting bacteria and SL7207 is one of its attenuated strains. SL7207 was first made for bacterial vaccine development and its therapeutic efficacy and safety for hepatocellular carcinoma has not been characterized. In this study, the inhibitory ability of SL7207-lux on human hepatoma HepG2 cells was tested in vitro and in vivo. A bacterial luminescent gene cluster (lux CDABE was transfected into SL7207 to better monitor the invasion of the bacteria. The results show that SL7207-lux can rapidly enter HepG2 cells and localize in the cytoplasm. This invasion represses cell proliferation and induces apoptosis. In vivo real-time invasion studies showed that the bacteria gradually accumulate in the tumor. This enrichment was confirmed by anatomic observation at 5 days after inoculation. About 40% of tumor growth was inhibited by SL7207-lux at 34 days post-treatment without significant loss of body weight. The area of necrosis of tumor tissue was clearly increased in the treated group. Bacterial quantification showed that the number of colony-forming units per gram of bacteria within tumor tissue was approximately 1000-fold higher than that of liver and spleen. These data suggest that attenuated S. typhimurium strain SL7207 has potential for the treatment of cancers.

Murid herpesvirus-4 exploits dendritic cells to infect B cells.

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Miguel Gaspar

2011-11-01

Full Text Available Dendritic cells (DCs play a central role in initiating immune responses. Some persistent viruses infect DCs and can disrupt their functions in vitro. However, these viruses remain strongly immunogenic in vivo. Thus what role DC infection plays in the pathogenesis of persistent infections is unclear. Here we show that a persistent, B cell-tropic gamma-herpesvirus, Murid Herpesvirus-4 (MuHV-4, infects DCs early after host entry, before it establishes a substantial infection of B cells. DC-specific virus marking by cre-lox recombination revealed that a significant fraction of the virus latent in B cells had passed through a DC, and a virus attenuated for replication in DCs was impaired in B cell colonization. In vitro MuHV-4 dramatically altered the DC cytoskeleton, suggesting that it manipulates DC migration and shape in order to spread. MuHV-4 therefore uses DCs to colonize B cells.

Alteration of cell cycle progression by Sindbis virus infection

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Yi, Ruirong; Saito, Kengo [Department of Molecular Virology, Graduate School of Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670 (Japan); Isegawa, Naohisa [Laboratory Animal Center, Graduate School of Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670 (Japan); Shirasawa, Hiroshi, E-mail: sirasawa@faculty.chiba-u.jp [Department of Molecular Virology, Graduate School of Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chiba 260-8670 (Japan)

2015-07-10

We examined the impact of Sindbis virus (SINV) infection on cell cycle progression in a cancer cell line, HeLa, and a non-cancerous cell line, Vero. Cell cycle analyses showed that SINV infection is able to alter the cell cycle progression in both HeLa and Vero cells, but differently, especially during the early stage of infection. SINV infection affected the expression of several cell cycle regulators (CDK4, CDK6, cyclin E, p21, cyclin A and cyclin B) in HeLa cells and caused HeLa cells to accumulate in S phase during the early stage of infection. Monitoring SINV replication in HeLa and Vero cells expressing cell cycle indicators revealed that SINV which infected HeLa cells during G{sub 1} phase preferred to proliferate during S/G{sub 2} phase, and the average time interval for viral replication was significantly shorter in both HeLa and Vero cells infected during G{sub 1} phase than in cells infected during S/G{sub 2} phase. - Highlights: • SINV infection was able to alter the cell cycle progression of infected cancer cells. • SINV infection can affect the expression of cell cycle regulators. • SINV infection exhibited a preference for the timing of viral replication among the cell cycle phases.

Alteration of cell cycle progression by Sindbis virus infection

International Nuclear Information System (INIS)

Yi, Ruirong; Saito, Kengo; Isegawa, Naohisa; Shirasawa, Hiroshi

2015-01-01

We examined the impact of Sindbis virus (SINV) infection on cell cycle progression in a cancer cell line, HeLa, and a non-cancerous cell line, Vero. Cell cycle analyses showed that SINV infection is able to alter the cell cycle progression in both HeLa and Vero cells, but differently, especially during the early stage of infection. SINV infection affected the expression of several cell cycle regulators (CDK4, CDK6, cyclin E, p21, cyclin A and cyclin B) in HeLa cells and caused HeLa cells to accumulate in S phase during the early stage of infection. Monitoring SINV replication in HeLa and Vero cells expressing cell cycle indicators revealed that SINV which infected HeLa cells during G 1 phase preferred to proliferate during S/G 2 phase, and the average time interval for viral replication was significantly shorter in both HeLa and Vero cells infected during G 1 phase than in cells infected during S/G 2 phase. - Highlights: • SINV infection was able to alter the cell cycle progression of infected cancer cells. • SINV infection can affect the expression of cell cycle regulators. • SINV infection exhibited a preference for the timing of viral replication among the cell cycle phases

Variables affecting the tumor localization of 131I-antiferritin in experimental hepatoma

International Nuclear Information System (INIS)

Rostock, R.A.; Klein, J.L.; Kopher, K.A.; Order, S.E.

1984-01-01

Ferritin is both a normal tissue- and tumor-associated protein. The in vivo localization of 131 I-radiolabeled antitumor ferritin and normal IgG antibodies in the H-4-II-E rat hepatoma model was investigated in both tumor and normal tissues over a dose range of 0.67 micrograms to 5 mg of normal and antiferritin IgG and at labeling ratios (microCi 131 I per micrograms IgG) of 15:1, 5:1, and 1:10. The total dose from nonpenetrating radiation in rads was calculated and demonstrated a maximum of 2.9 times greater dose deposition (rads) of antiferritin than normal IgG in hepatoma without specific increase in binding in normal tissues. The maximum tumor targeting achieved was dependent on the amount of injected IgG and not on the labeling ratio or procedure. The binding in tumor could be inhibited by unlabeled antiferritin but not by unlabeled normal rabbit IgG and demonstrated the requirement of specificity for tumor binding. Normal tissues did not target with antiferritin. Most normal tissues have a capacity to bind normal and antiferritin IgG nonspecifically that is linear in relationship to the amount of injected IgG. The results demonstrate that 131 I-antiferritin selectively targets ferritin-secreting hepatoma over normal tissues and that the amount of targeting is dependent on the amount of antiferritin injected. The physiologic reasons for such selective localization is not known, but the term ''biologic window'' has been used to describe the differential availability of tumor ferritin for binding

Hepatoma-derived growth factor-related protein-3 is a novel angiogenic factor.

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Michelle E LeBlanc

Full Text Available Hepatoma-derived growth factor-related protein-3 (Hdgfrp3 or HRP-3 was recently reported as a neurotrophic factor and is upregulated in hepatocellular carcinoma to promote cancer cell survival. Here we identified HRP-3 as a new endothelial ligand and characterized its in vitro and in vivo functional roles and molecular signaling. We combined open reading frame phage display with multi-round in vivo binding selection to enrich retinal endothelial ligands, which were systematically identified by next generation DNA sequencing. One of the identified endothelial ligands was HRP-3. HRP-3 expression in the retina and brain was characterized by Western blot and immunohistochemistry. Cell proliferation assay showed that HRP-3 stimulated the growth of human umbilical vein endothelial cells (HUVECs. HRP-3 induced tube formation of HUVECs in culture. Wound healing assay indicated that HRP-3 promoted endothelial cell migration. HRP-3 was further confirmed for its in vitro angiogenic activity by spheroid sprouting assay. HRP-3 extrinsically activated the extracellular-signal-regulated kinase ½ (ERK1/2 pathway in endothelial cells. The angiogenic activity of HRP-3 was independently verified by mouse cornea pocket assay. Furthermore, in vivo Matrigel plug assay corroborated HRP-3 activity to promote new blood vessel formation. These results demonstrated that HRP-3 is a novel angiogenic factor.

Suppression of cytochrome P450 reductase (POR) expression in hepatoma cells replicates the hepatic lipidosis observed in hepatic POR-null mice.

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Porter, Todd D; Banerjee, Subhashis; Stolarczyk, Elzbieta I; Zou, Ling

2011-06-01

Cytochrome P450 reductase (POR) is a microsomal electron transport protein essential to cytochrome P450-mediated drug metabolism and sterol and bile acid synthesis. The conditional deletion of hepatic POR gene expression in mice results in a marked decrease in plasma cholesterol levels counterbalanced by the accumulation of triglycerides in lipid droplets in hepatocytes. To evaluate the role of cholesterol and bile acid synthesis in this hepatic lipidosis, as well as the possible role of lipid transport from peripheral tissues, we developed a stable, small interfering RNA (siRNA)-mediated cell culture model for the suppression of POR. POR mRNA and protein expression were decreased by greater than 50% in McArdle-RH7777 rat hepatoma cells 10 days after transfection with a POR-siRNA expression plasmid, and POR expression was nearly completely extinguished by day 20. Immunofluorescent analysis revealed a marked accumulation of lipid droplets in cells by day 15, accompanied by a nearly 2-fold increase in cellular triglyceride content, replicating the lipidosis seen in hepatic POR-null mouse liver. In contrast, suppression of CYP51A1 (lanosterol demethylase) did not result in lipid accumulation, indicating that loss of cholesterol synthesis is not the basis for this lipidosis. Indeed, addition of cholesterol to the medium appeared to augment the lipidosis in POR-suppressed cells, whereas removal of lipids from the medium reversed the lipidosis. Oxysterols did not accumulate in POR-suppressed cells, discounting a role for liver X receptor in stimulating triglyceride synthesis, but addition of chenodeoxycholate significantly repressed lipid accumulation, suggesting that the absence of bile acids and loss of farnesoid X receptor stimulation lead to excessive triglyceride synthesis.

Caffeic acid, a coffee-related organic acid, inhibits infection by severe fever with thrombocytopenia syndrome virus in vitro.

Science.gov (United States)

Ogawa, Motohiko; Shirasago, Yoshitaka; Ando, Shuji; Shimojima, Masayuki; Saijo, Masayuki; Fukasawa, Masayoshi

2018-04-05

Severe fever with thrombocytopenia syndrome (SFTS) virus (SFTSV) causes tick-borne hemorrhagic fever in East Asia. The disease is characterized by high morbidity and mortality. Here, we evaluated the effects of caffeic acid (CA), a coffee-related organic acid with antiviral effects, against SFTSV infection. CA dose-dependently inhibited SFTSV infection in permissive human hepatoma Huh7.5.1-8 cells when SFTSV was added into the culture medium with CA. However, quinic acid (QA), another coffee-related organic acid, did not inhibit SFTSV infection. The 50% inhibitory concentration (IC 50 ) of CA against SFTSV was 0.048 mM, whereas its 50% cytotoxic concentration was 7.6 mM. The selectivity index (SI) was 158. Pre-incubation of SFTSV with CA for 4 h resulted in a greater inhibition of SFTSV infection (IC 50  = 0.019 mM; SI = 400). The pre-incubation substantially decreased viral attachment to the cells. CA treatment of the SFTSV-infected cells also inhibited the infection, albeit less effectively. CA activity after cell infection with SFTSV was more pronounced at a low multiplicity of infection (MOI) of 0.01 per cell (IC 50  = 0.18 mM) than at a high MOI of 1 per cell (IC 50  > 1 mM). Thus, CA inhibited virus spread by acting directly on the virus rather than on the infected cells. In conclusion, CA acted on SFTSV and inhibited viral infection and spread, mainly by inhibiting the binding of SFTSV to the cells. We therefore demonstrated CA to be a potential anti-SFTSV drug for preventing and treating SFTS. Copyright © 2018 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Alpha-feto protein radioimmunoassay and its values for the diagnosis of hepatoma among normal Japanese population and those with chronic schistosomiasis

International Nuclear Information System (INIS)

Lio, Masahiro; Yamada, Hideo; Luchi, Masahiko

1972-01-01

Since its first report made by Ablevet al., α-feto protein which appears both in fetal serum and serum in the cases with hepatoma drew attention of hepatologist in order to establish an qualitative method to diagnose the presence of hepatoma. Since conventional liver scintigraphy is not able to make the diagnosis of the nature of lesion which appears as space occupying lesion need for the more qualitative method facilitated the establishment of the method to detect the presence of α-feto protein in serum. α-feto protein in the serum of hepatoma cases was reported to be identical to fetal α-feto protein by the immunological method. At first, assay of the α-feto protein was performed using the precipitation reaction in agar gel and became quite popular among clinical hepatologist. Detection sensitivity of this conventional method, however, is approximately 10 μgr/ml and α-feto protein was detected in 80% of hepatoma patient. However detection percentage of the hepatoma cases associated with chronic schistosomiasis was found to be as poor as 30% which was measured by our group at Kofu municipal Hospital. Because of the frequent incidences of deformed and small liver by scintigraphy among cases with chronic schistosomiasis and these abnormal liver scintigraphy easily lead one to middiagnose the presence of hepatoma, establishment of more sensitive assay method is needed. After the success of purification and crystallization of the α-feto protein by the group of Univ. of Hokkaido, the radioimmunoassay technique was developed in Japan and α-feto-125 Kit is now became available commercially. Initial trial of this new promising and this double antibody technique enabled us to measure 2 mμgr/ml of α-feto protein which was almost 500-1,000 times more sensitive than those of the conventional method.

CYP1A1 and CYP1A2 expression: Comparing 'humanized' mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines

International Nuclear Information System (INIS)

Uno, Shigeyuki; Endo, Kaori; Ishida, Yuji; Tateno, Chise; Makishima, Makoto; Yoshizato, Katsutoshi; Nebert, Daniel W.

2009-01-01

Human and rodent cytochrome P450 (CYP) enzymes sometimes exhibit striking species-specific differences in substrate preference and rate of metabolism. Human risk assessment of CYP substrates might therefore best be evaluated in the intact mouse by replacing mouse Cyp genes with human CYP orthologs; however, how 'human-like' can human gene expression be expected in mouse tissues? Previously a bacterial-artificial-chromosome-transgenic mouse, carrying the human CYP1A1 C YP1A2 locus and lacking the mouse Cyp1a1 and Cyp1a2 orthologs, was shown to express robustly human dioxin-inducible CYP1A1 and basal versus inducible CYP1A2 (mRNAs, proteins, enzyme activities) in each of nine mouse tissues examined. Chimeric mice carrying humanized liver have also been generated, by transplanting human hepatocytes into a urokinase-type plasminogen activator(+/+) s evere-combined-immunodeficiency (uPA/SCID) line with most of its mouse hepatocytes ablated. Herein we compare basal and dioxin-induced CYP1A mRNA copy numbers, protein levels, and four enzymes (benzo[a]pyrene hydroxylase, ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, methoxyresorufin O-demethylase) in liver of these two humanized mouse lines versus wild-type mice; we also compare these same parameters in mouse Hepa-1c1c7 and human HepG2 hepatoma-derived established cell lines. Most strikingly, mouse liver CYP1A1-specific enzyme activities are between 38- and 170-fold higher than human CYP1A1-specific enzyme activities (per unit of mRNA), whereas mouse versus human CYP1A2 enzyme activities (per unit of mRNA) are within 2.5-fold of one another. Moreover, both the mouse and human hepatoma cell lines exhibit striking differences in CYP1A mRNA levels and enzyme activities. These findings are relevant to risk assessment involving human CYP1A1 and CYP1A2 substrates, when administered to mice as environmental toxicants or drugs.

Genotoxicity and induction of DNA damage responsive genes by food-borne heterocyclic aromatic amines in human hepatoma HepG2 cells.

Science.gov (United States)

Pezdirc, Marko; Žegura, Bojana; Filipič, Metka

2013-09-01

Heterocyclic aromatic amines (HAAs) are potential human carcinogens formed in well-done meats and fish. The most abundant are 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-Amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline (4,8-DiMeIQx) and 2-Amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ). HAAs exert genotoxic activity after metabolic transformation by CYP1A enzymes, that is well characterized, however the genomic and intervening responses are not well explored. We have examined cellular and genomic responses of human hepatoma HepG2 cells after 24h exposure to HAAs. Comet assay revealed increase in formation of DNA strand breaks by PhIP, MeIQx and IQ but not 4,8-DiMeIQx, whereas increased formation of micronuclei was not observed. The four HAAs up-regulated expression of genes encoding metabolic enzymes CYP1A1, CYP1A2 and UGT1A1 and expression of TP53 and its downstream regulated genes CDKN1A, GADD45α and BAX. Consistent with the up-regulation of CDKN1A and GADD45α the cell-cycle analysis showed arrest in S-phase by PhIP and IQ, and in G1-phase by 4,8-DiMeIQx and MeIQx. The results indicate that upon exposure to HAAs the cells respond with the cell-cycle arrest, which enables cells to repair the damage or eliminate them by apoptosis. However, elevated expression of BCL2 and down-regulation of BAX may indicate that HAAs could suppress apoptosis meaning higher probability of damaged cells to survive and mutate. Copyright © 2013 Elsevier Ltd. All rights reserved.

Measuring and modeling of binary mixture effects of pharmaceuticals and nickel on cell viability/cytotoxicity in the human hepatoma derived cell line HepG2

International Nuclear Information System (INIS)

Rudzok, S.; Schlink, U.; Herbarth, O.; Bauer, M.

2010-01-01

The interaction of drugs and non-therapeutic xenobiotics constitutes a central role in human health risk assessment. Still, available data are rare. Two different models have been established to predict mixture toxicity from single dose data, namely, the concentration addition (CA) and independent action (IA) model. However, chemicals can also act synergistic or antagonistic or in dose level deviation, or in a dose ratio dependent deviation. In the present study we used the MIXTOX model (EU project ENV4-CT97-0507), which incorporates these algorithms, to assess effects of the binary mixtures in the human hepatoma cell line HepG2. These cells possess a liver-like enzyme pattern and a variety of xenobiotic-metabolizing enzymes (phases I and II). We tested binary mixtures of the metal nickel, the anti-inflammatory drug diclofenac, and the antibiotic agent irgasan and compared the experimental data to the mathematical models. Cell viability was determined by three different methods the MTT-, AlamarBlue (registered) and NRU assay. The compounds were tested separately and in combinations. We could show that the metal nickel is the dominant component in the mixture, affecting an antagonism at low-dose levels and a synergism at high-dose levels in combination with diclofenac or irgasan, when using the NRU and the AlamarBlue assay. The dose-response surface of irgasan and diclofenac indicated a concentration addition. The experimental data could be described by the algorithms with a regression of up to 90%, revealing the HepG2 cell line and the MIXTOX model as valuable tool for risk assessment of binary mixtures for cytotoxic endpoints. However the model failed to predict a specific mode of action, the CYP1A1 enzyme activity.

Phosphorus NMR of isolated perfused morris hepatomas

International Nuclear Information System (INIS)

Graham, R.A.; Meyer, R.A.; Brown, T.R.; Sauer, L.A.

1986-01-01

The authors are developing techniques for the study of perfused solid tumors by NMR. Tissue-isolated solid hepatomas were grown to 1-2 cm diameter as described previously. The arterial supply was isolated and the tumors perfused (0.5 - 1.0 ml/min) in vitro at 25 C with a 15% suspension of red blood cells in Krebs-Henseliet solution. 31 P-NMR spectra were acquired at 162 MHz in a specially-designed NMR probe using a solenoidal coil. Intracellular pH (monitored from the chemical shift of inorganic phosphate) and ATP levels were stable for up to 6 hrs during perfusion. During 30 min of global ischemia, ATP decreased by 75% and pH fell from 7.0 to 6.7. These changes were reversed by 1 hr reperfusion. In addition to ATP and phosphate, the spectra included a large resonance due to phosphomonoesters, as well as peaks consistent with glycerylphosphocholine, glyceryl-phosphoethanolamine, phosphocreatine, NAD, and UDPG. However, the most novel feature of the spectra was the presence of an unidentified peak in the phosphonate region (+ 16.9 ppm). The peak was not present in spectra of muscle, liver, brain, kidney, or fat tissues excised from the same animals. They are presently attempting to identify the compound that gives rise to this peak and to establish its metabolic origin

H32, a non-quinone sulfone analog of vitamin K3, inhibits human hepatoma cell growth by inhibiting Cdc25 and activating ERK.

Science.gov (United States)

Kar, Siddhartha; Wang, Meifang; Ham, Seung Wook; Carr, Brian I

2006-10-01

We previously synthesized a K-vitamin derivative, Cpd 5, which was a potent growth inhibitor of human tumor cells, including Hep3B hepatoma cells. However, being a quinone compound, Cpd 5 has the potential for generating toxic reactive oxygen species (ROS). We therefore synthesized a nonquinone sulfone derivative, H32, which has a sufone group substituting the quinone. The IC50 of H32 for Hep3B cells was found to be 2.5 microM, which was 2.5 and 3.2 times more potent than Cpd 5 and vitamin K3 respectively. It induced apoptosis in Hep3B cells but did not generate ROS when compared to Cpd 5. Interestingly, under similar culture conditions, normal rat hepatocytes were 14-fold more and 7-fold more resistant to the growth inhibitory effects of H32 than Hep3B and PLC/PRF5 cells respectively. H32 preferentially inhibited the activities of the cell cycle controlling Cdc25A phosphatase likely by binding to its catalytic cysteine. As a consequence, it induced inhibitory tyrosine phosphorylation of the Cdc25 substrate kinases Cdk2 and Cdk4 in Hep3B cells and the cells undergo an arrest in the G1 phase of the cell cycle. H32 also induced persistent phosphorylation of the MAPK protein ERK1/2, but marginal JNK1/2 and p38 phosphorylation. The ERK inhibitor U0126, added at least 30 min prior to H32, antagonized the growth inhibition induced by H32. However, the JNK and p38 inhibitors, JNKI-II and SB203580, were not able to antagonize H32 induced growth inhibition. Thus, H32 differentially inhibited growth of normal and liver tumor cells by preferentially inhibiting the actions of Cdc25 phosphatases and inducing persistent ERK phosphorylation.

Mycobacterium tuberculosis infection induces non-apoptotic cell death of human dendritic cells

LENUS (Irish Health Repository)

Ryan, Ruth CM

2011-10-24

Abstract Background Dendritic cells (DCs) connect innate and adaptive immunity, and are necessary for an efficient CD4+ and CD8+ T cell response after infection with Mycobacterium tuberculosis (Mtb). We previously described the macrophage cell death response to Mtb infection. To investigate the effect of Mtb infection on human DC viability, we infected these phagocytes with different strains of Mtb and assessed viability, as well as DNA fragmentation and caspase activity. In parallel studies, we assessed the impact of infection on DC maturation, cytokine production and bacillary survival. Results Infection of DCs with live Mtb (H37Ra or H37Rv) led to cell death. This cell death proceeded in a caspase-independent manner, and without nuclear fragmentation. In fact, substrate assays demonstrated that Mtb H37Ra-induced cell death progressed without the activation of the executioner caspases, 3\\/7. Although the death pathway was triggered after infection, the DCs successfully underwent maturation and produced a host-protective cytokine profile. Finally, dying infected DCs were permissive for Mtb H37Ra growth. Conclusions Human DCs undergo cell death after infection with live Mtb, in a manner that does not involve executioner caspases, and results in no mycobactericidal effect. Nonetheless, the DC maturation and cytokine profile observed suggests that the infected cells can still contribute to TB immunity.

Knockdown of hepatoma-derived growth factor-related protein-3 induces apoptosis of H1299 cells via ROS-dependent and p53-independent NF-κB activation

International Nuclear Information System (INIS)

Yun, Hong Shik; Baek, Jeong-Hwa; Yim, Ji-Hye; Lee, Su-Jae; Lee, Chang-Woo; Song, Jie-Young; Um, Hong-Duck; Park, Jong Kuk; Park, In-Chul; Hwang, Sang-Gu

2014-01-01

Highlights: • HRP-3 is a radiation- and anticancer drug-responsive protein in H1299 cells. • Depletion of HRP-3 induces apoptosis of radio- and chemoresistant H1299 cells. • Depletion of HRP-3 promotes ROS generation via inhibition of the Nrf2/HO-1 pathway. • ROS generation enhances NF-κB activity, which acts as an upstream signal in the c-Myc/Noxa apoptotic pathway. - Abstract: We previously identified hepatoma-derived growth factor-related protein-3 (HRP-3) as a radioresistant biomarker in p53 wild-type A549 cells and found that p53-dependent induction of the PUMA pathway was a critical event in regulating the radioresistant phenotype. Here, we found that HRP-3 knockdown regulates the radioresistance of p53-null H1299 cells through a distinctly different molecular mechanism. HRP-3 depletion was sufficient to cause apoptosis of H1299 cells by generating substantial levels of reactive oxygen species (ROS) through inhibition of the Nrf2/HO-1 antioxidant pathway. Subsequent, ROS-dependent and p53-independent NF-κB activation stimulated expression of c-Myc and Noxa proteins, thereby inducing the apoptotic machinery. Our results thus extend the range of targets for the development of new drugs to treat both p53 wild-type or p53-null radioresistant lung cancer cells

Coronavirus infection of polarized epithelial cells

NARCIS (Netherlands)

Rossen, J W; Horzinek, M C; Rottier, P J

1995-01-01

Epithelial cells are the first host cells to be infected by incoming c oronaviruses. Recent observations in vitro show that coronaviruses are released from a specific side of these polarized cells, and this polarized release might be important for the spread of the infection in vivo. Mechanisms for

Polypeptide synthesis in alphavirus-infected aedes albopictus cells during the establishment of persistent infection

International Nuclear Information System (INIS)

Richardson, M.A.; Boulton, R.W.; Raghow, R.S.; Dalgarno, L.

1980-01-01

Polypeptide synthesis was examined in mosquito cells during the establishment of a persistent infection with two alphaviruses, Ross River virus (RRV) and Semliki Forest virus (SFV), and in vertebrate cells cytopathically-infected with the same viruses. In Aedes albopictus cells, RRV reached peak titres at 34-48 hours p.i. At 12 hours 85 per cent of cells assayed as infected by infective centre assay; by 48 hours when persistence was established, virus production was reduced and <5 per cent of cells assayed as infected. There was not shutdown of host polypeptide synthesis during infection. Viral polypeptide synthesis was maximal between 10 and 24 hours p.i. The major viral polypeptides labelled were nucleocapsid protein and envelope protein(s).The precursor polypeptide p95 which was prominent in infected BHK cells was not detected in mosquito cells. Similar results were obtained on SFV infection. During the establishment of persistence there was a coordinate decline in the synthesis of RRV polypeptides, reaching undetectable levels by 72 hours p.i. Subculturing persistently-infected cells led to a small increase in viral polypeptide synthesis and virus titre. In contrast, during RRV growth in BHK cells host protein synthesis was severely inhibited and by 9-11 hours p.i. virus-specific polypeptide synthesis represented more than 90 per cent of total protein synthetic activity. (author)

Genotoxicity assessment of membrane concentrates of landfill leachate treated with Fenton reagent and UV-Fenton reagent using human hepatoma cell line

Energy Technology Data Exchange (ETDEWEB)

Wang, Guifang [Department of Chemistry, Jinan University, Guangzhou 510632 (China); Lu, Gang [Key Laboratory of Water/Soil Toxic Pollutants Control and Bioremediation of Guangdong Higher Education Institutes, Department of Environmental Engineering, Jinan University, Guangzhou 510632 (China); Yin, Pinghe, E-mail: tyinph@jnu.edu.cn [Research Center of Analysis and Test, Jinan University, Guangzhou 510632 (China); Zhao, Ling, E-mail: zhaoling@jnu.edu.cn [Key Laboratory of Water/Soil Toxic Pollutants Control and Bioremediation of Guangdong Higher Education Institutes, Department of Environmental Engineering, Jinan University, Guangzhou 510632 (China); Jimmy Yu, Qiming [Griffith School of Engineering, Griffith University, Nathan Campus, Brisbane, Queensland 4111 (Australia)

2016-04-15

Highlights: • Membrane concentrates have a threat to human health and environment. • Untreated membrane concentrates induces cytotoxic and genotoxic to HepG2 cells. • Both methods were effective method for degradation of BPA and NP in concentrates. • Both methods were efficient in reducing genotoxic effects of concentrates. • UV-Fenton reagent had higher removal efficiency and provides toxicological safety. - Abstract: Membrane concentrates of landfill leachates contain organic and inorganic contaminants that could be highly toxic and carcinogenic. In this paper, the genotoxicity of membrane concentrates before and after Fenton and UV-Fenton reagent was assessed. The cytotoxicity and genotoxicity was determined by using the methods of methyltetrazolium (MTT), cytokinesis-block micronucleus (CBMN) and comet assay in human hepatoma cells. MTT assay showed a cytotoxicity of 75% after 24 h of exposure to the highest tested concentration of untreated concentrates, and no cytotoxocity for UV-Fenton and Fenton treated concentrates. Both CBMN and comet assays showed increased levels of genotoxicity in cells exposed to untreated concentrates, compared to those occurred in cells exposed to UV-Fenton and Fenton reagent treated concentrates. There was no significant difference between negative control and UV-Fenton treated concentrates for micronucleus and comet assay parameters. UV-Fenton and Fenton treatment, especially the former, were effective methods for degradation of bisphenol A and nonylphenol in concentrates. These findings showed UV-Fenton and Fenton reaction were effective methods for treatment of such complex concentrates, UV-Fenton reagent provided toxicological safety of the treated effluent, and the genotoxicity assays were found to be feasible tools for assessment of toxicity risks of complex concentrates.

Genotoxicity assessment of membrane concentrates of landfill leachate treated with Fenton reagent and UV-Fenton reagent using human hepatoma cell line

International Nuclear Information System (INIS)

Wang, Guifang; Lu, Gang; Yin, Pinghe; Zhao, Ling; Jimmy Yu, Qiming

2016-01-01

Highlights: • Membrane concentrates have a threat to human health and environment. • Untreated membrane concentrates induces cytotoxic and genotoxic to HepG2 cells. • Both methods were effective method for degradation of BPA and NP in concentrates. • Both methods were efficient in reducing genotoxic effects of concentrates. • UV-Fenton reagent had higher removal efficiency and provides toxicological safety. - Abstract: Membrane concentrates of landfill leachates contain organic and inorganic contaminants that could be highly toxic and carcinogenic. In this paper, the genotoxicity of membrane concentrates before and after Fenton and UV-Fenton reagent was assessed. The cytotoxicity and genotoxicity was determined by using the methods of methyltetrazolium (MTT), cytokinesis-block micronucleus (CBMN) and comet assay in human hepatoma cells. MTT assay showed a cytotoxicity of 75% after 24 h of exposure to the highest tested concentration of untreated concentrates, and no cytotoxocity for UV-Fenton and Fenton treated concentrates. Both CBMN and comet assays showed increased levels of genotoxicity in cells exposed to untreated concentrates, compared to those occurred in cells exposed to UV-Fenton and Fenton reagent treated concentrates. There was no significant difference between negative control and UV-Fenton treated concentrates for micronucleus and comet assay parameters. UV-Fenton and Fenton treatment, especially the former, were effective methods for degradation of bisphenol A and nonylphenol in concentrates. These findings showed UV-Fenton and Fenton reaction were effective methods for treatment of such complex concentrates, UV-Fenton reagent provided toxicological safety of the treated effluent, and the genotoxicity assays were found to be feasible tools for assessment of toxicity risks of complex concentrates.

HIV Cell-to-Cell Spread Results in Earlier Onset of Viral Gene Expression by Multiple Infections per Cell.

Directory of Open Access Journals (Sweden)

Mikaël Boullé

2016-11-01

Full Text Available Cell-to-cell spread of HIV, a directed mode of viral transmission, has been observed to be more rapid than cell-free infection. However, a mechanism for earlier onset of viral gene expression in cell-to-cell spread was previously uncharacterized. Here we used time-lapse microscopy combined with automated image analysis to quantify the timing of the onset of HIV gene expression in a fluorescent reporter cell line, as well as single cell staining for infection over time in primary cells. We compared cell-to-cell spread of HIV to cell-free infection, and limited both types of transmission to a two-hour window to minimize differences due to virus transit time to the cell. The mean time to detectable onset of viral gene expression in cell-to-cell spread was accelerated by 19% in the reporter cell line and by 35% in peripheral blood mononuclear cells relative to cell-free HIV infection. Neither factors secreted by infected cells, nor contact with infected cells in the absence of transmission, detectably changed onset. We recapitulated the earlier onset by infecting with multiple cell-free viruses per cell. Surprisingly, the acceleration in onset of viral gene expression was not explained by cooperativity between infecting virions. Instead, more rapid onset was consistent with a model where the fastest expressing virus out of the infecting virus pool sets the time for infection independently of the other co-infecting viruses.

Brucella abortus-infected B cells induce osteoclastogenesis.

Science.gov (United States)

Pesce Viglietti, Ayelén Ivana; Arriola Benitez, Paula Constanza; Giambartolomei, Guillermo Hernán; Delpino, María Victoria

2016-09-01

Brucella abortus is an intracellular bacterium that establishes lifelong infections in livestock and humans although the mechanisms of its chronicity are poorly understood. Activated B cells have long lifespan and B. abortus infection activates B cells. Our results indicate that the direct infection of B cells with B. abortus induced matrix metalloproteinase-9 (MMP-9), receptor activator for NF κB ligand (RANKL), tumor necrosis factor (TNF)-α and interleukin (IL)-6 secretion. In addition, supernatants from B. abortus-infected B cells induced bone marrow-derived monocytes to undergo osteoclastogenesis. Using osteoprotegerin, RANKL's decoy receptor, we determined that RANKL is involved in osteoclastogenesis induced by supernatants from B. abortus-infected B cells. The results presented here shed light on how the interactions of B. abortus with B cells may have a role in the pathogenesis of brucellar osteoarticular disease. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Yeast endoribonuclease stimulated by Novikoff Hepatoma small nuclear RNAS U1 and U2

International Nuclear Information System (INIS)

Stevens, A.

1982-01-01

Using [ 3 H]m 7 Gppp[ 14 C]RNA-poly(A) from yeast as a substrate, an endoribonuclease has been detected in enzyme fractions derived from a high salt wash of ribonucleoprotein particles of Saccharomyces cerevisiae. The [ 3 H]m 7 Gppp[ 14 C]RNA-poly(A) seems to be a preferred substrate since other polyribonucleotides are hydrolyzed more slowly, if at all. The enzyme is inhibited by ethidium bromide, but fully double-stranded polyribonucleotides are not hydrolyzed. The hydrolysis of [ 3 H]m 7 Gppp[ 14 C]RNA-poly(A) is stimulated about 2.5-fold by the addition of small nuclear RNAs U1 and U2 of Novikoff hepatoma cells. Results show that the stimulation involves an interaction of the labeled RNA with the small nuclear RNA

Demonstration of NK cell-mediated lysis of varicella-zoster virus (VZV)-infected cells: characterization of the effector cells

Energy Technology Data Exchange (ETDEWEB)

Tilden, A.B.; Cauda, R.; Grossi, C.E.; Balch, C.M.; Lakeman, A.D.; Whitley, R.J.

1986-06-01

Infection with varicella-zoster virus (VZV) rendered RAJI cells more susceptible to lysis by non-adherent blood lymphocytes. At an effector to target ratio of 80:1 the mean percentage of /sup 51/Cr release of VZV-infected RAJI cells was 41 +/- 12%, whereas that of uninfected RAJI cells was 15 +/- 6%. The increased susceptibility to lysis was associated with increased effector to target conjugate formation in immunofluorescence binding assays. The effector cells cytotoxic for VZV-infected RAJI cells were predominantly Leu-11a/sup +/ Leu-4/sup -/ granular lymphocytes as demonstrated by fluorescence-activated cell sorting. The effector cell active against VZV-infected RAJI cells appeared similar to those active against herpes simplex virus (HSV)-infected cells, because in cold target competition experiments the lysis of /sup 51/Cr-labeled VZV-infected RAJI cells was efficiently inhibited by either unlabeled VZV-infected RAJI cells (mean 71% inhibition, 2:1 ratio unlabeled to labeled target) or HSV-infected RAJI cells (mean 69% inhibition) but not by uninfected RAJI cells (mean 10% inhibition). In contrast, competition experiments revealed donor heterogeneity in the overlap between effector cells for VZV- or HSV-infected RAJI vs K-562 cells.

Mast cells in viral infections

Directory of Open Access Journals (Sweden)

Piotr Witczak

2012-04-01

Full Text Available  There are some premises suggesting that mast cells are involved in the mechanisms of anti-virus defense and in viral disease pathomechanisms. Mast cells are particularly numerous at the portals of infections and thus may have immediate and easy contact with the external environment and invading pathogens. These cells express receptors responsible for recognition of virus-derived PAMP molecules, mainly Toll-like receptors (TLR3, TLR7/8 and TLR9, but also RIG-I-like and NOD-like molecules. Furthermore, mast cells generate various mediators, cytokines and chemokines which modulate the intensity of inflammation and regulate the course of innate and adaptive anti-viral immunity. Indirect evidence for the role of mast cells in viral infections is also provided by clinical observations and results of animal studies. Currently, more and more data indicate that mast cells can be infected by some viruses (dengue virus, adenoviruses, hantaviruses, cytomegaloviruses, reoviruses, HIV-1 virus. It is also demonstrated that mast cells can release pre formed mediators as well as synthesize de novo eicosanoids in response to stimulation by viruses. Several data indicate that virus-stimulated mast cells secrete cytokines and chemokines, including interferons as well as chemokines with a key role in NK and Tc lymphocyte influx. Moreover, some information indicates that mast cell stimulation via TLR3, TLR7/8 and TLR9 can affect their adhesion to extracellular matrix proteins and chemotaxis, and influence expression of some membrane molecules. Critical analysis of current data leads to the conclusion that it is not yet possible to make definitive statements about the role of mast cells in innate and acquired defense mechanisms developing in the course of viral infection and/or pathomechanisms of viral diseases.

188Re-SSS lipiodol: radiolabelling and biodistribution following injection into the hepatic artery of rats bearing hepatoma.

Science.gov (United States)

Garin, Etienne; Denizot, Benoit; Noiret, Nicolas; Lepareur, Nicolas; Roux, Jerome; Moreau, Myriam; Herry, Jean-Yves; Bourguet, Patrick; Benoit, Jean-Pierre; Lejeune, Jean-Jacques

2004-10-01

Although intra-arterial radiation therapy with 131I-lipiodol is a useful therapeutic approach to the treatment of hepatocellular carcinoma, various disadvantages limit its use. To describe the development of a method for the labelling of lipiodol with 188Re-SSS (188Re (S2CPh)(S3CPh)2 complex) and to investigate its biodistribution after injection into the hepatic artery of rats with hepatoma. 188Re-SSS lipiodol was obtained after dissolving a chelating agent, previously labelled with 188Re, in cold lipiodol. The radiochemical purity (RCP) of labelling was checked immediately. The 188Re-SSS lipiodol was injected into the hepatic artery of nine rats with a Novikoff hepatoma. They were sacrificed 1, 24 and 48 h after injection, and used for ex vivo counting. Labelling of 188Re-SSS lipiodol was achieved with a yield of 97.3+/-2.1%. The immediate RCP was 94.1+/-1.7%. Ex vivo counting confirmed a predominantly hepatic uptake, with a good tumoral retention of 188Re-SSS lipiodol, a weak pulmonary uptake and a very faint digestive uptake. The 'tumour/non-tumoral liver' ratio was high at 1, 24 and 48 h after injection (2.9+/-1.5, 4.1+/-/4.1 and 4.1+/-0.7, respectively). Using the method described here, 188Re-SSS lipiodol can be obtained with a very high yield and a satisfactory RCP. The biodistribution in rats with hepatoma indicates a good tumoral retention of 188Re-SSS lipiodol associated with a predominant hepatic uptake, a weak pulmonary uptake and a very faint digestive uptake. This product should be considered for intra-arterial radiation therapy in human hepatoma.

pH-Responsive Hyaluronic Acid-Based Mixed Micelles for the Hepatoma-Targeting Delivery of Doxorubicin

Directory of Open Access Journals (Sweden)

Jing-Liang Wu

2016-03-01

Full Text Available The tumor targetability and stimulus responsivity of drug delivery systems are crucial in cancer diagnosis and treatment. In this study, hepatoma-targeting mixed micelles composed of a hyaluronic acid–glycyrrhetinic acid conjugate and a hyaluronic acid-l-histidine conjugate (HA–GA/HA–His were prepared through ultrasonic dispersion. The formation and characterization of the mixed micelles were confirmed via 1H-NMR, particle size, and ζ potential measurements. The in vitro cellular uptake of the micelles was evaluated using human liver carcinoma (HepG2 cells. The antitumor effect of doxorubicin (DOX-loaded micelles was investigated in vitro and in vivo. Results indicated that the DOX-loaded HA–GA/HA–His micelles showed a pH-dependent controlled release and were remarkably absorbed by HepG2 cells. Compared with free DOX, the DOX-loaded HA–GA/HA–His micelles showed a higher cytotoxicity to HepG2 cells. Moreover, the micelles effectively inhibited tumor growth in H22 cell-bearing mice. These results suggest that the HA–GA/HA–His mixed micelles are a good candidate for drug delivery in the prevention and treatment of hepatocarcinoma.

Combination of anti-retroviral drugs and radioimmunotherapy specifically kills infected cells from HIV infected individuals

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Dina Tsukrov

2016-09-01

Full Text Available Eliminating virally infected cells is an essential component of any HIV eradication strategy. Radioimmunotherapy (RIT, a clinically established method for killing cells using radiolabeled antibodies, was recently applied to target HIV-1 gp41 antigen expressed on the surface of infect-ed cells. Since gp41 expression by infected cells is likely down-regulated in patients on an-tiretroviral therapy (ART, we evaluated the ability of RIT to kill ART-treated infected cells us-ing both in vitro models and lymphocytes isolated from HIV-infected subjects. Human peripheral blood mononuclear cells (PBMCs were infected with HIV and cultured in the presence of two clinically relevant ART combinations. Scatchard analysis of the 2556 human monoclonal anti-body to HIV gp41 binding to the infected and ART-treated cells demonstrated sufficient residual expression of gp41 on the cell surface to warrant subsequent RIT. This is the first time the quantification of gp41 post-ART is being reported. Cells were then treated with Bismuth-213-labeled 2556 antibody. conjugated to the human monoclonal antibody 2556, which binds to HIV gp41. Cell survival was quantified by Trypan blue and residual viremia by p24 ELISA. Cell surface gp41 expression was assessed by Scatchard analysis. The experiments were repeated using PBMCs isolated from blood specimens obtained from 15 HIV-infected individuals: ten on ART and five ART-naive. We found that 213Bi-2556 killed ART-treated infected PBMCs and reduced viral production to undetectable levels. ART and RIT co-treatment was more effective at reducing viral load in vitro than either therapy alone, indicating that gp41 expression under ART was sufficient to allow 213Bi-2556 to deliver cytocidal doses of radiation to infected cells. This study provides proof of concept that 213Bi-2556 may represent an innovative and effective targeting method for killing HIV-infected cells treated with ART, and supports continued development of 213Bi

The diagnostic application of Ga-67 scintigraphy in primary lung cancer, hepatoma and abdominal abscess

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Oshiumi, Yoshihiko

1983-01-01

It is difficult to detect tumor lesions by 67 Gascintigraphy, though it is named as a tumor scintigraphy. Recently, 67 Ga-scintigraphy is well used in the detection of inflammatic lesions. This paper is to discuss the detectability of lesions and diagnostic limitation on operability by 67 Ga-scintigraphy to primary lung cancer, minute hepatoma and abdominal absccess. Primary lung cancer: In detecting metastatic hilar lymph nodes, the sensitivity is 54%, and the specificity is 78%. In detecting metastatic mediastinal lymph nodes, the sensitivity is 32% and the specificity is 89%. Minute hepatoma : The detectability depended on the size of the lesion. It is hard to detect lesions with under 2 cm by 67 Ga-scintigraphy. Abdominal abscess : The sensitivity is 88%, and the specificity is 92%. (author)

Fungal cell gigantism during mammalian infection.

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Oscar Zaragoza

2010-06-01

Full Text Available The interaction between fungal pathogens with the host frequently results in morphological changes, such as hyphae formation. The encapsulated pathogenic fungus Cryptococcus neoformans is not considered a dimorphic fungus, and is predominantly found in host tissues as round yeast cells. However, there is a specific morphological change associated with cryptococcal infection that involves an increase in capsule volume. We now report another morphological change whereby gigantic cells are formed in tissue. The paper reports the phenotypic characterization of giant cells isolated from infected mice and the cellular changes associated with giant cell formation. C. neoformans infection in mice resulted in the appearance of giant cells with cell bodies up to 30 microm in diameter and capsules resistant to stripping with gamma-radiation and organic solvents. The proportion of giant cells ranged from 10 to 80% of the total lung fungal burden, depending on infection time, individual mice, and correlated with the type of immune response. When placed on agar, giant cells budded to produce small daughter cells that traversed the capsule of the mother cell at the speed of 20-50 m/h. Giant cells with dimensions that approximated those in vivo were observed in vitro after prolonged culture in minimal media, and were the oldest in the culture, suggesting that giant cell formation is an aging-dependent phenomenon. Giant cells recovered from mice displayed polyploidy, suggesting a mechanism by which gigantism results from cell cycle progression without cell fission. Giant cell formation was dependent on cAMP, but not on Ras1. Real-time imaging showed that giant cells were engaged, but not engulfed by phagocytic cells. We describe a remarkable new strategy for C. neoformans to evade the immune response by enlarging cell size, and suggest that gigantism results from replication without fission, a phenomenon that may also occur with other fungal pathogens.

Fungal cell gigantism during mammalian infection.

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Zaragoza, Oscar; García-Rodas, Rocío; Nosanchuk, Joshua D; Cuenca-Estrella, Manuel; Rodríguez-Tudela, Juan Luis; Casadevall, Arturo

2010-06-17

The interaction between fungal pathogens with the host frequently results in morphological changes, such as hyphae formation. The encapsulated pathogenic fungus Cryptococcus neoformans is not considered a dimorphic fungus, and is predominantly found in host tissues as round yeast cells. However, there is a specific morphological change associated with cryptococcal infection that involves an increase in capsule volume. We now report another morphological change whereby gigantic cells are formed in tissue. The paper reports the phenotypic characterization of giant cells isolated from infected mice and the cellular changes associated with giant cell formation. C. neoformans infection in mice resulted in the appearance of giant cells with cell bodies up to 30 microm in diameter and capsules resistant to stripping with gamma-radiation and organic solvents. The proportion of giant cells ranged from 10 to 80% of the total lung fungal burden, depending on infection time, individual mice, and correlated with the type of immune response. When placed on agar, giant cells budded to produce small daughter cells that traversed the capsule of the mother cell at the speed of 20-50 m/h. Giant cells with dimensions that approximated those in vivo were observed in vitro after prolonged culture in minimal media, and were the oldest in the culture, suggesting that giant cell formation is an aging-dependent phenomenon. Giant cells recovered from mice displayed polyploidy, suggesting a mechanism by which gigantism results from cell cycle progression without cell fission. Giant cell formation was dependent on cAMP, but not on Ras1. Real-time imaging showed that giant cells were engaged, but not engulfed by phagocytic cells. We describe a remarkable new strategy for C. neoformans to evade the immune response by enlarging cell size, and suggest that gigantism results from replication without fission, a phenomenon that may also occur with other fungal pathogens.

Intracellular Events and Cell Fate in Filovirus Infection

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Elena Ryabchikova

2011-08-01

Full Text Available Marburg and Ebola viruses cause a severe hemorrhagic disease in humans with high fatality rates. Early target cells of filoviruses are monocytes, macrophages, and dendritic cells. The infection spreads to the liver, spleen and later other organs by blood and lymph flow. A hallmark of filovirus infection is the depletion of non-infected lymphocytes; however, the molecular mechanisms leading to the observed bystander lymphocyte apoptosis are poorly understood. Also, there is limited knowledge about the fate of infected cells in filovirus disease. In this review we will explore what is known about the intracellular events leading to virus amplification and cell damage in filovirus infection. Furthermore, we will discuss how cellular dysfunction and cell death may correlate with disease pathogenesis.

Kinetics of liver macrophages (Kupffer cells) in SIV-infected macaques

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Ahsan, Muhammad H.; Gill, Amy F.; Alvarez, Xavier; Lackner, Andrew A.; Veazey, Ronald S.

2013-01-01

Since the liver drains antigens from the intestinal tract, and since the intestinal tract is a major site of viral replication, we examined the dynamics of liver macrophages (Kupffer cells) throughout SIV infection. Absolute numbers of Kupffer cells increased in the livers in acute infection, and in animals with AIDS. Significantly higher percentages of proliferating (BrdU+) Kupffer cells were detected in acute infection and in AIDS with similar trends in blood monocytes. Significantly higher percentages of apoptotic (AC3+) Kupffer cells were also found in acute and AIDS stages. However, productively infected cells were not detected in liver of 41/42 animals examined, despite abundant infected cells in gut and lymph nodes of all animals. Increased rates of Kupffer cell proliferation resulting in an increase in Kupffer cells without productive infection indicate SIV infection affects Kupffer cells, but the liver does not appear to be a major site of productive viral replication. - Highlights: • Kupffer cells increase in the liver of SIV-infected macaques. • Increased proliferation and apoptosis of Kupffer cells occurs in SIV infection. • Productively infected cells are rarely detected in the liver. • The liver is not a major site for SIV replication

NKT cell depletion in humans during early HIV infection.

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Fernandez, Caroline S; Kelleher, Anthony D; Finlayson, Robert; Godfrey, Dale I; Kent, Stephen J

2014-08-01

Natural killer T (NKT) cells bridge across innate and adaptive immune responses and have an important role in chronic viral infections such as human immunodeficiency virus (HIV). NKT cells are depleted during chronic HIV infection, but the timing, drivers and implications of this NKT cell depletion are poorly understood. We studied human peripheral blood NKT cell levels, phenotype and function in 31 HIV-infected subjects not on antiretroviral treatment from a mean of 4 months to 2 years after HIV infection. We found that peripheral CD4(+) NKT cells were substantially depleted and dysfunctional by 4 months after HIV infection. The depletion of CD4(+) NKT cells was more marked than the depletion of total CD4(+) T cells. Further, the early depletion of NKT cells correlated with CD4(+) T-cell decline, but not HIV viral levels. Levels of activated CD4(+) T cells correlated with the loss of NKT cells. Our studies suggest that the early loss of NKT cells is associated with subsequent immune destruction during HIV infection.

A yeast endoribonuclease stimulated by Novikoff hepatoma small nuclear RNAs U1 and U2

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Stevens, A.

1982-01-01

Using [ 3 H]m 7 Gppp[ 14 C]RNA-poly(A) from yeast as a substrate, an endoribonuclease has been detected in enzyme fractions derived from a high salt wash of ribonucleoprotein particles of Saccharomyces cerevisiae. The [ 3 H]m 7 Gppp[ 14 C]RNA-poly(A) seems to be a preferred substrate since other polyribonucleotides are hydrolyzed more slowly, if at all. The enzyme is inhibited by ethidium bromide, but fully double-stranded polyribonucleotides are not hydrolyzed. The hydrolysis of [ 3 H]m 7 Gppp[ 14 C]RNA-poly(A) is stimulated about 2.5-fold by the addition of small nuclear RNAs U1 and U2 of Novikoff hepatoma cells. Results show that the stimulation involves an interaction of the labeled RNA with the small nuclear RNA

Alemtuzumab-induced elimination of HIV-1-infected immune cells.

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Ruxrungtham, Kiat; Sirivichayakul, Sunee; Buranapraditkun, Supranee; Krause, Werner

2016-01-01

Currently, there is no drug known that is able to eradicate either HIV or HIV-infected host cells. The effectiveness of all available treatments is based on the prevention of viral replication. We investigated whether the monoclonal, CD52 receptor-targeting antibody, alemtuzumab, which is currently approved for the treatment of multiple sclerosis, is able to eliminate HIV-infected immune cells. In blood samples from healthy donors and from HIV-1-infected subjects who were either treatment-naïve or resistant to HAART, we studied whether the CD52 expression on T cells and their subsets (CD3, CD4, CD8), B cells (CD19), dendritic cells (CD123) and monocytes (CD11c) is retained in HIV-1 infection and whether alemtuzumab is able to eradicate infected cells, using four-colour flow cytometry. We found that CD52 expression on immune cells is retained in HIV-1 infection regardless of CD4 cell count, viral load and treatment status, and is amenable to alemtuzumab-induced depletion. For the first time it could be shown in vitro that HIV-1-infected immune cells can be eliminated by using the monoclonal antibody alemtuzumab.

Nipah virus infection and glycoprotein targeting in endothelial cells

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Maisner Andrea

2010-11-01

Full Text Available Abstract Background The highly pathogenic Nipah virus (NiV causes fatal respiratory and brain infections in animals and humans. The major hallmark of the infection is a systemic endothelial infection, predominantly in the CNS. Infection of brain endothelial cells allows the virus to overcome the blood-brain-barrier (BBB and to subsequently infect the brain parenchyma. However, the mechanisms of NiV replication in endothelial cells are poorly elucidated. We have shown recently that the bipolar or basolateral expression of the NiV surface glycoproteins F and G in polarized epithelial cell layers is involved in lateral virus spread via cell-to-cell fusion and that correct sorting depends on tyrosine-dependent targeting signals in the cytoplasmic tails of the glycoproteins. Since endothelial cells share many characteristics with epithelial cells in terms of polarization and protein sorting, we wanted to elucidate the role of the NiV glycoprotein targeting signals in endothelial cells. Results As observed in vivo, NiV infection of endothelial cells induced syncytia formation. The further finding that infection increased the transendothelial permeability supports the idea of spread of infection via cell-to-cell fusion and endothelial cell damage as a mechanism to overcome the BBB. We then revealed that both glycoproteins are expressed at lateral cell junctions (bipolar, not only in NiV-infected primary endothelial cells but also upon stable expression in immortalized endothelial cells. Interestingly, mutation of tyrosines 525 and 542/543 in the cytoplasmic tail of the F protein led to an apical redistribution of the protein in endothelial cells whereas tyrosine mutations in the G protein had no effect at all. This fully contrasts the previous results in epithelial cells where tyrosine 525 in the F, and tyrosines 28/29 in the G protein were required for correct targeting. Conclusion We conclude that the NiV glycoprotein distribution is responsible for

Stem Cell Transplant Patients and Fungal Infections

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... Foodborne, Waterborne, and Environmental Diseases Mycotic Diseases Branch Stem Cell Transplant Patients and Fungal Infections Recommend on Facebook ... Mold . Top of Page Preventing fungal infections in stem cell transplant patients Fungi are difficult to avoid because ...

Establishment of human papillomavirus infection requires cell cycle progression.

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Dohun Pyeon

2009-02-01

Full Text Available Human papillomaviruses (HPVs are DNA viruses associated with major human cancers. As such there is a strong interest in developing new means, such as vaccines and microbicides, to prevent HPV infections. Developing the latter requires a better understanding of the infectious life cycle of HPVs. The HPV infectious life cycle is closely linked to the differentiation state of the stratified epithelium it infects, with progeny virus only made in the terminally differentiating suprabasal compartment. It has long been recognized that HPV must first establish its infection within the basal layer of stratified epithelium, but why this is the case has not been understood. In part this restriction might reflect specificity of expression of entry receptors. However, this hypothesis could not fully explain the differentiation restriction of HPV infection, since many cell types can be infected with HPVs in monolayer cell culture. Here, we used chemical biology approaches to reveal that cell cycle progression through mitosis is critical for HPV infection. Using infectious HPV16 particles containing the intact viral genome, G1-synchronized human keratinocytes as hosts, and early viral gene expression as a readout for infection, we learned that the recipient cell must enter M phase (mitosis for HPV infection to take place. Late M phase inhibitors had no effect on infection, whereas G1, S, G2, and early M phase cell cycle inhibitors efficiently prevented infection. We conclude that host cells need to pass through early prophase for successful onset of transcription of the HPV encapsidated genes. These findings provide one reason why HPVs initially establish infections in the basal compartment of stratified epithelia. Only this compartment of the epithelium contains cells progressing through the cell cycle, and therefore it is only in these cells that HPVs can establish their infection. By defining a major condition for cell susceptibility to HPV infection, these

Poliovirus mutants excreted by a chronically infected hypogammaglobulinemic patient establish persistent infections in human intestinal cells

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Labadie, Karine; Pelletier, Isabelle; Saulnier, Aure; Martin, Javier; Colbere-Garapin, Florence

2004-01-01

Immunodeficient patients whose gut is chronically infected by vaccine-derived poliovirus (VDPV) may excrete large amounts of virus for years. To investigate how poliovirus (PV) establishes chronic infections in the gut, we tested whether it is possible to establish persistent VDPV infections in human intestinal Caco-2 cells. Four type 3 VDPV mutants, representative of the viral evolution in the gut of a hypogammaglobulinemic patient over almost 2 years [J. Virol. 74 (2000) 3001], were used to infect both undifferentiated, dividing cells, and differentiated, polarized enterocytes. A VDPV mutant excreted 36 days postvaccination by the patient was lytic in both types of intestinal cell cultures, like the parental Sabin 3 (S3) strain. In contrast, three VDPVs excreted 136, 442, and 637 days postvaccination, established persistent infections both in undifferentiated cells and in enterocytes. Thus, viral determinants selected between day 36 and 136 conferred on VDPV mutants the capacity to infect intestinal cells persistently. The percentage of persistently VDPV-infected cultures was higher in enterocytes than in undifferentiated cells, implicating cellular determinants involved in the differentiation of enterocytes in persistent VDPV infections. The establishment of persistent infections in enterocytes was not due to poor replication of VDPVs in these cells, but was associated with reduced viral adsorption to the cell surface

Infection of endothelial cells by common human viruses.

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Friedman, H M

1989-01-01

Common human viruses were evaluated for their ability to replicate in the endothelial cells of human umbilical vein and bovine thoracic aorta in vitro. Infection occurred with most viruses. The susceptibilities of endothelial cells derived from bovine aorta, pulmonary artery, and vena cava were compared. Among the viruses studied, no differences were noted in the ability to grow in endothelial cells from these three large vessels. One virus, herpes simplex virus type 1, was evaluated for its ability to produce persistent infection of endothelial cells. Infection developed and persisted for up to 3 months. After the first week, productive infection was found in less than 1% of cells. Nevertheless, the infection markedly affected the growth and morphology of the endothelial monolayer. Infection with any of several different viruses was noted to alter endothelial cell functions, including adherence of granulocytes, production of colony-stimulating factor, and synthesis of matrix protein. In addition, herpes simplex virus type 1 induced receptors for the Fc portion of IgG and for complement component C3b. These findings indicate that common human viruses can profoundly affect the biology of the endothelium.

Early events associated with infection of Epstein-Barr virus infection of primary B-cells.

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Sabyasachi Halder

2009-09-01

Full Text Available Epstein Barr virus (EBV is closely associated with the development of a vast number of human cancers. To develop a system for monitoring early cellular and viral events associated with EBV infection a self-recombining BAC containing 172-kb of the Epstein Barr virus genome BAC-EBV designated as MD1 BAC (Chen et al., 2005, J.Virology was used to introduce an expression cassette of green fluorescent protein (GFP by homologous recombination, and the resultant BAC clone, BAC-GFP-EBV was transfected into the HEK 293T epithelial cell line. The resulting recombinant GFP EBV was induced to produce progeny virus by chemical inducer from the stable HEK 293T BAC GFP EBV cell line and the virus was used to immortalize human primary B-cell as monitored by green fluorescence and outgrowth of the primary B cells. The infection, B-cell activation and cell proliferation due to GFP EBV was monitored by the expression of the B-cell surface antigens CD5, CD10, CD19, CD23, CD39, CD40 , CD44 and the intercellular proliferation marker Ki-67 using Flow cytometry. The results show a dramatic increase in Ki-67 which continues to increase by 6-7 days post-infection. Likewise, CD40 signals showed a gradual increase, whereas CD23 signals were increased by 6-12 hours, maximally by 3 days and then decreased. Monitoring the viral gene expression pattern showed an early burst of lytic gene expression. This up-regulation of lytic gene expression prior to latent genes during early infection strongly suggests that EBV infects primary B-cell with an initial burst of lytic gene expression and the resulting progeny virus is competent for infecting new primary B-cells. This process may be critical for establishment of latency prior to cellular transformation. The newly infected primary B-cells can be further analyzed for investigating B cell activation due to EBV infection.

Effects of achilline on lipid metabolism gene expression in cell culture

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A. V. Ratkin

2016-01-01

Full Text Available Objective. Evaluation in vitro of the mechanisms of the hypolipidemic effect of sesquiterpene γ-lactone achilline in the hepatoma tissue culture (HTC.Materials and methods.The influence of sesquiterpene γ-lactone achilline and gemfibrozil (comparison drug on the viability, lipid content and expression of key genes of lipid metabolism in the hepatoma tissue culture. The lipid content was assessed by fluorescent method with the vital dye Nile Red, the cell viability was assessed using MTT assay.Results. Cultivation of of cell cultures of rat’s hepatoma cell line HTC for 48 h with achilline in a concentration of from 0.25 to 1.0 mm and gemfibrozil from 0,25 to 0,5 mm did not change cell viability compared to control. In these same concentrations of the test substance reduced the lipid content in the cells, assessed by fluorescent method with the vital dye Nile Red. To study the mechanism of hypolipidemicaction of achillinedetermined the expression of key genes of lipid metabolism in cell culture lines HTC. The possible mechanism of hypolipidemic action of achilline can be attributed to the increased transport and oxidation of long-chain fatty acids in mitochondria, as evidenced by the increase in the gene expression of carnitine-palmitoyltransferase 2 (Cpt2. The decrease in cholesterol level may be due to increased synthesis of bile acids from cholesterol, due to increased gene expression of 7-alphahydroxylase (Cyp7a1. Conclusion. In cell cultures of rat’s hepatoma cell line HTC sesquiterpene γ-lactone achilline reduces the accumulation of lipids in cells, as evidenced by the decrease in the fluorescence of Nile Red, increased gene expression of the carnitine-palmitoyltransferase 2 (Cpt2 gene and 7-alpha-hydroxylase (Cyp7a1.

Human innate lymphoid cells (ILCs) in filarial infections.

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Bonne-Année, S; Nutman, T B

2018-02-01

Filarial infections are characteristically chronic and can cause debilitating diseases governed by parasite-induced innate and adaptive immune responses. Filarial parasites traverse or establish niches in the skin (migrating infective larvae), in nonmucosal tissues (adult parasite niche) and in the blood or skin (circulating microfilariae) where they intersect with the host immune response. While several studies have demonstrated that filarial parasites and their antigens can modulate myeloid cells (monocyte, macrophage and dendritic cell subsets), T- and B-lymphocytes and skin resident cell populations, the role of innate lymphoid cells during filarial infections has only recently emerged. Despite the identification and characterization of innate lymphoid cells (ILCs) in murine helminth infections, little is actually known about the role of human ILCs during parasitic infections. The focus of this review will be to highlight the composition of ILCs in the skin, lymphatics and blood; where the host-parasite interaction is well-defined and to examine the role of ILCs during filarial infections. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

Semen CD4+ T Cells and Macrophages Are Productively Infected at All Stages of SIV infection in Macaques

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Bernard-Stoecklin, Sibylle; Gommet, Céline; Corneau, Aurélien B.; Guenounou, Sabrina; Torres, Claire; Dejucq-Rainsford, Nathalie; Cosma, Antonio; Dereuddre-Bosquet, Nathalie; Le Grand, Roger

2013-01-01

The mucosal events of HIV transmission have been extensively studied, but the role of infected cells present in the genital and rectal secretions, and in the semen, in particular, remains a matter of debate. As a prerequisite to a thorough in vivo investigation of the early transmission events through infected cells, we characterized in detail by multi-parameter flow cytometry the changes in macaque seminal leukocytes during SIVmac251 infection, focusing on T cells, macrophages and dendritic cells. Using immunocytofluorescence targeting SIV proteins and real-time quantitative PCR targeting SIV DNA, we investigated the nature of the infected cells on sorted semen leukocytes from macaques at different stages of infection. Finally, we cocultured semen CD4+ T cells and macrophages with a cell line permissive to SIV infection to assess their infectivity in vitro. We found that primary infection induced strong local inflammation, which was associated with an increase in the number of leukocytes in semen, both factors having the potential to favor cell-associated virus transmission. Semen CD4+ T cells and macrophages were productively infected at all stages of infection and were infectious in vitro. Lymphocytes had a mucosal phenotype and expressed activation (CD69 & HLA-DR) and migration (CCR5, CXCR4, LFA-1) markers. CD69 expression was increased in semen T cells by SIV infection, at all stages of infection. Macrophages predominated at all stages and expressed CD4, CCR5, MAC-1 and LFA-1. Altogether, we demonstrated that semen contains the two major SIV-target cells (CD4+ T cells and macrophages). Both cell types can be productively infected at all stages of SIV infection and are endowed with markers that may facilitate transmission of infection during sexual exposure. PMID:24348253

Infectious mononucleosis accompanied by clonal proliferation of EBV-infected cells and infection of CD8-positive cells.

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Arai, Ayako; Yamaguchi, Takeshi; Komatsu, Honami; Imadome, Ken-Ichi; Kurata, Morito; Nagata, Kaoru; Miura, Osamu

2014-01-01

A 22-year-old male was admitted for a sustained fever of 2 months, lymphadenopathy, and liver dysfunction. Anti-VCA-IgM antibody was positive, with elevated Epstein-Barr virus (EBV)-DNA load in the peripheral blood. Liver biopsy revealed infiltration of CD8-positive and EBV-positive cells. Most peripheral blood mononuclear cells (PBMCs) were also positive for CD8, and showed detectable levels of EBV-DNA. Monoclonal proliferation of EBV-infected cells was detected in the PBMCs by Southern blotting for EBV-terminal repeat (EBV-TR). Although EBV-positive T-cell lymphoproliferative disease (EBV-T-LPD) was suspected, the symptoms spontaneously resolved within 12 months. Anti-VCA-IgM antibody and the clonal band of EBV-TR were negative 1 year after the onset, while anti-EBNA antibody was positive. The final diagnosis was thus confirmed as infectious mononucleosis (IM). Our results indicate that EBV-infected CD8-positive cells and clonal proliferation of EBV-infected cells may be temporally detected in IM. EBV-T-LPDs should be carefully excluded in such cases.

Apoptosis and changes in glucose transport early after treatment of Morris hepatoma with gemcitabine

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Haberkorn, U.; Bellemann, M.E.; Brix, G.; Kamencic, H.; Traut, U.; Kinscherf, R.; Doll, J.; Blatter, J.

2001-01-01

Apoptosis has been described as an energy-consuming process. This combined in vivo/in vitro study investigated the effects of the antineoplastic agent gemcitabine on tumour metabolism and on the induction of apoptosis. Dynamic positron emission tomography (PET) measurements of fluorine-18 fluorodeoxyglucose (FDG) uptake were done in rats bearing Morris hepatoma prior to and after therapy with 90 mg gemcitabine/kg b.w. Furthermore, thymidine (TdR) incorporation into the DNA of these tumours was determined. In vitro measurements of FDG and TdR uptake were performed immediately and 24 h after the end of gemcitabine treatment, and the amount of apoptotic cells was determined using the TUNEL reaction. In vivo an increase in FDG transport and phosphorylation occurred early after gemcitabine treatment, although TdR incorporation into the DNA of the tumours declined. In vitro, an enhanced glucose transport, an increase in TdR uptake in the cytoplasm and a decrease in TdR incorporation in the nucleic acid fraction early after treatment occurred. Inhibition of glucose transport caused an increase in the amount of apoptotic cells. The increase in glucose uptake and TdR metabolism early after therapy is interpreted as a stress reaction of the tumour cells, protecting the cells from apoptosis during this early period after exposure to cytotoxic drugs like gemcitabine. (orig.)

Apoptosis and changes in glucose transport early after treatment of Morris hepatoma with gemcitabine

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Haberkorn, U. [Heidelberg Univ. (Germany). Abt. fuer Klinische Nuklearmedizin; Clinical Cooperation Unit Nuclear Medicine, German Cancer Research Center, Heidelberg (Germany); Bellemann, M.E. [Department of Biomedical Engineering, University of Applied Sciences, Jena (Germany); Brix, G. [Department of Medical Radiation Hygiene, Federal Office for Radiation Protection, Neuherberg (Germany); Kamencic, H.; Traut, U.; Kinscherf, R. [Heidelberg Univ. (Germany). Inst. fuer Anatomie und Zellbiologie; Morr, I.; Altmann, A. [Clinical Cooperation Unit Nuclear Medicine, German Cancer Research Center, Heidelberg (Germany); Doll, J. [Dept. of Medical Physics, German Cancer Research Center, Heidelberg (Germany); Blatter, J. [Lilly GmbH Germany, Bad Homburg (Germany)

2001-04-01

Apoptosis has been described as an energy-consuming process. This combined in vivo/in vitro study investigated the effects of the antineoplastic agent gemcitabine on tumour metabolism and on the induction of apoptosis. Dynamic positron emission tomography (PET) measurements of fluorine-18 fluorodeoxyglucose (FDG) uptake were done in rats bearing Morris hepatoma prior to and after therapy with 90 mg gemcitabine/kg b.w. Furthermore, thymidine (TdR) incorporation into the DNA of these tumours was determined. In vitro measurements of FDG and TdR uptake were performed immediately and 24 h after the end of gemcitabine treatment, and the amount of apoptotic cells was determined using the TUNEL reaction. In vivo an increase in FDG transport and phosphorylation occurred early after gemcitabine treatment, although TdR incorporation into the DNA of the tumours declined. In vitro, an enhanced glucose transport, an increase in TdR uptake in the cytoplasm and a decrease in TdR incorporation in the nucleic acid fraction early after treatment occurred. Inhibition of glucose transport caused an increase in the amount of apoptotic cells. The increase in glucose uptake and TdR metabolism early after therapy is interpreted as a stress reaction of the tumour cells, protecting the cells from apoptosis during this early period after exposure to cytotoxic drugs like gemcitabine. (orig.)

Susceptibility of different leukocyte cell types to Vaccinia virus infection

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Sánchez-Puig Juana M

2004-11-01

Full Text Available Abstract Background Vaccinia virus, the prototype member of the family Poxviridae, was used extensively in the past as the Smallpox vaccine, and is currently considered as a candidate vector for new recombinant vaccines. Vaccinia virus has a wide host range, and is known to infect cultures of a variety of cell lines of mammalian origin. However, little is known about the virus tropism in human leukocyte populations. We report here that various cell types within leukocyte populations have widely different susceptibility to infection with vaccinia virus. Results We have investigated the ability of vaccinia virus to infect human PBLs by using virus recombinants expressing green fluorescent protein (GFP, and monoclonal antibodies specific for PBL subpopulations. Flow cytometry allowed the identification of infected cells within the PBL mixture 1–5 hours after infection. Antibody labeling revealed that different cell populations had very different infection rates. Monocytes showed the highest percentage of infected cells, followed by B lymphocytes and NK cells. In contrast to those cell types, the rate of infection of T lymphocytes was low. Comparison of vaccinia virus strains WR and MVA showed that both strains infected efficiently the monocyte population, although producing different expression levels. Our results suggest that MVA was less efficient than WR in infecting NK cells and B lymphocytes. Overall, both WR and MVA consistently showed a strong preference for the infection of non-T cells. Conclusions When infecting fresh human PBL preparations, vaccinia virus showed a strong bias towards the infection of monocytes, followed by B lymphocytes and NK cells. In contrast, very poor infection of T lymphocytes was detected. These finding may have important implications both in our understanding of poxvirus pathogenesis and in the development of improved smallpox vaccines.

Dendritic cells during Epstein Barr virus infection

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Christian eMunz

2014-06-01

Full Text Available Epstein Barr virus (EBV causes persistent infection in more than 90% of the human adult population and is associated with 2% of all tumors in humans. This -herpesvirus infects primarily human B and epithelial cells, but has been reported to be sensed by dendritic cells (DCs during primary infection. These activated DCs are thought to contribute to innate restriction of EBV infection and initiate EBV specific adaptive immune responses via cross-priming. The respective evidence and their potential importance for EBV specific vaccine development will be discussed in this review.

Immune regulation in Chandipura virus infection: characterization of CD4+ T regulatory cells from infected mice

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Shahir Prajakta

2011-05-01

Full Text Available Abstract Back ground Chandipura virus produces acute infection in mice. During infection drastic reduction of CD4+, CD8+ and CD19 + cell was noticed. Depletion of lymphocytes also noticed in spleen. The reduction may be due to the regulatory mechanism of immune system to prevent the bystander host tissue injury. There are several mechanisms like generation of regulatory cells, activation induced cell death (ACID etc were indicated to control the activation and maintain cellular homeostasis. Role of regulatory cells in homeostasis has been described in several viral diseases. This study was undertaken to characterize CD4+T regulatory cells from the infected mice. Method In this study we purified the CD4+ T cells from Chandipura virus infected susceptible Balb/c mice. CD4+ T regulatory cells were identified by expression of cell surface markers CD25, CD127 and CTLA-4 and intracellular markers Foxp3, IL-10 and TGF-beta. Antigen specificity and ability to suppress the proliferation of other lymphocytes were studied in vitro by purified CD4+CD25+T regulatory cells from infected mice. The proliferation was calculated by proliferation module of Flow Jo software. Expression of death receptors on regulatory cells were studied by flowcytometer. Results The CD4+ T cells isolated from infected mice expressed characteristic markers of regulatory phenotype at all post infective hours tested. The CD4+ T regulatory cells were proliferated when stimulated with Chandipura virus antigen. The regulatory cells did not suppress the proliferation of splenocytes stimulated with anti CD3 antibody when co cultured with them. Interesting observation was, while purification of CD4+ T cells by negative selection, the population of cells negative for CD4 also co purified along with CD4+ T cell. Flow cytometry analysis and light microscopy revealed that CD4 negative cells were of different size and shape (atypical compared to the normal lymphocytes. Greater percentage of

MAIT cells are activated in acute Dengue virus infection and after in vitro Zika virus infection.

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Dominic Paquin-Proulx

2018-01-01

Full Text Available Dengue virus (DENV and Zika virus (ZIKV are members of the Flaviviridae and are predominantly transmitted via mosquito bites. Both viruses are responsible for a growing number of infections in tropical and subtropical regions. DENV infection can cause lethargy with severe morbidity and dengue shock syndrome leading to death in some cases. ZIKV is now linked with Guillain-Barré syndrome and fetal malformations including microcephaly and developmental disorders (congenital Zika syndrome. The protective and pathogenic roles played by the immune response in these infections is unknown. Mucosal-associated invariant T (MAIT cells are a population of innate T cells with potent anti-bacterial activity. MAIT cells have also been postulated to play a role in the immune response to viral infections. In this study, we evaluated MAIT cell frequency, phenotype, and function in samples from subjects with acute and convalescent DENV infection. We found that in acute DENV infection, MAIT cells had elevated co-expression of the activation markers CD38 and HLA-DR and had a poor IFNγ response following bacterial stimulation. Furthermore, we found that MAIT cells can produce IFNγ in response to in vitro infection with ZIKV. This MAIT cell response was independent of MR1, but dependent on IL-12 and IL-18. Our results suggest that MAIT cells may play an important role in the immune response to Flavivirus infections.

Transient Oral Human Cytomegalovirus Infections Indicate Inefficient Viral Spread from Very Few Initially Infected Cells.

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Mayer, Bryan T; Krantz, Elizabeth M; Swan, David; Ferrenberg, James; Simmons, Karen; Selke, Stacy; Huang, Meei-Li; Casper, Corey; Corey, Lawrence; Wald, Anna; Schiffer, Joshua T; Gantt, Soren

2017-06-15

Cytomegalovirus (CMV) is acquired by the oral route in children, and primary infection is associated with abundant mucosal replication, as well as the establishment of latency in myeloid cells that results in lifelong infection. The efficiency of primary CMV infection in humans following oral exposure, however, is unknown. We consistently detected self-limited, low-level oral CMV shedding events, which we termed transient CMV infections, in a prospective birth cohort of 30 highly exposed CMV-uninfected infants. We estimated the likelihood of transient oral CMV infections by comparing their observed frequency to that of established primary infections, characterized by persistent high-level shedding, viremia, and seroconversion. We developed mathematical models of viral dynamics upon initial oral CMV infection and validated them using clinical shedding data. Transient infections comprised 76 to 88% of oral CMV shedding events. For this high percentage of transient infections to occur, we identified two mathematical prerequisites: a very small number of initially infected oral cells (1 to 4) and low viral infectivity (<1.5 new cells infected/cell). These observations indicate that oral CMV infection in infants typically begins with a single virus that spreads inefficiently to neighboring cells. Thus, although the incidence of CMV infection is high during infancy, our data provide a mechanistic framework to explain why multiple CMV exposures are typically required before infection is successfully established. These findings imply that a sufficiently primed immune response could prevent CMV from establishing latent infection in humans and support the achievability of a prophylactic CMV vaccine. IMPORTANCE CMV infects the majority of the world's population and is a major cause of birth defects. Developing a vaccine to prevent CMV infection would be extremely valuable but would be facilitated by a better understanding of how natural human CMV infection is acquired. We

Role of Bruton's tyrosine kinase inhibitors in HIV-1-infected cells.

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Guendel, Irene; Iordanskiy, Sergey; Sampey, Gavin C; Van Duyne, Rachel; Calvert, Valerie; Petricoin, Emanuel; Saifuddin, Mohammed; Kehn-Hall, Kylene; Kashanchi, Fatah

2015-06-01

Many cellular cofactors have been documented to be critical for various stages of viral replication. Using high-throughput proteomic assays, we have previously identified Bruton's tyrosine kinase (BTK) as a host protein that was uniquely upregulated in the plasma membrane of human immunodeficiency virus (HIV-1)-infected T cells. Here, we have further characterized the BTK expression in HIV-1 infection and show that this cellular factor is specifically expressed in infected myeloid cells. Significant upregulation of the phosphorylated form of BTK was observed in infected cells. Using size exclusion chromatography, we found BTK to be virtually absent in the uninfected U937 cells; however, new BTK protein complexes were identified and distributed in both high molecular weight (∼600 kDa) and a small molecular weight complex (∼60-120 kDa) in the infected U1 cells. BTK levels were highest in cells either chronically expressing virus or induced/infected myeloid cells and that BTK translocated to the membrane following induction of the infected cells. BTK knockdown in HIV-1-infected cells using small interfering RNA (siRNA) resulted in selective death of infected, but not uninfected, cells. Using BTK-specific antibody and small-molecule inhibitors including LFM-A13 and a FDA-approved compound, ibrutinib (PCI-32765), we have found that HIV-1-infected cells are sensitive to apoptotic cell death and result in a decrease in virus production. Overall, our data suggests that HIV-1-infected cells are sensitive to treatments targeting BTK expressed in infected cells.

Retroviral infection of non-dividing cells: Old and new perspectives

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Yamashita, Masahiro; Emerman, Michael

2006-01-01

The dependence of retroviral replication on cell proliferation was described as early as 1958, although different classes of retroviruses are able to infect non-dividing cells with different efficiencies. For example, the human immunodeficiency virus (HIV) and other lentiviruses infect most non-dividing cells nearly as well as dividing cells, while the gammaretroviruses such as the murine leukemia virus (MLV) cannot infect non-dividing cells, and other retroviruses have intermediate phenotypes. One exception to the ability of HIV to infect non-dividing cells involves resting CD4+ T cells in vitro where there are multiple restrictions. However, recent data show that there is massive infection of non-activated CD4+ T cell during acute infection which suggests that the situation is different in vivo. Finally, much work trying to explain the difference between HIV and MLV in non-dividing cells has focused on describing the ability of HIV to enter the nucleus during interphase. However, we suggest that events in the viral lifecycle other than nuclear import may be more important in determining the ability of a given retrovirus to infect non-dividing cells

T Follicular Helper Cells and B Cell Dysfunction in Aging and HIV-1 Infection.

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Pallikkuth, Suresh; de Armas, Lesley; Rinaldi, Stefano; Pahwa, Savita

2017-01-01

T follicular helper (Tfh) cells are a subset of CD4 T cells that provide critical signals to antigen-primed B cells in germinal centers to undergo proliferation, isotype switching, and somatic hypermutation to generate long-lived plasma cells and memory B cells during an immune response. The quantity and quality of Tfh cells therefore must be tightly controlled to prevent immune dysfunction in the form of autoimmunity and, on the other hand, immune deficiency. Both Tfh and B cell perturbations appear during HIV infection resulting in impaired antibody responses to vaccines such as seasonal trivalent influenza vaccine, also seen in biologic aging. Although many of the HIV-associated defects improve with antiretroviral therapy (ART), excess immune activation and antigen-specific B and T cell responses including Tfh function are still impaired in virologically controlled HIV-infected persons on ART. Interestingly, HIV infected individuals experience increased risk of age-associated pathologies. This review will discuss Tfh and B cell dysfunction in HIV infection and highlight the impact of chronic HIV infection and aging on Tfh-B cell interactions.

Lysis of endogenously infected CD4+ T cell blasts by rIL-2 activated autologous natural killer cells from HIV-infected viremic individuals.

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Manuela Fogli

2008-07-01

Full Text Available Understanding the cellular mechanisms that ensure an appropriate innate immune response against viral pathogens is an important challenge of biomedical research. In vitro studies have shown that natural killer (NK cells purified from healthy donors can kill heterologous cell lines or autologous CD4+ T cell blasts exogenously infected with several strains of HIV-1. However, it is not known whether the deleterious effects of high HIV-1 viremia interferes with the NK cell-mediated cytolysis of autologous, endogenously HIV-1-infected CD4+ T cells. Here, we stimulate primary CD4+ T cells, purified ex vivo from HIV-1-infected viremic patients, with PHA and rIL2 (with or without rIL-7. This experimental procedure allows for the significant expansion and isolation of endogenously infected CD4+ T cell blasts detected by intracellular staining of p24 HIV-1 core antigen. We show that, subsequent to the selective down-modulation of MHC class-I (MHC-I molecules, HIV-1-infected p24(pos blasts become partially susceptible to lysis by rIL-2-activated NK cells, while uninfected p24(neg blasts are spared from killing. This NK cell-mediated killing occurs mainly through the NKG2D activation pathway. However, the degree of NK cell cytolytic activity against autologous, endogenously HIV-1-infected CD4+ T cell blasts that down-modulate HLA-A and -B alleles and against heterologous MHC-I(neg cell lines is particularly low. This phenomenon is associated with the defective surface expression and engagement of natural cytotoxicity receptors (NCRs and with the high frequency of the anergic CD56(neg/CD16(pos subsets of highly dysfunctional NK cells from HIV-1-infected viremic patients. Collectively, our data demonstrate that the chronic viral replication of HIV-1 in infected individuals results in several phenotypic and functional aberrancies that interfere with the NK cell-mediated killing of autologous p24(pos blasts derived from primary T cells.

Networked T cell death following macrophage infection by Mycobacterium tuberculosis.

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Stephen H-F Macdonald

Full Text Available BACKGROUND: Depletion of T cells following infection by Mycobacterium tuberculosis (Mtb impairs disease resolution, and interferes with clinical test performance that relies on cell-mediated immunity. A number of mechanisms contribute to this T cell suppression, such as activation-induced death and trafficking of T cells out of the peripheral circulation and into the diseased lungs. The extent to which Mtb infection of human macrophages affects T cell viability however, is not well characterised. METHODOLOGY/PRINCIPAL FINDINGS: We found that lymphopenia (<1.5 × 10(9 cells/l was prevalent among culture-positive tuberculosis patients, and lymphocyte counts significantly improved post-therapy. We previously reported that Mtb-infected human macrophages resulted in death of infected and uninfected bystander macrophages. In the current study, we sought to examine the influence of infected human alveolar macrophages on T cells. We infected primary human alveolar macrophages (the primary host cell for Mtb or PMA-differentiated THP-1 cells with Mtb H37Ra, then prepared cell-free supernatants. The supernatants of Mtb-infected macrophages caused dose-dependent, caspase-dependent, T cell apoptosis. This toxic effect of infected macrophage secreted factors did not require TNF-α or Fas. The supernatant cytotoxic signal(s were heat-labile and greater than 50 kDa in molecular size. Although ESAT-6 was toxic to T cells, other Mtb-secreted factors tested did not influence T cell viability; nor did macrophage-free Mtb bacilli or broth from Mtb cultures. Furthermore, supernatants from Mycobacterium bovis Bacille de Calmette et Guerin (BCG- infected macrophages also elicited T cell death suggesting that ESAT-6 itself, although cytotoxic, was not the principal mediator of T cell death in our system. CONCLUSIONS: Mtb-Infected macrophages secrete heat-labile factors that are toxic to T cells, and may contribute to the immunosuppression seen in tuberculosis as well as

Diagnostic application of Ga-67 scintigraphy in primary lung cancer, hepatoma and abdominal abscess. On surgical operation

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Oshiumi, Yoshihiko (National Central Hospital of Fukuoka (Japan))

1983-05-01

It is difficult to detect tumor lesions by /sup 67/Ga-scintigraphy, though it is named as a tumor scintigraphy. Recently, /sup 67/Ga-scintigraphy is well used in the detection of inflammatic lesions. This paper is to discuss the detectability of lesions and diagnostic limitation on operability by /sup 67/Ga-scintigraphy to primary lung cancer, minute hepatoma and abdominal abscess. Primary lung cancer: In detecting metastatic hilar lymph nodes, the sensitivity is 54%, and the specificity is 78%. In detecting metastatic mediastinal lymph nodes, the sensitivity is 32% and the specificity is 89%. Minute hepatoma : The detectability depended on the size of the lesion. It is hard to detect lesions with under 2 cm by /sup 67/Ga-scintigraphy. Abdominal abscess : The sensitivity is 88%, and the specificity is 92%.

Study on radiation regulation of hypoxia inducible factor-1α expression and its correlation with hepatoma radiosensitivity

International Nuclear Information System (INIS)

Jin Wensen; Kong Zhaolu; Shen Zhifen; Tong Shungao; Ji Huajun; Jin Yizun

2008-01-01

Objective: To study the regulation of hypoxia inducible factor-1α (HIF-1α) expression in hepatoma cells after irradiation and the expression of HIF-1α effect on the radiosensitivity of heptoma cells. Methods: HepG2 cells were pretreated by Cobalt chloride (COCl 2 ), a chemical hypoxia agent, to induce and stabilize the expression of HIF-1α, and then exposed to different γ-irradiation doses. Clonogenic assay was used to evaluate HepG2 cell survival fraction (SF) after irradiation under normoxia and chemical hypoxia. Reverse transcriptase polymerase chain reaction (RT-PCR) and immunoblot assay (Western blot) were utilized to detect the changes of intracellular HIF-1α on the level of transcripation and translation. Results: Cell survival level was elevated by chemical hypoxia and there was a statistical difference between chemical hypoxic group and normoxic group. The ratios of SF(SF co /SF o 2 )on two different conditions were increased with irradiation doses. Meanwhile, the irradiation induced up-regulation of HIF-1α in dose-dependent manner. The expression of HIF-1α was correlated with HepG2 cell survival level to some extent. Conclusions: Irradiation could up-regulate the level of HIF-1α expression in HepG2 cells under chemical hypoxic condition. The cells survival level might be influenced by the changes in HIF-1α expression. (authors)

Mycoplasma agalactiae Induces Cytopathic Effects in Infected Cells Cultured In Vitro.

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Shrilakshmi Hegde

Full Text Available Mycoplasma agalactiae is the etiological agent of the contagious agalactia syndrome in sheep and goats and causes significant economic losses worldwide. Yet the mechanism of pathogenesis is largely unknown. Even whole-genome sequence analysis of its pathogenic type strain did not lead to any conclusions regarding its virulence or pathogenicity factors. Although inflammation and tissue destruction at the local site of M. agalactiae infection are largely considered as effects of the host immune response, the direct effect of the agent on host cells is not completely understood. The aim of this study was to investigate the effect of M. agalactiae infection on the quality and viability of host cells in vitro. Changes in cell morphology including cell elongation, cytoplasm shrinkage and membrane blebbing were observed in infected HeLa cells. Chromatin condensation and increased caspase-3 cleavage in infected HeLa cells 48 h after infection suggests an apoptosis-like phenomenon in M. agalactiae-infected cells. In compliance with these results, decreased viability and cell lysis of M. agalactiae-infected HeLa cells was also observed. Measurement of the amount of LDH released after M. agalactiae infection revealed a time- and dose-dependent increase in HeLa cell lysis. A significant decrease in LDH released after gentamicin treatment of infected cells confirmed the major role of cytadherent M. agalactiae in inducing host cell lysis. This is the first study illustrating M. agalactiae's induction of cytopathic effects in infected HeLa cells. Further detailed investigation of infected host tissue for apoptotic markers might demonstrate the association between M. agalactiae-induced host cell lysis and the tissue destruction observed during M. agalactiae natural infection.

Quantitative comparison of HTLV-1 and HIV-1 cell-to-cell infection with new replication dependent vectors.

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Dmitriy Mazurov

2010-02-01

Full Text Available We have developed an efficient method to quantify cell-to-cell infection with single-cycle, replication dependent reporter vectors. This system was used to examine the mechanisms of infection with HTLV-1 and HIV-1 vectors in lymphocyte cell lines. Effector cells transfected with reporter vector, packaging vector, and Env expression plasmid produced virus-like particles that transduced reporter gene activity into cocultured target cells with zero background. Reporter gene expression was detected exclusively in target cells and required an Env-expression plasmid and a viral packaging vector, which provided essential structural and enzymatic proteins for virus replication. Cell-cell fusion did not contribute to infection, as reporter protein was rarely detected in syncytia. Coculture of transfected Jurkat T cells and target Raji/CD4 B cells enhanced HIV-1 infection two fold and HTLV-1 infection ten thousand fold in comparison with cell-free infection of Raji/CD4 cells. Agents that interfere with actin and tubulin polymerization strongly inhibited HTLV-1 and modestly decreased HIV-1 cell-to-cell infection, an indication that cytoskeletal remodeling was more important for HTLV-1 transmission. Time course studies showed that HTLV-1 transmission occurred very rapidly after cell mixing, whereas slower kinetics of HIV-1 coculture infection implies a different mechanism of infectious transmission. HTLV-1 Tax was demonstrated to play an important role in altering cell-cell interactions that enhance virus infection and replication. Interestingly, superantigen-induced synapses between Jurkat cells and Raji/CD4 cells did not enhance infection for either HTLV-1 or HIV-1. In general, the dependence on cell-to-cell infection was determined by the virus, the effector and target cell types, and by the nature of the cell-cell interaction.

Autoreactive T Cells and Chronic Fungal Infection Drive Esophageal Carcinogenesis

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Zhu, Feng; Willette-Brown, Jami; Song, Na-Young; Lomada, Dakshayani; Song, Yongmei; Xue, Liyan; Gray, Zane; Zhao, Zitong; Davis, Sean R.; Sun, Zhonghe; Zhang, Peilin; Wu, Xiaolin; Zhan, Qimin; Richie, Ellen R.; Hu, Yinling

2018-01-01

SUMMARY Humans with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a T cell–driven autoimmune disease caused by impaired central tolerance, are susceptible to developing chronic fungal infection and esophageal squamous cell carcinoma (ESCC). However, the relationship between autoreactive T cells and chronic fungal infection in ESCC development remains unclear. We find that kinase-dead Ikkα knockin mice develop phenotypes reminiscent of APECED, including impaired central tolerance, autoreactive T cells, chronic fungal infection, and ESCCs expressing specific human ESCC markers. Using this model, we investigated the potential link between ESCC and fungal infection. Autoreactive CD4 T cells permit fungal infection and incite tissue injury and inflammation. Antifungal treatment or depletion of autoreactive CD4 T cells rescues, whereas oral fungal administration promotes, ESCC development. Inhibition of inflammation or EGFR activity decreases fungal burden. Importantly, fungal infection is highly associated with ESCCs in non-autoimmune human patients. Therefore, autoreactive T cells and chronic fungal infection, fostered by inflammation and epithelial injury, promote ESCC development. PMID:28407484

Regulation of NKT Cell Localization in Homeostasis and Infection

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Slauenwhite, Drew; Johnston, Brent

2015-01-01

Natural killer T (NKT) cells are a specialized subset of T lymphocytes that regulate immune responses in the context of autoimmunity, cancer, and microbial infection. Lipid antigens derived from bacteria, parasites, and fungi can be presented by CD1d molecules and recognized by the canonical T cell receptors on NKT cells. Alternatively, NKT cells can be activated through recognition of self-lipids and/or pro-inflammatory cytokines generated during infection. Unlike conventional T cells, only a small subset of NKT cells traffic through the lymph nodes under homeostatic conditions, with the largest NKT cell populations localizing to the liver, lungs, spleen, and bone marrow. This is thought to be mediated by differences in chemokine receptor expression profiles. However, the impact of infection on the tissue localization and function of NKT remains largely unstudied. This review focuses on the mechanisms mediating the establishment of peripheral NKT cell populations during homeostasis and how tissue localization of NKT cells is affected during infection. PMID:26074921

Regulation of NKT Cell Localization in Homeostasis and Infection.

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Slauenwhite, Drew; Johnston, Brent

2015-01-01

Natural killer T (NKT) cells are a specialized subset of T lymphocytes that regulate immune responses in the context of autoimmunity, cancer, and microbial infection. Lipid antigens derived from bacteria, parasites, and fungi can be presented by CD1d molecules and recognized by the canonical T cell receptors on NKT cells. Alternatively, NKT cells can be activated through recognition of self-lipids and/or pro-inflammatory cytokines generated during infection. Unlike conventional T cells, only a small subset of NKT cells traffic through the lymph nodes under homeostatic conditions, with the largest NKT cell populations localizing to the liver, lungs, spleen, and bone marrow. This is thought to be mediated by differences in chemokine receptor expression profiles. However, the impact of infection on the tissue localization and function of NKT remains largely unstudied. This review focuses on the mechanisms mediating the establishment of peripheral NKT cell populations during homeostasis and how tissue localization of NKT cells is affected during infection.

Differential pulmonic NK and NKT cell responses in Schistosoma japonicum-infected mice.

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Cha, Hefei; Qin, Wenjuan; Yang, Quan; Xie, Hongyan; Qu, Jiale; Wang, Mei; Chen, Daixiong; Wang, Fang; Dong, Nuo; Chen, Longhua; Huang, Jun

2017-02-01

Natural killer cells (NK cells) and natural killer T cells (NKT cells) play a role in anti-infection, anti-tumor, transplantation immunity, and autoimmune regulation. However, the role of NK and NKT cells during Schistosoma japonicum (S. japonicum) infection has not been widely reported, especially regarding lung infections. The aim of this study was to research the NK and NKT cell response to S. japonicum infection in the lungs of mice. Using immunofluorescent histological analysis, NK and NKT cells were found near pulmonary granulomas. Moreover, flow cytometry revealed that the percentage and number of pulmonic NK cells in S. japonicum-infected mice were significantly increased (P cell number of NKT cells were decreased compared to those of normal mice (P NKT cells was increased after infection (P NKT cells (P cells (P NKT cells significantly increased (P NKT cells (P NKT cell activation during S. japonicum infection.

NK cell-like behavior of Valpha14i NK T cells during MCMV infection.

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Johnna D Wesley

2008-07-01

Full Text Available Immunity to the murine cytomegalovirus (MCMV is critically dependent on the innate response for initial containment of viral replication, resolution of active infection, and proper induction of the adaptive phase of the anti-viral response. In contrast to NK cells, the Valpha14 invariant natural killer T cell response to MCMV has not been examined. We found that Valpha14i NK T cells become activated and produce significant levels of IFN-gamma, but do not proliferate or produce IL-4 following MCMV infection. In vivo treatment with an anti-CD1d mAb and adoptive transfer of Valpha14i NK T cells into MCMV-infected CD1d(-/- mice demonstrate that CD1d is dispensable for Valpha14i NK T cell activation. In contrast, both IFN-alpha/beta and IL-12 are required for optimal activation. Valpha14i NK T cell-derived IFN-gamma is partially dependent on IFN-alpha/beta but highly dependent on IL-12. Valpha14i NK T cells contribute to the immune response to MCMV and amplify NK cell-derived IFN-gamma. Importantly, mortality is increased in CD1d(-/- mice in response to high dose MCMV infection when compared to heterozygote littermate controls. Collectively, these findings illustrate the plasticity of Valpha14i NK T cells that act as effector T cells during bacterial infection, but have NK cell-like behavior during the innate immune response to MCMV infection.

Defective Natural Killer cell antiviral capacity in paediatric HBV infection

DEFF Research Database (Denmark)

Heiberg, Ida Louise; Laura J., Pallett; Winther, Thilde Nordmann

2015-01-01

Natural Killer (NK) cells exhibit dysregulated effector function in adult chronic HBV infection (CHB), which may contribute to virus persistence. The role of NK cells in children infected perinatally with HBV is less studied. Access to a unique cohort enabled the cross-sectional evaluation of NK...... cell frequency, phenotype and function in HBV-infected children relative to uninfected children. We observed a selective defect in NK cell IFN-γ production, with conserved cytolytic function, mirroring the functional dichotomy observed in adult infection. Reduced expression of NKp30 on NK cells...

Invariant NKT cells: regulation and function during viral infection.

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Jennifer A Juno

Full Text Available Natural killer T cells (NKT cells represent a subset of T lymphocytes that express natural killer (NK cell surface markers. A subset of NKT cells, termed invariant NKT cells (iNKT, express a highly restricted T cell receptor (TCR and respond to CD1d-restricted lipid ligands. iNKT cells are now appreciated to play an important role in linking innate and adaptive immune responses and have been implicated in infectious disease, allergy, asthma, autoimmunity, and tumor surveillance. Advances in iNKT identification and purification have allowed for the detailed study of iNKT activity in both humans and mice during a variety of chronic and acute infections. Comparison of iNKT function between non-pathogenic simian immunodeficiency virus (SIV infection models and chronic HIV-infected patients implies a role for iNKT activity in controlling immune activation. In vitro studies of influenza infection have revealed novel effector functions of iNKT cells including IL-22 production and modulation of myeloid-derived suppressor cells, but ex vivo characterization of human iNKT cells during influenza infection are lacking. Similarly, as recent evidence suggests iNKT involvement in dengue virus pathogenesis, iNKT cells may modulate responses to a number of emerging pathogens. This Review will summarize current knowledge of iNKT involvement in responses to viral infections in both human and mouse models and will identify critical gaps in knowledge and opportunities for future study. We will also highlight recent efforts to harness iNKT ligands as vaccine adjuvants capable of improving vaccination-induced cellular immune responses.

Human induced pluripotent stem cell-derived models to investigate human cytomegalovirus infection in neural cells.

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Leonardo D'Aiuto

Full Text Available Human cytomegalovirus (HCMV infection is one of the leading prenatal causes of congenital mental retardation and deformities world-wide. Access to cultured human neuronal lineages, necessary to understand the species specific pathogenic effects of HCMV, has been limited by difficulties in sustaining primary human neuronal cultures. Human induced pluripotent stem (iPS cells now provide an opportunity for such research. We derived iPS cells from human adult fibroblasts and induced neural lineages to investigate their susceptibility to infection with HCMV strain Ad169. Analysis of iPS cells, iPS-derived neural stem cells (NSCs, neural progenitor cells (NPCs and neurons suggests that (i iPS cells are not permissive to HCMV infection, i.e., they do not permit a full viral replication cycle; (ii Neural stem cells have impaired differentiation when infected by HCMV; (iii NPCs are fully permissive for HCMV infection; altered expression of genes related to neural metabolism or neuronal differentiation is also observed; (iv most iPS-derived neurons are not permissive to HCMV infection; and (v infected neurons have impaired calcium influx in response to glutamate.

Dynamics of human T-cell lymphotropic virus I (HTLV-I) infection of CD4+ T-cells.

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Katri, Patricia; Ruan, Shigui

2004-11-01

Stilianakis and Seydel (Bull. Math. Biol., 1999) proposed an ODE model that describes the T-cell dynamics of human T-cell lymphotropic virus I (HTLV-I) infection and the development of adult T-cell leukemia (ATL). Their model consists of four components: uninfected healthy CD4+ T-cells, latently infected CD4+ T-cells, actively infected CD4+ T-cells, and ATL cells. Mathematical analysis that completely determines the global dynamics of this model has been done by Wang et al. (Math. Biosci., 2002). In this note, we first modify the parameters of the model to distinguish between contact and infectivity rates. Then we introduce a discrete time delay to the model to describe the time between emission of contagious particles by active CD4+ T-cells and infection of pure cells. Using the results in Culshaw and Ruan (Math. Biosci., 2000) in the analysis of time delay with respect to cell-free viral spread of HIV, we study the effect of time delay on the stability of the endemically infected equilibrium. Numerical simulations are presented to illustrate the results.

Induction of cancer cell death by proton beam in tumor hypoxic region

International Nuclear Information System (INIS)

Hur, T. R.; Lee, Y. M.; Park, J. W.; Sohn, E. J.

2006-05-01

The physical properties of charged particles such as protons are uniquely suited to target the radiation dose precisely in the tumor. In proton therapy, the Bragg peak is spread out by modulating or degrading the energy of the particles to cover a well defined target volume at a given depth. Due to heterogeneity in the various tumors and end-points as well as in the physical properties of the beams considered, it is difficult to fit the various results into a clear general description of the biological effect of proton in tumor therapy. Tumor hypoxia is a main obstacle to radiotherapy, including gamma-ray. Survived tumor cells under hypoxic region are resistant to radiation and more aggressive to be metastasized. To investigate the dose of proton beam to induce cell death of various tumor cells and hypoxic tumor cells at the Bragg peak in vitro, we used 3 kinds of tumor cells, lung cancer, leukemia and hepatoma cells. Proton beam induces apoptosis in Lewis lung carcinoma cells dose dependently and, slightly in leukemia but not in hepatoma cells at all. Above 1000 gray of proton beam, 60% of cells died even the hypoxic cells in Lewis lung carcinoma cells. But the Molt-4 leukemia cells showed milder effect, 20% cell death by the above 1000 Gray of proton beam and typical resistant pattern (5-10%) of hypoxia in desferrioxamine treated cells. Hepatoma cells (HepG2) were not responsive to proton beam even in rather higher dose (4000G). However, by the gamma-irradiation, Molt-4 was more sensitive than hepatoma or lung cancer cells, but still showed hypoxic resistance. The cell death by proton beam in Lewis lung carcinoma cells was confirmed by PARP cleavage and may be mediated by increased p53. Pro-caspases were also activated and cleaved by the proton beam irradiations for lung cancer cell death. In conclusion, high dose of proton beam (above 1000 gray) may be a good therapeutic radiation even in hypoxic region at the Bragg peak, but further investigations about the

Detection of bacteriophage-infected cells of Lactococcus lactis using flow cytometry

DEFF Research Database (Denmark)

Michelsen, Ole; Cuesta-Dominguez, Álvaro; Albrektsen, Bjarne

2007-01-01

Bacteriophage infection in dairy fermentation constitutes a serious problem worldwide. We have studied bacteriophage infection in Lactococcus lactis by using the flow cytometer. The first effect of the infection of the bacterium is a change from cells in chains toward single cells. We interpret...... describe a new method for detection of phage infection in Lactococcus lactis dairy cultures. The method is based on flow cytometric detection of cells with low-density cell walls. The method allows fast and early detection of phage-infected bacteria, independently of which phage has infected the culture...

Role of Bruton’s Tyrosine Kinase inhibitors in HIV-1 infected cells

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Guendel, Irene; Iordanskiy, Sergey; Sampey, Gavin C; Van Duyne, Rachel; Calvert, Valerie; Petricoin, Emanuel; Saifuddin, Mohammed; Kehn-Hall, Kylene; Kashanchi, Fatah

2015-01-01

Many cellular cofactors have been documented to be critical for various stages of viral replication. Using high throughput proteomic assays, we have previously identified Bruton’s tyrosine kinase (BTK) as a host protein that was uniquely up-regulated in the plasma membrane of HIV-1 infected T-cells. Here, we have further characterized the BTK expression in HIV-1 infection and show that this cellular factor is specifically expressed in infected myeloid cells. Significant up-regulation of the phosphorylated form of BTK was observed in infected cells. Using size exclusion chromatography, we found BTK to be virtually absent in the uninfected U937 cells, however new BTK protein complexes were identified and distributed in both high molecular weight (~600 kDa) and a small molecular weight complex (~60–120 kDa) in the infected U1 cells. BTK levels were highest in cells either chronically expressing virus or induced/infected myeloid cells and that BTK translocated to the membrane following induction of the infected cells. BTK knockdown in HIV-1 infected cells using siRNA resulted in selective death of infected, but not uninfected, cells. Using BTK specific antibody and small molecule inhibitors including LFM-A13 and a FDA approved compound, Ibrutinib (PCI – 32765), we have found that HIV-1 infected cells are sensitive to apoptotic cell death and result in a decrease in virus production. Overall, our data suggests that HIV-1 infected cells are sensitive to treatments targeting BTK expressed in infected cells. PMID:25672887

The homeostasis of Plasmodium falciparum-infected red blood cells.

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Jakob M A Mauritz

2009-04-01

Full Text Available The asexual reproduction cycle of Plasmodium falciparum, the parasite responsible for severe malaria, occurs within red blood cells. A merozoite invades a red cell in the circulation, develops and multiplies, and after about 48 hours ruptures the host cell, releasing 15-32 merozoites ready to invade new red blood cells. During this cycle, the parasite increases the host cell permeability so much that when similar permeabilization was simulated on uninfected red cells, lysis occurred before approximately 48 h. So how could infected cells, with a growing parasite inside, prevent lysis before the parasite has completed its developmental cycle? A mathematical model of the homeostasis of infected red cells suggested that it is the wasteful consumption of host cell hemoglobin that prevents early lysis by the progressive reduction in the colloid-osmotic pressure within the host (the colloid-osmotic hypothesis. However, two critical model predictions, that infected cells would swell to near prelytic sphericity and that the hemoglobin concentration would become progressively reduced, remained controversial. In this paper, we are able for the first time to correlate model predictions with recent experimental data in the literature and explore the fine details of the homeostasis of infected red blood cells during five model-defined periods of parasite development. The conclusions suggest that infected red cells do reach proximity to lytic rupture regardless of their actual volume, thus requiring a progressive reduction in their hemoglobin concentration to prevent premature lysis.

A selective splicing variant of hepcidin mRNA in hepatocellular carcinoma cell lines

International Nuclear Information System (INIS)

Toki, Yasumichi; Sasaki, Katsunori; Tanaka, Hiroki; Yamamoto, Masayo; Hatayama, Mayumi; Ito, Satoshi; Ikuta, Katsuya; Shindo, Motohiro; Hasebe, Takumu; Nakajima, Shunsuke; Sawada, Koji; Fujiya, Mikihiro; Torimoto, Yoshihiro; Ohtake, Takaaki; Kohgo, Yutaka

2016-01-01

Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. By RT-PCR/agarose gel electrophoresis of hepcidin mRNA in a hepatocellular carcinoma cell line HLF, a smaller mRNA band was shown in addition to the wild-type hepcidin mRNA. From sequencing analysis, this additional band was a selective splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene, producing the transcript that encodes truncated peptide lacking 20 amino acids at the middle of preprohepcidin. In the present study, we used the digital PCR, because such a small amount of variant mRNA was difficult to quantitate by the conventional RT-PCR amplification. Among seven hepatoma-derived cell lines, six cell lines have significant copy numbers of this variant mRNA, but not in one cell line. In the transient transfection analysis of variant-type hepcidin cDNA, truncated preprohepcidin has a different character comparing with native preprohepcidin: its product is insensitive to digestion, and secreted into the medium as a whole preprohepcidin form without maturation. Loss or reduction of function of HAMP gene by aberrantly splicing may be a suitable phenomenon to obtain the proliferating advantage of hepatoma cells. - Highlights: • An aberrant splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene. • Absolute quantification of hepcidin mRNA by digital PCR amplification. • Hepatoma-derived cell lines have significant copies of variant-type hepcidin mRNA. • Truncated preprohepcidin is secreted from cells without posttranslational cleavage.

A selective splicing variant of hepcidin mRNA in hepatocellular carcinoma cell lines

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Toki, Yasumichi [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Sasaki, Katsunori, E-mail: k-sasaki@asahikawa-med.ac.jp [Department of Gastrointestinal Immunology and Regenerative Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Tanaka, Hiroki [Department of Legal Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Yamamoto, Masayo; Hatayama, Mayumi; Ito, Satoshi; Ikuta, Katsuya; Shindo, Motohiro; Hasebe, Takumu; Nakajima, Shunsuke; Sawada, Koji; Fujiya, Mikihiro [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Torimoto, Yoshihiro [Oncology Center, Asahikawa Medical University Hospital, Hokkaido 078-8510 (Japan); Ohtake, Takaaki; Kohgo, Yutaka [Department of Gastroenterology, International University of Health and Welfare Hospital, Tochigi 329-2763 (Japan)

2016-08-05

Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. By RT-PCR/agarose gel electrophoresis of hepcidin mRNA in a hepatocellular carcinoma cell line HLF, a smaller mRNA band was shown in addition to the wild-type hepcidin mRNA. From sequencing analysis, this additional band was a selective splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene, producing the transcript that encodes truncated peptide lacking 20 amino acids at the middle of preprohepcidin. In the present study, we used the digital PCR, because such a small amount of variant mRNA was difficult to quantitate by the conventional RT-PCR amplification. Among seven hepatoma-derived cell lines, six cell lines have significant copy numbers of this variant mRNA, but not in one cell line. In the transient transfection analysis of variant-type hepcidin cDNA, truncated preprohepcidin has a different character comparing with native preprohepcidin: its product is insensitive to digestion, and secreted into the medium as a whole preprohepcidin form without maturation. Loss or reduction of function of HAMP gene by aberrantly splicing may be a suitable phenomenon to obtain the proliferating advantage of hepatoma cells. - Highlights: • An aberrant splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene. • Absolute quantification of hepcidin mRNA by digital PCR amplification. • Hepatoma-derived cell lines have significant copies of variant-type hepcidin mRNA. • Truncated preprohepcidin is secreted from cells without posttranslational cleavage.

Production and characterization of high-titer serum-free cell culture grown hepatitis C virus particles of genotype 1-6

DEFF Research Database (Denmark)

Mathiesen, Christian K; Jensen, Tanja B; Prentoe, Jannick

2014-01-01

Recently, cell culture systems producing hepatitis C virus particles (HCVcc) were developed. Establishment of serum-free culture conditions is expected to facilitate development of a whole-virus inactivated HCV vaccine. We describe generation of genotype 1-6 serum-free HCVcc (sf-HCVcc) from Huh7.......5 hepatoma cells cultured in adenovirus expression medium. Compared to HCVcc, sf-HCVcc showed 0.6-2.1 log10 higher infectivity titers (4.7-6.2 log10 Focus Forming Units/mL), possibly due to increased release and specific infectivity of sf-HCVcc. In contrast to HCVcc, sf-HCVcc had a homogeneous single......-peak density profile. Entry of sf-HCVcc depended on HCV co-receptors CD81, LDLr, and SR-BI, and clathrin-mediated endocytosis. HCVcc and sf-HCVcc were neutralized similarly by chronic-phase patient sera and by human monoclonal antibodies targeting conformational epitopes. Thus, we developed serum-free culture...

T-cell-dependent control of acute Giardia lamblia infections in mice.

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Singer, S M; Nash, T E

2000-01-01

We have studied immune mechanisms responsible for control of acute Giardia lamblia and Giardia muris infections in adult mice. Association of chronic G. lamblia infection with hypogammaglobulinemia and experimental infections of mice with G. muris have led to the hypothesis that antibodies are required to control these infections. We directly tested this hypothesis by infecting B-cell-deficient mice with either G. lamblia or G. muris. Both wild-type mice and B-cell-deficient mice eliminated the vast majority of parasites between 1 and 2 weeks postinfection with G. lamblia. G. muris was also eliminated in both wild-type and B-cell-deficient mice. In contrast, T-cell-deficient and scid mice failed to control G. lamblia infections, as has been shown previously for G. muris. Treatment of wild-type or B-cell-deficient mice with antibodies to CD4 also prevented elimination of G. lamblia, confirming a role for T cells in controlling infections. By infecting mice deficient in either alphabeta- or gammadelta-T-cell receptor (TCR)-expressing T cells, we show that the alphabeta-TCR-expressing T cells are required to control parasites but that the gammadelta-TCR-expressing T cells are not. Finally, infections in mice deficient in production of gamma interferon or interleukin 4 (IL-4) and mice deficient in responding to IL-4 and IL-13 revealed that neither the Th1 nor the Th2 subset is absolutely required for protection from G. lamblia. We conclude that a T-cell-dependent mechanism is essential for controlling acute Giardia infections and that this mechanism is independent of antibody and B cells.

Natural Killer Cell Function and Dysfunction in Hepatitis C Virus Infection

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Kayla A. Holder

2014-01-01

Full Text Available Viruses must continually adapt against dynamic innate and adaptive responses of the host immune system to establish chronic infection. Only a small minority (~20% of those exposed to hepatitis C virus (HCV spontaneously clear infection, leaving approximately 200 million people worldwide chronically infected with HCV. A number of recent research studies suggest that establishment and maintenance of chronic HCV infection involve natural killer (NK cell dysfunction. This relationship is illustrated in vitro by disruption of typical NK cell responses including both cell-mediated cytotoxicity and cytokine production. Expression of a number of activating NK cell receptors in vivo is also affected in chronic HCV infection. Thus, direct in vivo and in vitro evidence of compromised NK function in chronic HCV infection in conjunction with significant epidemiological associations between the outcome of HCV infection and certain combinations of NK cell regulatory receptor and class I human histocompatibility linked antigen (HLA genotypes indicate that NK cells are important in the immune response against HCV infection. In this review, we highlight evidence suggesting that selective impairment of NK cell activity is related to establishment of chronic HCV infection.

Potentiating effect of graphene nanomaterials on aromatic environmental pollutant-induced cytochrome P450 1A expression in the topminnow fish hepatoma cell line PLHC-1.

Science.gov (United States)

Lammel, Tobias; Boisseaux, Paul; Navas, José M

2015-09-01

Graphene and its derivatives are an emerging class of carbon nanomaterial with great potential for a broad range of industrial and consumer applications. However, their increasing production and use is expected to result in release of nano-sized graphene platelets into the environment, where they may interact with chemical pollutants modifying their fate and toxic potential. The objective of this study was to assess whether graphene nanoplatelets can act as vector for aromatic environmental pollutants increasing their cellular uptake and associated hazardous effects in vitro. For this purpose, cell cultures of the topminnow fish (Poeciliopsis lucida) hepatoma cell line PLHC-1 were simultaneously (and successively) exposed to graphene nanoplatelets (graphene oxide (GO) or carboxyl graphene (CXYG)) and an aryl hydrocarbon receptor (AhR) agonist (β-naphthoflavone (β-NF), benzo(k)fluoranthene (BkF) or 3,3',4,4',5,5'-hexachlorobiphenyl (PCB169)). Following exposure cytochrome P450 1A (Cyp1A) induction was assessed by measuring cyp1A mRNA expression levels using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and Cyp1A-dependent ethoxyresorufin-O-deethylase (EROD) activity. It was observed that pre- and co-exposure of cells to GO and CXYG nanoplatelets had a potentiating effect on β-NF, BkF, and PCB169-dependent Cyp1A induction suggesting that graphene nanoplatelets increase the effective concentration of AhR agonists by facilitating their passive diffusion into the cells by damaging the cells' plasma membrane and/or by transporting them over the plasma membrane via a Trojan horse-like mechanism. The results demonstrate the existence of combination effects between nanomaterials and environmental pollutants and stress the importance of considering these effects when evaluating their respective hazard. © 2014 Wiley Periodicals, Inc.

Electron Microscopy of Ebola Virus-Infected Cells.

Science.gov (United States)

Noda, Takeshi

2017-01-01

Ebola virus (EBOV) replicates in host cells, where both viral and cellular components show morphological changes during the process of viral replication from entry to budding. These steps in the replication cycle can be studied using electron microscopy (EM), including transmission electron microscopy (TEM) and scanning electron microscopy (SEM), which is one of the most useful methods for visualizing EBOV particles and EBOV-infected cells at the ultrastructural level. This chapter describes conventional methods for EM sample preparation of cultured cells infected with EBOV.

Human neuronal cell protein responses to Nipah virus infection

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Hassan Sharifah

2007-06-01

Full Text Available Abstract Background Nipah virus (NiV, a recently discovered zoonotic virus infects and replicates in several human cell types. Its replication in human neuronal cells, however, is less efficient in comparison to other fully susceptible cells. In the present study, the SK-N-MC human neuronal cell protein response to NiV infection is examined using proteomic approaches. Results Method for separation of the NiV-infected human neuronal cell proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE was established. At least 800 protein spots were resolved of which seven were unique, six were significantly up-regulated and eight were significantly down-regulated. Six of these altered proteins were identified using mass spectrometry (MS and confirmed using MS/MS. The heterogenous nuclear ribonucleoprotein (hnRNP F, guanine nucleotide binding protein (G protein, voltage-dependent anion channel 2 (VDAC2 and cytochrome bc1 were present in abundance in the NiV-infected SK-N-MC cells in contrast to hnRNPs H and H2 that were significantly down-regulated. Conclusion Several human neuronal cell proteins that are differentially expressed following NiV infection are identified. The proteins are associated with various cellular functions and their abundance reflects their significance in the cytopathologic responses to the infection and the regulation of NiV replication. The potential importance of the ratio of hnRNP F, and hnRNPs H and H2 in regulation of NiV replication, the association of the mitochondrial protein with the cytopathologic responses to the infection and induction of apoptosis are highlighted.

Adrenaline-induced mobilization of T cells in HIV-infected patients

DEFF Research Database (Denmark)

Søndergaard, S R; Cozzi-Lepri, A; Ullum, H

2000-01-01

The present study aimed to investigate lymphocyte mobilization from peripheral cell reservoirs in HIV-infected patients. Nine HIV-infected patients on stable highly active anti-retroviral therapy (HAART), eight treatment-naive HIV-infected patients and eight HIV- controls received a 1-h adrenaline...... infusion. The adrenaline infusion induced a three-fold increase in the concentration of lymphocytes in all three groups. All HIV-infected patients mobilized significantly higher numbers of CD8+ cells but less CD4+ cells. All subjects mobilized CD45RA+CD62L+ and CD8+CD28+ cells to a lesser extent than CD45......RO+CD45RA- and CD8+CD28-cells. Furthermore, high numbers of CD8+CD38+ cells were mobilized only in the HIV-infected patients. It was therefore predominantly T cells with an activated phenotype which were mobilized after adrenaline stimulation. It is concluded that the HIV-associated immune defect...

New World hantaviruses activate IFNlambda production in type I IFN-deficient vero E6 cells.

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Joseph Prescott

Full Text Available Hantaviruses indigenous to the New World are the etiologic agents of hantavirus cardiopulmonary syndrome (HCPS. These viruses induce a strong interferon-stimulated gene (ISG response in human endothelial cells. African green monkey-derived Vero E6 cells are used to propagate hantaviruses as well as many other viruses. The utility of the Vero E6 cell line for virus production is thought to owe to their lack of genes encoding type I interferons (IFN, rendering them unable to mount an efficient innate immune response to virus infection. Interferon lambda, a more recently characterized type III IFN, is transcriptionally controlled much like the type I IFNs, and activates the innate immune system in a similar manner.We show that Vero E6 cells respond to hantavirus infection by secreting abundant IFNlambda. Three New World hantaviruses were similarly able to induce IFNlambda expression in this cell line. The IFNlambda contained within virus preparations generated with Vero E6 cells independently activates ISGs when used to infect several non-endothelial cell lines, whereas innate immune responses by endothelial cells are specifically due to viral infection. We show further that Sin Nombre virus replicates to high titer in human hepatoma cells (Huh7 without inducing ISGs.Herein we report that Vero E6 cells respond to viral infection with a highly active antiviral response, including secretion of abundant IFNlambda. This cytokine is biologically active, and when contained within viral preparations and presented to human epithelioid cell lines, results in the robust activation of innate immune responses. We also show that both Huh7 and A549 cell lines do not respond to hantavirus infection, confirming that the cytoplasmic RNA helicase pathways possessed by these cells are not involved in hantavirus recognition. We demonstrate that Vero E6 actively respond to virus infection and inhibiting IFNlambda production in these cells might increase their utility

Case of double cancer of the liver (hepatoma and cholangioma) caused by accumulation of thorotrast

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Masai, K; Watanabe, T [Yanai-Byoin National Sanatorium, (Japan)

1977-03-01

Fairly many cases of delayed disorders caused by thorotrast have been reported. The authors experienced a case of a 65 year old male who had received an infusion of thorotrast solution at the old army hospital 38 years ago and died from liver cirrhosis and hepatic cancer (double cancer of hepatoma and cholangiocarcinoma). By the biopsy after his death, alpha rays were demonstrated in granules of foreign body in hepatic tissues, and in corporation with its contrast, it was certified that a chain of abnormal shadows mainly seen in the liver and the spleen, on the scout film of the abdomen before death, was caused by thorotrast. From pathological findings in hepatic cancers, hepatoma was recognized in the right lobe and cholangioma, in the left lobe, and there was no pathological relationship between the two cancers. Namely, it was double cancer within the same organ. Case of double cancer in the liver such as this case were thought to be very rare in Japan, and a few literature considerations were also reported.

Molecular dynamics study of lipid bilayers modeling the plasma membranes of mouse hepatocytes and hepatomas.

Science.gov (United States)

Andoh, Yoshimichi; Aoki, Noriyuki; Okazaki, Susumu

2016-02-28

Molecular dynamics (MD) calculations of lipid bilayers modeling the plasma membranes of normal mouse hepatocytes and hepatomas in water have been performed under physiological isothermal-isobaric conditions (310.15 K and 1 atm). The changes in the membrane properties induced by hepatic canceration were investigated and were compared with previous MD calculations included in our previous study of the changes in membrane properties induced by murine thymic canceration. The calculated model membranes for normal hepatocytes and hepatomas comprised 23 and 24 kinds of lipids, respectively. These included phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. We referred to previously published experimental values for the mole fraction of the lipids adopted in the present calculations. The calculated structural and dynamic properties of the membranes such as lateral structure, order parameters, lateral self-diffusion constants, and rotational correlation times all showed that hepatic canceration causes plasma membranes to become more ordered laterally and less fluid. Interestingly, this finding contrasts with the less ordered structure and increased fluidity of plasma membranes induced by thymic canceration observed in our previous MD study.

Infections and endothelial cells

NARCIS (Netherlands)

Keller, Tymen T.; Mairuhu, Albert T. A.; de Kruif, Martijn D.; Klein, Saskia K.; Gerdes, Victor E. A.; ten Cate, Hugo; Brandjes, Dees P. M.; Levi, Marcel; van Gorp, Eric C. M.

2003-01-01

Systemic infection by various pathogens interacts with the endothelium and may result in altered coagulation, vasculitis and atherosclerosis. Endothelium plays a role in the initiation and regulation of both coagulation and fibrinolysis. Exposure of endothelial cells may lead to rapid activation of

Identification of a Monocyte Receptor on Herpesvirus-Infected Endothelial Cells

Science.gov (United States)

Etingin, Orli R.; Silverstein, Roy L.; Hajjar, David P.

1991-08-01

The adhesion of circulating blood cells to vascular endothelium may be an initial step in atherosclerosis, inflammation, and wound healing. One mechanism for promoting cell-cell adhesion involves the expression of adhesion molecules on the surface of the target cell. Herpes simplex virus infection of endothelium induces arterial injury and has been implicated in the development of human atherosclerosis. We now demonstrate that HSV-infected endothelial cells express the adhesion molecule GMP140 and that this requires cell surface expression of HSV glycoprotein C and local thrombin generation. Monocyte adhesion to HSV-infected endothelial cells was completely inhibited by anti-GMP140 antibodies but not by antibodies to other adhesion molecules such as VCAM and ELAM-1. The induction of GMP140 expression on HSV-infected endothelium may be an important pathophysiological mechanism in virus-induced cell injury and inflammation.

Lymphocytes and macrophages are infected by Theileria equi, but T cells and B cells are not required to establish infection in vivo.

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Joshua D Ramsay

Full Text Available Theileria equi has a biphasic life cycle in horses, with a period of intraleukocyte development followed by patent erythrocytic parasitemia that causes acute and sometimes fatal hemolytic disease. Unlike Theileria spp. that infect cattle (Theileria parva and Theileria annulata, the intraleukocyte stage (schizont of Theileria equi does not cause uncontrolled host cell proliferation or other significant pathology. Nevertheless, schizont-infected leukocytes are of interest because of their potential to alter host cell function and because immune responses directed against this stage could halt infection and prevent disease. Based on cellular morphology, Theileria equi has been reported to infect lymphocytes in vivo and in vitro, but the specific phenotype of schizont-infected cells has yet to be defined. To resolve this knowledge gap in Theileria equi pathogenesis, peripheral blood mononuclear cells were infected in vitro and the phenotype of infected cells determined using flow cytometry and immunofluorescence microscopy. These experiments demonstrated that the host cell range of Theileria equi was broader than initially reported and included B lymphocytes, T lymphocytes and monocyte/macrophages. To determine if B and T lymphocytes were required to establish infection in vivo, horses affected with severe combined immunodeficiency (SCID, which lack functional B and T lymphocytes, were inoculated with Theileria equi sporozoites. SCID horses developed patent erythrocytic parasitemia, indicating that B and T lymphocytes are not necessary to complete the Theileria equi life cycle in vivo. These findings suggest that the factors mediating Theileria equi leukocyte invasion and intracytoplasmic differentiation are common to several leukocyte subsets and are less restricted than for Theileria annulata and Theileria parva. These data will greatly facilitate future investigation into the relationships between Theileria equi leukocyte tropism and pathogenesis

Differences between Mycobacterium-Host Cell Relationships in Latent Tuberculous Infection of Mice Ex Vivo and Mycobacterial Infection of Mouse Cells In Vitro

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Elena Ufimtseva

2016-01-01

Full Text Available The search for factors that account for the reproduction and survival of mycobacteria, including vaccine strains, in host cells is the priority for studies on tuberculosis. A comparison of BCG-mycobacterial loads in granuloma cells obtained from bone marrow and spleens of mice with latent tuberculous infection and cells from mouse bone marrow and peritoneal macrophage cultures infected with the BCG vaccine in vitro has demonstrated that granuloma macrophages each normally contained a single BCG-Mycobacterium, while those acutely infected in vitro had increased mycobacterial loads and death rates. Mouse granuloma cells were observed to produce the IFNγ, IL-1α, GM-CSF, CD1d, CD25, CD31, СD35, and S100 proteins. None of these activation markers were found in mouse cell cultures infected in vitro or in intact macrophages. Lack of colocalization of lipoarabinomannan-labeled BCG-mycobacteria with the lysosomotropic LysoTracker dye in activated granuloma macrophages suggests that these macrophages were unable to destroy BCG-mycobacteria. However, activated mouse granuloma macrophages could control mycobacterial reproduction in cells both in vivo and in ex vivo culture. By contrast, a considerable increase in the number of BCG-mycobacteria was observed in mouse bone marrow and peritoneal macrophages after BCG infection in vitro, when no expression of the activation-related molecules was detected in these cells.

Infection and Proliferation of Giant Viruses in Amoeba Cells.

Science.gov (United States)

Takemura, Masaharu

2016-01-01

Acanthamoeba polyphaga mimivirus, the first discovered giant virus with genome size and particle size much larger than previously discovered viruses, possesses several genes for translation and CRISPER Cas system-like defense mechanism against virophages, which co-infect amoeba cells with the giant virus and which inhibit giant virus proliferation. Mimiviruses infect amoeba cells by phagocytosis and release their DNA into amoeba cytoplasm through their stargate structure. After infection, giant virion factories (VFs) form in amoeba cytoplasm, followed by DNA replication and particle formation at peripheral regions of VF. Marseilleviruses, the smallest giant viruses, infect amoeba cells by phagocytosis or endocytosis, form larger VF than Mimivirus's VF in amoeba cytoplasm, and replicate their particles. Pandoraviruses found in 2013 have the largest genome size and particle size among all viruses ever found. Pandoraviruses infect amoeba cells by phagocytosis and release their DNA into amoeba cytoplasm through their mouth-like apical pores. The proliferation of Pandoraviruses occurs along with nucleus disruption. New virions form at the periphery of the region formerly occupied by the amoeba cell nucleus.

Value of an hepatobiliary imaging agent for diagnosing hepatoma. Example of diethyl-IDA

Energy Technology Data Exchange (ETDEWEB)

Bourguet, P; Estable, P; Herry, J Y

1985-01-01

A comparative study was performed using two hepatic tracers, a Tc 99m labelled colloid and an hepatobiliary agent Tc 99m labelled diethyl-IDA. In some patients with isolated primary hepatocarcinoma the uptake of the hepatobiliary agent was observed but the colloid was not taken up. In the contrary, the hepatobiliary agent has proved to be of limited value for the diagnosis of hepatomas coexisting with cirrhosis and for the detection of secondary hepatocarcinoma.

Potential Cellular Signatures of Viral Infections in Human Hematopoietic Cells

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J. Mikovits

2001-01-01

Full Text Available Expression profiling of cellular genes was performed using a 10,000 cDNA human gene array in order to identify expression changes following chronic infection of human hematopoietic cells with Kapsosi’s Sarcoma -associated Virus (KSHV also known as Human Herpesvirus 8 (HHV8 and Human T cell leukemia virus-1 (HTLV-1. We performed cell-free {\\it in vitro} infection of primary bone marrow derived CD34+ cells using semi-purified HHV8 and a mature IL-2 dependent T cell line, KIT 225, using highly concentrated viral stocks prepared from an infectious molecular clone of HTLV-1. Thirty days post infection, mRNA was isolated from infected cultures and uninfected controls and submitted for microarray analysis. More than 400 genes were differentially expressed more than two-fold following HHV8 infection of primary bone marrow derived CD34+ cells. Of these 400, interferon regulatory factor 4 (IRF4, cyclin B2, TBP-associated factor, eukaryotic elongation factor and pim 2 were up-regulated more than 3.5 fold. In contrast, less than 100 genes were differentially expressed more than two-fold following chronic infection of a mature T cell line with HTLV-1. Of these, only cdc7 was up-regulated more than 3.5 fold. These data may provide insight into cellular signatures of infection useful for diagnosis of infection as well as potential targets for therapeutic intervention.

Alterations in the nuclear proteome of HIV-1 infected T-cells

International Nuclear Information System (INIS)

DeBoer, Jason; Jagadish, Teena; Haverland, Nicole A.; Madson, Christian J.; Ciborowski, Pawel; Belshan, Michael

2014-01-01

Virus infection of a cell involves the appropriation of host factors and the innate defensive response of the cell. The identification of proteins critical for virus replication may lead to the development of novel, cell-based inhibitors. In this study we mapped the changes in T-cell nuclei during human immunodeficiency virus type 1 (HIV-1) at 20 hpi. Using a stringent data threshold, a total of 13 and 38 unique proteins were identified in infected and uninfected cells, respectively, across all biological replicates. An additional 15 proteins were found to be differentially regulated between infected and control nuclei. STRING analysis identified four clusters of protein–protein interactions in the data set related to nuclear architecture, RNA regulation, cell division, and cell homeostasis. Immunoblot analysis confirmed the differential expression of several proteins in both C8166-45 and Jurkat E6-1 T-cells. These data provide a map of the response in host cell nuclei upon HIV-1 infection. - Highlights: • We identify changes in the expression of nuclear proteins during HIV-1 infection. • 163 nuclear proteins were found differentially regulated during HIV-1 infection. • Bioinformatic analysis identified several nuclear pathways altered by HIV infection. • Candidate factors were validated in two independent cell lines

Alterations in the nuclear proteome of HIV-1 infected T-cells

Energy Technology Data Exchange (ETDEWEB)

DeBoer, Jason [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); Jagadish, Teena; Haverland, Nicole A. [Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198 (United States); Madson, Christian J. [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); Ciborowski, Pawel [Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198 (United States); The Nebraska Center for Virology, University of Nebraska, Lincoln 68583 (United States); Belshan, Michael, E-mail: michaelbelshan@creighton.edu [Department of Medical Microbiology and Immunology, Creighton University, 2500 California Plaza, Omaha, NE 68178 (United States); The Nebraska Center for Virology, University of Nebraska, Lincoln 68583 (United States)

2014-11-15

Virus infection of a cell involves the appropriation of host factors and the innate defensive response of the cell. The identification of proteins critical for virus replication may lead to the development of novel, cell-based inhibitors. In this study we mapped the changes in T-cell nuclei during human immunodeficiency virus type 1 (HIV-1) at 20 hpi. Using a stringent data threshold, a total of 13 and 38 unique proteins were identified in infected and uninfected cells, respectively, across all biological replicates. An additional 15 proteins were found to be differentially regulated between infected and control nuclei. STRING analysis identified four clusters of protein–protein interactions in the data set related to nuclear architecture, RNA regulation, cell division, and cell homeostasis. Immunoblot analysis confirmed the differential expression of several proteins in both C8166-45 and Jurkat E6-1 T-cells. These data provide a map of the response in host cell nuclei upon HIV-1 infection. - Highlights: • We identify changes in the expression of nuclear proteins during HIV-1 infection. • 163 nuclear proteins were found differentially regulated during HIV-1 infection. • Bioinformatic analysis identified several nuclear pathways altered by HIV infection. • Candidate factors were validated in two independent cell lines.

6K2-induced vesicles can move cell to cell during turnip mosaic virus infection

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Romain eGrangeon

2013-12-01

Full Text Available To successfully infect plants, viruses replicate in an initially infected cell and then move to neighboring cells through plasmodesmata (PDs. However, the nature of the viral entity that crosses over the cell barrier into non-infected ones is not clear. The membrane-associated 6K2 protein of turnip mosaic virus (TuMV induces the formation of vesicles involved in the replication and intracellular movement of viral RNA. This study shows that 6K2-induced vesicles trafficked towards the plasma membrane and were associated with plasmodesmata (PD. We demonstrated also that 6K2 moved cell-to-cell into adjoining cells when plants were infected with TuMV. 6K2 was then fused to photo-activable GFP (6K2:PAGFP to visualize how 6K2 move intercellularly during TuMV infection. After activation, 6K2:PAGFP-tagged vesicles moved to the cell periphery and across the cell wall into adjacent cells. These vesicles were shown to contain the viral RNA-dependent RNA polymerase and viral RNA. Symplasmic movement of TuMV may thus be achieved in the form of a membrane-associated viral RNA complex induced by 6K2.

Susceptibility of Primary Human Choroid Plexus Epithelial Cells and Meningeal Cells to Infection by JC Virus.

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O'Hara, Bethany A; Gee, Gretchen V; Atwood, Walter J; Haley, Sheila A

2018-04-15

JC polyomavirus (JCPyV) establishes a lifelong persistence in roughly half the human population worldwide. The cells and tissues that harbor persistent virus in vivo are not known, but renal tubules and other urogenital epithelial cells are likely candidates as virus is shed in the urine of healthy individuals. In an immunosuppressed host, JCPyV can become reactivated and cause progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the central nervous system. Recent observations indicate that JCPyV may productively interact with cells in the choroid plexus and leptomeninges. To further study JCPyV infection in these cells, primary human choroid plexus epithelial cells and meningeal cells were challenged with virus, and their susceptibility to infection was compared to the human glial cell line, SVG-A. We found that JCPyV productively infects both choroid plexus epithelial cells and meningeal cells in vitro Competition with the soluble receptor fragment LSTc reduced virus infection in these cells. Treatment of cells with neuraminidase also inhibited both viral infection and binding. Treatment with the serotonin receptor antagonist, ritanserin, reduced infection in SVG-A and meningeal cells. We also compared the ability of wild-type and sialic acid-binding mutant pseudoviruses to transduce these cells. Wild-type pseudovirus readily transduced all three cell types, but pseudoviruses harboring mutations in the sialic acid-binding pocket of the virus failed to transduce the cells. These data establish a novel role for choroid plexus and meninges in harboring virus that likely contributes not only to meningoencephalopathies but also to PML. IMPORTANCE JCPyV infects greater than half the human population worldwide and causes central nervous system disease in patients with weakened immune systems. Several recent reports have found JCPyV in the choroid plexus and leptomeninges of patients with encephalitis. Due to their role in forming the blood

Microsporidia infection impacts the host cell's cycle and reduces host cell apoptosis

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Higes, Mariano; Sagastume, Soledad; Juarranz, Ángeles; Dias-Almeida, Joyce; Budge, Giles E.; Meana, Aránzazu; Boonham, Neil

2017-01-01

Intracellular parasites can alter the cellular machinery of host cells to create a safe haven for their survival. In this regard, microsporidia are obligate intracellular fungal parasites with extremely reduced genomes and hence, they are strongly dependent on their host for energy and resources. To date, there are few studies into host cell manipulation by microsporidia, most of which have focused on morphological aspects. The microsporidia Nosema apis and Nosema ceranae are worldwide parasites of honey bees, infecting their ventricular epithelial cells. In this work, quantitative gene expression and histology were studied to investigate how these two parasites manipulate their host’s cells at the molecular level. Both these microsporidia provoke infection-induced regulation of genes involved in apoptosis and the cell cycle. The up-regulation of buffy (which encodes a pro-survival protein) and BIRC5 (belonging to the Inhibitor Apoptosis protein family) was observed after infection, shedding light on the pathways that these pathogens use to inhibit host cell apoptosis. Curiously, different routes related to cell cycle were modified after infection by each microsporidia. In the case of N. apis, cyclin B1, dacapo and E2F2 were up-regulated, whereas only cyclin E was up-regulated by N. ceranae, in both cases promoting the G1/S phase transition. This is the first report describing molecular pathways related to parasite-host interactions that are probably intended to ensure the parasite’s survival within the cell. PMID:28152065

Interleukin-10 regulates hepcidin in Plasmodium falciparum malaria

KAUST Repository

Huang, Honglei; Lamikanra, Abigail A.; Alkaitis, Matthew S.; Thé zé nas, Marie L.; Ramaprasad, Abhinay; Moussa, Ehab; Roberts, David J.; Casals-Pascual, Climent

2014-01-01

. falciparum malaria. Methods: We have measured secretion of hepcidin by primary macrophages and the hepatoma cell line HepG2 stimulated with IL-10, IL-6 and Plasmodium falciparum-infected erythrocytes. Findings: We have observed that IL-10 and IL-6 production

Dengue virus-induced regulation of the host cell translational machinery

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C.S.A. Villas-Bôas

2009-11-01

Full Text Available Dengue virus (DV-induced changes in the host cell protein synthesis machinery are not well understood. We investigated the transcriptional changes related to initiation of protein synthesis. The human hepatoma cell line, HepG2, was infected with DV serotype 2 for 1 h at a multiplicity of infection of one. RNA was extracted after 6, 24 and 48 h. Microarray results showed that 36.5% of the translation factors related to initiation of protein synthesis had significant differential expression (Z-score ≥ ±2.0. Confirmation was obtained by quantitative real-time reverse transcription-PCR. Of the genes involved in the activation of mRNA for cap-dependent translation (eIF4 factors, eIF4A, eIF4G1 and eIF4B were up-regulated while the negative regulator of translation eIF4E-BP3 was down-regulated. This activation was transient since at 24 h post-infection levels were not significantly different from control cells. However, at 48 h post-infection, eIF4A, eIF4E, eIF4G1, eIF4G3, eIF4B, and eIF4E-BP3 were down-regulated, suggesting that cap-dependent translation could be inhibited during the progression of infection. To test this hypothesis, phosphorylation of p70S6K and 4E-BP1, which induce cap-dependent protein synthesis, was assayed. Both proteins remained phosphorylated when assayed at 6 h after infection, while infection induced dephosphorylation of p70S6K and 4E-BP1 at 24 and 48 h of infection, respectively. Taken together, these results provide biological evidence suggesting that in HepG2 cells DV sustains activation of the cap-dependent machinery at early stages of infection, but progression of infection switches protein synthesis to a cap-independent process.

Ureaplasma parvum infection alters filamin a dynamics in host cells

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Brown Mary B

2011-04-01

Full Text Available Abstract Background Ureaplasmas are among the most common bacteria isolated from the human urogenital tract. Ureaplasmas can produce asymptomatic infections or disease characterized by an exaggerated inflammatory response. Most investigations have focused on elucidating the pathogenic potential of Ureaplasma species, but little attention has been paid to understanding the mechanisms by which these organisms are capable of establishing asymptomatic infection. Methods We employed differential proteome profiling of bladder tissues from rats experimentally infected with U. parvum in order to identify host cell processes perturbed by colonization with the microbe. Tissues were grouped into four categories: sham inoculated controls, animals that spontaneously cleared infection, asymptomatic urinary tract infection (UTI, and complicated UTI. One protein that was perturbed by infection (filamin A was used to further elucidate the mechanism of U. parvum-induced disruption in human benign prostate cells (BPH-1. BPH-1 cells were evaluated by confocal microscopy, immunoblotting and ELISA. Results Bladder tissue from animals actively colonized with U. parvum displayed significant alterations in actin binding proteins (profilin 1, vinculin, α actinin, and filamin A that regulate both actin polymerization and cell cytoskeletal function pertaining to focal adhesion formation and signal transduction (Fisher's exact test, P U. parvum perturbed the regulation of filamin A. Specifically, infected BPH-1 cells exhibited a significant increase in filamin A phosphorylated at serine2152 (P ≤ 0.01, which correlated with impaired proteolysis of the protein and its normal intracellular distribution. Conclusion Filamin A dynamics were perturbed in both models of infection. Phosphorylation of filamin A occurs in response to various cell signaling cascades that regulate cell motility, differentiation, apoptosis and inflammation. Thus, this phenomenon may be a useful

IL-15 STIMULATED NATURAL KILLER CELLS CLEAR HIV-1 INFECTED CELLS FOLLOWING LATENCY REVERSAL EX VIVO.

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Garrido, Carolina; Abad-Fernandez, Maria; Tuyishime, Marina; Pollara, Justin J; Ferrari, Guido; Soriano-Sarabia, Natalia; Margolis, David M

2018-03-28

Current efforts towards HIV eradication include approaches to augment immune recognition and elimination of persistently infected cells following latency reversal. Natural killer (NK) cells, the main effectors of the innate immune system, recognize and clear targets using different mechanisms than CD8 + T cells, offering an alternative or complementary approach for HIV clearance strategies. We assessed the impact of IL-15 treatment on NK cell function and the potential of stimulated NK cells to clear the HIV reservoir. We measured NK cell receptor expression, antibody-dependent cell-dependent cytotoxicity (ADCC), cytotoxicity, IFN-γ production and antiviral activity in autologous HIV replication systems. All NK cell functions were uniformly improved by IL-15, and more importantly, IL-15-treated NK cells were able to clear latently HIV infected cells after exposure to vorinostat, a clinically relevant latency reversing agent. We also demonstrate that NK cells from HIV infected individuals aviremic on antiretroviral therapy can be efficiently stimulated with IL-15. Our work opens a promising line of investigation towards future immunotherapies to clear persistent HIV infection using NK cells. IMPORTANCE In the search for an HIV cure, strategies to enhance immune function to allow recognition and clearance of HIV infected cells following latency reversal are being evaluated. Natural killer (NK) cells possess characteristics that can be exploited for immunotherapy against persistent HIV infection. We demonstrate that NK cells from HIV-positive donors can be strongly stimulated with IL-15, improving their antiviral and cytotoxic potential, and more importantly, clearing HIV infected cells after latency reversal with a clinically relevant drug. Our results encourage further investigation to design NK cell-based immunotherapies to achieve HIV eradication. Copyright © 2018 American Society for Microbiology.

Impact of persistent cytomegalovirus infection on human neuroblastoma cell gene expression

International Nuclear Information System (INIS)

Hoever, Gerold; Vogel, Jens-Uwe; Lukashenko, Polina; Hofmann, Wolf-Karsten; Komor, Martina; Doerr, Hans Wilhelm; Cinatl, Jindrich

2005-01-01

In a model of human neuroblastoma (NB) cell lines persistently infected with human cytomegalovirus (HCMV) we previously showed that persistent HCMV infection is associated with an increased malignant phenotype, enhanced drug resistance, and invasive properties. To gain insights into the mechanisms of increased malignancy we analyzed the global changes in cellular gene expression induced by persistent HCMV infection of human neuroblastoma cells by use of high-density oligonucleotide microarrays (HG-U133A, Affymetrix) and RT-PCR. Comparing the gene expression of different NB cell lines with persistently infected cell sub-lines revealed 11 host cell genes regulated in a similar manner throughout all infected samples. Nine of these 11 genes may contribute to the previously observed changes in malignant phenotype of persistently HCMV infected NB cells by influencing invasive growth, apoptosis, angiogenesis, and proliferation. Thus, this work provides the basis for further functional studies

Cell death by pyroptosis drives CD4 T-cell depletion in HIV-1 infection

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Doitsh, Gilad; Galloway, Nicole L. K.; Geng, Xin; Yang, Zhiyuan; Monroe, Kathryn M.; Zepeda, Orlando; Hunt, Peter W.; Hatano, Hiroyu; Sowinski, Stefanie; Muñoz-Arias, Isa; Greene, Warner C.

2014-01-01

The pathway causing CD4 T-cell death in HIV-infected hosts remains poorly understood although apoptosis has been proposed as a key mechanism. We now show that caspase-3-mediated apoptosis accounts for the death of only a small fraction of CD4 T cells corresponding to those that are both activated and productively infected. The remaining over 95% of quiescent lymphoid CD4 T cells die by caspase-1-mediated pyroptosis triggered by abortive viral infection. Pyroptosis corresponds to an intensely inflammatory form of programmed cell death in which cytoplasmic contents and pro-inflammatory cytokines, including IL-1β, are released. This death pathway thus links the two signature events in HIV infection--CD4 T-cell depletion and chronic inflammation--and creates a pathogenic vicious cycle in which dying CD4 T cells release inflammatory signals that attract more cells to die. This cycle can be broken by caspase 1 inhibitors shown to be safe in humans, raising the possibility of a new class of `anti-AIDS' therapeutics targeting the host rather than the virus.

Role of Bunyamwera Orthobunyavirus NSs protein in infection of mosquito cells.

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Szemiel, Agnieszka M; Failloux, Anna-Bella; Elliott, Richard M

2012-01-01

Bunyamwera orthobunyavirus is both the prototype and study model of the Bunyaviridae family. The viral NSs protein seems to contribute to the different outcomes of infection in mammalian and mosquito cell lines. However, only limited information is available on the growth of Bunyamwera virus in cultured mosquito cells other than the Aedes albopictus C6/36 line. To determine potential functions of the NSs protein in mosquito cells, replication of wild-type virus and a recombinant NSs deletion mutant was compared in Ae. albopictus C6/36, C7-10 and U4.4 cells, and in Ae. aegypti Ae cells by monitoring N protein production and virus yields at various times post infection. Both viruses established persistent infections, with the exception of NSs deletion mutant in U4.4 cells. The NSs protein was nonessential for growth in C6/36 and C7-10 cells, but was important for productive replication in U4.4 and Ae cells. Fluorescence microscopy studies using recombinant viruses expressing green fluorescent protein allowed observation of three stages of infection, early, acute and late, during which infected cells underwent morphological changes. In the absence of NSs, these changes were less pronounced. An RNAi response efficiently reduced virus replication in U4.4 cells transfected with virus specific dsRNA, but not in C6/36 or C7/10 cells. Lastly, Ae. aegypti mosquitoes were exposed to blood-meal containing either wild-type or NSs deletion virus, and at various times post-feeding, infection and disseminated infection rates were measured. Compared to wild-type virus, infection rates by the mutant virus were lower and more variable. If the NSs deletion virus was able to establish infection, it was detected in salivary glands at 6 days post-infection, 3 days later than wild-type virus. Bunyamwera virus NSs is required for efficient replication in certain mosquito cell lines and in Ae. aegypti mosquitoes.

Apoptosis transcriptional mechanism of feline infectious peritonitis virus infected cells.

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Shuid, Ahmad Naqib; Safi, Nikoo; Haghani, Amin; Mehrbod, Parvaneh; Haron, Mohd Syamsul Reza; Tan, Sheau Wei; Omar, Abdul Rahman

2015-11-01

Apoptosis has been postulated to play an important role during feline infectious peritonitis virus (FIPV) infection; however, its mechanism is not well characterized. This study is focused on apoptosis and transcriptional profiling of FIPV-infected cells following in vitro infection of CRFK cells with FIPV 79-1146 WSU. Flow cytometry was used to determine mode of cell death in first 42 h post infection (hpi). FIPV infected cells underwent early apoptosis at 9 hpi (p apoptosis at 12 hpi (p apoptosis cluster (80 down-regulated and 51 up-regulated) along with increase of apoptosis, p53, p38 MAPK, VEGF and chemokines/cytokines signaling pathways were probably involved in apoptosis process. Six of the de-regulated genes expression (RASSF1, BATF2, MAGEB16, PDCD5, TNFα and TRAF2) and TNFα protein concentration were analyzed by RT-qPCR and ELISA, respectively, at different time-points. Up-regulations of both pro-apoptotic (i.e. PDCD5) and anti-apoptotic (i.e. TRAF2) were detected from first hpi and continuing to deregulate during apoptosis process in the infected cells.

Exogenous regucalcin suppresses the growth of human liver cancer HepG2 cells in vitro.

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Yamaguchi, Masayoshi; Murata, Tomiyasu

2018-04-05

Regucalcin, which its gene is localized on the X chromosome, plays a pivotal role as a suppressor protein in signal transduction in various types of cells and tissues. Regucalcin gene expression has been demonstrated to be suppressed in various tumor tissues of animal and human subjects, suggesting a potential role of regucalcin in carcinogenesis. Regucalcin, which is produced from the tissues including liver, is found to be present in the serum of human subjects and animals. This study was undertaken to determine the effects of exogenous regucalcin on the proliferation in cloned human hepatoma HepG2 cells in vitro. Proliferation of HepG2 cells was suppressed after culture with addition of regucalcin (0.01 – 10 nM) into culture medium. Exogenous regucalcin did not reveal apoptotic cell death in HepG2 cells in vitro. Suppressive effects of regucalcin on cell proliferation were not enhanced in the presence of various signaling inhibitors including tumor necrosis factor-α (TNF-α), Bay K 8644, PD98059, staurosporine, worthomannin, 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) or gemcitabine, which were found to suppress the proliferation. In addition, exogenous regucalcin suppressed the formation of colonies of cultured hepatoma cells in vitro. These findings demonstrated that exogenous regucalcin exhibits a suppressive effect on the growth of human hepatoma HepG2 cells, proposing a strategy with the gene therapy for cancer treatment.

Value of an hepatobiliary imaging agent for diagnosing hepatoma. Example of diethyl-IDA

International Nuclear Information System (INIS)

Bourguet, P.; Estable, P.; Herry, J.Y.

1985-01-01

A comparative study was performed using two hepatic tracers, a Tc 99m labelled colloid and an hepatobiliary agent Tc 99m labelled diethyl-IDA. In some patients with isolated primary hepatocarcinoma the uptake of the hepatobiliary agent was observed but the colloid was not taken up. In the contrary, the hepatobiliary agent has proved to be of limited value for the diagnosis of hepatomas coexisting with cirrhosis and for the detection of secondary hepatocarcinoma [fr

Cells in Dengue Virus Infection In Vivo

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Sansanee Noisakran

2010-01-01

Full Text Available Dengue has been recognized as one of the most important vector-borne emerging infectious diseases globally. Though dengue normally causes a self-limiting infection, some patients may develop a life-threatening illness, dengue hemorrhagic fever (DHF/dengue shock syndrome (DSS. The reason why DHF/DSS occurs in certain individuals is unclear. Studies in the endemic regions suggest that the preexisting antibodies are a risk factor for DHF/DSS. Viremia and thrombocytopenia are the key clinical features of dengue virus infection in patients. The amounts of virus circulating in patients are highly correlated with severe dengue disease, DHF/DSS. Also, the disturbance, mainly a transient depression, of hematological cells is a critical clinical finding in acute dengue patients. However, the cells responsible for the dengue viremia are unresolved in spite of the intensive efforts been made. Dengue virus appears to replicate and proliferate in many adapted cell lines, but these in vitro properties are extremely difficult to be reproduced in primary cells or in vivo. This paper summarizes reports on the permissive cells in vitro and in vivo and suggests a hematological cell lineage for dengue virus infection in vivo, with the hope that a new focus will shed light on further understanding of the complexities of dengue disease.

Apoptosis in HEp-2 cells infected with Ureaplasma diversum.

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Amorim, Aline Teixeira; Marques, Lucas Miranda; Santos, Angelita Maria Oliveira Gusmão; Martins, Hellen Braga; Barbosa, Maysa Santos; Rezende, Izadora Souza; Andrade, Ewerton Ferraz; Campos, Guilherme Barreto; Lobão, Tássia Neves; Cortez, Beatriz Araujo; Monezi, Telma Alvez; Machado-Santelli, Glaucia Maria; Timenetsky, Jorge

2014-09-04

Bacterial pathogens have many strategies for infecting and persisting in host cells. Adhesion, invasion and intracellular life are important features in the biology of mollicutes. The intracellular location of Ureaplasma diversum may trigger disturbances in the host cell. This includes activation or inhibition of pro and anti-apoptotic factors, which facilitate the development of host damage. The aim of the present study was to associate U. diversum infection in HEp-2 cells and apoptosis induction. Cells were infected for 72hs with four U. diversum clinical isolates and an ATCC strain. The U. diversum invasion was analyzed by Confocal Laser Scanning Microscopy and gentamicin invasion assay. The apoptosis was evaluated using pro-apoptotic and anti-apoptotic gene expression, and FITC Annexin V/Dead Cell Apoptosis Kit. The number of internalized ureaplasma in HEp-2 cells increased significantly throughout the infection. The flow cytometry analysis with fluorochromes to detect membrane depolarization and gene expression for caspase 2, 3 and 9 increased in infected cells after 24 hours. However, after 72 hours a considerable decrease of apoptotic cells was observed. The data suggests that apoptosis may be initially induced by some isolates in association with HEp-2 cells, but over time, there was no evidence of apoptosis in the presence of ureaplasma and HEp-2 cells. The initial increase and then decrease in apoptosis could be related to bacterial pathogen-associated molecular pattern (PAMPS). Moreover, the isolates of U. diversum presented differences in the studied parameters for apoptosis. It was also observed that the amount of microorganisms was not proportional to the induction of apoptosis in HEp-2 cells.

TIM-1 Promotes Hepatitis C Virus Cell Attachment and Infection.

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Wang, Jing; Qiao, Luhua; Hou, Zhouhua; Luo, Guangxiang

2017-01-15

Human TIM and TAM family proteins were recently found to serve as phosphatidylserine (PS) receptors which promote infections by many different viruses, including dengue virus, West Nile virus, Ebola virus, Marburg virus, and Zika virus. In the present study, we provide substantial evidence demonstrating that TIM-1 is important for efficient infection by hepatitis C virus (HCV). The knockdown of TIM-1 expression significantly reduced HCV infection but not HCV RNA replication. Likewise, TIM-1 knockout in Huh-7.5 cells remarkably lowered HCV cell attachment and subsequent HCV infection. More significantly, the impairment of HCV infection in the TIM-1 knockout cells could be restored completely by ectopic expression of TIM-1 but not TIM-3 or TIM-4. Additionally, HCV infection and cell attachment were inhibited by PS but not by phosphatidylcholine (PC), demonstrating that TIM-1-mediated enhancement of HCV infection is PS dependent. The exposure of PS on the HCV envelope was confirmed by immunoprecipitation of HCV particles with a PS-specific monoclonal antibody. Collectively, these findings demonstrate that TIM-1 promotes HCV infection by serving as an attachment receptor for binding to PS exposed on the HCV envelope. TIM family proteins were recently found to enhance infections by many different viruses, including several members of the Flaviviridae family. However, their importance in HCV infection has not previously been examined experimentally. The TIM family proteins include three members in humans: TIM-1, TIM-3, and TIM-4. The findings derived from our studies demonstrate that TIM-1, but not TIM-3 or TIM-4, promotes HCV infection by functioning as an HCV attachment factor. Knockout of the TIM-1 gene resulted in a remarkable reduction of HCV cell attachment and infection. PS-containing liposomes blocked HCV cell attachment and subsequent HCV infection. HCV particles could also be precipitated with a PS-specific monoclonal antibody. These findings suggest that TIM-1

In vivo infection of IgG-containing cells by Jembrana disease virus during acute infection

International Nuclear Information System (INIS)

Desport, Moira; Tenaya, I.W. Masa; McLachlan, Alexander; McNab, Tegan J.; Rachmat, Judhi; Hartaningsih, Nining; Wilcox, Graham E.

2009-01-01

Jembrana disease virus (JDV) is an unusual bovine lentivirus which causes a non-follicular proliferation of lymphocytes, a transient immunosuppression and a delayed humoral response in infected Bali cattle in Indonesia. A double-immunofluorescent labeling method was developed to identify the subset of mononuclear cells in which the viral capsid protein could be detected. Viral antigen was present in pleomorphic centroblast-like cells which were identified as IgG-containing cells, including plasma cells, in lymphoid tissues. There was no evidence of infection of CD3 + T-cells or MAC387 + monocytes in tissues but large vacuolated cells with a macrophage-like morphology in the lung were found to contain viral antigen although they could not be shown conclusively to be infected. The tropism of JDV for mature IgG-containing cells may be relevant to understanding the pathogenesis of Jembrana disease, the delayed antibody responses and the genetic composition of this atypical lentivirus.

Dendritic Cells Activate and Mature after Infection with Mycobacterium tuberculosis

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Mamo Gezahagne

2011-07-01

Full Text Available Abstract Background Dendritic cells (DCs can take up an array of different antigens, including microorganisms which they can process and present more effectively than any other antigen presenting cell. However, whether the interaction between the human DC and Mycobacterium tuberculosis represents a defense mechanism by the invaded host, or helping the invader to evade the defense mechanism of the host is still not clearly understood. Findings To analyze the interactions between M. tuberculosis and immune cells, human peripheral blood monocyte-derived immature DCs were infected with M. tuberculosis H37Rv wild type strain and flow cytometry was used to analyse cell surface expression markers. The ability of the M. tuberculosis infected DC to induce T cell proliferation using 5 and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE dilution technique was also investigated. DCs were found to internalize the mycobacteria and show dose dependent infection and necrosis with different multiplicity of infection. Flow cytometry analysis of cell surface expression markers CD40, CD54, CD80, CD83, CD86 and HLA DR in infected DC revealed significant (p M. tuberculosis in comparison to immature DC with no stimulation. Lipopolysaccharide (LPS from Salmonella abortus equi, a known DC maturation agent, was used as a positive control and showed a comparable up regulation of cell surface markers as observed with M. tuberculosis infected DC. It was revealed that the M. tuberculosis infected DC induced T cell proliferation. Conclusion These data clearly demonstrate that M. tuberculosis induces activation and maturation of human monocyte-derived immature DC as well as induces T cell proliferation in vitro.

Unconventional Pro-inflammatory CD4+ T Cell Response in B Cell-Deficient Mice Infected with Trypanosoma cruzi

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Melisa Gorosito Serrán

2017-11-01

Full Text Available Chagas disease, caused by the parasite Trypanosoma cruzi, is endemic in Latin America but has become a global public health concern by migration of infected people. It has been reported that parasite persistence as well as the intensity of the inflammatory immune response are determinants of the clinical manifestations of the disease. Even though inflammation is indispensable for host defense, when deregulated, it can contribute to tissue injury and organ dysfunction. Here, we report the importance of B cells in conditioning T cell response in T. cruzi infection. Mice deficient in mature B cells (muMT mice infected with T. cruzi exhibited an increase in plasma TNF concentration, TNF-producing CD4+ T cells, and mortality. The increase in TNF-producing CD4+ T cells was accompanied by a reduction in IFNγ+CD4+ T cells and a decrease of the frequency of regulatory Foxp3+, IL-10+, and IL17+CD4+ T cells populations. The CD4+ T cell population activated by T. cruzi infection, in absence of mature B cells, had a high frequency of Ly6C+ cells and showed a lower expression of inhibitory molecules such as CTLA-4, PD-1, and LAG3. CD4+ T cells from infected muMT mice presented a high frequency of CD62LhiCD44− cells, which is commonly associated with a naïve phenotype. Through transfer experiments we demonstrated that CD4+ T cells from infected muMT mice were able to condition the CD4+ T cells response from infected wild-type mice. Interestingly, using Blimp-flox/flox-CD23icre mice we observed that in absence of plasmablast/plasma cell T. cruzi-infected mice exhibited a higher number of TNF-producing CD4+ T cells. Our results showed that the absence of B cells during T. cruzi infection affected the T cell response at different levels and generated a favorable scenario for unconventional activation of CD4+ T cell leading to an uncontrolled effector response and inflammation. The product of B cell differentiation, the plasmablast/plasma cells, could be able

Targeted sorting of single virus-infected cells of the coccolithophore Emiliania huxleyi.

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Martínez Martínez, Joaquín; Poulton, Nicole J; Stepanauskas, Ramunas; Sieracki, Michael E; Wilson, William H

2011-01-01

Discriminating infected from healthy cells is the first step to understanding the mechanisms and ecological implications of viral infection. We have developed a method for detecting, sorting, and performing molecular analysis of individual, infected cells of the important microalga Emiliania huxleyi, based on known physiological responses to viral infection. Of three fluorescent dyes tested, FM 1-43 (for detecting membrane blebbing) gave the most unequivocal and earliest separation of cells. Furthermore, we were able to amplify the genomes of single infected cells using Multiple Displacement Amplification. This novel method to reliably discriminate infected from healthy cells in cultures will allow researchers to answer numerous questions regarding the mechanisms and implications of viral infection of E. huxleyi. The method may be transferable to other virus-host systems.

Preferential infection and depletion of Mycobacterium tuberculosis-specific CD4 T cells after HIV-1 infection

NARCIS (Netherlands)

Geldmacher, Christof; Ngwenyama, Njabulo; Schuetz, Alexandra; Petrovas, Constantinos; Reither, Klaus; Heeregrave, Edwin J.; Casazza, Joseph P.; Ambrozak, David R.; Louder, Mark; Ampofo, William; Pollakis, Georgios; Hill, Brenna; Sanga, Erica; Saathoff, Elmar; Maboko, Leonard; Roederer, Mario; Paxton, William A.; Hoelscher, Michael; Koup, Richard A.

2010-01-01

HIV-1 infection results in the progressive loss of CD4 T cells. In this study, we address how different pathogen-specific CD4 T cells are affected by HIV infection and the cellular parameters involved. We found striking differences in the depletion rates between CD4 T cells to two common

Equine infectious anemia virus-infected dendritic cells retain antigen presentation capability

International Nuclear Information System (INIS)