Necrosis - the dominant form of cell death after phototoxicity impact of hypericin in colon adenocarcinoma cells HT29

International Nuclear Information System (INIS)

Mikes, J.; Kleban, J.; Sackova, V.; Solar, P.; Fedorocko, P.

2006-01-01

Photodynamic therapy (PDT) is a therapeutical approach for the treatment of malignant as well as non-malignant disorders based on administration of nontoxic/weakly toxic photosensitive compound and its activation with light. Hypericin, one of the promising photosensitizers, is known to induce apoptosis with high efficiency in various cell line models. However, here we report the prevalence of necrosis in colon adenocarcinoma HT-29 cells exposed to an extensive range of PDT doses evoked by variations in two variables - hypericin concentration and light dose. Necrosis was the principal mode of cell death despite different PDT doses and the absence of anti-apoptotic Bcl-2 expression. It is likely that the mutation in p53 plays a crucial role in cell death signaling in HT-29. Data indicating proliferation shifting in HT29 cells, incidence of cell death (apoptosis, necrosis and secondary necrosis) and comparison of cytotoxicity and caspase-3 activity of HT29 with HeLa cells are presented. (authors)

BDNF/TrkB signaling protects HT-29 human colon cancer cells from EGFR inhibition

International Nuclear Information System (INIS)

Brunetto de Farias, Caroline; Heinen, Tiago Elias; Pereira dos Santos, Rafael; Abujamra, Ana Lucia; Schwartsmann, Gilberto

2012-01-01

Highlights: ► BDNF protected HT-29 colorectal cancer cells from the antitumor effect of cetuximab. ► TrkB inhibition potentiated the antitumor effect of cetuximab. ► BDNF/TrkB signaling might be involved in resistance to anti-EGFR therapy. -- Abstract: The clinical success of targeted treatment of colorectal cancer (CRC) is often limited by resistance to anti-epidermal growth factor receptor (EGFR) therapy. The neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor TrkB have recently emerged as anticancer targets, and we have previously shown increased BDNF levels in CRC tumor samples. Here we report the findings from in vitro experiments suggesting that BDNF/TrkB signaling can protect CRC cells from the antitumor effects of EGFR blockade. The anti-EGFR monoclonal antibody cetuximab reduced both cell proliferation and the mRNA expression of BDNF and TrkB in human HT-29 CRC cells. The inhibitory effect of cetuximab on cell proliferation and survival was counteracted by the addition of human recombinant BDNF. Finally, the Trk inhibitor K252a synergistically enhanced the effect of cetuximab on cell proliferation, and this effect was blocked by BDNF. These results provide the first evidence that increased BDNF/TrkB signaling might play a role in resistance to EGFR blockade. Moreover, it is possible that targeting TrkB could potentiate the anticancer effects of anti-EGFR therapy.

Mechanisms underlying apoptosis-inducing effects of Kaempferol in HT-29 human colon cancer cells.

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Lee, Hyun Sook; Cho, Han Jin; Yu, Rina; Lee, Ki Won; Chun, Hyang Sook; Park, Jung Han Yoon

2014-02-17

We previously noted that kaempferol, a flavonol present in vegetables and fruits, reduced cell cycle progression of HT-29 cells. To examine whether kaempferol induces apoptosis of HT-29 cells and to explore the underlying molecular mechanisms, cells were treated with various concentrations (0-60 μmol/L) of kaempferol and analyzed by Hoechst staining, Annexin V staining, JC-1 labeling of the mitochondria, immunoprecipitation, in vitro kinase assays, Western blot analyses, and caspase-8 assays. Kaempferol increased chromatin condensation, DNA fragmentation and the number of early apoptotic cells in HT-29 cells in a dose-dependent manner. In addition, kaempferol increased the levels of cleaved caspase-9, caspase-3 and caspase-7 as well as those of cleaved poly (ADP-ribose) polymerase. Moreover, it increased mitochondrial membrane permeability and cytosolic cytochrome c concentrations. Further, kaempferol decreased the levels of Bcl-xL proteins, but increased those of Bik. It also induced a reduction in Akt activation and Akt activity and an increase in mitochondrial Bad. Additionally, kaempferol increased the levels of membrane-bound FAS ligand, decreased those of uncleaved caspase-8 and intact Bid and increased caspase-8 activity. These results indicate that kaempferol induces the apoptosis of HT-29 cells via events associated with the activation of cell surface death receptors and the mitochondrial pathway.

Cyclin D1 negatively regulates the expression of differentiation genes in HT-29 M6 mucus-secreting colon cancer cells.

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Mayo, Clara; Mayol, Xavier

2009-08-28

HT-29 M6 colon cancer cells differentiate to a mucus-secreting phenotype in culture. We found that the pattern of cyclin D1 expression in HT-29 M6 cells did not correlate with instances of cell proliferation but was specifically induced during a dedifferentiation process following disaggregation of epithelial cell layers, even under conditions that did not allow cell cycle reentrance. Interestingly, ectopic expression of cyclin D1 in differentiated cells led to the inhibition of the transcriptional activity of differentiation gene promoters, such as the mucin MUC1. We thus propose that the overexpression of cyclin D1 found in colon cancer favours tumour dedifferentiation as one mechanism of tumour progression.

Protein expression profile of HT-29 human colon cancer cells after treatment with a cytotoxic daunorubicin-GnRH-III derivative bioconjugate.

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Verena Natalie Schreier

Full Text Available Targeted delivery of chemotherapeutic agents is a new approach for the treatment of cancer, which provides increased selectivity and decreased systemic toxicity. We have recently developed a promising drug delivery system, in which the anticancer drug daunorubicin (Dau was attached via oxime bond to a gonadotropin-releasing hormone-III (GnRH-III derivative used as a targeting moiety (Glp-His-Trp-Lys(Ac-His-Asp-Trp-Lys(Da  = Aoa-Pro-Gly-NH2; Glp = pyroglutamic acid, Ac = acetyl; Aoa = aminooxyacetyl. This bioconjugate exerted in vitro cytostatic/cytotoxic effect on human breast, prostate and colon cancer cells, as well as significant in vivo tumor growth inhibitory effect on colon carcinoma bearing mice. In our previous studies, H-Lys(Dau = Aoa-OH was identified as the smallest metabolite produced in the presence of rat liver lysosomal homogenate, which was able to bind to DNA in vitro. To get a deeper insight into the mechanism of action of the bioconjugate, changes in the protein expression profile of HT-29 human colon cancer cells after treatment with the bioconjugate or free daunorubicin were investigated by mass spectrometry-based proteomics. Our results indicate that several metabolism-related proteins, molecular chaperons and proteins involved in signaling are differently expressed after targeted chemotherapeutic treatment, leading to the conclusion that the bioconjugate exerts its cytotoxic action by interfering with multiple intracellular processes.

The natural triterpene maslinic acid induces apoptosis in HT29 colon cancer cells by a JNK-p53-dependent mechanism

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Reyes-Zurita, Fernando J; Pachón-Peña, Gisela; Lizárraga, Daneida; Rufino-Palomares, Eva E; Cascante, Marta; Lupiáñez, José A

2011-01-01

Maslinic acid, a pentacyclic triterpene found in the protective wax-like coating of the leaves and fruit of Olea europaea L., is a promising agent for the prevention of colon cancer. We have shown elsewhere that maslinic acid inhibits cell proliferation to a significant extent and activates mitochondrial apoptosis in colon cancer cells. In our latest work we have investigated further this compound's apoptotic molecular mechanism. We used HT29 adenocarcinoma cells. Changes genotoxicity were analyzed by single-cell gel electrophoresis (comet assay). The cell cycle was determined by flow cytometry. Finally, changes in protein expression were examined by western blotting. Student's t-test was used for statistical comparison. HT29 cells treated with maslinic acid showed significant increases in genotoxicity and cell-cycle arrest during the G0/G1 phase after 72 hours' treatment and an apoptotic sub-G0/G1 peak after 96 hours. Nevertheless, the molecular mechanism for this cytotoxic effect of maslinic acid has never been properly explored. We show here that the anti-tumoral activity of maslinic acid might proceed via p53-mediated apoptosis by acting upon the main signaling components that lead to an increase in p53 activity and the induction of the rest of the factors that participate in the apoptotic pathway. We found that in HT29 cells maslinic acid activated the expression of c-Jun NH2-terminal kinase (JNK), thus inducing p53. Treatment of tumor cells with maslinic acid also resulted in an increase in the expression of Bid and Bax, repression of Bcl-2, release of cytochrome-c and an increase in the expression of caspases -9, -3, and -7. Moreover, maslinic acid produced belated caspase-8 activity, thus amplifying the initial mitochondrial apoptotic signaling. All these results suggest that maslinic acid induces apoptosis in human HT29 colon-cancer cells through the JNK-Bid-mediated mitochondrial apoptotic pathway via the activation of p53. Thus we propose

Cytotoxic effects of Urtica dioica radix on human colon (HT29) and gastric (MKN45) cancer cells mediated through oxidative and apoptotic mechanisms.

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Ghasemi, S; Moradzadeh, M; Mousavi, S H; Sadeghnia, H R

2016-10-15

Defects in the apoptotic pathways are responsible for both the colorectal cancer pathogenesis and resistance to therapy. In this study, we examined the level of cellular oxidants, cytotoxicity and apoptosis induced by hydroalcoholic extract of U. dioica radix (0-2000 µg/mL) and oxaliplatin (0-1000 µg/mL, as positive control) in human gastric (MKN45) and colon (HT29) cancer, as well as normal human foreskin fibroblast (HFF) cells. Exposure to U. dioica or oxaliplatin showed a concentration dependent suppression in cell survival with IC50 values of 24.7, 249.9 and 857.5 µg/mL for HT29, MKN45 and HFF cells after 72 h treatment, respectively. ROS formation and lipid peroxidation were also concentration-dependently increased following treatment with U. dioica, similar to oxaliplatin. In addition, the number of apoptotic cells significantly increased concomitantly with concentration of U. dioica as compared with control cells, which is similar to oxaliplatin and serum-deprived cancer cells. In conclusion, the present study demonstrated that U. dioica inhibited proliferation of gastric and colorectal cancer cells while posing no significant toxic effect on normal cells. U. dioica not only increased levels of oxidants, but also induced concomitant increase of apoptosis. The precise signaling pathway by which U. dioica induce apoptosis needs further research.

BDNF/TrkB signaling protects HT-29 human colon cancer cells from EGFR inhibition

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Brunetto de Farias, Caroline [Cancer Research Laboratory, University Hospital Research Center (CPE-HCPA), Federal University of Rio Grande do Sul, 90035-003 Porto Alegre, RS (Brazil); Children' s Cancer Institute, 90420-140 Porto Alegre, RS (Brazil); Laboratory of Neuropharmacology and Neural Tumor Biology, Department of Pharmacology, Institute for Basic Health Sciences, Federal University of Rio Grande do Sul, 90050-170 Porto Alegre, RS (Brazil); National Institute for Translational Medicine (INCT-TM), 90035-003 Porto Alegre, RS (Brazil); Heinen, Tiago Elias; Pereira dos Santos, Rafael [Cancer Research Laboratory, University Hospital Research Center (CPE-HCPA), Federal University of Rio Grande do Sul, 90035-003 Porto Alegre, RS (Brazil); Laboratory of Neuropharmacology and Neural Tumor Biology, Department of Pharmacology, Institute for Basic Health Sciences, Federal University of Rio Grande do Sul, 90050-170 Porto Alegre, RS (Brazil); National Institute for Translational Medicine (INCT-TM), 90035-003 Porto Alegre, RS (Brazil); Abujamra, Ana Lucia [Cancer Research Laboratory, University Hospital Research Center (CPE-HCPA), Federal University of Rio Grande do Sul, 90035-003 Porto Alegre, RS (Brazil); Children' s Cancer Institute, 90420-140 Porto Alegre, RS (Brazil); National Institute for Translational Medicine (INCT-TM), 90035-003 Porto Alegre, RS (Brazil); Schwartsmann, Gilberto [Cancer Research Laboratory, University Hospital Research Center (CPE-HCPA), Federal University of Rio Grande do Sul, 90035-003 Porto Alegre, RS (Brazil); National Institute for Translational Medicine (INCT-TM), 90035-003 Porto Alegre, RS (Brazil); Department of Internal Medicine, School of Medicine, Federal University of Rio Grande do Sul, 90035-003 Porto Alegre, RS (Brazil); and others

2012-08-24

Highlights: Black-Right-Pointing-Pointer BDNF protected HT-29 colorectal cancer cells from the antitumor effect of cetuximab. Black-Right-Pointing-Pointer TrkB inhibition potentiated the antitumor effect of cetuximab. Black-Right-Pointing-Pointer BDNF/TrkB signaling might be involved in resistance to anti-EGFR therapy. -- Abstract: The clinical success of targeted treatment of colorectal cancer (CRC) is often limited by resistance to anti-epidermal growth factor receptor (EGFR) therapy. The neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor TrkB have recently emerged as anticancer targets, and we have previously shown increased BDNF levels in CRC tumor samples. Here we report the findings from in vitro experiments suggesting that BDNF/TrkB signaling can protect CRC cells from the antitumor effects of EGFR blockade. The anti-EGFR monoclonal antibody cetuximab reduced both cell proliferation and the mRNA expression of BDNF and TrkB in human HT-29 CRC cells. The inhibitory effect of cetuximab on cell proliferation and survival was counteracted by the addition of human recombinant BDNF. Finally, the Trk inhibitor K252a synergistically enhanced the effect of cetuximab on cell proliferation, and this effect was blocked by BDNF. These results provide the first evidence that increased BDNF/TrkB signaling might play a role in resistance to EGFR blockade. Moreover, it is possible that targeting TrkB could potentiate the anticancer effects of anti-EGFR therapy.

Sp1 is a transcription repressor to stanniocalcin-1 expression in TSA-treated human colon cancer cells, HT29.

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Law, Alice Y S; Yeung, B H Y; Ching, L Y; Wong, Chris K C

2011-08-01

Our previous study demonstrated that, stanniocalcin-1 (STC1) was a target of histone deacetylase (HDAC) inhibitors and was involved in trichostatin A (TSA) induced apoptosis in the human colon cancer cells, HT29. In this study, we reported that the transcriptional factor, specificity protein 1 (Sp1) in association with retinoblastoma (Rb) repressed STC1 gene transcription in TSA-treated HT29 cells. Our data demonstrated that, a co-treatment of the cells with TSA and Sp1 inhibitor, mithramycin A (MTM) led to a marked synergistic induction of STC1 transcript levels, STC1 promoter (1 kb)-driven luciferase activity and an increase of apoptotic cell population. The knockdown of Sp1 gene expression in TSA treated cells, revealed the repressor role of Sp1 in STC1 transcription. Using a protein phosphatase inhibitor okadaic acid (OKA), an increase of Sp1 hyperphosphorylation and so a reduction of its transcriptional activity, led to a significant induction of STC1 gene expression. Chromatin immunoprecipitation (ChIP) assay revealed that Sp1 binding on STC1 proximal promoter in TSA treated cells. The binding of Sp1 to STC1 promoter was abolished by the co-treatment of MTM or OKA in TSA-treated cells. Re-ChIP assay illustrated that Sp1-mediated inhibition of STC1 transcription was associated with the recruitment of another repressor molecule, Rb. Collectively our findings identify STC1 is a downstream target of Sp1. Copyright © 2011 Wiley-Liss, Inc.

Curcumin induces apoptosis and cell cycle arrest via the activation of reactive oxygen species-independent mitochondrial apoptotic pathway in Smad4 and p53 mutated colon adenocarcinoma HT29 cells.

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Agarwal, Ayushi; Kasinathan, Akiladdevi; Ganesan, Ramamoorthi; Balasubramanian, Akhila; Bhaskaran, Jahnavi; Suresh, Samyuktha; Srinivasan, Revanth; Aravind, K B; Sivalingam, Nageswaran

2018-03-01

Curcumin is a natural dietary polyphenol compound that has various pharmacological activities such as antiproliferative and cancer-preventive activities on tumor cells. Indeed, the role reactive oxygen species (ROS) generated by curcumin on cell death and cell proliferation inhibition in colon cancer is poorly understood. In the present study, we hypothesized that curcumin-induced ROS may promote apoptosis and cell cycle arrest in colon cancer. To test this hypothesis, the apoptosis-inducing potential and cell cycle inhibition effect of ROS induced by curcumin was investigated in Smd4 and p53 mutated HT-29 colon adenocarcinoma cells. We found that curcumin treatment significantly increased the level of ROS in HT-29 cells in a dose- and time-dependent manner. Furthermore, curcumin treatment markedly decreased the cell viability and proliferation potential of HT-29 cells in a dose- and time-dependent manner. Conversely, generation of ROS and inhibitory effect of curcumin on HT-29 cells were abrogated by N-acetylcysteine treatment. In addition, curcumin treatment did not show any cytotoxic effects on HT-29 cells. Furthermore, curcumin-induced ROS generation caused the DNA fragmentation, chromatin condensation, and cell nuclear shrinkage and significantly increased apoptotic cells in a dose- and time-dependent manner in HT-29 cells. However, pretreatment of N-acetylcysteine inhibited the apoptosis-triggering effect of curcumin-induced ROS in HT-29 cells. In addition, curcumin-induced ROS effectively mediated cell cycle inhibition in HT-29 cells. In conclusion, our data provide the first evidence that curcumin induces ROS independent apoptosis and cell cycle arrest in colon cancer cells that carry mutation on Smad4 and p53. Copyright © 2018. Published by Elsevier Inc.

Adrenaline promotes cell proliferation and increases chemoresistance in colon cancer HT29 cells through induction of miR-155

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Pu, Jun; Bai, Danna; Yang, Xia; Lu, Xiaozhao; Xu, Lijuan; Lu, Jianguo

2012-01-01

Highlights: ► Adrenaline increases colon cancer cell proliferation and its resistance to cisplatin. ► Adrenaline activates NFκB in a dose dependent manner. ► NFκB–miR-155 pathway contributes to cell proliferation and resistance to cisplatin. -- Abstract: Recently, catecholamines have been described as being involved in the regulation of cancer genesis and progression. Here, we reported that adrenaline increased the cell proliferation and decreased the cisplatin induced apoptosis in HT29 cells. Further study found that adrenaline increased miR-155 expression in an NFκB dependent manner. HT29 cells overexpressing miR-155 had a higher cell growth rate and more resistance to cisplatin induced apoptosis. In contrast, HT29 cells overexpressing miR-155 inhibitor displayed decreased cell proliferation and sensitivity to cisplatin induced cell death. In summary, our study here revealed that adrenaline–NFκB–miR-155 pathway at least partially contributes to the psychological stress induced proliferation and chemoresistance in HT29 cells, shedding light on increasing the therapeutic strategies of cancer chemotherapy.

Induction of G1 and G2/M cell cycle arrests by the dietary compound 3,3'-diindolylmethane in HT-29 human colon cancer cells

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Choi Hyun

2009-05-01

Full Text Available Abstract Background 3,3'-Diindolylmethane (DIM, an indole derivative produced in the stomach after the consumption of broccoli and other cruciferous vegetables, has been demonstrated to exert anti-cancer effects in both in vivo and in vitro models. We have previously determined that DIM (0 – 30 μmol/L inhibited the growth of HT-29 human colon cancer cells in a concentration-dependent fashion. In this study, we evaluated the effects of DIM on cell cycle progression in HT-29 cells. Methods HT-29 cells were cultured with various concentrations of DIM (0 – 30 μmol/L and the DNA was stained with propidium iodide, followed by flow cytometric analysis. [3H]Thymidine incorporation assays, Western blot analyses, immunoprecipitation and in vitro kinase assays for cyclin-dependent kinase (CDK and cell division cycle (CDC2 were conducted. Results The percentages of cells in the G1 and G2/M phases were dose-dependently increased and the percentages of cells in S phase were reduced within 12 h in DIM-treated cells. DIM also reduced DNA synthesis in a dose-dependent fashion. DIM markedly reduced CDK2 activity and the levels of phosphorylated retinoblastoma proteins (Rb and E2F-1, and also increased the levels of hypophosphorylated Rb. DIM reduced the protein levels of cyclin A, D1, and CDK4. DIM also increased the protein levels of CDK inhibitors, p21CIP1/WAF1 and p27KIPI. In addition, DIM reduced the activity of CDC2 and the levels of CDC25C phosphatase and cyclin B1. Conclusion Here, we have demonstrated that DIM induces G1 and G2/M phase cell cycle arrest in HT-29 cells, and this effect may be mediated by reduced CDK activity.

Induction of G1 and G2/M cell cycle arrests by the dietary compound 3,3'-diindolylmethane in HT-29 human colon cancer cells.

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Choi, Hyun Ju; Lim, Do Young; Park, Jung Han Yoon

2009-05-29

3,3'-Diindolylmethane (DIM), an indole derivative produced in the stomach after the consumption of broccoli and other cruciferous vegetables, has been demonstrated to exert anti-cancer effects in both in vivo and in vitro models. We have previously determined that DIM (0 - 30 micromol/L) inhibited the growth of HT-29 human colon cancer cells in a concentration-dependent fashion. In this study, we evaluated the effects of DIM on cell cycle progression in HT-29 cells. HT-29 cells were cultured with various concentrations of DIM (0 - 30 micromol/L) and the DNA was stained with propidium iodide, followed by flow cytometric analysis. [3H]Thymidine incorporation assays, Western blot analyses, immunoprecipitation and in vitro kinase assays for cyclin-dependent kinase (CDK) and cell division cycle (CDC)2 were conducted. The percentages of cells in the G1 and G2/M phases were dose-dependently increased and the percentages of cells in S phase were reduced within 12 h in DIM-treated cells. DIM also reduced DNA synthesis in a dose-dependent fashion. DIM markedly reduced CDK2 activity and the levels of phosphorylated retinoblastoma proteins (Rb) and E2F-1, and also increased the levels of hypophosphorylated Rb. DIM reduced the protein levels of cyclin A, D1, and CDK4. DIM also increased the protein levels of CDK inhibitors, p21CIP1/WAF1 and p27KIPI. In addition, DIM reduced the activity of CDC2 and the levels of CDC25C phosphatase and cyclin B1. Here, we have demonstrated that DIM induces G1 and G2/M phase cell cycle arrest in HT-29 cells, and this effect may be mediated by reduced CDK activity.

Metabolism and apoptotic properties of elevated ceramide in HT29(rev) cells

NARCIS (Netherlands)

Veldman, R J; Klappe, K; Hoekstra, D; Kok, J W

1998-01-01

Ceramide (Cer) has been implicated in the regulation of apoptosis. In this study, we elevated cellular Cer levels in human colon-carcinoma (HT29(rev)) cells by incubating the cells in the presence of bacterial sphingomyelinase (bSMase) or, alternatively, in the presence of C2-Cer, a short-chain

Adrenaline promotes cell proliferation and increases chemoresistance in colon cancer HT29 cells through induction of miR-155

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Pu, Jun [Department of General Surgery, Tangdu Hospital of the Fourth Military Medical University, Xi' an 710038 (China); Bai, Danna [Department of Cardiology, 323 Hospital of PLA, Xi' an 710054 (China); Yang, Xia [Department of Teaching and Medical Administration, Tangdu Hospital of the Fourth Military Medical University, Xi' an 710038 (China); Lu, Xiaozhao [Department of Nephrology, The 323 Hospital of PLA, Xi' an 710054 (China); Xu, Lijuan, E-mail: 13609296272@163.com [Department of Nephrology, The 323 Hospital of PLA, Xi' an 710054 (China); Lu, Jianguo, E-mail: lujianguo029@yahoo.com.cn [Department of General Surgery, Tangdu Hospital of the Fourth Military Medical University, Xi' an 710038 (China)

2012-11-16

Highlights: Black-Right-Pointing-Pointer Adrenaline increases colon cancer cell proliferation and its resistance to cisplatin. Black-Right-Pointing-Pointer Adrenaline activates NF{kappa}B in a dose dependent manner. Black-Right-Pointing-Pointer NF{kappa}B-miR-155 pathway contributes to cell proliferation and resistance to cisplatin. -- Abstract: Recently, catecholamines have been described as being involved in the regulation of cancer genesis and progression. Here, we reported that adrenaline increased the cell proliferation and decreased the cisplatin induced apoptosis in HT29 cells. Further study found that adrenaline increased miR-155 expression in an NF{kappa}B dependent manner. HT29 cells overexpressing miR-155 had a higher cell growth rate and more resistance to cisplatin induced apoptosis. In contrast, HT29 cells overexpressing miR-155 inhibitor displayed decreased cell proliferation and sensitivity to cisplatin induced cell death. In summary, our study here revealed that adrenaline-NF{kappa}B-miR-155 pathway at least partially contributes to the psychological stress induced proliferation and chemoresistance in HT29 cells, shedding light on increasing the therapeutic strategies of cancer chemotherapy.

Bovine lactoferrin and lactoferricin exert antitumor activities on human colorectal cancer cells (HT-29) by activating various signaling pathways.

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Jiang, Rulan; Lönnerdal, Bo

2017-02-01

Lactoferrin (Lf) is an iron-binding glycoprotein that is present at high concentrations in milk. Bovine lactoferricin (LfcinB) is a peptide fragment generated by pepsin proteolysis of bovine lactoferrin (bLf). LfcinB consists of amino acid residues 17-41 proximal to the N-terminus of bLf and a disulfide bond between residues 19 and 36, forming a loop. Both bLf and LfcinB have been demonstrated to have antitumor activities. Colorectal cancer is the second most common cause of cancer death in developed countries. We hypothesized that bLf and LfcinB exert antitumor activities on colon cancer cells (HT-29) by triggering various signaling pathways. bLf and LfcinB significantly induced apoptosis in HT-29 cells but not in normal human intestinal epithelial cells, as revealed by the ApoTox-Glo Triplex Assay. The LIVE/DEAD cell viability assay showed that both bLf and LfcinB reduced the viability of HT-29 cells. Transcriptome analysis indicated that bLf, cyclic LfcinB, and linear LfcinB exerted antitumor activities by differentially activating diverse signaling pathways, including p53, apoptosis, and angiopoietin signaling. Immunoblotting results confirmed that both bLf and LfcinBs increased expression of caspase-8, p53, and p21, critical proteins in tumor suppression. These results provide valuable information regarding bLf and LfcinB for potential clinical applications in colon cancer therapy.

Dietary compounds that induce cancer preventive phase 2 enzymes activate apoptosis at comparable doses in HT29 colon carcinoma cells.

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Kirlin, W G; Cai, J; DeLong, M J; Patten, E J; Jones, D P

1999-10-01

Dietary agents that induce glutathione S-transferases and related detoxification systems (Phase 2 enzyme inducers) are thought to prevent cancer by enhancing elimination of chemical carcinogens. The present study shows that compounds of this group (benzyl isothiocyanate, allyl sulfide, dimethyl fumarate, butylated hydroxyanisole) activated apoptosis in human colon carcinoma (HT29) cells in culture over the same concentration ranges that elicited increases in enzyme activity (5-25, 25-100, 10-100, 15-60 micromol/L, respectively). Pretreatment of cells with sodium butyrate, an agent that induces HT29 cell differentiation, resulted in parallel increases in Phase 2 enzyme activities and induction of apoptosis in response to the inducers. Cell death characteristics included apoptotic morphological changes, appearance of cells at sub-G1 phase on flow cytometry, caspase activation, DNA fragmentation and TUNEL-positive staining. The results suggest that dietary Phase 2 inducers may protect against cancer by a mechanism distinct from and in addition to that associated with enhanced elimination of carcinogens. If this occurs in vivo, diets high in such compounds could eliminate precancerous cells by apoptosis at time points well after initial exposure to chemical mutagens and carcinogens.

5-HT3 and 5-HT4 antagonists inhibit peristaltic contractions in guinea-pig distal colon by mechanisms independent of endogenous 5-HT

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Tiong Cheng Sia

2013-08-01

Full Text Available Recent studies have shown that endogenous serotonin is not required for colonic peristalsis in vitro, nor gastrointestinal (GI transit in vivo. However, antagonists of 5-Hydroxytryptamine (5-HT receptors can inhibit peristalsis and GI-transit in mammals, including humans. This raises the question of how these antagonists inhibit GI-motility and transit, if depletion of endogenous 5-HT does not cause any significant inhibitory changes to either GI-motility or transit ? We investigated the mechanism by which 5-HT3 and 5-HT4 antagonists inhibit distension-evoked peristaltic contractions in guinea-pig distal colon. In control animals, repetitive peristaltic contractions of the circular muscle were evoked in response to fixed fecal pellet distension. Distension-evoked peristaltic contractions were unaffected in animals with mucosa and submucosal plexus removed, that were also treated with reserpine (to deplete neuronal 5-HT. In control animals, peristaltic contractions were blocked temporarily by ondansetron (1-10µM and SDZ-205-557 (1-10µM in many animals. Interestingly, after this temporary blockade, and whilst in the continued presence of these antagonists, peristaltic contractions recovered, with characteristics no different from controls. Surprisingly, similar effects were seen in mucosa-free preparations, which had no detectable 5-HT, as detected by mass spectrometry. In summary, distension-evoked peristaltic reflex contractions of the circular muscle layer of the guinea-pig colon can be inhibited temporarily, or permanently, in the same preparation by selective 5-HT3 and 5-HT4 antagonists, depending on the concentration of the antagonists applied. These effects also occur in preparations that lack any detectable 5-HT. We suggest caution should be exercised when interpreting the effects of 5-HT3 and 5-HT4 antagonists; and the role of endogenous 5-HT, in the generation of distension-evoked colonic peristalsis.

Boletus edulis ribonucleic acid - a potent apoptosis inducer in human colon adenocarcinoma cells.

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Lemieszek, Marta Kinga; Ribeiro, Miguel; Guichard Alves, Helena; Marques, Guilhermina; Nunes, Fernando Milheiro; Rzeski, Wojciech

2016-07-13

Despite the large popularity of the Boletus edulis mushroom, little is known about its influence on human health and the possibilities of its therapeutic use. Nevertheless, several reports revealed the usefulness of biopolymers isolated from it in cancer treatment. Our previous studies have shown that B. edulis water soluble biopolymers are not toxic against normal colon epithelial cells (CCD841 CoTr) and at the same concentration range elicited a very prominent antiproliferative effect in colon cancer cells (LS180) which was accompanied with cell cycle arrest in the G0/G1 phase. The purpose of the present study was to verify the proapoptotic properties of a selected fraction from B. edulis - BE3, as well as determine its chemical nature. The BE3 fraction was extracted with hot water and purified by anion-exchange chromatography. Further chemical examinations revealed that BE3 consists mainly of ribonucleic acid (59.1%). The ability of BE3 to induce programmed cell death was examined in human colon cancer cell lines LS180 and HT-29 by measuring caspase activation, DNA fragmentation and expression of BAX, BCL2, TP53 and CDKN1A genes. The sensitivity of colon cancer cells with silenced BAX, TP53 and CDKN1A expression to BE3 treatment was also evaluated. We have demonstrated for the first time that the BE3 fraction is a potent apoptosis inducer in human colon cancer cells. The revealed mechanism of apoptosis triggering was dependent on the presence of functional p53 and consequently was a little different in investigated cell lines. Our results indicated that BE3 stimulated proapoptotic genes BAX (LS180, HT-29), TP53 (LS180) and CDKN1A (HT-29) while at the same time silenced the expression of the key prosurvival gene BCL2 (LS180, HT-29). The obtained results indicate the high therapeutic potential of the BE3 fraction against colon cancer, yet it is necessary to further confirm fraction efficacy and safety in animal and clinical studies.

Short communication: Antiproliferative effect of wild Lactobacillus strains isolated from fermented foods on HT-29 cells.

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Tuo, Y F; Zhang, L W; Yi, H X; Zhang, Y C; Zhang, W Q; Han, X; Du, M; Jiao, Y H; Wang, S M

2010-06-01

In vitro studies, animal models, epidemiology, and human intervention studies provide evidence that some lactic acid bacteria can reduce the risk of certain cancers. In this study, heat-killed bacterial cells, genomic DNA, and cell wall of 7 wild Lactobacillus strains isolated from traditional fermented foods in western China were tested in vitro for cytotoxicity on colonic cancer cell line HT-29 by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The heat-killed bacterial cells, genomic DNA, and cell wall of the 7 strains exhibited direct antiproliferative activities against HT-29 cells. Among the strains, the cellular components of Lactobacillus coryniformis ssp. torquens T3L exerted marked antiproliferative activities against HT-29 cells. The maximum inhibition rates of HT-29 cells by the heat-killed bacterial cells (1x10(7) cfu/mL), cell wall (20 microg of protein/mL) and genomic DNA (100 microg/mL) of L. coryniformis ssp. torquens T3L were 30, 44.9, and 35.9%, respectively. The results indicate that the heat-killed bacterial cells, cell wall, and genomic DNA of the 7 wild Lactobacillus strains could inhibit the growth of HT-29 cells. 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Cell Free DNA of Tumor Origin Induces a 'Metastatic' Expression Profile in HT-29 Cancer Cell Line.

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István Fűri

Full Text Available Epithelial cells in malignant conditions release DNA into the extracellular compartment. Cell free DNA of tumor origin may act as a ligand of DNA sensing mechanisms and mediate changes in epithelial-stromal interactions.To evaluate and compare the potential autocrine and paracrine regulatory effect of normal and malignant epithelial cell-related DNA on TLR9 and STING mediated pathways in HT-29 human colorectal adenocarcinoma cells and normal fibroblasts.DNA isolated from normal and tumorous colonic epithelia of fresh frozen surgically removed tissue samples was used for 24 and 6 hour treatment of HT-29 colon carcinoma and HDF-α fibroblast cells. Whole genome mRNA expression analysis and qRT-PCR was performed for the elements/members of TLR9 signaling pathway. Immunocytochemistry was performed for epithelial markers (i.e. CK20 and E-cadherin, DNA methyltransferase 3a (DNMT3a and NFκB (for treated HDFα cells.Administration of tumor derived DNA on HT29 cells resulted in significant (p<0.05 mRNA level alteration in 118 genes (logFc≥1, p≤0.05, including overexpression of metallothionein genes (i.e. MT1H, MT1X, MT1P2, MT2A, metastasis-associated genes (i.e. TACSTD2, MACC1, MALAT1, tumor biomarker (CEACAM5, metabolic genes (i.e. INSIG1, LIPG, messenger molecule genes (i.e. DAPP, CREB3L2. Increased protein levels of CK20, E-cadherin, and DNMT3a was observed after tumor DNA treatment in HT-29 cells. Healthy DNA treatment affected mRNA expression of 613 genes (logFc≥1, p≤0.05, including increased expression of key adaptor molecules of TLR9 pathway (e.g. MYD88, IRAK2, NFκB, IL8, IL-1β, STING pathway (ADAR, IRF7, CXCL10, CASP1 and the FGF2 gene.DNA from tumorous colon epithelium, but not from the normal epithelial cells acts as a pro-metastatic factor to HT-29 cells through the overexpression of pro-metastatic genes through TLR9/MYD88 independent pathway. In contrast, DNA derived from healthy colonic epithelium induced TLR9 and STING signaling

Human-derived gut microbiota modulates colonic secretion in mice by regulating 5-HT3 receptor expression via acetate production.

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Bhattarai, Yogesh; Schmidt, Bradley A; Linden, David R; Larson, Eric D; Grover, Madhusudan; Beyder, Arthur; Farrugia, Gianrico; Kashyap, Purna C

2017-07-01

Serotonin [5-hydroxytryptamine (5-HT)], an important neurotransmitter and a paracrine messenger in the gastrointestinal tract, regulates intestinal secretion by its action primarily on 5-HT 3 and 5-HT 4 receptors. Recent studies highlight the role of gut microbiota in 5-HT biosynthesis. In this study, we determine whether human-derived gut microbiota affects host secretory response to 5-HT and 5-HT receptor expression. We used proximal colonic mucosa-submucosa preparation from age-matched Swiss Webster germ-free (GF) and humanized (HM; ex-GF colonized with human gut microbiota) mice. 5-HT evoked a significantly greater increase in short-circuit current (Δ I sc ) in GF compared with HM mice. Additionally, 5-HT 3 receptor mRNA and protein expression was significantly higher in GF compared with HM mice. Ondansetron, a 5-HT 3 receptor antagonist, inhibited 5-HT-evoked Δ I sc in GF mice but not in HM mice. Furthermore, a 5-HT 3 receptor-selective agonist, 2-methyl-5-hydroxytryptamine hydrochloride, evoked a significantly higher Δ I sc in GF compared with HM mice. Immunohistochemistry in 5-HT 3A -green fluorescent protein mice localized 5-HT 3 receptor expression to enterochromaffin cells in addition to nerve fibers. The significant difference in 5-HT-evoked Δ I sc between GF and HM mice persisted in the presence of tetrodotoxin (TTX) but was lost after ondansetron application in the presence of TTX. Application of acetate (10 mM) significantly lowered 5-HT 3 receptor mRNA in GF mouse colonoids. We conclude that host secretory response to 5-HT may be modulated by gut microbiota regulation of 5-HT 3 receptor expression via acetate production. Epithelial 5-HT 3 receptor may function as a mediator of gut microbiota-driven change in intestinal secretion. NEW & NOTEWORTHY We found that gut microbiota alters serotonin (5-HT)-evoked intestinal secretion in a 5-HT 3 receptor-dependent mechanism and gut microbiota metabolite acetate alters 5-HT 3 receptor expression in

Biogenic Synthesis, Characterization and Evaluation of Silver Nanoparticles from Aspergillus niger JX556221 Against Human Colon Cancer Cell Line HT-29.

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Chengzheng, Wang; Jiazhi, Wen; Shuangjiang, Chen; Swamy, Mallappa Kumara; Sinniah, Uma Rani; Akhtar, Mohd Sayeed; Umar, Ahmad

2018-05-01

Nanobiotechnology has emerged as a promising technology to develop new therapeutically active nanomaterials. The present study was aimed to biosynthesize AgNPs extracellularly using Aspergillus niger JX556221 fungal extract and to evaluate their anticancer potential against colon cancer cell line, HT-29. UV-visible spectral characterization of the synthesized AgNPs showed higher absorption peak at 440 nm wavelength. Transmission Electron Microscopy (TEM) analysis revealed the monodispersed nature of synthesized AgNPs occurring in spherical shape with a size in the range of 20-25 nm. Further, characterization using Energy Dispersive Spectroscopy (EDX) confirmed the face-centred cubic crystalline structure of metallic AgNPs. FTIR data revealed the occurrence of various phytochemicals in the cell free fungal extract which substantiated the fungal extract mediated AgNPs synthesis. The cytotoxic effect of AgNPs was studied by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results evidenced the cytotoxic effect of AgNPs on HT-29 cell lines in a dose dependent manner. The highest activity was found at 100 μg/ml concentration after 24 h of incubation. Use of propidium iodide staining examination method confirmed the cytotoxic effect of AgNPs through inducing cell apoptosis. AgNPs cytotoxicity was found to be through elevating reactive oxygen species (ROS), and caspase-3 activation resulting in induced apoptosis. Therefore, this research finding provides an insight towards the development of novel anticancer agents using biological sources.

A redox-based mechanism for induction of interleukin-1 production by nitric oxide in a human colonic epithelial cell line (HT29-Cl.16E).

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Vallette, G; Jarry, A; Branka, J E; Laboisse, C L

1996-01-01

We evaluated the effects of two NO donors, sodium nitroprusside (SNP) and 3-morpholino-sydnonimine (SIN-1), characterized by alternative redox states, i.e. nitrosonium ion (NO+) and nitric oxide (NO.) respectively, on intracellular interleukin-1 (IL-1) production, by a human colonic epithelial cell line (HT29-Cl.16E). SNP was able to induce intracellular IL-1 alpha production up to 10 h incubation, in a dose-dependent manner. Several experiments provide evidence that the NO+ redox form, and not the free radical NO., is implicated in the IL-1 alpha production: (i) SIN-1, devoid of any NO+ character, led to a very weak IL-1 production as compared with SNP; (ii) the reductive action of a thiol such as cysteine on NO+ led to a dose-dependent increase in NO, concentration, measured as NO2-/NO3- accumulation, and to large decrease in IL-1 production. Dibutyryl cGMP had no effect on IL-1 production, this finding supporting the concept that a cGMP-independent pathway is involved in the intracellular signalling of NO+. Together these results point out that NO, depending on its redox form, is able to modulate IL-1 production in cultured colonic epithelial cells. PMID:8546706

Carcino-embryonic antigen in monitoring the growth of human colon adenocarcinoma tumour cells SK-CO-1 and HT-29 in vitro and in nude mice

DEFF Research Database (Denmark)

Sölétormos, G; Fogh, J M; Sehested-Hansen, B

1997-01-01

A set of experimental model systems were designed to investigate (a) the inter-relationship between growth of two human cancer cell lines (SK-CO-1, HT-29) and carcino-embryonic antigen (CEA) kinetics; and (b) whether neoplastic growth or CEA concentration is modulated by human growth hormone (hGH...

CD44 regulates cell migration in human colon cancer cells via Lyn kinase and AKT phosphorylation.

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Subramaniam, Venkateswaran; Vincent, Isabella R; Gardner, Helena; Chan, Emily; Dhamko, Helena; Jothy, Serge

2007-10-01

Colon cancer is among the leading causes of cancer death in North America. CD44, an adhesion and antiapoptotic molecule is overexpressed in colon cancer. Cofilin is involved in the directional motility of cells. In the present study, we looked at how CD44 might modulate cell migration in human colon cancer via cofilin. We used a human colon cancer cell line, HT29, which expresses CD44, HT29 where CD44 expression was knocked down by siRNA, SW620, a human colon cancer cell line which does not express CD44, stably transfected exons of CD44 in SW620 cells and the colon from CD44 knockout and wild-type mouse. Western blot analysis of siRNA CD44 lysates showed increased level of AKT phosphorylation and decreased level of cofilin expression. Similar results were also observed with SW620 cells and CD44 knockout mouse colon lysates. Experiments using the AKT phosphorylation inhibitor LY294002 indicate that AKT phosphorylation downregulates cofilin. Immunoprecipitation studies showed CD44 complex formation with Lyn, providing an essential link between CD44 and AKT phosphorylation. LY294002 also stabilized Lyn from phosphorylated AKT, suggesting an interaction between Lyn and AKT phosphorylation. Immunocytochemistry showed that cofilin and Lyn expression were downregulated in siRNA CD44 cells and CD44 knockout mouse colon. siRNA CD44 cells had significantly less migration compared to HT29 vector. Given the well-defined roles of CD44, phosphorylated AKT in apoptosis and cancer, these results indicate that CD44-induced cell migration is dependent on its complex formation with Lyn and its consequent regulation of AKT phosphorylation and cofilin expression.

DNA Tetrahedron Delivery Enhances Doxorubicin-Induced Apoptosis of HT-29 Colon Cancer Cells

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Zhang, Guiyu; Zhang, Zhiyong; Yang, Junen

2017-08-01

As a nano-sized drug carrier with the advantage of modifiability and proper biocompatibility, DNA tetrahedron (DNA tetra) delivery is hopeful to enhance the inhibitory efficiency of nontargeted anticancer drugs. In this investigation, doxorubicin (Dox) was assembled to a folic acid-modified DNA tetra via click chemistry to prepare a targeted antitumor agent. Cellular uptake efficiency was measured via fluorescent imaging. Cytotoxicity, inhibition efficiency, and corresponding mechanism on colon cancer cell line HT-29 were evaluated by MTT assay, cell proliferation curve, western blot, and flow cytometry. No cytotoxicity was induced by DNA tetra, but the cellular uptake ratio increased obviously resulting from the DNA tetra-facilitated penetration through cellular membrane. Accordingly, folic acid-DNA tetra-Dox markedly increased the antitumor efficiency with increased apoptosis levels. In details, 100 μM was the effective concentration and a 6-h incubation period was needed for apoptosis induction. In conclusion, nano-sized DNA tetrahedron was a safe and effective delivery system for Dox and correspondingly enhanced the anticancer efficiency.

Traditional Chinese Medicine Curcumin Sensitizes Human Colon Cancer to Radiation by Altering the Expression of DNA Repair-related Genes.

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Yang, Guangen; Qiu, Jianming; Wang, Dong; Tao, Yong; Song, Yihuan; Wang, Hongtao; Tang, Juping; Wang, Xing; Sun, Y U; Yang, Zhijian; Hoffman, Robert M

2018-01-01

The aim of the present study was to investigate the radio-sensitizing efficacy of curcumin, a traditional Chinese medicine (TCM) on colon cancer cells in vitro and in vivo. Human colon cancer HT-29 cells were treated with curcumin (2.5 μM), irradiation (10 Gy) and the combination of irradiation and curcumin. Cell proliferation was assessed using the MTT assay. Apoptotic cells were detected by Annexin V-PE/7-AAD analysis. PCR was performed to determine differential-expression profiling of 95 DNA-repair genes in irradiated cells and cells treated with both irradiation and curcumin. Differentially-expressed genes were confirmed by Western blotting. In vivo radio-sensitizing efficacy of curcumin was assessed in a xenograft mouse model of HT-29 colon cancer. Curcumin was administrated daily by intraperitoneal injection at 20 mg/kg/dose. Mice received irradiation (10 Gy) twice weekly. Apoptosis of the cancer cells following treatment was determined by TUNEL staining. Irradiation induced proliferation inhibition and apoptosis of HT-29 cells in vitro. Concurrent curcumin treatment sensitized the HT-29 tumor to irradiation (pcurcumin and irradiation compared with irradiation alone (pcurcumin and irradiation resulted in a significantly greater tumor-growth inhibition and apoptosis compared to irradiation treatment alone (pCurcumin sensitizes human colon cancer in vitro and in vivo to radiation. Downregulation of LIG4 and PNKP and upregulation of XRCC5 and CCNH DNA-repair-related genes were involved in the radio-sensitizing efficacy of curcumin in colon cancer. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Recombinant Lactococcus lactis NZ9000 secretes a bioactive kisspeptin that inhibits proliferation and migration of human colon carcinoma HT-29 cells.

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Zhang, Bo; Li, Angdi; Zuo, Fanglei; Yu, Rui; Zeng, Zhu; Ma, Huiqin; Chen, Shangwu

2016-06-10

Proteinaceous bioactive substances and pharmaceuticals are most conveniently administered orally. However, the facing problems are the side effects of proteolytic degradation and denaturation in the gastrointestinal tract. In recent years, lactic acid bacteria (LAB) have been verified to be a promising delivery vector for susceptible functional proteins and drugs. KiSS-1 peptide, a cancer suppressor, plays a critical role in inhibiting cancer metastasis and its activity has been confirmed by direct administration. However, whether this peptide can be functionally expressed in LAB and exert activity on cancer cells, thus providing a potential alternative administration manner in the future, has not been demonstrated. A recombinant Lactococcus lactis strain NZ9000-401-kiss1 harboring a plasmid containing the gene of the tumor metastasis-inhibiting peptide KiSS1 was constructed. After optimization of the nisin induction conditions, the recombinant strain efficiently secreted KiSS1 with a maximum detectable amount of 27.9 μg/ml in Dulbecco's Modified Eagle medium. The 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide and would healing assays, respectively, indicated that the secreted KiSS1 peptide remarkably inhibited HT-29 cell proliferation and migration. Furthermore, the expressed KiSS1 was shown to induce HT-29 cell morphological changes, apoptosis and reduce the expression of matrix metalloproteinase 9 (MMP-9) at both mRNA and protein levels. A recombinant L. lactis NZ9000-401-kiss1 successfully expressing the human kiss1 was constructed. The secreted KiSS1 peptide inhibited human HT-29 cells' proliferation and migration probably by invoking, or mediating the cell-apoptosis pathway and by down regulating MMP-9 expression, respectively. Our results suggest that L. lactis is an ideal cell factory for secretory expression of tumor metastasis-inhibiting peptide KiSS1, and the KiSS1-producing L. lactis strain may serve as a new tool for cancer therapy in

1-(2,6-Dihydroxy-4-methoxyphenyl-2-(4-hydroxyphenyl Ethanone-Induced Cell Cycle Arrest in G1/G0 in HT-29 Cells Human Colon Adenocarcinoma Cells

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Ma Ma Lay

2014-01-01

Full Text Available 1-(2,6-Dihydroxy-4-methoxyphenyl-2-(4-hydroxyphenyl ethanone (DMHE was isolated from the ethyl acetate fraction of Phaleria macrocarpa (Scheff. Boerl fruits and the structure confirmed by GC-MS (gas chromatography-mass spectrometry and NMR (nuclear magnetic resonance analysis. This compound was tested on the HT-29 human colon adenocarcinoma cell line using MTT (method of transcriptional and translational cell proliferation assay. The results of MTT assay showed that DMHE exhibited good cytotoxic effect on HT-29 cells in a dose- and time-dependent manner but no cytotoxic effect on the MRC-5 cell line after 72 h incubation. Morphological features of apoptotic cells upon treatment by DMHE, e.g., cell shrinkage and membrane blebbing, were examined by an inverted and phase microscope. Other features, such as chromatin condension and nuclear fragmentation were studied using acridine orange and propidium iodide staining under the fluorescence microscope. Future evidence of apoptosis/necrosis was provided by result fromannexin V-FITC/PI (fluorescein-isothiocyanate/propidium iodide staining revealed the percentage of early apoptotic, late apoptotic, necrotic and live cells in a dose- and time-dependent manner using flow cytometry. Cell cycle analysis showed G0/G1 arrest in a time-dependent manner. A western blot analysis indicated that cell death might be associated with the up-regulation of the pro-apoptotic proteins Bax PUMA. However, the anit-apotptic proteins Bcl-2, Bcl-xL, and Mcl-1 were also found to increase in a time-dependent manner. The expression of the pro-apoptotic protein Bak was not observed.

Time- and dose-dependent effects of curcumin on gene expression in human colon cancer cells

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van Erk Marjan J

2004-05-01

Full Text Available Abstract Background Curcumin is a spice and a coloring food compound with a promising role in colon cancer prevention. Curcumin protects against development of colon tumors in rats treated with a colon carcinogen, in colon cancer cells curcumin can inhibit cell proliferation and induce apoptosis, it is an anti-oxidant and it can act as an anti-inflammatory agent. The aim of this study was to elucidate mechanisms and effect of curcumin in colon cancer cells using gene expression profiling. Methods Gene expression changes in response to curcumin exposure were studied in two human colon cancer cell lines, using cDNA microarrays with four thousand human genes. HT29 cells were exposed to two different concentrations of curcumin and gene expression changes were followed in time (3, 6, 12, 24 and 48 hours. Gene expression changes after short-term exposure (3 or 6 hours to curcumin were also studied in a second cell type, Caco-2 cells. Results Gene expression changes (>1.5-fold were found at all time points. HT29 cells were more sensitive to curcumin than Caco-2 cells. Early response genes were involved in cell cycle, signal transduction, DNA repair, gene transcription, cell adhesion and xenobiotic metabolism. In HT29 cells curcumin modulated a number of cell cycle genes of which several have a role in transition through the G2/M phase. This corresponded to a cell cycle arrest in the G2/M phase as was observed by flow cytometry. Functional groups with a similar expression profile included genes involved in phase-II metabolism that were induced by curcumin after 12 and 24 hours. Expression of some cytochrome P450 genes was downregulated by curcumin in HT29 and Caco-2 cells. In addition, curcumin affected expression of metallothionein genes, tubulin genes, p53 and other genes involved in colon carcinogenesis. Conclusions This study has extended knowledge on pathways or processes already reported to be affected by curcumin (cell cycle arrest, phase

Neurotensin-induced Erk1/2 phosphorylation and growth of human colonic cancer cells are independent from growth factors receptors activation

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Massa, Fabienne; Tormo, Aurelie; Beraud-Dufour, Sophie; Coppola, Thierry [Institut de Pharmacologie Moleculaire et Cellulaire, Universite de Nice-Sophia Antipolis, CNRS UMR 6097, 660 route des Lucioles, 06560 Valbonne (France); Mazella, Jean, E-mail: mazella@ipmc.cnrs.fr [Institut de Pharmacologie Moleculaire et Cellulaire, Universite de Nice-Sophia Antipolis, CNRS UMR 6097, 660 route des Lucioles, 06560 Valbonne (France)

2011-10-14

Highlights: {yields} We compare intracellular pathways of NT and EGF in HT29 cells. {yields} NT does not transactivate EGFR. {yields} Transactivation of EGFR is not a general rule in cancer cell growth. -- Abstract: Neurotensin (NT) promotes the proliferation of human colonic cancer cells by undefined mechanisms. We already demonstrated that, in the human colon adenocarcinoma cell line HT29, the effects of NT were mediated by a complex formed between the NT receptor-1 (NTSR1) and-3 (NTSR3). Here we examined cellular mechanisms that led to NT-induced MAP kinase phosphorylation and growth factors receptors transactivation in colonic cancer cells and proliferation in HT29 cells. With the aim to identify upstream signaling involved in NT-elicited MAP kinase activation, we found that the stimulatory effects of the peptide were totally independent from the activation of the epidermal growth factor receptor (EGFR) both in the HT29 and the HCT116 cells. NT was unable to promote phosphorylation of EGFR and to compete with EGF for its binding to the receptor. Pharmacological approaches allowed us to differentiate EGF and NT signaling in HT29 cells since only NT activation of Erk1/2 was shown to be sensitive to PKC inhibitors and since only NT increased the intracellular level of calcium. We also observed that NT was not able to transactivate Insulin-like growth factor receptor. Our findings indicate that, in the HT29 and HCT116 cell lines, NT stimulates MAP kinase phosphorylation and cell growth by a pathway which does not involve EGF system but rather NT receptors which transduce their own intracellular effectors. These results indicate that depending on the cell line used, blocking EGFR is not the general rule to inhibit NT-induced cancer cell proliferation.

Micropropagation effect on the anti-carcinogenic activitiy of polyphenolics from Mexican oregano (Poliomintha glabrescens Gray) in human colon cancer cells HT-29.

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García-Pérez, Enrique; Noratto, Giuliana D; García-Lara, Silverio; Gutiérrez-Uribe, Janet A; Mertens-Talcott, Susanne U

2013-06-01

Phenolic extracts obtained from spices are known to have anti-carcinogenic activities but little is known about the effect of micropropagation on these beneficial effects. The main objective of this study was to evaluate the cytotoxic activity of flavonoid-enriched extracts (FEE) from the leaves of wild (WT), in vitro (IN), and ex vitro (EX) grown oregano plants in colon cancer cells HT-29 and the non-cancer cells CCD-18Co. Cell proliferation of HT-29 cells was reduced to 50 % by WT, IN, and EX at concentrations of 4.01, 1.32, and 4.84 mg of gallic acid equivalents (GAE)/L, respectively. In contrast, in CCD-18Co cells, higher concentrations were required for the same cytotoxic effect. At 6 mg GAE/L, WT and IN reduced the production of reactive oxygen species (ROS) of lipopolysaccharides (LPS)-stimulated control cells to 59.89 and 59.43 %, respectively, and EX to 73.89 %. The mRNA of Caspase-3 was increased 1.53-fold when cells were treated with 4 mg GAE/L of IN extract, and tumor necrosis factor receptor superfamily, member 6 (FAS), and BCL2-associated X protein (BAX) mRNA increased 2.55 and 1.53 fold, respectively. Results on protein expression corroborated the apoptotic effects with a significant decrease of B-cell lymphoma 2 (BCL2) expression for all treatments but more remarkable for EX that also showed the most intense signal of BAX. Overall, FEE extracts derived from micropropagation had increased pro-apoptotic effects, however extracts from the in vitro plants produced more efficacy at the transcriptional level while extracts from the ex vitro plant were superior at the traductional level.

Cathelicidin suppresses colon cancer development by inhibition of cancer associated fibroblasts

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Cheng M

2014-12-01

Full Text Available Michelle Cheng,1,* Samantha Ho,1,* Jun Hwan Yoo,1,2,* Deanna Hoang-Yen Tran,1,* Kyriaki Bakirtzi,1 Bowei Su,1 Diana Hoang-Ngoc Tran,1 Yuzu Kubota,1 Ryan Ichikawa,1 Hon Wai Koon1 1Center for Inflammatory Bowel Diseases, Division of Digestive Diseases, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA; 2Digestive Disease Center, CHA University Bundang Medical Center, Seongnam, Republic of Korea *These authors share co-first authorship Background: Cathelicidin (LL-37 in humans and mCRAMP in mice represents a family of endogenous antimicrobial and anti-inflammatory peptides. Cancer-associated fibroblasts can promote the proliferation of colon cancer cells and growth of colon cancer tumors. Methods: We examined the role of cathelicidin in the development of colon cancer, using subcutaneous human HT-29 colon-cancer-cell-derived tumor model in nude mice and azoxymethane- and dextran sulfate-mediated colon cancer model in C57BL/6 mice. We also determined the indirect antitumoral mechanism of cathelicidin via the inhibition of epithelial–mesenchymal transition (EMT of colon cancer cells and fibroblast-supported colon cancer cell proliferation. Results: Intravenous administration of cathelicidin expressing adeno-associated virus significantly reduced the size of tumors, tumor-derived collagen expression, and tumor-derived fibroblast expression in HT-29-derived subcutaneous tumors in nude mice. Enema administration of the mouse cathelicidin peptide significantly reduced the size and number of colonic tumors in azoxymethane- and dextran sulfate-treated mice without inducing apoptosis in tumors and the adjacent normal colonic tissues. Cathelicidin inhibited the collagen expression and vimentin-positive fibroblast expression in colonic tumors. Cathelicidin did not directly affect HT-29 cell viability, but did significantly reduce tumor growth factor-ß1-induced EMT of colon cancer cells. Media conditioned by the

In Vitro Evaluation of the Impact of the Probiotic E. coli Nissle 1917 on Campylobacter jejuni's Invasion and Intracellular Survival in Human Colonic Cells.

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Helmy, Yosra A; Kassem, Issmat I; Kumar, Anand; Rajashekara, Gireesh

2017-01-01

Campylobacter jejuni is a leading cause of bacterial food poisoning in humans. Due to the rise in antibiotic-resistant Campylobacter , there exists a need to develop antibiotic-independent interventions to control infections in humans. Here, we evaluated the impact of Escherichia coli Nissle 1917 (EcN), a probiotic strain, on C. jejuni's invasion and intracellular survival in polarized human colonic cells (HT-29). To further understand how EcN mediates its impact, the expression of 84 genes associated with tight junctions and cell adhesion was profiled in HT-29 cells after treatment with EcN and challenge with C. jejuni . The pre-treatment of polarized HT-29 cells with EcN for 4 h showed a significant effect on C. jejuni 's invasion (∼2 log reduction) of the colonic cells. Furthermore, no intracellular C. jejuni were recovered from EcN pre-treated HT-29 cells at 24 h post-infection. Other probiotic strains tested had no significant impact on C. jejuni invasion and intracellular survival. C. jejuni decreased the expression of genes associated with epithelial cells permeability and barrier function in untreated HT-29 cells. However, EcN positively affected the expression of genes that are involved in enhanced intestinal barrier function, decreased cell permeability, and increased tight junction integrity. The results suggest that EcN impedes C. jejuni invasion and subsequent intracellular survival by affecting HT-29 cells barrier function and tight junction integrity. We conclude that EcN might be a viable alternative for controlling C. jejuni infections.

Cytotoxicity effect of Zataria multiflora Boiss. on two human colon carcinoma cell lines

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F. Sharififar

2017-10-01

Full Text Available Background and objectives: Natural products are one of the major sources for investigations of novel medicines. Zataria multiflora Boiss (ZM has shown pharmacological activities especially in gastrointestinal tract; however, there are limited studies about its cytotoxicity effects. In this study, the effect of Zataria multiflora was examined on two colon cancer cell lines (SW-48 and HT-29. Methods: Hydro-alcoholic extract of ZM and its fractions including chloroform, petroleum ether and methanol extract were prepared by warm maceration method. Different concentrations were prepared and examined on SW-48 and HT-29 cell lines using 2-(4, 5-dimethylthiazol-2-yl 2, 5-diphenyltetrazolium bromide (MTT assay. Results: The results of the present study have shown the cytotoxic effect of some fractions of ZM. The most considerable cytotoxic effect was shown against HT-29 cell line. Also, total ZM extract and the petroleum ether fraction demonstrated cytotoxic effects with IC50 values of 44.22 and 33.42 µg/ml on SW-48 and HT-29 cell lines, respectively. Conclusion: Zataria multiflora was cytotoxic to against colon cancer cell lines HT-29 and SW-48.

Combined ginger extract & Gelam honey modulate Ras/ERK and PI3K/AKT pathway genes in colon cancer HT29 cells.

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Tahir, Analhuda Abdullah; Sani, Nur Fathiah Abdul; Murad, Noor Azian; Makpol, Suzana; Ngah, Wan Zurinah Wan; Yusof, Yasmin Anum Mohd

2015-04-01

The interconnected Ras/ERK and PI3K/AKT pathways play a central role in colorectal tumorigenesis, and they are targets for elucidating mechanisms involved in attempts to induce colon cancer cell death. Both ginger (Zingiber officinale) and honey have been shown to exhibit anti-tumor and anti-inflammation properties against many types of cancer, including colorectal cancer. However, there are currently no reports showing the combined effect of these two dietary compounds in cancer growth inhibition. The aim of this study was to evaluate the synergistic effect of crude ginger extract and Gelam honey in combination as potential cancer chemopreventive agents against the colorectal cancer cell line HT29. The cells were divided into 4 groups: the first group represents HT29 cells without treatment, the second and third groups were cells treated singly with either ginger or Gelam honey, respectively, and the last group represents cells treated with ginger and Gelam honey combined. The results of MTS assay showed that the IC50 of ginger and Gelam honey alone were 5.2 mg/ml and 80 mg/ml, respectively, whereas the IC50 of the combination treatment was 3 mg/ml of ginger plus 27 mg/ml of Gelam honey with a combination index of ginger and Gelam honey treatment was associated with the stimulation of early apoptosis (upregulation of caspase 9 and IκB genes) accompanied by downregulation of the KRAS, ERK, AKT, Bcl-xL, NFkB (p65) genes in a synergistic manner. In conclusion, the combination of ginger and Gelam honey may be an effective chemopreventive and therapeutic strategy for inducing the death of colon cancer cells.

Piper betle leaf extract enhances the cytotoxicity effect of 5-fluorouracil in inhibiting the growth of HT29 and HCT116 colon cancer cells*

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Ng, Pek Leng; Rajab, Nor Fadilah; Then, Sue Mian; Mohd Yusof, Yasmin Anum; Wan Ngah, Wan Zurinah; Pin, Kar Yong; Looi, Mee Lee

2014-01-01

Objective: The combination effect of Piper betle (PB) and 5-fluorouracil (5-FU) in enhancing the cytotoxic potential of 5-FU in inhibiting the growth of colon cancer cells was investigated. Methods: HT29 and HCT116 cells were subjected to 5-FU or PB treatment. 5-FU and PB were then combined and their effects on both cell lines were observed after 24 h of treatment. PB-5-FU interaction was elucidated by isobologram analysis. Apoptosis features of the treated cells were revealed by annexin V/PI stain. High-performance liquid chromatography (HPLC) was performed to exclude any possible chemical interaction between the compounds. Results: In the presence of PB extract, the cytotoxicity of 5-FU was observed at a lower dose (IC50 12.5 μmol/L) and a shorter time (24 h) in both cell lines. Both cell lines treated with 5-FU or PB alone induced a greater apoptosis effect compared with the combination treatment. Isobologram analysis indicated that PB and 5-FU interacted synergistically and antagonistically in inhibiting the growth of HT29 and HCT116 cells, respectively. Conclusions: In the presence of PB, a lower dosage of 5-FU is required to achieve the maximum drug effect in inhibiting the growth of HT29 cells. However, PB did not significantly reduce 5-FU dosage in HCT116 cells. Our result showed that this interaction may not solely contribute to the apoptosis pathway. PMID:25091987

Piper betle leaf extract enhances the cytotoxicity effect of 5-fluorouracil in inhibiting the growth of HT29 and HCT116 colon cancer cells.

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Ng, Pek Leng; Rajab, Nor Fadilah; Then, Sue Mian; Mohd Yusof, Yasmin Anum; Wan Ngah, Wan Zurinah; Pin, Kar Yong; Looi, Mee Lee

2014-08-01

The combination effect of Piper betle (PB) and 5-fluorouracil (5-FU) in enhancing the cytotoxic potential of 5-FU in inhibiting the growth of colon cancer cells was investigated. HT29 and HCT116 cells were subjected to 5-FU or PB treatment. 5-FU and PB were then combined and their effects on both cell lines were observed after 24 h of treatment. PB-5-FU interaction was elucidated by isobologram analysis. Apoptosis features of the treated cells were revealed by annexin V/PI stain. High-performance liquid chromatography (HPLC) was performed to exclude any possible chemical interaction between the compounds. In the presence of PB extract, the cytotoxicity of 5-FU was observed at a lower dose (IC50 12.5 µmol/L) and a shorter time (24 h) in both cell lines. Both cell lines treated with 5-FU or PB alone induced a greater apoptosis effect compared with the combination treatment. Isobologram analysis indicated that PB and 5-FU interacted synergistically and antagonistically in inhibiting the growth of HT29 and HCT116 cells, respectively. In the presence of PB, a lower dosage of 5-FU is required to achieve the maximum drug effect in inhibiting the growth of HT29 cells. However, PB did not significantly reduce 5-FU dosage in HCT116 cells. Our result showed that this interaction may not solely contribute to the apoptosis pathway.

Modulation of pathogen-induced CCL20 secretion from HT-29 human intestinal epithelial cells by commensal bacteria.

LENUS (Irish Health Repository)

Sibartie, Shomik

2009-01-01

BACKGROUND: Human intestinal epithelial cells (IECs) secrete the chemokine CCL20 in response to infection by various enteropathogenic bacteria or exposure to bacterial flagellin. CCL20 recruits immature dendritic cells and lymphocytes to target sites. Here we investigated IEC responses to various pathogenic and commensal bacteria as well as the modulatory effects of commensal bacteria on pathogen-induced CCL20 secretion. HT-29 human IECs were incubated with commensal bacteria (Bifidobacterium infantis or Lactobacillus salivarius), or with Salmonella typhimurium, its flagellin, Clostridium difficile, Mycobacterium paratuberculosis, or Mycobacterium smegmatis for varying times. In some studies, HT-29 cells were pre-treated with a commensal strain for 2 hr prior to infection or flagellin stimulation. CCL20 and interleukin (IL)-8 secretion and nuclear factor (NF)-kappaB activation were measured using enzyme-linked immunosorbent assays. RESULTS: Compared to untreated cells, S. typhimurium, C. difficile, M. paratuberculosis, and flagellin activated NF-kappaB and stimulated significant secretion of CCL20 and IL-8 by HT-29 cells. Conversely, B. infantis, L. salivarius or M. smegmatis did not activate NF-kappaB or augment CCL20 or IL-8 production. Treatment with B. infantis, but not L. salivarius, dose-dependently inhibited the baseline secretion of CCL20. In cells pre-treated with B. infantis, C. difficile-, S. typhimurium-, and flagellin-induced CCL20 were significantly attenuated. B. infantis did not limit M. Paratuberculosis-induced CCL20 secretion. CONCLUSION: This study is the first to demonstrate that a commensal strain can attenuate CCL20 secretion in HT-29 IECs. Collectively, the data indicate that M. paratuberculosis may mediate mucosal damage and that B. infantis can exert immunomodulatory effects on IECs that mediate host responses to flagellin and flagellated enteric pathogens.

Inhibition of human MCF-7 breast cancer cells and HT-29 colon cancer cells by rice-produced recombinant human insulin-like growth binding protein-3 (rhIGFBP-3.

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Stanley C K Cheung

Full Text Available BACKGROUND: Insulin-like growth factor binding protein-3 (IGFBP-3 is a multifunctional molecule which is closely related to cell growth, apoptosis, angiogenesis, metabolism and senescence. It combines with insulin-like growth factor-I (IGF-I to form a complex (IGF-I/IGFBP-3 that can treat growth hormone insensitivity syndrome (GHIS and reduce insulin requirement in patients with diabetes. IGFBP-3 alone has been shown to have anti-proliferation effect on numerous cancer cells. METHODOLOGY/PRINCIPAL FINDINGS: We reported here an expression method to produce functional recombinant human IGFBP-3 (rhIGFBP-3 in transgenic rice grains. Protein sorting sequences, signal peptide and endoplasmic reticulum retention tetrapeptide (KDEL were included in constructs for enhancing rhIGFBP-3 expression. Western blot analysis showed that only the constructs with signal peptide were successfully expressed in transgenic rice grains. Both rhIGFBP-3 proteins, with or without KDEL sorting sequence inhibited the growth of MCF-7 human breast cancer cells (65.76 ± 1.72% vs 45.00 ± 0.86%, p < 0.05; 50.84 ± 1.97% vs 45.00 ± 0.86%, p < 0.01 respectively and HT-29 colon cancer cells (65.14 ± 3.84% vs 18.01 ± 13.81%, p < 0.05 and 54.7 ± 9.44% vs 18.01 ± 13.81%, p < 0.05 respectively when compared with wild type rice. CONCLUSION/SIGNIFICANCE: These findings demonstrated the feasibility of producing biological active rhIGFBP-3 in rice using a transgenic approach, which will definitely encourage more research on the therapeutic use of hIGFBP-3 in future.

Pharmacological profile of DA-6886, a novel 5-HT4 receptor agonist to accelerate colonic motor activity in mice.

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Lee, Min Jung; Cho, Kang Hun; Park, Hyun Min; Sung, Hyun Jung; Choi, Sunghak; Im, Weonbin

2014-07-15

DA-6886, the gastrointestinal prokinetic benzamide derivative is a novel 5-HT4 receptor agonist being developed for the treatment of constipation-predominant irritable bowel syndrome (IBS-C). The purpose of this study was to characterize in vitro and in vivo pharmacological profile of DA-6886. We used various receptor binding assay, cAMP accumulation assay, organ bath experiment and colonic transit assay in normal and chemically constipated mice. DA-6886 exhibited high affinity and selectivity to human 5-HT4 receptor splice variants, with mean pKi of 7.1, 7.5, 7.9 for the human 5-HT4a, 5-HT4b and 5-HT4d, respectively. By contrast, DA-6886 did not show significant affinity for several receptors including dopamine D2 receptor, other 5-HT receptors except for 5-HT2B receptor (pKi value of 6.2). The affinity for 5-HT4 receptor was translated into functional agonist activity in Cos-7 cells expressing 5-HT4 receptor splice variants. Furthermore, DA-6886 induced relaxation of the rat oesophagus preparation (pEC50 value of 7.4) in a 5-HT4 receptor antagonist-sensitive manner. The evaluation of DA-6886 in CHO cells expressing hERG channels revealed that it inhibited hERG channel current with an pIC50 value of 4.3, indicating that the compound was 1000-fold more selective for the 5-HT4 receptor over hERG channels. In the normal ICR mice, oral administration of DA-6886 (0.4 and 2mg/kg) resulted in marked stimulation of colonic transit. Furthermore, in the loperamide-induced constipation mouse model, 2mg/kg of DA-6886 significantly improved the delay of colonic transit, similar to 10mg/kg of tegaserod. Taken together, DA-6886 is a highly potent and selective 5-HT4 receptor agonist to accelerate colonic transit in mice, which might be therapeutic agent having a favorable safety profile in the treatment of gastrointestinal motor disorders such as IBS-C and chronic constipation. Copyright © 2014 Elsevier B.V. All rights reserved.

Musa paradisiaca inflorescence induces human colon cancer cell death by modulating cascades of transcriptional events.

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K B, Arun; Madhavan, Aravind; T R, Reshmitha; Thomas, Sithara; Nisha, P

2018-01-24

Colorectal cancer (CRC) is one of the leading causes of cancer death, and diet plays an important role in the etiology of CRC. Traditional medical practitioners in many South Asian countries use plantain inflorescence to treat various gastro-intestinal ailments. The aim of the present study was to investigate the anticancer effects of extracts of inflorescence of Musa paradisiaca against HT29 human colon cancer cells and elucidate the mechanism of these effects by studying the modulation of cascades of transcriptional events. In vitro assays depicted that methanol extract of Musa paradisiaca inflorescence (PIMET) was cytotoxic to HT29 cells. PIMET induced DNA damage and arrested the cell cycle at the G2/M phase. Expression studies showed that PIMET pretreatment upregulates pro-apoptotic Bcl2 and downregulates anti-apoptotic Bax proteins. Different assays showed that the deregulation of pro/antiapoptotic proteins reduces the mitochondrial membrane potential and ATP production; moreover, it enhances cytochrome c release, which triggers the apoptotic pathway, and further cleaves caspase 3 and PARP proteins, resulting in apoptosis. Changes in the protein expression profile of HT29 cells after PIMET treatment were analyzed using mass-spectrometry-based proteomics. PIMET treatment significantly altered the expression of HT29 protein; interestingly, X-linked inhibitor of apoptosis protein was also downregulated. Alteration in the expression of this protein has significant effects, leading to HT29 cell death.

2-Dodecylcyclobutanone, a radiolytic product of palmitic acid, is genotoxic in primary human colon cells and in cells from preneoplastic lesions

International Nuclear Information System (INIS)

Knoll, Nadine; Weise, Anja; Claussen, Uwe; Sendt, Wolfgang; Marian, Brigitte; Glei, Michael; Pool-Zobel, Beatrice L.

2006-01-01

The irradiation of fat results in the formation of 2-alkylcyclobutanones, a new class of food contaminants. Results of previous in vitro studies with primary human colon cells and in vivo experiments with rats fed with 2-alkylcyclobutanones indicated that these radiolytic derivatives may be genotoxic and enhance the progression of colon tumors. The underlying mechanisms of these effects, however, are not clearly understood. Therefore we performed additional investigations to elucidate the genotoxic potential of 2-dodecylcyclobutanone (2dDCB) that is generated from palmitic acid. In particular, we explored the relative sensitivities of human colon cells, representing different stages of tumor development and healthy colon tissues, respectively. HT29clone19A cells, LT97 adenoma cells and primary human epithelial cells were exposed to 2dDCB (150-2097 μM). We determined cytotoxic effects using trypan blue exclusion. Genotoxicity, reflected as strand breaks, was assessed using the alkaline version of the comet assay and chromosomal abnormalities were investigated by 24-color fluorescence-in-situ-hybridization. 2dDCB was cytotoxic in a time- and dose-dependent manner in LT97 adenoma cells and in freshly isolated primary cells but not in the human colon tumor cell line. Associated with this was a marked induction of DNA damage by 2dDCB in LT97 adenoma cells and in freshly isolated colonocytes, whereas in the HT29clone19A cells no strand breaks were detectable. A long-term incubation of LT97 adenoma cells with lower concentrations of 2dDCB revealed cytogenetic effects. In summary, 2dDCB was clearly genotoxic in healthy human colon epithelial cells and in cells representing preneoplastic colon adenoma. These findings provide additional evidence that this compound may be regarded as a possible risk factor for processes in colon carcinogenesis related to initiation and progression

Inactivation of Adenomatous Polyposis Coli Reduces Bile Acid/Farnesoid X Receptor Expression through Fxr gene CpG Methylation in Mouse Colon Tumors and Human Colon Cancer Cells.

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Selmin, Ornella I; Fang, Changming; Lyon, Adam M; Doetschman, Tom C; Thompson, Patricia A; Martinez, Jesse D; Smith, Jeffrey W; Lance, Peter M; Romagnolo, Donato F

2016-02-01

The farnesoid X receptor (FXR) regulates bile acid (BA) metabolism and possesses tumor suppressor functions. FXR expression is reduced in colorectal tumors of subjects carrying inactivated adenomatous polyposis coli (APC). Identifying the mechanisms responsible for this reduction may offer new molecular targets for colon cancer prevention. We investigated how APC inactivation influences the regulation of FXR expression in colonic mucosal cells. We hypothesized that APC inactivation would epigenetically repress nuclear receptor subfamily 1, group H, member 4 (FXR gene name) expression through increased CpG methylation. Normal proximal colonic mucosa and normal-appearing adjacent colonic mucosa and colon tumors were collected from wild-type C57BL/6J and Apc-deficient (Apc(Min) (/+)) male mice, respectively. The expression of Fxr, ileal bile acid-binding protein (Ibabp), small heterodimer partner (Shp), and cyclooxygenase-2 (Cox-2) were determined by real-time polymerase chain reaction. In both normal and adjacent colonic mucosa and colon tumors, we measured CpG methylation of Fxr in bisulfonated genomic DNA. In vitro, we measured the impact of APC inactivation and deoxycholic acid (DCA) treatment on FXR expression in human colon cancer HCT-116 cells transfected with silencing RNA for APC and HT-29 cells carrying inactivated APC. In Apc(Min) (/+) mice, constitutive CpG methylation of the Fxrα3/4 promoter was linked to reduced (60-90%) baseline Fxr, Ibabp, and Shp and increased Cox-2 expression in apparently normal adjacent mucosa and colon tumors. Apc knockdown in HCT-116 cells increased cellular myelocytomatosis (c-MYC) and lowered (∼50%) FXR expression, which was further reduced (∼80%) by DCA. In human HCT-116 but not HT-29 colon cancer cells, DCA induced FXR expression and lowered CpG methylation of FXR. We conclude that the loss of APC function favors the silencing of FXR expression through CpG hypermethylation in mouse colonic mucosa and human colon cells

Development of a serum-free co-culture of human intestinal epithelium cell-lines (Caco-2/HT29-5M21)

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Nollevaux, Géraldine; Devillé, Christelle; El Moualij, Benaïssa; Zorzi, Willy; Deloyer, Patricia; Schneider, Yves-Jacques; Peulen, Olivier; Dandrifosse, Guy

2006-01-01

Background The absorptive and goblet cells are the main cellular types encountered in the intestine epithelium. The cell lineage Caco-2 is a model commonly used to reproduce the features of the bowel epithelium. However, there is a strong debate regarding the value of Caco-2 cell culture to mimick in vivo situation. Indeed, some authors report in Caco-2 a low paracellular permeability and an ease of access of highly diffusible small molecules to the microvilli, due to an almost complete lack of mucus. The HT29-5M21 intestinal cell lineage is a mucin-secreting cellular population. A co-culture system carried out in a serum-free medium and comprising both Caco-2 and HT29-5M21 cells was developed. The systematic use of a co-culture system requires the characterization of the monolayer under a given experimental procedure. Results In this study, we investigated the activity and localization of the alkaline phosphatase and the expression of IAP and MUC5AC genes to determine a correlation between these markers and the cellular composition of a differentiated monolayer obtained from a mixture of Caco-2 and HT29-5M21 cells. We observed that the culture conditions used (serum-free medium) did not change the phenotype of each cell type, and produced a reproducible model. The alkaline phosphatase expression characterizing Caco-2 cells was influenced by the presence of HT29-5M21 cells. Conclusion The culture formed by 75% Caco-2 and 25% HT29-5M21 produce a monolayer containing the two main cell types of human intestinal epithelium and characterized by a reduced permeability to macromolecules. PMID:16670004

Plant Polyphenols and Oxidative Metabolites of the Herbal Alkenylbenzene Methyleugenol Suppress Histone Deacetylase Activity in Human Colon Carcinoma Cells

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Isabel Anna Maria Groh

2013-01-01

Full Text Available Evidence has been provided that diet and environmental factors directly influence epigenetic mechanisms associated with cancer development in humans. The inhibition of histone deacetylase (HDAC activity and the disruption of the HDAC complex have been recognized as a potent strategy for cancer therapy and chemoprevention. In the present study, we investigated whether selected plant constituents affect HDAC activity or HDAC1 protein status in the human colon carcinoma cell line HT29. The polyphenols (−-epigallocatechin-3-gallate (EGCG and genistein (GEN as well as two oxidative methyleugenol (ME metabolites were shown to inhibit HDAC activity in intact HT29 cells. Concomitantly, a significant decrease of the HDAC1 protein level was observed after incubation with EGCG and GEN, whereas the investigated ME metabolites did not affect HDAC1 protein status. In conclusion, dietary compounds were found to possess promising HDAC-inhibitory properties, contributing to epigenetic alterations in colon tumor cells, which should be taken into account in further risk/benefit assessments of polyphenols and alkenylbenzenes.

Aqueous Fraction of Nephelium ramboutan-ake Rind Induces Mitochondrial-Mediated Apoptosis in HT-29 Human Colorectal Adenocarcinoma Cells

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Muhamad Noor Alfarizal Kamarudin

2012-05-01

Full Text Available The aim of this study was to investigate the cytotoxic and apoptotic effects of Nephelium ramboutan-ake (pulasan rind in selected human cancer cell lines. The crude ethanol extract and fractions (ethyl acetate and aqueous of N. ramboutan-ake inhibited the growth of HT-29, HCT-116, MDA-MB-231, Ca Ski cells according to MTT assays. The N. ramboutan-ake aqueous fraction (NRAF was found to exert the greatest cytotoxic effect against HT-29 in a dose-dependent manner. Evidence of apoptotic cell death was revealed by features such as chromatin condensation, nuclear fragmentation and apoptotic body formation. The result from a TUNEL assay strongly suggested that NRAF brings about DNA fragmentation in HT-29 cells. Phosphatidylserine (PS externalization on the outer leaflet of plasma membranes was detected with annexin V-FITC/PI binding, confirming the early stage of apoptosis. The mitochondrial permeability transition is an important step in the induction of cellular apoptosis, and the results clearly suggested that NRAF led to collapse of mitochondrial transmembrane potential in HT-29 cells. This attenuation of mitochondrial membrane potential (Δψm was accompanied by increased production of ROS and depletion of GSH, an increase of Bax protein expression, and induced-activation of caspase-3/7 and caspase-9. These combined results suggest that NRAF induces mitochondrial-mediated apoptosis.

Control de la diferenciación celular in vitro en células HT-29 de cáncer colorectal

OpenAIRE

Mayo de las Casas, Clara de la Caridad

2005-01-01

La línea celular HT-29 M6 es una línea tumoral humana derivada de adenocarcinoma de colon, con capacidad de diferenciación in vitro hacia un fenotipo mucosecretor, obtenida por selección con 10-7 M y 10-6 M de metotrexato, en tratamientos sucesivos, de la línea parental indiferenciada HT-29 (Lesuffleur T, et al, 1990). Nosotros utilizamos esta línea celular como modelo para estudiar el proceso de diferenciación in vitro que ocurre de manera espontánea durante el crecimiento hacia confluencia....

Anti-proliferative effects of Bifidobacterium adolescentis SPM0212 extract on human colon cancer cell lines

International Nuclear Information System (INIS)

Lee, Do Kyung; Jang, Seok; Kim, Mi Jin; Kim, Jung Hyun; Chung, Myung Jun; Kim, Kyung Jae; Ha, Nam Joo

2008-01-01

Lactic acid bacteria (LAB) are beneficial probiotic organisms that contribute to improved nutrition, microbial balance, and immuno-enhancement of the intestinal tract, as well as anti-tumor activity. The aim of the present work was to study the growth inhibition of tumor cells by butanol extract of Bifidobacterium adolescentis isolated from healthy young Koreans. The anti-proliferative activity of B. adolescentis isolates was assessed by XTT assays on three human colon cancer cell lines (Caco-2, HT-29, and SW480). The effects of B. adolescentis SPM0212 butanol extract on tumor necrosis factor-α (TNF-α) and nitric oxide (NO) production were tested using the murine macrophage RAW 264.7 cell line. The butanol extract of B. adolescentis SPM0212 dose-dependently inhibited the growth of Caco-2, HT-29, and SW480 cells by 70%, 30%, and 40%, respectively, at 200 μg/mL. Additionally, the butanol extract of B. adolescentis SPM0212 induced macrophage activation and significantly increased the production of TNF-α and NO, which regulate immune modulation and are cytotoxic to tumor cells. The butanol extract of B. adolescentis SPM0212 increased activity of the host immune system and may improve human health by helping to prevent colon cancer as a biological response modifier

Development of a serum-free co-culture of human intestinal epithelium cell-lines (Caco-2/HT29-5M21

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Schneider Yves-Jacques

2006-05-01

Full Text Available Abstract Background The absorptive and goblet cells are the main cellular types encountered in the intestine epithelium. The cell lineage Caco-2 is a model commonly used to reproduce the features of the bowel epithelium. However, there is a strong debate regarding the value of Caco-2 cell culture to mimick in vivo situation. Indeed, some authors report in Caco-2 a low paracellular permeability and an ease of access of highly diffusible small molecules to the microvilli, due to an almost complete lack of mucus. The HT29-5M21 intestinal cell lineage is a mucin-secreting cellular population. A co-culture system carried out in a serum-free medium and comprising both Caco-2 and HT29-5M21 cells was developed. The systematic use of a co-culture system requires the characterization of the monolayer under a given experimental procedure. Results In this study, we investigated the activity and localization of the alkaline phosphatase and the expression of IAP and MUC5AC genes to determine a correlation between these markers and the cellular composition of a differentiated monolayer obtained from a mixture of Caco-2 and HT29-5M21 cells. We observed that the culture conditions used (serum-free medium did not change the phenotype of each cell type, and produced a reproducible model. The alkaline phosphatase expression characterizing Caco-2 cells was influenced by the presence of HT29-5M21 cells. Conclusion The culture formed by 75% Caco-2 and 25% HT29-5M21 produce a monolayer containing the two main cell types of human intestinal epithelium and characterized by a reduced permeability to macromolecules.

Different molecular organization of two carotenoids, lutein and zeaxanthin, in human colon epithelial cells and colon adenocarcinoma cells

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Grudzinski, Wojciech; Piet, Mateusz; Luchowski, Rafal; Reszczynska, Emilia; Welc, Renata; Paduch, Roman; Gruszecki, Wieslaw I.

2018-01-01

Two cell lines, human normal colon epithelial cells (CCD 841 CoTr) and human colon adenocarcinoma cells (HT-29) were cultured in the presence of exogenous carotenoids, either zeaxanthin or lutein. Both carotenoids demonstrated cytotoxicity with respect to cancer cells but not to normal cells. Cells from both the cell lines were analyzed with application of fluorescence lifetime imaging microscopy and Raman scattering microscopy. Both imaging techniques show effective incorporation of carotenoid molecules into growing cells. Comparison of the Raman scattering and fluorescence lifetime characteristics reveals different molecular organization of carotenoids in the carcinoma and normal cells. The main difference consists in a carotenoid aggregation level which is substantially lower in the carcinoma cells as compared to the normal cells. Different molecular organization of carotenoids was interpreted in terms of a different metabolism of normal and carcinoma cells and has been concluded to provide a possibility of cancer diagnosis based on spectroscopic analyses.

Morusin induces apoptosis and suppresses NF-κB activity in human colorectal cancer HT-29 cells

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Lee, J.-C.; Won, S.-J.; Chao, C.-L.; Wu, F.-L.; Liu, H.-S.; Ling Pin; Lin, C.-N.; Su, C.-L.

2008-01-01

Morusin is a pure compound isolated from root bark of Morusaustralis (Moraceae). In this study, we demonstrated that morusin significantly inhibited the growth and clonogenicity of human colorectal cancer HT-29 cells. Apoptosis induced by morusin was characterized by accumulation of cells at the sub-G 1 phase, fragmentation of DNA, and condensation of chromatin. Morusin also inhibited the phosphorylation of IKK-α, IKK-β and IκB-α, increased expression of IκB-α, and suppressed nuclear translocation of NF-κB and its DNA binding activity. Dephosphorylation of NF-κB upstream regulators PI3K, Akt and PDK1 was also displayed. In addition, activation of caspase-8, change of mitochondrial membrane potential, release of cytochrome c and Smac/DIABLO, and activation of caspase-9 and -3 were observed at the early time point. Downregulation in the expression of Ku70 and XIAP was exhibited afterward. Caspase-8 or wide-ranging caspase inhibitor suppressed morusin-induced apoptosis. Therefore, the antitumor mechanism of morusin in HT-29 cells may be via activation of caspases and inhibition of NF-κB

Kynurenine promotes the goblet cell differentiation of HT-29 colon carcinoma cells by modulating Wnt, Notch and AhR signals.

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Park, Joo-Hung; Lee, Jeong-Min; Lee, Eun-Jin; Kim, Da-Jeong; Hwang, Won-Bhin

2018-04-01

Various amino acids regulate cell growth and differentiation. In the present study, we examined the ability of HT-29 cells to differentiate into goblet cells in RPMI and DMEM which are largely different in the amounts of numerous amino acids. Most of the HT-29 cells differentiated into goblet cells downregulating the stem cell marker Lgr5 when cultured in DMEM, but remained undifferentiated in RPMI. The goblet cell differentiation in DMEM was inhibited by 1-methyl-tryptophan (1-MT), an inhibitor of indoleamine 2,3 dioxygenase-1 which is the initial enzyme in tryptophan metabolism along the kynurenine (KN) pathway, whereas tryptophan and KN induced goblet cell differentiation in RPMI. The levels of Notch1 and its activation product Notch intracytoplasmic domain in HT-29 cells were lower in DMEM than those in RPMI and were increased by 1-MT in both media. HT-29 cells grown in both media expressed β-catenin at the same level on day 2 when goblet cell differentiation was not observed. β-catenin expression, which was increased by 1-MT in both media, was decreased by KN. DMEM reduced Hes1 expression while enhancing Hath1 expression. Finally, aryl hydrocarbon receptor (AhR) activation moderately induced goblet cell differentiation. Our results suggest that KN promotes goblet cell differentiation by regulating Wnt, Notch, and AhR signals and expression of Hes1 and Hath1.

LA-12 overcomes confluence-dependent resistance of HT-29 colon cancer cells to Pt (II) compounds

Czech Academy of Sciences Publication Activity Database

Šindlerová, Lenka; Foltinová, V.; Vaculová, Alena; Horváth, Viktor; Souček, Karel; Sova, P.; Hofmanová, Jiřina; Kozubík, Alois

2010-01-01

Roč. 30, č. 4 (2010), s. 1183-1188 ISSN 0250-7005 R&D Projects: GA ČR(CZ) GA301/07/1557 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : LA-12 * resistance * HT-29 Subject RIV: BO - Biophysics Impact factor: 1.656, year: 2010

Oral and fecal Campylobacter concisus strains perturb barrier function by apoptosis induction in HT-29/B6 intestinal epithelial cells.

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Hans Linde Nielsen

Full Text Available Campylobacter concisus infections of the gastrointestinal tract can be accompanied by diarrhea and inflammation, whereas colonization of the human oral cavity might have a commensal nature. We focus on the pathophysiology of C. concisus and the effects of different clinical oral and fecal C. concisus strains on human HT-29/B6 colon cells. Six oral and eight fecal strains of C. concisus were isolated. Mucus-producing HT-29/B6 epithelial monolayers were infected with the C. concisus strains. Transepithelial electrical resistance (R(t and tracer fluxes of different molecule size were measured in Ussing chambers. Tight junction (TJ protein expression was determined by Western blotting, and subcellular TJ distribution was analyzed by confocal laser-scanning microscopy. Apoptosis induction was examined by TUNEL-staining and Western blot of caspase-3 activation. All strains invaded confluent HT-29/B6 cells and impaired epithelial barrier function, characterized by a time- and dose-dependent decrease in R(t either after infection from the apical side but even more from the basolateral compartment. TJ protein expression changes were sparse, only in apoptotic areas of infected monolayers TJ proteins were redistributed. Solely the barrier-forming TJ protein claudin-5 showed a reduced expression level to 66±8% (P<0.05, by expression regulation from the gene. Concomitantly, Lactate dehydrogenase release was elevated to 3.1±0.3% versus 0.7±0.1% in control (P<0.001, suggesting cytotoxic effects. Furthermore, oral and fecal C. concisus strains elevated apoptotic events to 5-fold. C. concisus-infected monolayers revealed an increased permeability for 332 Da fluorescein (1.74±0.13 vs. 0.56±0.17 10(-6 cm/s in control, P<0.05 but showed no difference in permeability for 4 kDa FITC-dextran (FD-4. The same was true in camptothecin-exposed monolayers, where camptothecin was used for apoptosis induction.In conclusion, epithelial barrier dysfunction by oral and

Efecto citotóxico del extracto metanólico de tres ecotipos de Lepidium peruvianum Chacón sobre líneas celulares HeLa y HT-29

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Libertad Alzamora

2013-05-01

Full Text Available La búsqueda de compuestos naturales con actividad citotóxica y antitumoral es una de las prioridades actuales de la lucha contra el cáncer; motivo por el cual el objetivo del presente trabajo fue evaluar la actividad citotóxica de los extractos metanólicos (EM de los ecotipos negro, morado y amarillo de Lepidium peruvianum, Chacón (conocida también como Lepidium meyenii Walp. (maca sobre las líneas celulares HeLa (Human Epithelial Carcinoma y HT-29 (Human Colon Adenocarcinoma. Se determinó que la concentración inhibitoria del 50% del crecimiento celular (IC50 para la línea celular HT-29, con los ecotipos negro, morado y amarillo fue de 8,32 mg/ml, 9,28 mg/ml y 0,487 mg/ml respectivamente, mientras que para la línea celular HeLa fue de 2,4 mg/ml, 1,93 mg/ml y 0,66 mg/ml respectivamente. Adicionalmente, se evaluó un EM del ecotipo amarillo con dos años de almacenamiento (10 ºC determinándose como IC50 4,29 mg/ml para HT-29 y 4,17 mg/ml para HeLa. Se concluye que el efecto citotóxico del ecotipo amarillo sobre HT-29 y HeLa fue superior al mostrado por los ecotipos negro y morado; que la línea celular más sensible a los ecotipos amarillo, negro y morado es HeLa, y que el EM del ecotipo amarillo conservó sus propiedades citotóxicas pese al tiempo de almacenamiento, aunque éstas disminuyeron.

Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines

Energy Technology Data Exchange (ETDEWEB)

Solmi, Rossella [Dipartimento di Istologia, Embriologia e Biologia Applicata, Università di Bologna, Via Belmeloro 8, 40126 Bologna (Italy); Montroni, Isacco [Dipartimento Emergenza/Urgenza, Chirurgia Generale e dei Trapianti, Università di Bologna, Bologna (Italy); Mattei, Gabriella [Dipartimento di Istologia, Embriologia e Biologia Applicata, Università di Bologna, Via Belmeloro 8, 40126 Bologna (Italy); Taffurelli, Mario [Dipartimento Emergenza/Urgenza, Chirurgia Generale e dei Trapianti, Università di Bologna, Bologna (Italy); Santini, Donatella [Dipartimento di Patologia, Università di Bologna, Bologna (Italy); Pezzetti, Furio [Dipartimento di Istologia, Embriologia e Biologia Applicata, Università di Bologna, Via Belmeloro 8, 40126 Bologna (Italy); Ruggeri, Alessandro [Dipartimento di Scienze Anatomiche Umane e Fisiopatologia dell' Apparato Locomotore, Università di Bologna, Bologna (Italy); Castellani, Gastone [Centro Interdipartimentale L. Galvani, Università di Bologna, Bologna (Italy); DIMORFIPA, Università di Bologna, Bologna (Italy); Guidotti, Lia [Dipartimento di Istologia, Embriologia e Biologia Applicata, Università di Bologna, Via Belmeloro 8, 40126 Bologna (Italy); Coppola, Domenico [H. Lee Moffit Cancer Center and Research Institute, University of South Florida, Tampa, FL (United States); Strippoli, Pierluigi; Lauriola, Mattia [Dipartimento di Istologia, Embriologia e Biologia Applicata, Università di Bologna, Via Belmeloro 8, 40126 Bologna (Italy); Francesconi, Mirko [Centro Interdipartimentale L. Galvani, Università di Bologna, Bologna (Italy); DIMORFIPA, Università di Bologna, Bologna (Italy); Martini, Désirée [Dipartimento di Scienze Anatomiche Umane e Fisiopatologia dell' Apparato Locomotore, Università di Bologna, Bologna (Italy); Voltattorni, Manuela [Laboratori di Biotecnologie, Via Beverara 123, Bologna (Italy); Ceccarelli, Claudio [Dipartimento di Patologia, Università di Bologna, Bologna (Italy); Ugolini, Giampaolo; Rosati, Giancarlo; Zanotti, Simone [Dipartimento Emergenza/Urgenza, Chirurgia Generale e dei Trapianti, Università di Bologna, Bologna (Italy)

2008-08-08

EGFR is frequently overexpressed in colon cancer. We characterized HT-29 and Caco-2, human colon cancer cell lines, untreated and treated with cetuximab or gefitinib alone and in combination with EGF. Cell growth was determined using a variation on the MTT assay. Cell-cycle analysis was conducted by flow cytometry. Immunohistochemistry was performed to evaluate EGFR expression and scanning electron microscopy (SEM) evidenced the ultrastructural morphology. Gene expression profiling was performed using hybridization of the microarray Ocimum Pan Human 40 K array A. Caco-2 and HT-29 were respectively 66.25 and 59.24 % in G0/G1. They maintained this level of cell cycle distribution after treatment, suggesting a predominantly differentiated state. Treatment of Caco-2 with EGF or the two EGFR inhibitors produced a significant reduction in their viability. SEM clearly showed morphological cellular transformations in the direction of cellular death in both cell lines treated with EGFR inhibitors. HT-29 and Caco-2 displayed an important reduction of the microvilli (which also lose their erect position in Caco-2), possibly invalidating microvilli absorption function. HT-29 treated with cetuximab lost their boundary contacts and showed filipodi; when treated with gefitinib, they showed some vesicles: generally membrane reshaping is evident. Both cell lines showed a similar behavior in terms of on/off switched genes upon treatment with cetuximab. The gefitinib global gene expression pattern was different for the 2 cell lines; gefitinib treatment induced more changes, but directly correlated with EGF treatment. In cetuximab or gefitinib plus EGF treatments there was possible summation of the morphological effects: cells seemed more weakly affected by the transformation towards apoptosis. The genes appeared to be less stimulated than for single drug cases. This is the first study to have systematically investigated the effect of cetuximab or gefitinib, alone and in combination

Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines

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Pezzetti Furio

2008-08-01

Full Text Available Abstract Background EGFR is frequently overexpressed in colon cancer. We characterized HT-29 and Caco-2, human colon cancer cell lines, untreated and treated with cetuximab or gefitinib alone and in combination with EGF. Methods Cell growth was determined using a variation on the MTT assay. Cell-cycle analysis was conducted by flow cytometry. Immunohistochemistry was performed to evaluate EGFR expression and scanning electron microscopy (SEM evidenced the ultrastructural morphology. Gene expression profiling was performed using hybridization of the microarray Ocimum Pan Human 40 K array A. Results Caco-2 and HT-29 were respectively 66.25 and 59.24 % in G0/G1. They maintained this level of cell cycle distribution after treatment, suggesting a predominantly differentiated state. Treatment of Caco-2 with EGF or the two EGFR inhibitors produced a significant reduction in their viability. SEM clearly showed morphological cellular transformations in the direction of cellular death in both cell lines treated with EGFR inhibitors. HT-29 and Caco-2 displayed an important reduction of the microvilli (which also lose their erect position in Caco-2, possibly invalidating microvilli absorption function. HT-29 treated with cetuximab lost their boundary contacts and showed filipodi; when treated with gefitinib, they showed some vesicles: generally membrane reshaping is evident. Both cell lines showed a similar behavior in terms of on/off switched genes upon treatment with cetuximab. The gefitinib global gene expression pattern was different for the 2 cell lines; gefitinib treatment induced more changes, but directly correlated with EGF treatment. In cetuximab or gefitinib plus EGF treatments there was possible summation of the morphological effects: cells seemed more weakly affected by the transformation towards apoptosis. The genes appeared to be less stimulated than for single drug cases. Conclusion This is the first study to have systematically investigated

Displayed correlation between gene expression profiles and submicroscopic alterations in response to cetuximab, gefitinib and EGF in human colon cancer cell lines

International Nuclear Information System (INIS)

Solmi, Rossella; Montroni, Isacco; Mattei, Gabriella; Taffurelli, Mario; Santini, Donatella; Pezzetti, Furio; Ruggeri, Alessandro; Castellani, Gastone; Guidotti, Lia; Coppola, Domenico; Strippoli, Pierluigi; Lauriola, Mattia; Francesconi, Mirko; Martini, Désirée; Voltattorni, Manuela; Ceccarelli, Claudio; Ugolini, Giampaolo; Rosati, Giancarlo; Zanotti, Simone

2008-01-01

EGFR is frequently overexpressed in colon cancer. We characterized HT-29 and Caco-2, human colon cancer cell lines, untreated and treated with cetuximab or gefitinib alone and in combination with EGF. Cell growth was determined using a variation on the MTT assay. Cell-cycle analysis was conducted by flow cytometry. Immunohistochemistry was performed to evaluate EGFR expression and scanning electron microscopy (SEM) evidenced the ultrastructural morphology. Gene expression profiling was performed using hybridization of the microarray Ocimum Pan Human 40 K array A. Caco-2 and HT-29 were respectively 66.25 and 59.24 % in G0/G1. They maintained this level of cell cycle distribution after treatment, suggesting a predominantly differentiated state. Treatment of Caco-2 with EGF or the two EGFR inhibitors produced a significant reduction in their viability. SEM clearly showed morphological cellular transformations in the direction of cellular death in both cell lines treated with EGFR inhibitors. HT-29 and Caco-2 displayed an important reduction of the microvilli (which also lose their erect position in Caco-2), possibly invalidating microvilli absorption function. HT-29 treated with cetuximab lost their boundary contacts and showed filipodi; when treated with gefitinib, they showed some vesicles: generally membrane reshaping is evident. Both cell lines showed a similar behavior in terms of on/off switched genes upon treatment with cetuximab. The gefitinib global gene expression pattern was different for the 2 cell lines; gefitinib treatment induced more changes, but directly correlated with EGF treatment. In cetuximab or gefitinib plus EGF treatments there was possible summation of the morphological effects: cells seemed more weakly affected by the transformation towards apoptosis. The genes appeared to be less stimulated than for single drug cases. This is the first study to have systematically investigated the effect of cetuximab or gefitinib, alone and in combination

Influence of ethacrynic acid on glutathione S-transferase pi transcript and protein half-lives in human colon cancer cells.

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Shen, H; Ranganathan, S; Kuzmich, S; Tew, K D

1995-10-12

Ethacrynic acid (EA) is a plant phenolic acid that is both an inhibitor and an inducer of glutathione S-transferase (GST) activity. To determine contributory factors in the increased GST activity caused by EA treatment, human colon carcinoma HT29 cells were compared with a cloned EA-resistant population (HT6-8) maintained in medium containing 72 microM EA. Several factors are involved in the increased expression of GST pi in HT6-8. For example, nuclear run-on experiments showed an approximately 2-fold increase in the rate of transcription of GST pi. In addition, the half-life of GST pi transcript was increased from 4.1 (wild type, HT29, HT4-1) to 8.4 hr. The half-life of GST pi protein was 1-2 hr in HT4-1 cells versus 8-9 hr in HT6-8 cells. When either human ovarian carcinoma cells (SKOV3) or human prostatic carcinoma cells (DU145) were treated with EA, the half-life of the GST pi transcript was also increased. The transcript half-lives of another thiol-metabolism enzyme, gamma-glutamylcysteine synthetase (gamma-GCS), and a phase II detoxification enzyme, dihydrodiol dehydrogenase (DDH), were also increased in HT6-8, SKOV3 and DU145 cells treated with EA. However, the half-lives of transcripts from "housekeeping genes," such as glyceraldehyde 3-phosphate dehydrogenase (G3PDH), beta-actin and beta-tubulin, were not changed in these cell lines following EA. Apparently, a number of coordinated factors are involved in EA-enhanced expression of GST pi and other detoxification enzymes.

A combination of indol-3-carbinol and genistein synergistically induces apoptosis in human colon cancer HT-29 cells by inhibiting Akt phosphorylation and progression of autophagy

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Watanabe Hirotsuna

2009-11-01

Full Text Available Abstract Background The chemopreventive effects of dietary phytochemicals on malignant tumors have been studied extensively because of a relative lack of toxicity. To achieve desirable effects, however, treatment with a single agent mostly requires high doses. Therefore, studies on effective combinations of phytochemicals at relatively low concentrations might contribute to chemopreventive strategies. Results Here we found for the first time that co-treatment with I3C and genistein, derived from cruciferous vegetables and soy, respectively, synergistically suppressed the viability of human colon cancer HT-29 cells at concentrations at which each agent alone was ineffective. The suppression of cell viability was due to the induction of a caspase-dependent apoptosis. Moreover, the combination effectively inhibited phosphorylation of Akt followed by dephosphorylation of caspase-9 or down-regulation of XIAP and survivin, which contribute to the induction of apoptosis. In addition, the co-treatment also enhanced the induction of autophagy mediated by the dephosphorylation of mTOR, one of the downstream targets of Akt, whereas the maturation of autophagosomes was inhibited. These results give rise to the possibility that co-treatment with I3C and genistein induces apoptosis through the simultaneous inhibition of Akt activity and progression of the autophagic process. This possibility was examined using inhibitors of Akt combined with inhibitors of autophagy. The combination effectively induced apoptosis, whereas the Akt inhibitor alone did not. Conclusion Although in vivo study is further required to evaluate physiological efficacies and toxicity of the combination treatment, our findings might provide a new insight into the development of novel combination therapies/chemoprevention against malignant tumors using dietary phytochemicals.

Effect of beta-carotene-rich tomato lycopene beta-cyclase ( tlcy-b) on cell growth inhibition in HT-29 colon adenocarcinoma cells.

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Palozza, Paola; Bellovino, Diana; Simone, Rossella; Boninsegna, Alma; Cellini, Francesco; Monastra, Giovanni; Gaetani, Sancia

2009-07-01

Lycopene beta-cyclase (tlcy-b) tomatoes, obtained by modulating carotenogenesis via genetic engineering, contain a large amount of beta-carotene, as clearly visible by their intense orange colour. In the present study we have subjected tlcy-b tomatoes to an in vitro simulated digestion and analysed the effects of digestate on cell proliferation. To this aim we used HT-29 human colon adenocarcinoma cells, grown in monolayers, as a model. Digested tomatoes were diluted (20 ml, 50 ml and 100 ml/l) in culture medium and added to the cells for different incubation times (24 h, 48 h and 72 h). Inhibition of cell growth by tomato digestate was dose-dependent and resulted from an arrest of cell cycle progression at the G0/G1 and G2/M phase and by apoptosis induction. A down-regulation of cyclin D1, Bcl-2 and Bcl-xl expression was observed. We also found that heat treatment of samples before digestion enhanced beta-carotene release and therefore cell growth inhibition. To induce with purified beta-carotene solubilised in tetrahydrofuran the same cell growth inhibition obtained with the tomato digestate, a higher amount of the carotenoid was necessary, suggesting that beta-carotene micellarised during digestion is utilised more efficiently by the cells, but also that other tomato molecules, reasonably made available during digestion, may be present and cooperate with beta-carotene in promoting cell growth arrest.

Comprehensive and Holistic Analysis of HT-29 Colorectal Cancer Cells and Tumor-Bearing Nude Mouse Model: Interactions Among Fractions Derived From the Chinese Medicine Formula Tian Xian Liquid in Effects on Human Colorectal Carcinoma.

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Leigh, Annballaw Bridget; Cheung, Ho Pan; Lin, Li-Zhu; Ng, Tzi Bun; Lao, Lixing; Zhang, Yanbo; Zhang, Zhang-Jin; Tong, Yao; Sze, Stephen Cho Wing

2017-09-01

The Chinese medicine formula Tian Xian Liquid (TXL) has been used clinically for cancer therapy in China for more than 25 years. However, the comprehensive and holistic effects of its bioactive fractions for various antitumor therapeutic effects have not been unraveled. This is the first study to scientifically elucidate the holistic effect of Chinese medicine formula for treating colon cancer, hence allowing a better understanding of the essence of Chinese medicine formula, through the comparison of the actions of TXL and its functional constituent fractions, including ethyl acetate (EA), butanol (BU), and aqueous (WA) fractions. Tissue-specific proliferative/antiproliferative effects of these fractions on human colorectal carcinoma HT-29 cells and splenocytes were studied by using the MTT assay. Their modulations on the expression of markers of antiproliferation, antimetastasis, reversion of multidrug resistance in treated HT-29 cells were examined with real-time polymerase chain reaction and Western blot analysis, and their modulations in a xenografted nude mouse model were examined by Western blot analysis. Results revealed that EA fraction slightly inhibited the proliferation of HT-29 cells, but tissue-specifically exerted the most potent antiproliferative effect on splenocytes. On the contrary, only TXL and BU fraction tissue-specifically contributed to the proliferation of splenocytes, but inhibited the proliferation of HT-29 cells. WA fraction exerted the most potent antiproliferative effect on HT-29 cells and also the strongest inhibitory action on tumor size in the nude mouse model in our previous study. In the HT-29 model, TXL and WA fraction exerted the most pronounced effect on upregulation of p21 mRNA and protein; TXL, and EA and WA fractions exerted the effect on downregulation of G1 phase cell cycle protein, cyclin D1 mRNA and protein; EA and BU fractions exerted the most prominent anti-invasive effect on anti-invasion via downregulation of MMP-1 m

Arctigenin induces apoptosis in colon cancer cells through ROS/p38MAPK pathway.

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Li, Qing-chun; Liang, Yun; Tian, Yuan; Hu, Guang-rui

2016-01-01

In the current study the antiproliferative effect of arctigenin, plant lignin, was evaluated on human colon cancer cell line HT-29. Furthermore, attempts were made to explore the signaling mechanism which may be responsible for its effect. Cell growth inhibition was assessed by MTT and LDH assays. Flow cytometric analysis was performed to determine cell arrest in the cell cycle phase and apoptosis. Furthermore, to confirm the apoptotic activity of arctigenin, caspase-9 and -3 activities analysis was performed. The levels of reactive oxygen species (ROS) and p38 mitogen activated protein kinase (MAPK) were investigated to determine their role in inducing apoptosis in arctigenin-treated HT-29 colon cancer cell line. MTT and LDH results demonstrated significant cell growth inhibitory effect of arctigenin on HT-29 cells in a dose-dependent manner. Furthermore, increase in cell number arrested at G2/M phase was observed in flow cytometric analysis upon arctigenin treatment. In addition, arctigenin increased the apoptotic ratio in a dose-dependent manner. The involvement of intrinsic apoptotic pathway was indicated by the activation of caspase-9 and -3. Moreover, increased ROS production, activation of p38 MAPK and changes in mitochondrial membrane potential (ΔΨm) also revealed the role of intrinsic apoptotic signaling pathway in cell growth inhibition after arctigenin exposure. Arctigenin induces apoptosis in HT-29 colon cancer cells by regulating ROS and p38 MAPK pathways.

Chemopreventive Potential of Powdered Red Wine Pomace Seasonings against Colorectal Cancer in HT-29 Cells.

Science.gov (United States)

Del Pino-García, Raquel; Rivero-Pérez, María D; González-SanJosé, María L; Ortega-Heras, Miriam; García Lomillo, Javier; Muñiz, Pilar

2017-01-11

This study evaluates the antiproliferative and antigenotoxic actions of powdered red wine pomace seasonings (Sk-S, seedless; W-S, whole; Sd-S, seeds). In vitro gastrointestinal digested and colonic fermented fractions of the seasonings were used as cell treatments. Phenolic acids from Sk-S showed the highest bioaccessibility in the small intestine, whereas polyphenols contained in Sd-S might be the most fermentable in the colon. Dietary fiber from Sk-S was the best substrate for short chain fatty acids production by gut microbiota. Colon cancerous (HT-29) cell viability was inhibited by 50% (IC 50 values) at treatment concentrations ranging from 845 (Sk-S) to 1085 (Sd-S) μg/mL prior digestion, but all digested fractions exhibited similar antiproliferative activities (mean IC 50 = 814 μg/mL). Oxidative DNA damage in cells was also attenuated by the treatments (200 μg/mL, 24 h preincubation), with all colonic fermented fractions displaying similar genoprotective action. These results suggest the potential of red wine pomace seasonings as chemopreventive agents in colorectal cancer.

Muscarinic receptor agonists stimulate matrix metalloproteinase 1-dependent invasion of human colon cancer cells

International Nuclear Information System (INIS)

Raufman, Jean-Pierre; Cheng, Kunrong; Saxena, Neeraj; Chahdi, Ahmed; Belo, Angelica; Khurana, Sandeep; Xie, Guofeng

2011-01-01

Highlights: ► Muscarinic receptor agonists stimulated robust human colon cancer cell invasion. ► Anti-matrix metalloproteinase1 antibody pre-treatment blocks cell invasion. ► Bile acids stimulate MMP1 expression, cell migration and MMP1-dependent invasion. -- Abstract: Mammalian matrix metalloproteinases (MMPs) which degrade extracellular matrix facilitate colon cancer cell invasion into the bloodstream and extra-colonic tissues; in particular, MMP1 expression correlates strongly with advanced colon cancer stage, hematogenous metastasis and poor prognosis. Likewise, muscarinic receptor signaling plays an important role in colon cancer; muscarinic receptors are over-expressed in colon cancer compared to normal colon epithelial cells. Muscarinic receptor activation stimulates proliferation, migration and invasion of human colon cancer cells. In mouse intestinal neoplasia models genetic ablation of muscarinic receptors attenuates carcinogenesis. In the present work, we sought to link these observations by showing that MMP1 expression and activation plays a mechanistic role in muscarinic receptor agonist-induced colon cancer cell invasion. We show that acetylcholine, which robustly increases MMP1 expression, stimulates invasion of HT29 and H508 human colon cancer cells into human umbilical vein endothelial cell monolayers – this was abolished by pre-incubation with atropine, a non-selective muscarinic receptor inhibitor, and by pre-incubation with anti-MMP1 neutralizing antibody. Similar results were obtained using a Matrigel chamber assay and deoxycholyltaurine (DCT), an amidated dihydroxy bile acid associated with colon neoplasia in animal models and humans, and previously shown to interact functionally with muscarinic receptors. DCT treatment of human colon cancer cells resulted in time-dependent, 10-fold increased MMP1 expression, and DCT-induced cell invasion was also blocked by pre-treatment with anti-MMP1 antibody. This study contributes to understanding

Muscarinic receptor agonists stimulate matrix metalloproteinase 1-dependent invasion of human colon cancer cells

Energy Technology Data Exchange (ETDEWEB)

Raufman, Jean-Pierre, E-mail: jraufman@medicine.umaryland.edu [Division of Gastroenterology and Hepatology, University of Maryland School of Medicine, Baltimore, MD (United States); Cheng, Kunrong; Saxena, Neeraj; Chahdi, Ahmed; Belo, Angelica; Khurana, Sandeep; Xie, Guofeng [Division of Gastroenterology and Hepatology, University of Maryland School of Medicine, Baltimore, MD (United States)

2011-11-18

Highlights: Black-Right-Pointing-Pointer Muscarinic receptor agonists stimulated robust human colon cancer cell invasion. Black-Right-Pointing-Pointer Anti-matrix metalloproteinase1 antibody pre-treatment blocks cell invasion. Black-Right-Pointing-Pointer Bile acids stimulate MMP1 expression, cell migration and MMP1-dependent invasion. -- Abstract: Mammalian matrix metalloproteinases (MMPs) which degrade extracellular matrix facilitate colon cancer cell invasion into the bloodstream and extra-colonic tissues; in particular, MMP1 expression correlates strongly with advanced colon cancer stage, hematogenous metastasis and poor prognosis. Likewise, muscarinic receptor signaling plays an important role in colon cancer; muscarinic receptors are over-expressed in colon cancer compared to normal colon epithelial cells. Muscarinic receptor activation stimulates proliferation, migration and invasion of human colon cancer cells. In mouse intestinal neoplasia models genetic ablation of muscarinic receptors attenuates carcinogenesis. In the present work, we sought to link these observations by showing that MMP1 expression and activation plays a mechanistic role in muscarinic receptor agonist-induced colon cancer cell invasion. We show that acetylcholine, which robustly increases MMP1 expression, stimulates invasion of HT29 and H508 human colon cancer cells into human umbilical vein endothelial cell monolayers - this was abolished by pre-incubation with atropine, a non-selective muscarinic receptor inhibitor, and by pre-incubation with anti-MMP1 neutralizing antibody. Similar results were obtained using a Matrigel chamber assay and deoxycholyltaurine (DCT), an amidated dihydroxy bile acid associated with colon neoplasia in animal models and humans, and previously shown to interact functionally with muscarinic receptors. DCT treatment of human colon cancer cells resulted in time-dependent, 10-fold increased MMP1 expression, and DCT-induced cell invasion was also blocked by pre

In vitro differentiation of HT-29 M6 mucus-secreting colon cancer cells involves a trychostatin A and p27(KIP1)-inducible transcriptional program of gene expression.

Science.gov (United States)

Mayo, Clara; Lloreta, Josep; Real, Francisco X; Mayol, Xavier

2007-07-01

Tumor cell dedifferentiation-such as the loss of cell-to-cell adhesion in epithelial tumors-is associated with tumor progression. To better understand the mechanisms that maintain carcinoma cells in a differentiated state, we have dissected in vitro differentiation pathways in the mucus-secretor HT-29 M6 colon cancer cell line, which spontaneously differentiates in postconfluent cultures. By lowering the extracellular calcium concentration to levels that prevent intercellular adhesion and epithelial polarization, our results reveal that differentiation is calcium-dependent and involves: (i) a process of cell cycle exit to G(0) and (ii) the induction of a transcriptional program of differentiation gene expression (i.e., mucins MUC1 and MUC5AC, and the apical membrane peptidase DPPIV). In calcium-deprived, non-differentiated postconfluent cultures, differentiation gene promoters are repressed by a trichostatin A (TSA)-sensitive mechanism, indicating that loss of gene expression by dedifferentiation is driven by histone deacetylases (HDAC). Since TSA treatment or extracellular calcium restoration allow gene promoter activation to similar levels, we suggest that induction of differentiation is one mechanism of HDAC inhibitor antitumor action. Moreover, transcriptional de-repression can also be induced in non-differentiating culture conditions by overexpressing the cyclin-dependent kinase inhibitor p27(KIP1), which is normally induced during spontaneous differentiation. Since p27(KIP1) downregulation in colon cancer is associated with poor prognosis independently of tumor cell division rates, we propose that p27 (KIP1) may prevent tumor progression by, at least in part, enhancing the expression of some differentiation genes. Therefore, the HT-29 M6 model allows the identification of some basic mechanisms of cancer cell differentiation control, so far revealing HDAC and p27(KIP1) as key regulatory factors of differentiation gene expression.

Effect of low frequency magnetic fields on the growth of MNP-treated HT29 colon cancer cells

Science.gov (United States)

Spyridopoulou, K.; Makridis, A.; Maniotis, N.; Karypidou, N.; Myrovali, E.; Samaras, T.; Angelakeris, M.; Chlichlia, K.; Kalogirou, O.

2018-04-01

Recent investigations have attempted to understand and exploit the impact of magnetic field-actuated internalized magnetic nanoparticles (MNPs) on the proliferation rate of cancer cells. Due to the complexity of the parameters governing magnetic field-exposure though, individual studies to date have raised contradictory results. In our approach we performed a comparative analysis of key parameters related to the cell exposure of cancer cells to magnetic field-actuated MNPs, and to the magnetic field, in order to better understand the factors affecting cellular responses to magnetic field-stimulated MNPs. We used magnetite MNPs with a hydrodynamic diameter of 100 nm and studied the proliferation rate of MNPs-treated versus untreated HT29 human colon cancer cells, exposed to either static or alternating low frequency magnetic fields with varying intensity (40-200 mT), frequency (0-8 Hz) and field gradient. All three parameters, field intensity, frequency, and field gradient affected the growth rate of cells, with or without internalized MNPs, as compared to control MNPs-untreated and magnetic field-untreated cells. We observed that the growth inhibitory effects induced by static and rotating magnetic fields were enhanced by pre-treating the cells with MNPs, while the growth promoting effects observed in alternating field-treated cells were weakened by MNPs. Compared to static, rotating magnetic fields of the same intensity induced a similar extend of cell growth inhibition, while alternating fields of varying intensity (70 or 100 mT) and frequency (0, 4 or 8 Hz) induced cell proliferation in a frequency-dependent manner. These results, highlighting the diverse effects of mode, intensity, and frequency of the magnetic field on cell growth, indicate that consistent and reproducible results can be achieved by controlling the complexity of the exposure of biological samples to MNPs and external magnetic fields, through monitoring crucial experimental parameters. We

Antiproliferative effect of a novel nitro-oxy derivative of celecoxib in human colon cancer cells: role of COX-2 and nitric oxide.

Science.gov (United States)

Bocca, Claudia; Bozzo, Francesca; Bassignana, Andrea; Miglietta, Antonella

2010-07-01

It has been shown previously that a novel nitrooxy derivative of celecoxib exerts antiproliferative and pro-apoptotic effects in human colon cancer cells. The aim of this study was to elucidate whether these biological properties depend on COX-2 inhibition and/or NO release. Therefore, the derivative was decomposed into the parent compound celecoxib and the NO donor benzyl nitrate and the biological role of each was tested in COX-2-positive (HT-29) and -negative (SW-480) colon cancer cells. The main findings were that the nitro-oxy derivative behaved like celecoxib in HT-29 cells in terms of COX-2 and ERK/MAPK inhibition, as well as induction of apoptosis, while the benzyl nitrate had no such effects. Interestingly, the beta-catenin system was activated by the nitro-oxy derivative as well as by benzyl nitrate alone more potently than by the parent compound celecoxib, suggesting a possible regulatory role for NO. In SW480 cells, these activities were substantially less pronounced, suggesting the presence of COX-2-dependent mechanisms in the modulation of these parameters.

Negligible colon cancer risk from food-borne acrylamide exposure in male F344 rats and nude (nu/nu mice-bearing human colon tumor xenografts.

Directory of Open Access Journals (Sweden)

Jayadev Raju

Full Text Available Acrylamide, a possible human carcinogen, is formed in certain carbohydrate-rich foods processed at high temperature. We evaluated if dietary acrylamide, at doses (0.5, 1.0 or 2.0 mg/kg diet reflecting upper levels found in human foods, modulated colon tumorigenesis in two rodent models. Male F344 rats were randomized to receive diets without (control or with acrylamide. 2-weeks later, rats in each group received two weekly subcutaneous injections of either azoxymethane (AOM or saline, and were killed 20 weeks post-injections; colons were assessed for tumors. Male athymic nude (nu/nu mice bearing HT-29 human colon adenocarcinoma cells-derived tumor xenografts received diets without (control or with acrylamide; tumor growth was monitored and mice were killed 4 weeks later. In the F344 rat study, no tumors were found in the colons of the saline-injected rats. However, the colon tumor incidence was 54.2% and 66.7% in the control and the 2 mg/kg acrylamide-treated AOM-injected groups, respectively. While tumor multiplicity was similar across all diet groups, tumor size and burden were higher in the 2 mg/kg acrylamide group compared to the AOM control. These results suggest that acrylamide by itself is not a "complete carcinogen", but acts as a "co-carcinogen" by exacerbating the effects of AOM. The nude mouse study indicated no differences in the growth of human colon tumor xenografts between acrylamide-treated and control mice, suggesting that acrylamide does not aid in the progression of established tumors. Hence, food-borne acrylamide at levels comparable to those found in human foods is neither an independent carcinogen nor a tumor promoter in the colon. However, our results characterize a potential hazard of acrylamide as a colon co-carcinogen in association with known and possibly other environmental tumor initiators/promoters.

Effect of carrageenans alone and in combination with casein or lipopolysaccharide on human epithelial intestinal HT-29 cells.

Science.gov (United States)

Sokolova, E V; Kuz'mich, A S; Byankina, A O; Yermak, I M

2017-10-01

The research described here was focused on the effect on human intestinal epithelial cell monolayers of sulfated red algal polysaccharides (κ-, λ-, and κ/β-carrageenans) alone and in combination with casein or lipopolysaccharide (LPS). HT-29 cells were investigated under normal and stress conditions; stress was induced by exposure to ethanol. Cell viability was monitored with a real-time system. The change in binding properties of negatively sulfated red algal polysaccharides assessed by the measurement of free carrageenans in mixtures with casein or McCoy's 5 A culture medium by means of toluidine blue O. Low sulfate content and the presence of 3,6-anhydogalactose are prerequisites for the recovery of ethanol-exposed HT-29 cells by carrageenans. Analysis of carrageenan binding ability confirmed that casein and LPS should affect carrageenan activity. Whether the combined action of the mucin-containing layer and carrageenans or the action of carrageenans alone was responsible for enhanced cell viability under stress conditions induced by ethanol is a subject for further research. © 2017 Wiley Periodicals Inc. J Biomed Mater Res Part A: 105A: 2843-2850, 2017. © 2017 Wiley Periodicals, Inc.

BG-4, a novel anticancer peptide from bitter gourd (Momordica charantia), promotes apoptosis in human colon cancer cells.

Science.gov (United States)

Dia, Vermont P; Krishnan, Hari B

2016-09-15

Momordica charantia is a perennial plant with reported health benefits. BG-4, a novel peptide from Momordica charantia, was isolated, purified and characterized. The trypsin inhibitory activity of BG-4 is 8.6 times higher than purified soybean trypsin inhibitor. The high trypsin inhibitory activity of BG-4 may be responsible for its capability to cause cytotoxicity to HCT-116 and HT-29 human colon cancer cells with ED50 values of 134.4 and 217.0 μg/mL after 48 h of treatment, respectively. The mechanism involved in the cytotoxic effect may be associated with induction of apoptosis as evidenced by increased percentage of HCT-116 and HT-29 colon cancer cells undergoing apoptosis from 5.4% (untreated) to 24.8% (BG-4 treated, 125 μg/mL for 16 h) and 8.5% (untreated) to 31.9% (BG-4 treated, 125 μg/mL for 16 h), respectively. The molecular mechanistic explanation in the apoptosis inducing property of BG-4 is due to reduced expression of Bcl-2 and increased expression of Bax leading to increased expression of caspase-3 and affecting the expression of cell cycle proteins p21 and CDK2. This is the first report on the anti-cancer potential of a novel bioactive peptide isolated from Momordica charantia in vitro supporting the potential therapeutic property of BG-4 against colon cancer that must be addressed using in vivo models of colon carcinogenesis.

Interferon-gamma sensitizes colonic epithelial cell lines to physiological and therapeutic inducers of colonocyte apoptosis.

LENUS (Irish Health Repository)

O'Connell, J

2012-02-03

Homeostasis in the colonic epithelium is achieved by a continuous cycle of proliferation and apoptosis, in which imbalances are associated with disease. Inflammatory bowel disease (IBD) and colon cancer are associated with either excessive or insufficient apoptosis of colonic epithelial cells, respectively. By using two colonic epithelial cell lines, HT29 and SW620, we investigated how the epithelial cell\\'s sensitivity to apoptosis was regulated by the proinflammatory cytokine interferon-gamma (IFN-gamma). We found that IFN-gamma sensitized HT29 cells, and to a lesser extent SW620, to diverse inducers of apoptosis of physiologic or therapeutic relevance to the colon. These apoptosis inducers included Fas (CD95\\/APO-1) ligand (FasL), short-chain fatty acids, and chemotherapeutic drugs. The extent of IFN-gamma-mediated apoptosis sensitization in these two cell lines correlated well with the degree of IFN-gamma-mediated upregulation of the proapoptotic protease caspase-1. Although IFN-gamma alone effectively sensitized HT29 cells to apoptosis, inclusion of the protein synthesis inhibitor cyclohexamide (CHX) during apoptotic challenge was necessary for maximal sensitization of SW620. The requirement of CHX to sensitize SW620 cells to apoptosis implies a need to inhibit translation of antiapoptotic proteins absent from HT29. In particular, the antiapoptotic protein Bcl-2 was strongly expressed in SW620 cells but absent from HT29. Our results indicate that IFN-gamma increases the sensitivity of colonic epithelial cells to diverse apoptotic stimuli in concert, via upregulation of caspase-1. Our findings implicate caspase-1 and Bcl-2 as important central points of control determining the general sensitivity of colonic epithelial cells to apoptosis.

Targeted inhibition of the phosphoinositide 3-kinase impairs cell proliferation, survival, and invasion in colon cancer.

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Yang, Fei; Gao, Jun-Yi; Chen, Hua; Du, Zhen-Hua; Zhang, Xue-Qun; Gao, Wei

2017-01-01

Colon cancer is the third most common cancer in the world, and its metastasis and drug resistance are challenging for its effective treatment. The PI3K/Akt/mTOR pathway plays a crucial role in the pathogenesis of colon cancer. The aim of this study was to investigate the targeting of PI3K in colon cancer cells HT-29 and HCT-116 in vitro. In HT-29 and HCT-116 cells, BEZ235, a dual inhibitor of PI3K/mTOR, and shRNAtarget to PI3KCA were used to inhibit PI3K/Akt/mTOR pathway. The inhibition efficiency of PI3K/Akt/mTOR pathway was detected by RT-PCR and Western blot. Cell proliferation, migration, invasion, and apoptosis were evaluated by Cell Counting Kit-8, Transwell, and flow cytometry assays. The expression of apoptosis-related proteins (cleavage caspase 3, Bcl-2, Bax, and Bim) were also detected. We found that in HT-29 and HCT-116 cells, the treatment of BEZ235 (1 μM) and PI3KCA knockdown inhibited the activation of PI3K/Akt/mTOR pathway and significantly suppressed cell proliferation, migration, and invasion of HT-29 and HCT-116 cells. In addition, we confirmed that knockdown of BEZ235 and PI3KCA induced cell apoptosis through the upregulated levels of cleavage caspase 3 and Bax and downregulated expression of Bcl-2 and Bim. Our results indicated that targeted inhibition of the PI3K/Akt/mTOR pathway impaired cell proliferation, survival, and invasion in human colon cancer.

E-selectin: sialyl Lewis, a dependent adhesion of colon cancer cells, is inhibited differently by antibodies against E-selectin ligands.

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Srinivas, U; Påhlsson, P; Lundblad, A

1996-09-01

Recent studies have demonstrated that selectins, a new family of cell-adhesion molecules with similar domain structures, mediate the adhesion of peripheral blood cells to interleukin-1 (IL-1)-activated endothelium. In the present study the authors evaluated the role of E-selectin-Sialyl Lewis x (SLe(x))/ Sialyl Lewis a (SLe(a)) interaction in mediating in vitro adhesion of two colon cancer cell lines, HT-29 and COLO 201, to human umbilical cord endothelial cells (HUVEC). Colon cancer cell lines had a strong expression of blood group-related carbohydrate epitopes as evaluated by fluorescence-activated cell sorter (FACS) analysis. It was established that adhesion of HT-29 and COLO 201 cells to IL-1 stimulated HUVEC was calcium dependent and could be inhibited by a monoclonal antibody directed against E-selectin. Prior incubation of cells with two different antibodies directed against SLe(x) and antibodies directed against related Lewis epitopes, Le(x) and Le(a), had no significant effect on adhesion. Three antibodies directed against SLe(a) differed in their capacity to inhibit the adhesion of HT-29 and COLO 201 cells to HUVEC. Only one antibody directed against the SLe(a) structure was effective in inhibiting adhesion of both COLO 201 and HT-29 cells. The difference could not be attributed to titre, the type or number of glycoproteins, or to a difference in the amount of SLe(a) present on individual proteins, suggesting that presence and right presentation of SLe(a) epitope might be important for adhesion of colon cancer cells. Finally, in the in vitro system used, adhesion of HT-29 and COLO 201 cells to activated HUVEC is mediated predominantly by E-selectin/SLe(a) interaction. SLe(x) and related epitopes, Le(x) and Le(a), seem to have limited relevance for colon cancer cell recognition of E-selectin.

New insights into the molecular mechanism of Boletus edulis ribonucleic acid fraction (BE3) concerning antiproliferative activity on human colon cancer cells.

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Lemieszek, Marta Kinga; Ribeiro, Miguel; Marques, Guilhermina; Nunes, Fernando Milheiro; Pożarowski, Piotr; Rzeski, Wojciech

2017-05-24

One of the relatively new and promising strategies of cancer treatment is chemoprevention, which involves the use of natural or synthetic compounds to block, inhibit or reverse carcinogenesis. A valuable and still untapped source of chemopreventive compounds seems to be edible mushrooms belonging to higher Basidiomycetes. Boletus edulis biopolymers extracted with hot water and purified by anion-exchange chromatography showed antiproliferative activity in colon cancer cells, but only fraction BE3, mostly composed of ribonucleic acids, was able to inhibit DNA synthesis in HT-29 cells. The present work aims to elucidate the molecular mechanism of this Boletus edulis ribonucleic acid fraction and in this sense flow cytometry and western blotting were applied to cell cycle analysis in HT-29 cells. We found that the antiproliferative ability of fraction BE3 observed in HT-29 cells was associated with the modulation of expression of cell cycle regulatory proteins (Cyclin D1, Cyclin A, p21 and p27) leading to cell accumulation in the S phase of the cell cycle. Furthermore, the BE3 fraction showed effective silencing of the signal transduction in an MAPK/Erk pathway in HT-29 and LS180 colon cancer cell lines. Thus, the previously and currently obtained results indicate that the BE3 fraction from Boletus edulis has great potential and needs to be further exploited through animal and clinical studies in order to develop a new efficient and safe therapeutic strategy for people who have been threatened by or suffered from colon cancer.

Portulaca oleracea extract can inhibit nodule formation of colon cancer stem cells by regulating gene expression of the Notch signal transduction pathway.

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Jin, Heiying; Chen, Li; Wang, Shuiming; Chao, Deng

2017-07-01

To investigate whether Portulaca oleracea extract affects tumor formation in colon cancer stem cells and its chemotherapy sensitivity. In addition, to analyze associated genetic changes within the Notch signal transduction pathway. Serum-free cultures of colon cancer cells (HT-29) and HT-29 cancer stem cells were treated with the chemotherapeutic drug 5-fluorouracil to assess sensitivity. Injections of the stem cells were also given to BALB/c mice to confirm tumor growth and note its characteristics. In addition, the effect of different concentrations of P. oleracea extract was tested on the growth of HT-29 colon cancer cells and HT-29 cancer stem cells, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. The effects of P. oleracea extract on the expression of β-catenin, Notch1, and Notch2 in the HT-29 cells were studied using reverse transcription polymerase chain reaction and Western blotting. The tumor volume of the HT29 cells was two times larger than that of HT29 cancer stem cells. Treatment with P. oleracea extract inhibited the proliferation of both HT-29 cancer cells and HT-29 cancer stem cells at doses from 0.07 to 2.25 µg/mL. Apoptosis of HT-29 cancer cells and HT-29 cancer stem cells was assessed by flow cytometry; it was enhanced by the addition of P. oleracea extract. Finally, treatment with P. oleracea extract significantly downregulated the expression of the Notch1 and β-catenin genes in both cell types. The results of this study show that P. oleracea extract inhibits the growth of colon cancer stem cells in a dose-dependent manner. Furthermore, it inhibits the expression of the Notch1 and β-catenin genes. Taken together, this suggests that it may elicit its effects through regulatory and target genes that mediate the Notch signal transduction pathway.

Resveratrol suppresses IGF-1 induced human colon cancer cell proliferation and elevates apoptosis via suppression of IGF-1R/Wnt and activation of p53 signaling pathways

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Radhakrishnan Sridhar

2010-05-01

Full Text Available Abstract Background Obesity is a global phenomenon and is associated with various types of cancer, including colon cancer. There is a growing interest for safe and effective bioactive compounds that suppress the risk for obesity-promoted colon cancer. Resveratrol (trans-3, 4', 5,-trihydroxystilbene, a stilbenoid found in the skin of red grapes and peanuts suppresses many types of cancers by regulating cell proliferation and apoptosis through a variety of mechanisms, however, resveratrol effects on obesity-promoted colon cancer are not clearly established. Methods We investigated the anti-proliferative effects of resveratrol on HT-29 and SW480 human colon cancer cells in the presence and absence of insulin like growth factor-1 (IGF-1; elevated during obesity and elucidated the mechanisms of action using IGF-1R siRNA in HT-29 cells which represents advanced colon carcinogenesis. Results Resveratrol (100-150 μM exhibited anti-proliferative properties in HT-29 cells even after IGF-1 exposure by arresting G0/G1-S phase cell cycle progression through p27 stimulation and cyclin D1 suppression. Treatment with resveratrol suppressed IGF-1R protein levels and concurrently attenuated the downstream Akt/Wnt signaling pathways that play a critical role in cell proliferation. Targeted suppression of IGF-1R using IGF-1R siRNA also affected these signaling pathways in a similar manner. Resveratrol treatment induced apoptosis by activating tumor suppressor p53 protein, whereas IGF-1R siRNA treatment did not affect apoptosis. Our data suggests that resveratrol not only suppresses cell proliferation by inhibiting IGF-1R and its downstream signaling pathways similar to that of IGF-1R siRNA but also enhances apoptosis via activation of the p53 pathway. Conclusions For the first time, we report that resveratrol suppresses colon cancer cell proliferation and elevates apoptosis even in the presence of IGF-1 via suppression of IGF-1R/Akt/Wnt signaling pathways and

Resveratrol suppresses IGF-1 induced human colon cancer cell proliferation and elevates apoptosis via suppression of IGF-1R/Wnt and activation of p53 signaling pathways

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Vanamala, Jairam; Reddivari, Lavanya; Radhakrishnan, Sridhar; Tarver, Chris

2010-01-01

Obesity is a global phenomenon and is associated with various types of cancer, including colon cancer. There is a growing interest for safe and effective bioactive compounds that suppress the risk for obesity-promoted colon cancer. Resveratrol (trans-3, 4', 5,-trihydroxystilbene), a stilbenoid found in the skin of red grapes and peanuts suppresses many types of cancers by regulating cell proliferation and apoptosis through a variety of mechanisms, however, resveratrol effects on obesity-promoted colon cancer are not clearly established. We investigated the anti-proliferative effects of resveratrol on HT-29 and SW480 human colon cancer cells in the presence and absence of insulin like growth factor-1 (IGF-1; elevated during obesity) and elucidated the mechanisms of action using IGF-1R siRNA in HT-29 cells which represents advanced colon carcinogenesis. Resveratrol (100-150 μM) exhibited anti-proliferative properties in HT-29 cells even after IGF-1 exposure by arresting G 0 /G 1 -S phase cell cycle progression through p27 stimulation and cyclin D1 suppression. Treatment with resveratrol suppressed IGF-1R protein levels and concurrently attenuated the downstream Akt/Wnt signaling pathways that play a critical role in cell proliferation. Targeted suppression of IGF-1R using IGF-1R siRNA also affected these signaling pathways in a similar manner. Resveratrol treatment induced apoptosis by activating tumor suppressor p53 protein, whereas IGF-1R siRNA treatment did not affect apoptosis. Our data suggests that resveratrol not only suppresses cell proliferation by inhibiting IGF-1R and its downstream signaling pathways similar to that of IGF-1R siRNA but also enhances apoptosis via activation of the p53 pathway. For the first time, we report that resveratrol suppresses colon cancer cell proliferation and elevates apoptosis even in the presence of IGF-1 via suppression of IGF-1R/Akt/Wnt signaling pathways and activation of p53, suggesting its potential role as a

Pathway of deoxynivalenol-induced apoptosis in human colon carcinoma cells

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Bensassi, Fatma; El Golli-Bennour, Emna; Abid-Essefi, Salwa; Bouaziz, Chayma; Hajlaoui, Mohamed Rabeh; Bacha, Hassen

2009-01-01

The mycotoxin, deoxynivalenol (DON), is generally detected in cereal grains and grain-based food products worldwide. Therefore, DON has numerous toxicological effects on animals and humans. The present investigation was conducted to determine the molecular aspects of DON toxicity on human colon carcinoma cells (HT 29). To this aim, we have monitored the effects of DON on (i) cell viability, (ii) Heat shock protein expressions as a parameter of protective and adaptive response, (iii) oxidative damage and (iv) cell death signalling pathway. Our results clearly showed that DON treatment inhibits cell proliferation, did not induce Hsp 70 protein expression and reactive oxygen species generation. We have also demonstrated that this toxin induced a DNA fragmentation followed by p53 and caspase-3 activations. Finally, our findings suggested that oxidative damage is not the major contributor to DON toxicity. This mycotoxin induces direct DNA lesions and could be considered by this fact as a genotoxic agent inducing cell death via an apoptotic process.

The effects of Bifidobacterium breve on immune mediators and proteome of HT29 cells monolayers.

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Sánchez, Borja; González-Rodríguez, Irene; Arboleya, Silvia; López, Patricia; Suárez, Ana; Ruas-Madiedo, Patricia; Margolles, Abelardo; Gueimonde, Miguel

2015-01-01

The use of beneficial microorganisms, the so-called probiotics, to improve human health is gaining popularity. However, not all of the probiotic strains trigger the same responses and they differ in their interaction with the host. In spite of the limited knowledge on mechanisms of action some of the probiotic effects seem to be exerted through maintenance of the gastrointestinal barrier function and modulation of the immune system. In the present work, we have addressed in vitro the response of the intestinal epithelial cell line HT29 to the strain Bifidobacterium breve IPLA20004. In the array of 84 genes involved in inflammation tested, the expression of 12 was modified by the bifidobacteria. The genes of chemokine CXCL6, the chemokine receptor CCR7, and, specially, the complement component C3 were upregulated. Indeed, HT29 cells cocultivated with B. breve produced significantly higher levels of protein C3a. The proteome of HT29 cells showed increased levels of cytokeratin-8 in the presence of B. breve. Altogether, it seems that B. breve IPLA20004 could favor the recruitment of innate immune cells to the mucosa reinforcing, as well as the physical barrier of the intestinal epithelium.

DK1 Induces Apoptosis via Mitochondria-Dependent Signaling Pathway in Human Colon Carcinoma Cell Lines In Vitro

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Yazmin Hussin

2018-04-01

Full Text Available Extensive research has been done in the search for innovative treatments against colon adenocarcinomas; however, the incidence rate of patients remains a major cause of cancer-related deaths in Malaysia. Natural bioactive compounds such as curcumin have been substantially studied as an alternative to anticancer drug therapies and have been surmised as a potent agent but, nevertheless, remain deficient due to its poor cellular uptake. Therefore, efforts now have shifted toward mimicking curcumin to synthesize novel compounds sharing similar effects. A synthetic analog, (Z-3-hydroxy-1-(2-hydroxyphenyl-3-phenylprop-2-ene-1-one (DK1, was recently synthesized and reported to confer improved bioavailability and selectivity toward human breast cancer cells. This study, therefore, aims to assess the anticancer mechanism of DK1 in relation to the induction of in vitro cell death in selected human colon cancer cell lines. Using the3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide(MTT assay, the cytotoxicity of DK1 towards HT29 and SW620 cell lines were investigated. Acridine orange/propidium iodide (AO/PI dual-staining assay and flow cytometry analyses (cell cycle analysis, Annexin/V-FITC and JC-1 assays were incorporated to determine the mode of cell death. To further determine the mechanism of cell death, quantitative real-time polymerase chain reaction (qRT-PCR and proteome profiling were conducted. Results from this study suggest that DK1 induced changes in cell morphology, leading to a decrease in cell viability and subsequent induction of apoptosis. DK1 treatment inhibited cell viability and proliferation 48 h post treatment with IC50 values of 7.5 ± 1.6 µM for HT29 cells and 14.5 ± 4.3 µM for SW620 cells, causing cell cycle arrest with increased accumulation of cell populations at the sub-G0/G1phaseof 74% and 23%, respectively. Flow cytometry analyses showed that DK1 treatment in cancer cells induced apoptosis, as indicated by DNA

Targeted inhibition of the phosphoinositide 3-kinase impairs cell proliferation, survival, and invasion in colon cancer

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Yang F

2017-09-01

Full Text Available Fei Yang,1,* Jun-Yi Gao,2,* Hua Chen,1 Zhen-Hua Du,1 Xue-Qun Zhang,3 Wei Gao4 1Department of Pathology, Jinan Central Hospital Affiliated to Shandong University, Jinan, 2Department of Clinical Medicine, Weifang Medical College, Weifang, 3Graduate School, Taishan Medical University, Xintai, 4Department of Oncology, Jinan Central Hospital Affiliated to Shandong University, Jinan, People’s Republic of China *These authors contributed equally to this work Background: Colon cancer is the third most common cancer in the world, and its metastasis and drug resistance are challenging for its effective treatment. The PI3K/Akt/mTOR pathway plays a crucial role in the pathogenesis of colon cancer. The aim of this study was to investigate the targeting of PI3K in colon cancer cells HT-29 and HCT-116 in vitro. Methods: In HT-29 and HCT-116 cells, BEZ235, a dual inhibitor of PI3K/mTOR, and shRNAtarget to PI3KCA were used to inhibit PI3K/Akt/mTOR pathway. The inhibition efficiency of PI3K/Akt/mTOR pathway was detected by RT-PCR and Western blot. Cell proliferation, migration, invasion, and apoptosis were evaluated by Cell Counting Kit-8, Transwell, and flow cytometry assays. The expression of apoptosis-related proteins (cleavage caspase 3, Bcl-2, Bax, and Bim were also detected. Results: We found that in HT-29 and HCT-116 cells, the treatment of BEZ235 (1 µM and PI3KCA knockdown inhibited the activation of PI3K/Akt/mTOR pathway and significantly suppressed cell proliferation, migration, and invasion of HT-29 and HCT-116 cells. In addition, we confirmed that knockdown of BEZ235 and PI3KCA induced cell apoptosis through the upregulated levels of cleavage caspase 3 and Bax and downregulated expression of Bcl-2 and Bim. Conclusion: Our results indicated that targeted inhibition of the PI3K/Akt/mTOR pathway impaired cell proliferation, survival, and invasion in human colon cancer. Keywords: human colon cancer, PI3K/Akt/mTOR pathway, BEZ235, PI3KCA knockdown

Differential expression of nanog1 and nanogp8 in colon cancer cells

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Ishiguro, Tatsuya; Sato, Ai; Ohata, Hirokazu; Sakai, Hiroaki; Nakagama, Hitoshi; Okamoto, Koji

2012-01-01

Highlights: ► Nanog is expressed in a majority of colon cancer cell lines examined. ► Both nanog1 and nanogp8 are expressed in colon cancer cells with varying ratios. ► Nanog mediates cell proliferation of colon cancer cells. ► Nanog predominantly localizes in cytoplasm of colon cancer cells. -- Abstract: Nanog, a homeodomain transcription factor, is an essential regulator for promotion of self-renewal of embryonic stem cells and inhibition of their differentiation. It has been demonstrated that nanog1 as well as nanogp8, a retrogene of nanog1, is preferentially expressed in advanced stages of several types of cancer, suggesting their involvement during cancer progression. Here, we investigated the expression of Nanog in well-characterized colon cancer cell lines. Expression of Nanog was detectable in 5 (HCT116, HT29, RKO, SW48, SW620) out of seven cell lines examined. RNA expression analyses of nanog1 and nanogp8 indicated that, while nanog1 was a major form in SW620 as well as in teratoma cells Tera-2, nanogp8 was preferentially expressed in HT29 and HCT116. In accordance with this, shRNA-mediated knockdown of nanog1 caused the reduction of Nanog in SW620 but not in HT29. Inhibition of Nanog in SW620 cells negatively affected cell proliferation and tumor formation in mouse xenograft. Biochemical subcellular fractionation and immunostaining analyses revealed predominant localization of Nanog in cytoplasm in SW620 and HT29, while it was mainly localized in nucleus in Tera-2. Our data indicate that nanog1 and nanogp8 are differentially expressed in colon cancer cells, and suggest that their expression contributes to proliferation of colon cancer cells.

Differential expression of nanog1 and nanogp8 in colon cancer cells

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Ishiguro, Tatsuya; Sato, Ai; Ohata, Hirokazu; Sakai, Hiroaki [Division of Cancer Differentiation, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Nakagama, Hitoshi, E-mail: hnakagam@ncc.go.jp [Division of Cancer Development System, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Okamoto, Koji, E-mail: kojokamo@ncc.go.jo [Division of Cancer Differentiation, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan)

2012-02-10

Highlights: Black-Right-Pointing-Pointer Nanog is expressed in a majority of colon cancer cell lines examined. Black-Right-Pointing-Pointer Both nanog1 and nanogp8 are expressed in colon cancer cells with varying ratios. Black-Right-Pointing-Pointer Nanog mediates cell proliferation of colon cancer cells. Black-Right-Pointing-Pointer Nanog predominantly localizes in cytoplasm of colon cancer cells. -- Abstract: Nanog, a homeodomain transcription factor, is an essential regulator for promotion of self-renewal of embryonic stem cells and inhibition of their differentiation. It has been demonstrated that nanog1 as well as nanogp8, a retrogene of nanog1, is preferentially expressed in advanced stages of several types of cancer, suggesting their involvement during cancer progression. Here, we investigated the expression of Nanog in well-characterized colon cancer cell lines. Expression of Nanog was detectable in 5 (HCT116, HT29, RKO, SW48, SW620) out of seven cell lines examined. RNA expression analyses of nanog1 and nanogp8 indicated that, while nanog1 was a major form in SW620 as well as in teratoma cells Tera-2, nanogp8 was preferentially expressed in HT29 and HCT116. In accordance with this, shRNA-mediated knockdown of nanog1 caused the reduction of Nanog in SW620 but not in HT29. Inhibition of Nanog in SW620 cells negatively affected cell proliferation and tumor formation in mouse xenograft. Biochemical subcellular fractionation and immunostaining analyses revealed predominant localization of Nanog in cytoplasm in SW620 and HT29, while it was mainly localized in nucleus in Tera-2. Our data indicate that nanog1 and nanogp8 are differentially expressed in colon cancer cells, and suggest that their expression contributes to proliferation of colon cancer cells.

Bifidobacterium breve - HT-29 cell line interaction: modulation of TNF-α induced gene expression.

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Boesten, R J; Schuren, F H J; Willemsen, L E M; Vriesema, A; Knol, J; De Vos, W M

2011-06-01

To provide insight in the molecular basis for intestinal host-microbe interactions, we determined the genome-wide transcriptional response of human intestinal epithelial cells following exposure to cells of Bifidobacterium breve. To select an appropriate test system reflecting inflammatory conditions, the responsiveness to TNF-α was compared in T84, Caco-2 and HT-29 cells. The highest TNF-α response was observed in HT-29 cells and this cell line was selected for exposure to the B. breve strains M-16V, NR246 and UCC2003. After one hour of bacterial pre-incubation followed by two hours of additional TNF-α stimulation, B. breve M-16V (86%), but to a much lesser extent strains NR246 (50%) or UCC2003 (32%), showed a strain-specific reduction of the HT-29 transcriptional response to the inflammatory treatment. The most important functional groups of genes that were transcriptionally suppressed by the presence of B. breve M-16V, were found to be involved in immune regulation and apoptotic processes. About 54% of the TNF-α induced genes were solely suppressed by the presence of B. breve M-16V. These included apoptosis-related cysteine protease caspase 7 (CASP7), interferon regulatory factor 3 (IRF3), amyloid beta (A4) precursor proteinbinding family A member 1 (APBA1), NADPH oxidase (NOX5), and leukemia inhibitory factor receptor (LIFR). The extracellular IL-8 concentration was determined by an immunological assay but did not change significantly, indicating that B. breve M-16V only partially modulates the TNF-α pathway. In conclusion, this study shows that B. breve strains modulate gene expression in HT-29 cells under inflammatory conditions in a strain-specific way.

Concomitant consumption of lycopene and fish oil inhibits tumor growth and progression in a mouse xenograft model of colon cancer

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Our previous report showed that concomitant supplementation of lycopene and eicosa-pentaenoic acid synergistically inhibited the proliferation of human colon cancer HT-29 cells in vitro. To validate our findings, the present study investigated whether consumption of lycopene and fish oil would help ...

The Effects of Bifidobacterium breve on Immune Mediators and Proteome of HT29 Cells Monolayers

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Borja Sánchez

2015-01-01

Full Text Available The use of beneficial microorganisms, the so-called probiotics, to improve human health is gaining popularity. However, not all of the probiotic strains trigger the same responses and they differ in their interaction with the host. In spite of the limited knowledge on mechanisms of action some of the probiotic effects seem to be exerted through maintenance of the gastrointestinal barrier function and modulation of the immune system. In the present work, we have addressed in vitro the response of the intestinal epithelial cell line HT29 to the strain Bifidobacterium breve IPLA20004. In the array of 84 genes involved in inflammation tested, the expression of 12 was modified by the bifidobacteria. The genes of chemokine CXCL6, the chemokine receptor CCR7, and, specially, the complement component C3 were upregulated. Indeed, HT29 cells cocultivated with B. breve produced significantly higher levels of protein C3a. The proteome of HT29 cells showed increased levels of cytokeratin-8 in the presence of B. breve. Altogether, it seems that B. breve IPLA20004 could favor the recruitment of innate immune cells to the mucosa reinforcing, as well as the physical barrier of the intestinal epithelium.

Title of paper: the induction of P-53 independent programmed cell death (apoptosis) with ionizing radiation and 5-fluorouracil (5-FU) in the HT-29 human colon carcinoma cell line

International Nuclear Information System (INIS)

Blackstock, A. Wm.; Gill, Misha; Hess, Suzanne M.; Fisher, Robert W.; Leadon, Steven A.; Tepper, Joel E.

1996-01-01

Purpose/Objective: The role of programmed cell death (apoptosis) as a cellular response to cancer therapy such as radiation or chemotherapy is the subject of much study, and manipulation of the apoptotic response in tumor cells may be valuable in the treatment of a variety of cancers. Both p53 dependent and independent apoptotic pathways have been identified; p53 is mutated in at least 50 % of human cancers and a majority of radiation resistant tumors contain p53 mutations. This study is designed to examine the induction of programmed cell death in a human colon carcinoma cell line that possesses two mutated p53 alleles. Ionizing radiation alone, or in combination with the chemotherapeutic drug 5-fluorouracil (5-FU), were used to elicit the apoptotic response. This study will focus on whether these treatments can induce a significant apoptotic response in cells that have mutated p53 alleles. Materials and Methods: HT-29 cells were assessed for clonogenic survival after being plated at a variety of densities, and treated with single graded doses of radiation (0, 1, 2, 4, 6, 8, 10 Gy) either alone or immediately prior to a 24 hour exposure to 5-FU (2 ug/ml). The extent of radiation and 5-FU-induced apoptosis was determined in the HT-29 cell line after single doses of 0, 2, 5, and 10 Gy either alone or immediately prior to a 24 hour incubation in 5-FU (2 ug/ml). Three separate assays were used to evaluate the apoptotic response. Cells undergoing apoptosis undergo gross morphological changes including a condensation of chromatin, membrane blebbing, and an eventual release of membrane bound cytoplasmic fragments. Hematoxylin and eosin staining were used to visualize some of these morphological changes. Another characteristic of the apoptotic response is the activation of an endonuclease that cleaves DNA into specific fragments. Accordingly, an ELISA cell death assay (Boehringer Mannheim, Indianapolis IN) was used to quantitate cytoplasmic histone-associated DNA

Colon-available raspberry polyphenols exhibit anti-cancer effects on in vitro models of colon cancer

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McDougall Gordon

2007-01-01

Full Text Available Abstract Background There is a probable association between consumption of fruit and vegetables and reduced risk of cancer, particularly cancer of the digestive tract. This anti-cancer activity has been attributed in part to anti-oxidants present in these foods. Raspberries in particular are a rich source of the anti-oxidant compounds, such as polyphenols, anthocyanins and ellagitannins. Methods A "colon-available" raspberry extract (CARE was prepared that contained phytochemicals surviving a digestion procedure that mimicked the physiochemical conditions of the upper gastrointestinal tract. The polyphenolic-rich extract was assessed for anti-cancer properties in a series of in vitro systems that model important stages of colon carcinogenesis, initiation, promotion and invasion. Results The phytochemical composition of CARE was monitored using liquid chromatography mass spectrometry. The colon-available raspberry extract was reduced in anthocyanins and ellagitannins compared to the original raspberry juice but enriched in other polyphenols and polyphenol breakdown products that were more stable to gastrointestinal digestion. Initiation – CARE caused significant protective effects against DNA damage induced by hydrogen peroxide in HT29 colon cancer cells measured using single cell microgelelectrophoresis. Promotion – CARE significantly decreased the population of HT29 cells in the G1 phase of the cell cycle, effectively reducing the number of cells entering the cell cycle. However, CARE had no effect on epithelial integrity (barrier function assessed by recording the trans-epithelial resistance (TER of CACO-2 cell monolayers. Invasion – CARE caused significant inhibition of HT115 colon cancer cell invasion using the matrigel invasion assay. Conclusion The results indicate that raspberry phytochemicals likely to reach the colon are capable of inhibiting several important stages in colon carcinogenesis in vitro.

Cytotoxic Activity of Kenaf Seed Oils from Supercritical Carbon Dioxide Fluid Extraction towards Human Colorectal Cancer (HT29) Cell Lines.

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Abd Ghafar, Siti Aisyah; Ismail, Maznah; Saiful Yazan, Latifah; Fakurazi, Sharida; Ismail, Norsharina; Chan, Kim Wei; Md Tahir, Paridah

2013-01-01

Kenaf (Hibiscus cannabinus) from the family Malvaceae, is a valuable fiber plant native to India and Africa and is currently planted as the fourth commercial crop in Malaysia. Kenaf seed oil contains alpha-linolenic acid, phytosterol such as β -sitosterol, vitamin E, and other antioxidants with chemopreventive properties. Kenaf seeds oil (KSO) was from supercritical carbon dioxide extraction fluid (SFE) at 9 different permutations of parameters based on range of pressures from 200 to 600 bars and temperature from 40 to 80°C. They were 200/40, 200/60, 200/80, 400/40, 400/60, 400/80, 600/40, 600/60, and 600/80. Extraction from 9 parameters of KSO-SFE was screened for cytotoxicity towards human colorectal cancer cell lines (HT29) and mouse embryonic fibroblast (NIH/3T3) cell lines using MTS assay. KSO-SFE at 600/40 showed the strongest cytotoxicity towards HT29 with IC50 of 200 µg/mL. The IC50 for NIH/3T3 was not detected even at highest concentration employed. Cell cycle analysis showed a significant increase in the accumulation of KSO-SFE-treated cells at sub-G1 phase, indicating the induction of apoptosis by KSO-SFE. Further apoptosis induction was confirmed by Annexin V/PI and AO/PI staining.

9-Hydroxystearic acid interferes with EGF signalling in a human colon adenocarcinoma

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Calonghi, Natalia; Pagnotta, Eleonora; Parolin, Carola; Tognoli, Cristina; Boga, Carla; Masotti, Lanfranco

2006-01-01

The epidermal growth factor has long been known to be strictly correlated with the highly proliferating activities of cancer cells and primary tumors. Moreover, in the nucleus, the epidermal growth factor/epidermal growth factor receptor complex (EGF/EGFR) functions as a transcriptional regulator that activates the cyclin D1 gene. 9-hydroxystearic acid (9-HSA) induces cell proliferation arrest and differentiation in HT29 colon cancer cells by inhibiting histone deacetylase 1 (HDAC1). 9-HSA-treated HT29, when stimulated with EGF, are not responsive and surprisingly undergo a further arrest. In order to understand the mechanisms of this effect, we analyzed the degree of internalization of the EGF/EGFR complex and its interactions with HDAC1. It appears that HDAC1, as modified by 9-HSA, is unable to associate with cyclin D1, interfering with the cell proliferation program, and sequesters the EGF/EGFR complex interrupting the transduction of the mitogenic signal

Hath1 inhibits proliferation of colon cancer cells probably through up-regulating expression of Muc2 and p27 and down-regulating expression of cyclin D1.

Science.gov (United States)

Zhu, Dai-Hua; Niu, Bai-Lin; Du, Hui-Min; Ren, Ke; Sun, Jian-Ming; Gong, Jian-Ping

2012-01-01

Previous studies showed that Math1 homologous to human Hath1 can cause mouse goblet cells to differentiate. In this context it is important that the majority of colon cancers have few goblet cells. In the present study, the potential role of Hath1 in colon carcinogenesis was investigated. Sections of paraffin-embedded tissues were used to investigate the goblet cell population of normal colon mucosa, mucosa adjacent colon cancer and colon cancer samples from 48 patients. Hath1 and Muc2 expression in these samples were tested by immunohistochemistry, quantitative real-time reverse transcription -PCR and Western blotting. After the recombinant plasmid, pcDNA3.1(+)-Hath1 had been transfected into HT29 colon cancer cells, three clones were selected randomly to test the levels of Hath1 mRNA, Muc2 mRNA, Hath1, Muc2, cyclin D1 and p27 by quantitative real-time reverse transcription-PCR and Western blotting. Moreover, the proliferative ability of HT29 cells introduced with Hath1 was assessed by means of colony formation assay and xenografting. Expression of Hath1, Muc2, cyclin D1 and p27 in the xenograft tumors was also detected by Western blotting. No goblet cells were to be found in colon cancer and levels of Hath1 mRNA and Hath1, Muc2 mRNA and Muc2 were significantly down-regulated. Hath1 could decrease cyclin D1, increase p27 and Muc2 in HT29 cells and inhibit their proliferation. Hath1 may be an anti-oncogene in colon carcinogenesis.

Influence of in vitro supplementation with lipids from conventional and Alpine milk on fatty acid distribution and cell growth of HT-29 cells

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Dänicke Sven

2011-08-01

Full Text Available Abstract Background To date, the influence of milk and dairy products on carcinogenesis remains controversial. However, lipids of ruminant origin such as conjugated linoleic acids (CLA are known to exhibit beneficial effects in vitro and in vivo. The aim of the present study was to determine the influence of milk lipids of different origin and varying quality presenting as free fatty acid (FFA solutions on cellular fatty acid distribution, cellular viability, and growth of human colon adenocarcinoma cells (HT-29. Methods FAME of conventional and Alpine milk lipids (MLcon, MLalp and cells treated with FFA derivatives of milk lipids were analyzed by means of GC-FID and Ag+-HPLC. Cellular viability and growth of the cells were determined by means of CellTiter-Blue®-assay and DAPI-assay (4',6-diamidino-2-phenylindole dihydrochloride, respectively. Results Supplementation with milk lipids significantly decreased viability and growth of HT-29 cells in a dose- and time-dependent manner. MLalp showed a lower SFA/MUFA ratio, a 8 fold increased CLA content, and different CLA profile compared to MLcon but did not demonstrate additional growth-inhibitory effects. In addition, total concentration and fatty acid distribution of cellular lipids were altered. In particular, treatment of the cells yielded highest amounts of two types of milk specific major fatty acids (μg FA/mg cellular protein after 8 h of incubation compared to 24 h; 200 μM of MLcon (C16:0, 206 ± 43, 200 μM of MLalp (C18:1 c9, (223 ± 19. Vaccenic acid (C18:1 t11 contained in milk lipids was converted to c9,t11-CLA in HT-29 cells. Notably, the ratio of t11,c13-CLA/t7,c9-CLA, a criterion for pasture feeding of the cows, was significantly changed after incubation for 8 h with lipids from MLalp (3.6 - 4.8, compared to lipids from MLcon (0.3 - 0.6. Conclusions Natural lipids from conventional and Alpine milk showed similar growth inhibitory effects. However, different changes in cellular

Synsepalum dulcificum extracts exhibit cytotoxic activity on human colorectal cancer cells and upregulate c-fos and c-jun early apoptotic gene expression

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Jichang Seong

2018-01-01

Full Text Available Objective: To explore cytotoxicity of Synsepalum dulcificum (S. dulcificum Daniell (Sapotaceae on human colon cancer (HCT-116 and HT-29, human monocytic leukemia (THP-1 and normal (HDFn cell lines, and its effect on the expression of early apoptotic genes, c-fos and c-jun. Methods: Leaf, stem and berry of S. dulcificum were separately extracted by using 2 solvents, 10% ethanol (EtOH and 80% methanol (MeOH. PrestoBlue® cell viability assay and qRT-PCR assay were conducted to examine the above objectives respectively. Results: Stem MeOH, stem EtOH, and berry EtOH extracts of S. dulcificum were cytotoxic to HCT-116 and HT-29 human colon cancer cells. For HCT-116, IC50 values of these 3 extracts were not significantly different (P>0.05 from that of the positive control bleomycin (IC50 of 33.57 μg/mL, while for HT-29, IC50 values of these 3 extracts were significantly lower (P<0.05 than that of bleomycin (IC50 of 25.24 μg/mL. None of the extracts were cytotoxic to the THP-1 monocytic leukemia cells and HDFn normal human dermal fibroblasts. For both HCT-116 and HT-29, these extracts significantly up-regulated (P<0.05 the expression of c-fos and c-jun compared to the untreated negative control. Conclusions: The results of this study suggest that cytotoxicity of stem MeOH, stem EtOH, and berry EtOH extracts of S. dulcificum on HCT-116 and HT-29 colon cancer cells is due to the induced apoptosis which is caused by the up-regulation of the expression of early apoptotic genes, c-fos and c-jun.

Lichen secondary metabolites are responsible for induction of apoptosis in HT-29 and A2780 human cancer cell lines.

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Bačkorová, M; Jendželovský, R; Kello, M; Bačkor, M; Mikeš, J; Fedoročko, P

2012-04-01

Lichens are a known source of approximately 800 unique secondary metabolites, many of which play important ecological roles, including regulating the equilibrium between symbionts. However, only a few of these compounds have been assessed for their effectiveness against various in vitro cancer models. Moreover, the mechanisms of biological activity of lichen secondary metabolites on living cells (including cancer cells) are still almost entirely unknown. In the present study, we investigated the mechanisms of cytotoxicity of four lichen secondary metabolites (parietin, atranorin, usnic acid and gyrophoric acid) on A2780 and HT-29 cancer cell lines. We found that usnic acid and atranorin were more effective anti-cancer compounds when compared to parietin and gyrophoric acid. Usnic acid and atranorin were capable of inducing a massive loss in the mitochondrial membrane potential, along with caspase-3 activation (only in HT-29 cells) and phosphatidylserine externalization in both tested cell lines. Induction of both ROS and especially RNS may be responsible, at least in part, for the cytotoxic effects of the tested compounds. Based on the detection of protein expression (PARP, p53, Bcl-2/Bcl-xL, Bax, p38, pp38) we found that usnic acid and atranorin are activators of programmed cell death in A2780 and HT-29, probably through the mitochondrial pathway. Copyright © 2012 Elsevier Ltd. All rights reserved.

Preparation and bioevaluation of {sup 177}Lu-labelled anti-CD44 for radioimmunotherapy of colon cancer

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Lee, So Young; Hong, Young Don; Jung, Sung Hee; Choi, Sun Ju [Radioisotope Research Division, Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

2015-12-15

CD44 is a particular adhesion molecule and facilitates both cell-cell and cell-matrix interactions. In particular, splice variants of CD44 are particularly overexpressed in a large number of malignancies and carcinomas. In this study, the {sup 177}Lu-labelled CD44 targeting antibody was prepared and bioevaluated in vitro and in vivo. Anti-CD44 was immunoconjugated with the equivalent molar ratio of cysteine-based dtPA-ncS and radioimmunoconjugated with {sup 177}Lu at room temperature within 15 minutes. the stability was tested in human serum. An in vitro study was carried out in Ht-29 human colon cancer cell lines. For the biodistribution study {sup 177}Lu-labelled anti-CD44 was injected in xenograft mice. Anti-CD44 was immunoconjugated with cysteinebased dtPA-ncS and purified by a centricon filter system having a molecular cut-off of 50 kda. radioimmunoconjugation with {sup 177}Lu was reacted for 15 min at room temperature. the radiolabeling yield was >99%, and it was stable in human serum without any fragmentation or degradation. The radioimmunoconjugate showed a high binding affinity on HT-29 colon cancer cell surfaces. In a biodistribution study, the tumor-to-blood ratio of the radioimmunoconjugate was 43 : 1 at 1 day post injection (p.i) in human colon cancer bearing mice. the anti-CD44 monoclonal antibody for the targeting of colon cancer was effectively radioimmunoconjugated with {sup 177}Lu. the in vitro high immunoactivity of this radioimmunoconjugate was determined by a cell binding assay. In addition, the antibody's tumor targeting ability was demonstrated with very high uptake in tumors. this radioimmunoconjugate is applicable to therapy in human colon cancer with highly expressed CD44.

Poly-γ-Glutamic Acid Induces Apoptosis via Reduction of COX-2 Expression in TPA-Induced HT-29 Human Colorectal Cancer Cells

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Eun Ju Shin

2015-04-01

Full Text Available Poly-γ-glutamic acid (PGA is one of the bioactive compounds found in cheonggukjang, a fast-fermented soybean paste widely utilized in Korean cooking. PGA is reported to have a number of beneficial health effects, and interestingly, it has been identified as a possible anti-cancer compound through its ability to promote apoptosis in cancer cells, although the precise molecular mechanisms remain unclear. Our findings demonstrate that PGA inhibits the pro-proliferative functions of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, a known chemical carcinogen in HT-29 human colorectal cancer cells. This inhibition was accompanied by hallmark apoptotic phenotypes, including DNA fragmentation and the cleavage of poly (ADP-ribose polymerase (PARP and caspase 3. In addition, PGA treatment reduced the expression of genes known to be overexpressed in colorectal cancer cells, including cyclooxygenase 2 (COX-2 and inducible nitric oxide synthase (iNOS. Lastly, PGA promoted activation of 5' adenosine monophosphate-activated protein (AMPK in HT-29 cells. Taken together, our results suggest that PGA treatment enhances apoptosis in colorectal cancer cells, in part by modulating the activity of the COX-2 and AMPK signaling pathways. These anti-cancer functions of PGA make it a promising compound for future study.

Cytotoxic Activity of Kenaf Seed Oils from Supercritical Carbon Dioxide Fluid Extraction towards Human Colorectal Cancer (HT29 Cell Lines

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Siti Aisyah Abd Ghafar

2013-01-01

Full Text Available Kenaf (Hibiscus cannabinus from the family Malvaceae, is a valuable fiber plant native to India and Africa and is currently planted as the fourth commercial crop in Malaysia. Kenaf seed oil contains alpha-linolenic acid, phytosterol such as β-sitosterol, vitamin E, and other antioxidants with chemopreventive properties. Kenaf seeds oil (KSO was from supercritical carbon dioxide extraction fluid (SFE at 9 different permutations of parameters based on range of pressures from 200 to 600 bars and temperature from 40 to 80°C. They were 200/40, 200/60, 200/80, 400/40, 400/60, 400/80, 600/40, 600/60, and 600/80. Extraction from 9 parameters of KSO-SFE was screened for cytotoxicity towards human colorectal cancer cell lines (HT29 and mouse embryonic fibroblast (NIH/3T3 cell lines using MTS assay. KSO-SFE at 600/40 showed the strongest cytotoxicity towards HT29 with IC50 of 200 µg/mL. The IC50 for NIH/3T3 was not detected even at highest concentration employed. Cell cycle analysis showed a significant increase in the accumulation of KSO-SFE-treated cells at sub-G1 phase, indicating the induction of apoptosis by KSO-SFE. Further apoptosis induction was confirmed by Annexin V/PI and AO/PI staining.

SILENCING THE NUCLEOCYTOPLASMIC O-GLCNAC TRANSFERASE REDUCES PROLIFERATION, ADHESION AND MIGRATION OF CANCER AND FETAL HUMAN COLON CELL LINES

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AGATA eSTEENACKERS

2016-05-01

Full Text Available The post-translational modification of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc is regulated by a unique couple of enzymes. O-GlcNAc transferase (OGT transfers the GlcNAc residue from UDP-GlcNAc, the final product of the hexosamine biosynthetic pathway (HBP, whereas O-GlcNAcase (OGA removes it. This study and others show that OGT and O-GlcNAcylation levels are increased in cancer cell lines. In that context we studied the effect of OGT silencing in the colon cancer cell lines HT29 and HCT116 and the primary colon cell line CCD841CoN. Herein we report that OGT silencing diminished proliferation, in vitro cell survival and adhesion of primary and cancer cell lines. SiOGT dramatically de-creased HT29 and CCD841CoN migration, CCD841CoN harboring high capabilities of mi-gration in Boyden chamber system when compared to HT29 and HCT116. The expression levels of actin and tubulin were unaffected by OGT knockdown but siOGT seemed to disor-ganize microfilament, microtubule and vinculin networks in CCD841CoN. While cancer cell lines harbor higher levels of OGT and O-GlcNAcylation to fulfill their proliferative and migra-tory properties, in agreement with their higher consumption of HBP main substrates glucose and glutamine, our data demonstrate that OGT expression is not only necessary for the biolog-ical properties of cancer cell lines but also for normal cells.

miR-320 enhances the sensitivity of human colon cancer cells to chemoradiotherapy in vitro by targeting FOXM1

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Wan, Lu-Ying; Deng, Jun; Xiang, Xiao-Jun; Zhang, Ling; Yu, Feng; Chen, Jun; Sun, Zhe; Feng, Miao; Xiong, Jian-Ping, E-mail: jpxiong@medmail.com.cn

2015-02-06

Highlights: • miR-320 plays a significant role in chemoresistance. • This role might be attribute to targeting FOXM1. • The Wnt/β-catenin pathway also involves in this chemotherapy sensitivity. - Abstract: miR-320 expression level is found to be down-regulated in human colon cancer. To date, however, its underlying mechanisms in the chemo-resistance remain largely unknown. In this study, we demonstrated that ectopic expression of miR-320 led to inhibit HCT-116 cell proliferation, invasion and hypersensitivity to 5-Fu and Oxaliplatin. Also, knockdown of miR-320 reversed these effects in HT-29 cells. Furthermore, we identified an oncogene, FOXM1, as a direct target of miR-320. In addition, miR-320 could inactive the activity of Wnt/β-catenin pathway. Finally, we found that miR-320 and FOXM1 protein had a negative correlation in colon cancer tissues and adjacent normal tissues. These findings implied that miR-320–FOXM1 axis may overcome chemo-resistance of colon cancer cells and provide a new therapeutic target for the treatment of colon cancer.

Troglitazone induced apoptosis via PPARγ activated POX-induced ROS formation in HT29 cells.

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Wang, Jing; Lv, XiaoWen; Shi, JiePing; Hu, XiaoSong; DU, YuGuo

2011-08-01

In order to investigate the potential mechanisms in troglitazone-induced apoptosis in HT29 cells, the effects of PPARγ and POX-induced ROS were explored. [3- (4, 5)-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay, Annexin V and PI staining using FACS, plasmid transfection, ROS formation detected by DCFH staining, RNA interference, RT-PCR & RT-QPCR, and Western blotting analyses were employed to investigate the apoptotic effect of troglitazone and the potential role of PPARγ pathway and POX-induced ROS formation in HT29 cells. Troglitazone was found to inhibit the growth of HT29 cells by induction of apoptosis. During this process, mitochondria related pathways including ROS formation, POX expression and cytochrome c release increased, which were inhibited by pretreatment with GW9662, a specific antagonist of PPARγ. These results illustrated that POX upregulation and ROS formation in apoptosis induced by troglitazone was modulated in PPARγ-dependent pattern. Furthermore, the inhibition of ROS and apoptosis after POX siRNA used in troglitazone-treated HT29 cells indicated that POX be essential in the ROS formation and PPARγ-dependent apoptosis induced by troglitazone. The findings from this study showed that troglitazone-induced apoptosis was mediated by POX-induced ROS formation, at least partly, via PPARγ activation. Copyright © 2011 The Editorial Board of Biomedical and Environmental Sciences. Published by Elsevier B.V. All rights reserved.

Spontaneous and cytokine induced expression and activity of matrix metalloproteinases in human colonic epithelium

DEFF Research Database (Denmark)

Pedersen, G; Saermark, T; Kirkegaard, T

2009-01-01

levels in cells from inflamed IBD mucosa. MMP-2 and -8 mRNA were expressed inconsistently and MMP-11, -13 and -14 mRNA undetectable. Proteolytic MMP activity was detected in CEC supernatants and the level was increased significantly in inflamed IBD epithelium. The enzyme activity was inhibited strongly......Matrix metalloproteinases (MMPs) have been implicated in tissue damage associated with inflammatory bowel disease (IBD).As the role of the intestinal epithelium in this process is unknown, we determined MMP expression and enzyme activity in human colonic epithelial cells (CEC). MMP mRNA expression...... was assessed by reverse transcription-polymerase chain reaction in HT-29 and DLD-1 cells and in CEC isolated from biopsies from IBD and control patients. Total MMP activity in the cells was measured by a functional assay, based on degradation of a fluorescent synthetic peptide containing the specific bond...

Investigations into the binding affinities of different human 5-HT4 receptor splice variants.

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Irving, Helen R; Tochon-Danguy, Nathalie; Chinkwo, Kenneth A; Li, Jian G; Grabbe, Carmen; Shapiro, Marina; Pouton, Colin W; Coupar, Ian M

2010-01-01

This study examined whether the drug-receptor-binding sites of 5 selected human 5-HT(4) receptor splice variants [h5-HT4(a), h5-HT4(b), h5-HT4(c), h5-HT4(d) and h5-HT4(g)] display preferential affinities towards agonists. The agonists selected on the basis of chemical diversity and clinical relevance were: 5-HT4 benzamides, renzapride, zacopride and prucalopride; the benzimidazolones, DAU 6236 and BIMU 1; the aromatic ketone, RS67333, and the indole carbazimidamide tegaserod. The rank order of affinities ranging across the splice variants was: tegaserod (pKi: 7.38-7.91) > or = Y-36912 (pKi: 7.03-7.85) = BIMU 1 (pKi: 6.92-7.78) > or = DAU 6236 (pKi: 6.79-7.99) > or = 5-HT (pKi: 5.82-7.29) > or = 5-MeOT (pKi: 5.64-6.83) > or = renzapride (pKi: 4.85-5.56). We obtained affinity values for the 5-HT4(b), (d) and (g) variants for RS67333 (pKi: 7:48-8.29), prucalopride (pKi: 6.86-7.37) and zacopride (pKi: 5.88-7.0). These results indicate that the ligands interact with the same conserved site in each splice variant. Some splice variants have a higher affinity for certain agonists and the direction of selectivity followed a common trend of lowest affinity at the (d) variant. However, this trend was not evident in functional experiments. Our findings suggest that it may be possible to design splice variant selective ligands, which may be of relevance for experimental drugs but may be difficult to develop clinically. 2010 S. Karger AG, Basel.

Targeting fatty acid amide hydrolase and transient receptor potential vanilloid-1 simultaneously to modulate colonic motility and visceral sensation in the mouse: A pharmacological intervention with N-arachidonoyl-serotonin (AA-5-HT).

Science.gov (United States)

Bashashati, M; Fichna, J; Piscitelli, F; Capasso, R; Izzo, A A; Sibaev, A; Timmermans, J-P; Cenac, N; Vergnolle, N; Di Marzo, V; Storr, M

2017-12-01

Endocannabinoid anandamide (AEA) inhibits intestinal motility and visceral pain, but it may also be proalgesic through transient receptor potential vanilloid-1 (TRPV1). AEA is degraded by fatty acid amide hydrolase (FAAH). This study explored whether dual inhibition of FAAH and TRPV1 reduces diarrhea and abdominal pain. Immunostaining was performed on myenteric plexus of the mouse colon. The effects of the dual FAAH/TRPV1 inhibitor AA-5-HT on electrically induced contractility, excitatory junction potential (EJP) and fast (f) and slow (s) inhibitory junction potentials (IJP) in the mouse colon, colonic propulsion and visceromotor response (VMR) to rectal distension were studied. The colonic levels of endocannabinoids and fatty acid amides were measured. CB1-positive neurons exhibited TRPV1; only some TRPV1 positive neurons did not express CB1. CB1 and FAAH did not colocalize. AA-5-HT (100 nM-10 μM) decreased colonic contractility by ~60%; this effect was abolished by TRPV1 antagonist 5'-IRTX, but not by CB1 antagonist, SR141716. AA-5-HT (1 μM-10 μM) inhibited EJP by ~30% and IJPs by ~50%. The effects of AA-5-HT on junction potentials were reversed by SR141716 and 5`-IRTX. AA-5-HT (20 mg/kg; i.p.) inhibited colonic propulsion by ~30%; SR141716 but not 5`-IRTX reversed this effect. AA-5-HT decreased VMR by ~50%-60%; these effects were not blocked by SR141716 or 5`-IRTX. AA-5-HT increased AEA in the colon. The effects of AA-5-HT on visceral sensation and colonic motility are differentially mediated by CB1, TRPV1 and non-CB1/TRPV1 mechanisms, possibly reflecting the distinct neuromodulatory roles of endocannabinoid and endovanilloid FAAH substrates in the mouse intestine. © 2017 John Wiley & Sons Ltd.

Induction of apoptosis in colon cancer cells treated with isorhamnetin glycosides from Opuntia ficus-indica pads.

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Antunes-Ricardo, Marilena; Moreno-García, Beatriz E; Gutiérrez-Uribe, Janet A; Aráiz-Hernández, Diana; Alvarez, Mario M; Serna-Saldivar, Sergio O

2014-12-01

(OFI) contains health-promoting compounds like flavonoids, being the isorhamnetin glycosides the most abundant. We evaluated the effect of OFI extracts with different isorhamnetin glycosides against two different human colon cancer cells (HT-29 and Caco2). The extracts were obtained by alkaline hydrolysis with NaOH at 40 °C during 15, 30 or 60 min. Tri and diglycosides were the most abundant isorhamnetin glycosides, therefore these compounds were isolated to compare their cytotoxic effect with the obtained from the extracts. The OFI extracts and purified isorhamnetin glycosides were more cytotoxic against HT-29 cells than Caco2 cells. OFI-30 exhibited the lowest IC50 value against HT-29 (4.9 ± 0.5 μg/mL) and against Caco2 (8.2 ± 0.3 μg/mL). Isorhamnetin diglycosides IG5 and IG6 were more cytotoxic than pure isorhamnetin aglycone or triglycosides when they were tested in HT-29 cells. Bioluminescent analysis revealed increased activity of caspase 3/7 in OFI extracts-treated cells, particularly for the extract with the highest concentration of isorhamnetin triglycosides. Flow cytometry analysis confirmed that OFI extract and isorhamnetin glycosides induced a higher percentage of apoptosis in HT-29 than in Caco2, while isorhamnetin was more apoptotic in Caco2. This research demonstrated that glycosilation affected antiproliferative effect of pure isorhamnetin glycosides or when they are mixed with other phytochemicals in an extract obtained from OFI.

5-HT modulation of pain perception in humans.

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Martin, Sarah L; Power, Andrea; Boyle, Yvonne; Anderson, Ian M; Silverdale, Monty A; Jones, Anthony K P

2017-10-01

Although there is clear evidence for the serotonergic regulation of descending control of pain in animals, little direct evidence exists in humans. The majority of our knowledge comes from the use of serotonin (5-HT)-modulating antidepressants as analgesics in the clinical management of chronic pain. Here, we have used an acute tryptophan depletion (ATD) to manipulate 5-HT function and examine its effects of ATD on heat pain threshold and tolerance, attentional manipulation of nociceptive processing and mood in human volunteers. Fifteen healthy participants received both ATD and balanced amino acid (BAL) drinks on two separate sessions in a double-blind cross-over design. Pain threshold and tolerance were determined 4 h post-drink via a heat thermode. Additional attention, distraction and temperature discrimination paradigms were completed using a laser-induced heat pain stimulus. Mood was assessed prior and throughout each session. Our investigation reported that the ATD lowered plasma TRP levels by 65.05 ± 7.29% and significantly reduced pain threshold and tolerance in response to the heat thermode. There was a direct correlation between the reduction in total plasma TRP levels and reduction in thermode temperature. In contrast, ATD showed no effect on laser-induced pain nor significant impact of the distraction-induced analgesia on pain perception but did reduce performance of the painful temperature discrimination task. Importantly, all findings were independent of any effects of ATD on mood. As far as we are aware, it is the first demonstration of 5-HT effects on pain perception which are not confounded by mood changes.

Saccharomyces boulardii Protease Inhibits the Effects of Clostridium difficile Toxins A and B in Human Colonic Mucosa

Science.gov (United States)

Castagliuolo, Ignazio; Riegler, Martin F.; Valenick, Leyla; LaMont, J. Thomas; Pothoulakis, Charalabos

1999-01-01

Saccharomyces boulardii is a nonpathogenic yeast used in the treatment of Clostridium difficile diarrhea and colitis. We have reported that S. boulardii inhibits C. difficile toxin A enteritis in rats by releasing a 54-kDa protease which digests the toxin A molecule and its brush border membrane (BBM) receptor (I. Castagliuolo, J. T. LaMont, S. T. Nikulasson, and C. Pothoulakis, Infect. Immun. 64:5225–5232, 1996). The aim of this study was to further evaluate the role of S. boulardii protease in preventing C. difficile toxin A enteritis in rat ileum and determine whether it protects human colonic mucosa from C. difficile toxins. A polyclonal rabbit antiserum raised against purified S. boulardii serine protease inhibited by 73% the proteolytic activity present in S. boulardii conditioned medium in vitro. The anti-protease immunoglobulin G (IgG) prevented the action of S. boulardii on toxin A-induced intestinal secretion and mucosal permeability to [3H]mannitol in rat ileal loops, while control rabbit IgG had no effect. The anti-protease IgG also prevented the effects of S. boulardii protease on digestion of toxins A and B and on binding of [3H]toxin A and [3H]toxin B to purified human colonic BBM. Purified S. boulardii protease reversed toxin A- and toxin B-induced inhibition of protein synthesis in human colonic (HT-29) cells. Furthermore, toxin A- and B-induced drops in transepithelial resistance in human colonic mucosa mounted in Ussing chambers were reversed by 60 and 68%, respectively, by preexposing the toxins to S. boulardii protease. We conclude that the protective effects of S. boulardii on C. difficile-induced inflammatory diarrhea in humans are due, at least in part, to proteolytic digestion of toxin A and B molecules by a secreted protease. PMID:9864230

Interaction of polyunsaturated fatty acids and sodium butyrate during apoptosis in HT-29 human colon adenocarcinoma cells

Czech Academy of Sciences Publication Activity Database

Hofmanová, Jiřina; Vaculová, Alena; Lojek, Antonín; Kozubík, Alois

2005-01-01

Roč. 44, č. 1 (2005), s. 40-51 ISSN 1436-6207 R&D Projects: GA ČR(CZ) GA525/01/0419 Institutional research plan: CEZ:AV0Z50040507 Keywords : colon cancer * diet * butyrate Subject RIV: BO - Biophysics Impact factor: 2.257, year: 2005

Pleiotropic effects of cancer cells' secreted factors on human stromal (mesenchymal) stem cells

DEFF Research Database (Denmark)

Al-toub, Mashael; Almusa, Abdulaziz; Almajed, Mohammed

2013-01-01

cells' secreted factors as represented by a panel of human cancer cell lines (breast (MCF7 and MDA-MB-231); prostate (PC-3); lung (NCI-H522); colon (HT-29) and head & neck (FaDu)) on the biological characteristics of MSCs. METHODS: Morphological changes were assessed using fluorescence microscopy......, but not from MCF7 and HT-29, developed an elongated, spindle-shaped morphology with bipolar processes. In association with phenotypic changes, genome-wide gene expression and bioinformatics analysis revealed an enhanced pro-inflammatory response of those MSCs. Pharmacological inhibitions of FAK and MAPKK...

Computer modelling of HT gas metabolism in humans

International Nuclear Information System (INIS)

Peterman, B.F.

1982-01-01

A mathematical model was developed to simulate the metabolism of HT gas in humans. The rate constants of the model were estimated by fitting the calculated curves to the experimental data by Pinson and Langham in 1957. The calculations suggest that the oxidation of HT gas (which probably occurs as a result of the enzymatic action of hydrogenase present in bacteria of human gut) occurs at a relatively low rate with a half-time of 10-12 hours. The inclusion of the dose due to the production of the HT oxidation product (HTO) in the soft tissues lowers the value of derived air concentration by about 50%. Furthermore the relationship between the concentration of HTO in urine and the dose to the lung from HT in the air in lungs is linear after short HT exposures, and hence HTO concentrations in urine can be used to estimate the upper limits on the lung dose from HT exposures. (author)

Screening of bifidobacteria and lactobacilli able to antagonise the cytotoxic effect of Clostridium difficile upon intestinal epithelial HT29 monolayer

Directory of Open Access Journals (Sweden)

Lorena eValdés-Varela

2016-04-01

Full Text Available Clostridium difficile is an opportunistic pathogen inhabiting the human gut, often being the aetiological agent of infections after a microbiota dysbiosis following, for example, an antibiotic treatment. C. difficile infections (CDI constitute a growing health problem with increasing rates of morbidity and mortality at groups of risk, such as elderly and hospitalized patients, but also in populations traditionally considered low-risk. This could be related to the occurrence of virulent strains which, among other factors, have high-level of resistance to fluoroquinolones, more efficient sporulation and markedly high toxin production. Several novel intervention strategies against CDI are currently under study, such as the use of probiotics to counteract the growth and/or toxigenic activity of C. difficile.In this work, we have analysed the capability of twenty Bifidobacterium and Lactobacillus strains, from human intestinal origin, to counteract the toxic effect of C. difficile LMG21717 upon the human intestinal epithelial cell line HT29. For this purpose, we incubated the bacteria together with toxigenic supernatants obtained from C. difficile. After this co-incubation new supernatants were collected in order to quantify the remnant A and B toxins, as well as to determine their residual toxic effect upon HT29 monolayers. To this end, the real time cell analyser (RTCA model, recently developed in our group to monitor C. difficile toxic effect, was used. Results obtained showed that strains of Bifidobacterium longum and Bifidobacterium breve were able to reduce the toxic effect of the pathogen upon HT29, the RTCA normalized cell-index values being inversely correlated with the amount of remnant toxin in the supernatant. The strain B. longum IPLA20022 showed the highest ability to counteract the cytotoxic effect of C. difficile acting directly against the toxin, also having the highest capability for removing the toxins from the clostridial

Differentiation Affects the Release of Exosomes from Colon Cancer Cells and Their Ability to Modulate the Behavior of Recipient Cells.

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Lucchetti, Donatella; Calapà, Federica; Palmieri, Valentina; Fanali, Caterina; Carbone, Federica; Papa, Alfredo; De Maria, Ruggero; De Spirito, Marco; Sgambato, Alessandro

2017-07-01

Exosomes are involved in intercellular communication. We previously reported that sodium butyrate-induced differentiation of HT29 colon cancer cells is associated with a reduced CD133 expression. Herein, we analyzed the role of exosomes in the differentiation of HT29 cells. Exosomes were prepared using ultracentrifugation. Gene expression levels were evaluated by real-time PCR. The cell proliferation rate was assessed by MTT assay and with the electric cell-substrate impedance sensing system, whereas cell motility was assessed using the scratch test and confocal microscopy. Sodium butyrate-induced differentiation of HT29 and Caco-2 cells increased the levels of released exosomes and their expression of CD133. Cell differentiation and the decrease of cellular CD133 expression levels were prevented by blocking multivesicular body maturation. Exosomes released by HT29 differentiating cells carried increased levels of miRNAs, induced an increased proliferation and motility of both colon cancer cells and normal fibroblasts, increased the colony-forming efficiency of cancer cells, and reduced the sodium butyrate-induced differentiation of HT29 cells. Such effects were associated with an increased phosphorylation level of both Src and extracellular signal regulated kinase proteins and with an increased expression of epithelial-to-mesenchymal transition-related genes. Release of exosomes is affected by differentiation of colon cancer cells; exosomes might be used by differentiating cells to get rid of components that are no longer necessary but might continue to exert their effects on recipient cells. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Catabolism of neurotensin by neural (neuroblastoma clone N1E115) and extraneural (HT29) cell lines

International Nuclear Information System (INIS)

Checler, F.; Amar, S.; Kitabgi, P.; Vincent, J.P.

1986-01-01

The mechanisms by which neurotensin (NT) was inactivated by differentiated neuroblastoma and HT29 cells were characterized. In both cell lines, the sites of primary cleavages of NT were Pro7-Arg8, Arg8-Arg9 and Pro10-Tyr11 bonds. The cleavage at the Pro7-Arg8 bond was totally inhibited by N-benzyloxycarbonyl-Prolyl-Prolinal and therefore resulted from the action of proline endopeptidase. This peptidase also contributed in a major way to the cleavage at the Pro10-Tyr11 bond. However the latter breakdown was partly due to an NT-degrading neutral metallopeptidase. Finally, we demonstrated the involvement of a recently purified rat brain soluble metalloendopeptidase at the Arg8-Arg9 site by the use of its specific inhibitor N-[1(R,S)-carboxy-2-Phenylethyl]-alanylalanylphenylalanine-p-amino benzoate. The secondary processing of NT degradation products revealed differences between HT29 and N1E115 cells. Angiotensin converting enzyme was shown to degrade NT1-10 and NT1-7 in N1E115 cells but was not detected in HT29 cells. A post-proline dipeptidyl aminopeptidase activity converted NT9-13 into NT11-13 in HT29 cells but not in N1E115 cells. Finally, bestatin-sensitive aminopeptidases rapidly broke down NT11-13 to Tyr in both cell lines. Models for the inactivation of NT in HT29 and N1E115 cells are proposed and compared to that previously described for purified rat brain synaptic membranes

Bacterial invasion of HT29-MTX-E12 monolayers: effects of human breast milk.

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Hall, Tim; Dymock, David; Corfield, Anthony P; Weaver, Gillian; Woodward, Mark; Berry, Monica

2013-02-01

The supramucosal gel, crucial for gut barrier function, might be compromised in necrotizing enterocolitis (NEC). Breast milk is associated with a reduced incidence of NEC. We compared the effects of human breast milk (BM) versus a neonatal formula, Nutriprem 1 (FF), on adherence, internalisation, and penetration of NEC-associated Escherichia coli through monolayers of mucus producing intestinal cells, HT29-MTX-E12 (E12). E12 cells were grown to confluence on membranes permeable to bacteria. E. coli, reference strain and isolated from a NEC-affected intestine, were cultured in LB broth, labelled with fluorescein and biotinylated. Bacteria were suspended in tissue culture medium (TC) or mixtures of TC with BM or FF and applied to the E12 cultures. Bacterial numbers were assessed by fluorescence. DyLight 650-labelled neutravidin, which cannot cross cell membrane, evaluated extracellular bacteria. Fluorescence of basolateral medium was measured to quantify translocation. Bacterial concentrations were compared using the Mann Whitney U test. After 1h exposure, E12 cultures adhered or internalised more NEC-derived bacteria than standard strain E. coli and more suspended in FF than BM (Pmilk was associated with relatively less adhesion and internalisation of NEC-associated E. coli to mucus covered E12s compared to formula milk. Copyright © 2013 Elsevier Inc. All rights reserved.

Polyphenols from evening primrose ( Oenothera paradoxa ) defatted seeds induce apoptosis in human colon cancer Caco-2 cells.

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Gorlach, Sylwia; Wagner, Waldemar; Podsedek, Anna; Sosnowska, Dorota; Dastych, Jarosław; Koziołkiewicz, Maria

2011-07-13

Polyphenols extracted from evening primrose seeds (industrial waste product) were studied as apoptosis inducers in human colorectal adenocarcinoma Caco-2 and HT-29 cell lines and in rat normal intestinal IEC-6 cells. The extract dose-dependently inhibited the growth of Caco-2, HT-29, and IEC-6 cells. However, nuclear DNA fragmentation characteristic of apoptosis was observed only in Caco-2. After 72 h of incubation with the extract at 150 μM gallic acid equivalents (44.1 μg extract/mL), Caco-2 cell numbers decreased to 19% of control and 48.8% of the cells were identified by flow cytometry as apoptotic. Under the same conditions only 8% of HT-29 cells and 12.6% of IEC-6 cells exhibited hypodiploid DNA content. The effects of the extract and its fractions on phosphatidylserine exposure and cell membrane integrity were assessed by high content screening image cytometry. The fractions strongly and dose-dependently reduced Caco-2 cell numbers, whereas HT-29 and IEC-6 cells were affected to lesser extents.

Characterization of four clones derived from human adenocarcinoma cell line, HT29, and analysis of their response to sodium butyrate

Czech Academy of Sciences Publication Activity Database

Štokrová, Jitka; Šloncová, Eva; Sovová, Vlasta; Kučerová, Dana; Žíla, Vojtěch; Turečková, Jolana; Vojtěchová, Martina; Korb, Jan; Tuháčková, Zdena

2006-01-01

Roč. 28, č. 2 (2006), s. 559-565 ISSN 1019-6439 R&D Projects: GA AV ČR(CZ) KJB5052302 Institutional research plan: CEZ:AV0Z50520514 Keywords : adenocarcinoma * HT29 cell line * ultrastructure analysis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.556, year: 2006

Cytotoxic effects of chloroform and hydroalcoholic extracts of aerial parts of Cuscuta chinensis and Cuscuta epithymum on Hela, HT29 and MDA-MB-468 tumor cells.

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Jafarian, A; Ghannadi, A; Mohebi, B

2014-01-01

Previous studies have indicated that some species of Cuscuta possess anticancer activity on various cell lines. Due to the lack of detailed researches on the cytotoxic effects of Cuscuta chinensis and Cuscuta epithymum, the aim of the present study was to evaluate cytotoxic effects of chloroform and hydroalcoholic extracts of these plants on the human breast carcinoma cell line (MDA-MB-468), human colorectal adenocarcinoma cell line (HT29) and human uterine cervical carcinoma (Hela). Using maceration method, different extracts of aerial parts of C. chinensis and C. epithymum were prepared. Extraction was performed using chloroform and ethanol/water (70/30). Total phenolic contents of the extracts were determined according to the Folin-Ciocalteu method. Using MTT assay, the cytotoxic activity of the extracts against HT29, Hela and MDA-MB-468 tumor cells was evaluated. Extracts were considered cytotoxic when more than 50% reduction on cell survival was observed. The poly-phenolic content of the hydroalcoholic and chloroform extracts of C. chinensis and C. epithymum were 56.08 ± 4.11, 21.49 ± 2.00, 10.64 ± 0.86 and 4.81 ± 0.38, respectively. Our findings showed that the chloroform extracts of C. chinensis and C. epithyum significantly reduced the viability of Hela, HT-29 and MDA-MB-468 cells. Also, hydroalcoholic extracts of C. chinensis significantly decreased the viability of HT29, Hela and MDA-MB-468 cells. However, in the case of hydroalcoholic extracts of C. epithymum only significant decrease in the viability of MDA-MB-468 cells was observed (IC50 = 340 μg/ml). From these findings it can be concluded that C. chinensis and C. epithymum are good candidates for further study to find new possible cytotoxic agents.

Behavior of LASL-made graphite, ZrC, and ZrC-containing coated particles in irradiation tests HT-28 and HT-29

International Nuclear Information System (INIS)

Reiswig, R.D.; Wagner, P.; Hollabaugh, C.M.; White, R.W.; O'Rourke, J.A.; Davidson, K.V.; Schell, D.H.

1976-01-01

Three types of materials, extruded graphite, hot-pressed ZrC, and particles with ZrC coatings, were irradiated in ORNL High Fluence Isotope Reactor Irradiation tests HT-28 and HT-29. The ZrC seemed unaffected. The graphite changed in dimensions, x-ray diffraction parameters, and thermal conductivity. The four types of coated particles tested all resisted the irradiation well, except one set of particles with double-graded C-ZrC-C coats. Overall, the results were considered encouraging for use of ZrC and extruded graphite fuel matrices. 16 fig

In vitro study of the effects of radio frequency generated for plasma in neoplastic cells HT-29

International Nuclear Information System (INIS)

Andrighetto, Daniela; Dornelles, Eduardo Bortoluzzi; Cruz, Ivana Beatrice Manica da; Lüdke, Everton

2014-01-01

The goal of this study is to develop an in vitro irradiation cell system with controllable irradiation intensities of 27 MHz produced by an argon plasma column with variable amplitude modulation in the 100-700 kHz range. This paper presents and discusses a proposed experiment, with toxicity analysis (DNA Picogreen®) and cell viability (MTT assay) in the radiation-induced HT-29 cell line (colon adenocarcinoma). The data allow us to observe that cellular toxicity effects may occur with exposure to fields produced by argon plasma with intensities on the order of at least 3.2 W / cm2 and exposure times above 3.5 hours continuously. An analysis of cell populations for cell toxicity tests using the Student's t-test did not show significant changes (p 0.34). Cytotoxic effects due to the destruction of cell wall by heating the samples were not detected in any of the tests

DNA microarray profiling of genes differentially regulated by the histone deacetylase inhibitors vorinostat and LBH589 in colon cancer cell lines

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Lenz Heinz-Josef

2009-11-01

Full Text Available Abstract Background Despite the significant progress made in colon cancer chemotherapy, advanced disease remains largely incurable and novel efficacious chemotherapies are urgently needed. Histone deacetylase inhibitors (HDACi represent a novel class of agents which have demonstrated promising preclinical activity and are undergoing clinical evaluation in colon cancer. The goal of this study was to identify genes in colon cancer cells that are differentially regulated by two clinically advanced hydroxamic acid HDACi, vorinostat and LBH589 to provide rationale for novel drug combination partners and identify a core set of HDACi-regulated genes. Methods HCT116 and HT29 colon cancer cells were treated with LBH589 or vorinostat and growth inhibition, acetylation status and apoptosis were analyzed in response to treatment using MTS, Western blotting and flow cytometric analyses. In addition, gene expression was analyzed using the Illumina Human-6 V2 BeadChip array and Ingenuity® Pathway Analysis. Results Treatment with either vorinostat or LBH589 rapidly induced histone acetylation, cell cycle arrest and inhibited the growth of both HCT116 and HT29 cells. Bioinformatic analysis of the microarray profiling revealed significant similarity in the genes altered in expression following treatment with the two HDACi tested within each cell line. However, analysis of genes that were altered in expression in the HCT116 and HT29 cells revealed cell-line-specific responses to HDACi treatment. In addition a core cassette of 11 genes modulated by both vorinostat and LBH589 were identified in both colon cancer cell lines analyzed. Conclusion This study identified HDACi-induced alterations in critical genes involved in nucleotide metabolism, angiogenesis, mitosis and cell survival which may represent potential intervention points for novel therapeutic combinations in colon cancer. This information will assist in the identification of novel pathways and targets

Modulation of Human Serotonin Transporter Expression by 5-HTTLPR in Colon Cells

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Tewin Tencomnao

2011-10-01

Full Text Available Serotonin (5-HT is a monoamine neurotransmitter and plays important roles in several of the human body’s systems. Known as a primary target for psychoactive drug development, the 5-HT transporter (5-HTT, SERT plays a critical role in the regulation of serotonergic function by reuptaking 5-HT. The allelic variation of 5-HTT expression is caused by functional gene promoter polymorphism with two principal variant alleles, 5-HTT gene-linked polymorphic region (5-HTTLPR. It has been demonstrated that 5-HTTLPR is associated with numerous neuropsychiatric disorders. The functional roles of 5-HTTLPR have been reported in human choriocarcinoma (JAR, lymphoblast and raphe cells. To date, the significance of 5-HTTLPR in gastrointestinal tract-derived cells has never been elucidated. Thus, the impact of 5-HTTLPR on 5-HTT transcription was studied in SW480 human colon carcinoma cells, which were shown to express 5-HTT. We found 42-bp fragment in long (L allele as compared to short (S allele, and this allelic difference resulted in 2-fold higher transcriptional efficiency of L allele (P < 0.05 as demonstrated using a functional reporter gene assay. Nevertheless, the transcriptional effect of estrogen and glucocorticoid on 5-HTT expression via 5-HTTLPR was not found in this cell line. Our study was the first to demonstrate the molecular role of this allelic variation in gastrointestinal tract cells.

The intraportal injection model: A practical animal model for hepatic metastases and tumor cell dissemination in human colon cancer

International Nuclear Information System (INIS)

Thalheimer, Andreas; Waaga-Gasser, Ana M; Otto, Christoph; Bueter, Marco; Illert, Bertram; Gattenlohner, Stefan; Gasser, Martin; Meyer, Detlef; Fein, Martin; Germer, Christoph T

2009-01-01

The development of new therapeutic strategies for treatment of metastasized colorectal carcinoma requires biologically relevant and adequate animal models that generate both reproducible metastasis and the dissemination of tumor cells in the form of so-called minimal residual disease (MRD), an expression of the systemic character of neoplastic disease. We injected immunoincompetent nude mice intraportally with different numbers (1 × 10 5 , 1 × 10 6 and 5 × 10 6 cells) of the human colon carcinoma cell lines HT-29 and SW-620 and investigated by histological studies and CK-20 RT-PCR the occurrence of hematogenous metastases and the dissemination of human tumor cells in bone marrow. Only the injection of 1 × 10 6 cells of each colon carcinoma cell line produced acceptable perioperative mortality with reproducible induction of hepatic metastases in up to 89% of all animals. The injection of 1 × 10 6 cells also generated tumor cell dissemination in the bone marrow in up to 63% of animals with hepatic metastases. The present intraportal injection model in immunoincompetent nude mice represents a biologically relevant and adequate animal model for the induction of both reproducible hepatic metastasis and tumor cell dissemination in the bone marrow as a sign of MRD

In vitro study of the effects of radio frequency generated for plasma in neoplastic cells HT-29; Estudo in vitro dos efeitos da radiofrequencia gerada por plasmas em celulas neoplasicas HT-29

Energy Technology Data Exchange (ETDEWEB)

Andrighetto, Daniela; Dornelles, Eduardo Bortoluzzi; Cruz, Ivana Beatrice Manica da; Lüdke, Everton, E-mail: daniela.andrighetto@hotmail.com, E-mail: dornellesedu@gmail.com, E-mail: ibmcruz@hotmail.com, E-mail: evertonludke@gmail.com [Universidade Federal de Santa Maria (UFSM), RS (BRazil)

2014-07-01

The goal of this study is to develop an in vitro irradiation cell system with controllable irradiation intensities of 27 MHz produced by an argon plasma column with variable amplitude modulation in the 100-700 kHz range. This paper presents and discusses a proposed experiment, with toxicity analysis (DNA Picogreen®) and cell viability (MTT assay) in the radiation-induced HT-29 cell line (colon adenocarcinoma). The data allow us to observe that cellular toxicity effects may occur with exposure to fields produced by argon plasma with intensities on the order of at least 3.2 W / cm2 and exposure times above 3.5 hours continuously. An analysis of cell populations for cell toxicity tests using the Student's t-test did not show significant changes (p <0.05) in the amount of DNA released by the action of radiofrequency, although it has been found that cell viability (MTT) is not significantly altered by long exposures to radiation induced plasma RF signals in 27 MHz (p> 0.34). Cytotoxic effects due to the destruction of cell wall by heating the samples were not detected in any of the tests.

Activation of neurotensin receptors and purinoceptors in human colonic adenocarcinoma cells detected with the microphysiometer.

Science.gov (United States)

Richards, M; van Giersbergen, P; Zimmermann, A; Lesur, B; Hoflack, J

1997-10-01

Activation of endogenous neurotensin (NT) receptors and P2-purinoceptors expressed by human colonic adenocarcinoma HT-29 cells increased extracellular acidification rates that were detected in the microphysiometer. NT (pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu), NT[8-13] (Arg-Arg-Pro-Tyr-Ile-Leu), NT[9-13] (Arg-Pro-Tyr-Ile-Leu), and NT1 (N alpha methyl-Arg-Lys-Pro-Trp-Tle-Leu [Tle = tert-leucine]) were full agonists, whereas XL 775 (N-[N-[2-[3-[[6-amino-1-oxo-2-[[(phenylmethoxy)carbonyl]-amino]hex yl]amino]phenyl]-3-(4-hydroxyphenyl)-1-oxo-2-propenyl]-L-isoleucyl]-L-le ucine) was a partial agonist for activating NT receptors expressed by HT-29 cells. Desensitization induced by NT was rapid and monophasic with 85% of the initial response lost by a 30-s exposure. Once initiated, the rate and extent of desensitization were similar for different concentrations of a given agonist, for agonists of different potencies, and for agonists of different efficacies, which suggests that desensitization may be independent of receptor occupancy or agonist efficacy. Resensitization was a much slower process, requiring 60 min before the full agonist response to NT was recovered. ATP, via P2-purinoceptors, also activated cellular acidification rates in a concentration-dependent manner. ATP induced a biphasic desensitization of purinoceptors with a loss of ca. 50% of the initial stimulation detectable between 30 and 90 s of exposure to the agonist. Desensitization of NT receptors did not influence the activation of P2-purinoceptors by ATP, suggesting there was no heterologous desensitization between the two types of receptors. Superfusion with NT receptor agonists for 15 min at concentrations that did not elicit changes in extracellular acidification rates blocked, in a concentration-dependent manner, the agonist response induced by 100 nM NT. This may reflect sequestration of the receptor. These results suggest that the high agonist affinity state of NT receptors may

Iron overload of human colon adenocarcinoma cells studied by synchrotron-based X-ray techniques

NARCIS (Netherlands)

Mihucz, Victor G.; Meirer, Florian; Polgári, Zsófia; Réti, Andrea; Pepponi, Giancarlo; Ingerle, Dieter; Szoboszlai, Norbert; Streli, Christina

2016-01-01

Fast- and slow-proliferating human adenocarcinoma colorectal cells, HT-29 and HCA-7, respectively, overloaded with transferrin (Tf), Fe(III) citrate, Fe(III) chloride and Fe(II) sulfate were studied by synchrotron radiation total-reflection X-ray spectrometry (TXRF), TXRF-X-ray absorption near edge

NADPH oxidase 1 supports proliferation of colon cancer cells by modulating reactive oxygen species-dependent signal transduction.

Science.gov (United States)

Juhasz, Agnes; Markel, Susan; Gaur, Shikha; Liu, Han; Lu, Jiamo; Jiang, Guojian; Wu, Xiwei; Antony, Smitha; Wu, Yongzhong; Melillo, Giovanni; Meitzler, Jennifer L; Haines, Diana C; Butcher, Donna; Roy, Krishnendu; Doroshow, James H

2017-05-12

Reactive oxygen species (ROS) play a critical role in cell signaling and proliferation. NADPH oxidase 1 (NOX1), a membrane-bound flavin dehydrogenase that generates O 2 ̇̄ , is highly expressed in colon cancer. To investigate the role that NOX1 plays in colon cancer growth, we used shRNA to decrease NOX1 expression stably in HT-29 human colon cancer cells. The 80-90% decrease in NOX1 expression achieved by RNAi produced a significant decline in ROS production and a G 1 /S block that translated into a 2-3-fold increase in tumor cell doubling time without increased apoptosis. The block at the G 1 /S checkpoint was associated with a significant decrease in cyclin D 1 expression and profound inhibition of mitogen-activated protein kinase (MAPK) signaling. Decreased steady-state MAPK phosphorylation occurred concomitant with a significant increase in protein phosphatase activity for two colon cancer cell lines in which NOX1 expression was knocked down by RNAi. Diminished NOX1 expression also contributed to decreased growth, blood vessel density, and VEGF and hypoxia-inducible factor 1α (HIF-1α) expression in HT-29 xenografts initiated from NOX1 knockdown cells. Microarray analysis, supplemented by real-time PCR and Western blotting, revealed that the expression of critical regulators of cell proliferation and angiogenesis, including c-MYC, c-MYB, and VEGF, were down-regulated in association with a decline in hypoxic HIF-1α protein expression downstream of silenced NOX1 in both colon cancer cell lines and xenografts. These studies suggest a role for NOX1 in maintaining the proliferative phenotype of some colon cancers and the potential of NOX1 as a therapeutic target in this disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Growth and adhesion to HT-29 cells inhibition of Gram-negatives by Bifidobacterium longum BB536 e Lactobacillus rhamnosus HN001 alone and in combination.

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Inturri, R; Stivala, A; Furneri, P M; Blandino, G

2016-12-01

The aim of this study was to test the inhibitory effect of supernatants of broth cultures of Bifidobacterium longum BB536 and Lactobacillus rhamnosus HN001, both individually and in combination, against Gram-negative strains (uropathogens, enteropathogens and a reference strain). Moreover, in vitro protection of B. longum BB536 and L. rhamnosus HN001, both individually and in combination, against pathogen adhesion to HT-29 cell line, was investigated. The inhibitory activity was performed by the agar diffusion test and in vitro antagonistic activity against pathogen adhesion to human epithelial intestinal HT-29 cells was performed using standardized culture techniques. The study showed that B. longum BB536 and L. rhamnosus HN001, individually and in combination have inhibitory activity against the majority of the Gram negative strains tested. Furthermore, the results showed that both probiotic strains have a good capacity to inhibit pathogenic adhesion to HT-29 cells. Moreover, the ability of B. longum BB536 and L. rhamnosus HN001 to inhibit pathogenic adhesion increased when they were used in combination. The combination of B. longum BB536 and L. rhamnosus HN001 showed inhibitory activity against Gram-negatives and an improved ability to reduce their adhesion properties and to compete with them. The simultaneous presence of the two-probiotic strains could promote competitive mechanisms able to reduce the adhesion properties of pathogen strains and have an important ecological role within the highly competitive environment of the human gut.

Comparison of intracellular accumulation and cytotoxicity of free mTHPC and mTHPC-loaded PLGA nanoparticles in human colon carcinoma cells

International Nuclear Information System (INIS)

Loew, Karin; Wagner, Sylvia; Briesen, Hagen von; Knobloch, Thomas; Wiehe, Arno; Engel, Andrea; Langer, Klaus

2011-01-01

The second generation photosensitizer mTHPC was approved by the European Medicines Agency (EMA) for the palliative treatment of advanced head and neck cancer in October 2001. It is known that mTHPC possesses a significant phototoxicity against a variety of human cancer cells in vitro but also exhibits dark toxicity and can cause adverse effects (especially skin photosensitization). Due to its poor water solubility, the administration of hydrophobic photosensitizer still presents several difficulties. To overcome the administration problems, the use of nanoparticles as drug carrier systems is much investigated. Nanoparticles based on poly(lactic-co-glycolic acid) (PLGA) have been extensively studied as delivery systems into tumours due to their biocompatibility and biodegradability. The goal of this study was the comparison of free mTHPC and mTHPC-loaded PLGA nanoparticles concerning cytotoxicity and intracellular accumulation in human colon carcinoma cells (HT29). The nanoparticles delivered the photosensitizer to the colon carcinoma cells and enabled drug release without losing its activity. The cytotoxicity assays showed a time- and concentration-dependent decrease in cell proliferation and viability after illumination. However, first and foremost mTHPC lost its dark toxic effects using the PLGA nanoparticles as a drug carrier system. Therefore, PLGA nanoparticles are a promising drug carrier system for the hydrophobic photosensitizer mTHPC.

Comparison of intracellular accumulation and cytotoxicity of free mTHPC and mTHPC-loaded PLGA nanoparticles in human colon carcinoma cells

Energy Technology Data Exchange (ETDEWEB)

Loew, Karin; Wagner, Sylvia; Briesen, Hagen von [Fraunhofer-Institute for Biomedical Engineering, D-66386 Strasse Ingbert (Germany); Knobloch, Thomas [Institute of Pharmaceutical Technology, Biocenter of Goethe-University, D-60438 Frankfurt (Germany); Wiehe, Arno [Biolitec AG, D-07745 Jena (Germany); Engel, Andrea; Langer, Klaus, E-mail: hagen.briesen@ibmt.fraunhofer.de [Institute of Pharmaceutical Technology and Biopharmacy, University of Muenster, D-48149 Muenster (Germany)

2011-06-17

The second generation photosensitizer mTHPC was approved by the European Medicines Agency (EMA) for the palliative treatment of advanced head and neck cancer in October 2001. It is known that mTHPC possesses a significant phototoxicity against a variety of human cancer cells in vitro but also exhibits dark toxicity and can cause adverse effects (especially skin photosensitization). Due to its poor water solubility, the administration of hydrophobic photosensitizer still presents several difficulties. To overcome the administration problems, the use of nanoparticles as drug carrier systems is much investigated. Nanoparticles based on poly(lactic-co-glycolic acid) (PLGA) have been extensively studied as delivery systems into tumours due to their biocompatibility and biodegradability. The goal of this study was the comparison of free mTHPC and mTHPC-loaded PLGA nanoparticles concerning cytotoxicity and intracellular accumulation in human colon carcinoma cells (HT29). The nanoparticles delivered the photosensitizer to the colon carcinoma cells and enabled drug release without losing its activity. The cytotoxicity assays showed a time- and concentration-dependent decrease in cell proliferation and viability after illumination. However, first and foremost mTHPC lost its dark toxic effects using the PLGA nanoparticles as a drug carrier system. Therefore, PLGA nanoparticles are a promising drug carrier system for the hydrophobic photosensitizer mTHPC.

Lactococcus lactis subsp. cremoris strain JFR1 attenuates Salmonella adhesion to human intestinal cells in vitro.

Science.gov (United States)

Zhang, Justina Su; Guri, Anilda; Corredig, Milena; Morales-Rayas, Rocio; Hassan, Ashraf; Griffiths, Mansel; LaPointe, Gisèle

2016-12-01

Lactococcus lactis subsp. cremoris JFR1 has been studied in reduced fat cheese due to its ability to produce exopolysaccharides (EPS) in situ, contributing to improved textural and organoleptic properties. In this study, the effect of strain JFR1 on virulence gene expression and attachment of Salmonella to HT-29 human colon carcinoma cells was investigated. Overnight cultures of L. lactis subsp. cremoris JFR1 containing EPS, grown in M17 media with 0.5% glucose supplementation, decreased attachment as well as down regulated virulence gene expression in Salmonella enterica subsp. enterica when tested on HT-29 cells. However, EPS isolated from milk fermented with L. lactis subsp. cremoris JFR1 did not affect Salmonella virulence gene expression or attachment to HT-29 cells. These results suggest that EPS does not contribute to the attachment of Salmonella to human intestinal cells. However, the possibility that the isolation process may have affected the structural features of EPS cannot be ruled out. Copyright © 2016 Elsevier Ltd. All rights reserved.

Cytotoxicity and analysis of apoptosis gene expression in colon cancer cell line treated with cell extract of Lactobacillus casei as indigenous probiotic bacterium

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Amir Mirzaie

2017-03-01

Full Text Available Background and aim: Nowadays, the probiotic bacteria such as lactobacilli are known as prevention factor for various disease especially cancer. The aim of this study was to investigate the cytotoxic effect of Lactobacillus casei PTCC 1608 cell extract as probiotic bacteria on colon cancer cell line (HT29 and analysis of Bax and Bcl2 apoptosis gene expression. Methods: In this experimental study, the cell extract of heat killed L. casei was prepared in 0.01, 0.1, 1, 10, 100 and 1000 µg/ml concentration and subsequently, the cytotoxicity of various cell extracts on HT29 and HEC293 cell lines were evaluated in 24 hours using MTT assay. Moreover, the Bax and Bcl2 apoptosis gene expression level in HT29 cell line was analyzed using Real Time PCR. The apoptotic effects of cell extract was determined using Flow-cytometry technique. Finally, the collected data were statistically analyzed using one-way anal­ysis of variance with the SPSS/18 software. Results: The results of MTT test show that cell extracts of L. casei is able to reduce the survival rate of HT29 cell line to 0.95±0.44, 73.45±0.21, 51.49±0.87, 39.5±0.45 and 19.7±0.55. In addition to, the Real Time PCR results indicated the expression level of Bax and Bcl2 was increased and decreased respectively, in HT29 cell line (2.76 ± 0.54 (P<0.05, 0.21 ± 0.43 (P< 0.05 in 24 h. Moreover, the flow cytometry results indicated the 35.62 % apoptosis in HT29 cell line treated with IC50 value. Conclusion: The results show that the cell extract of L. casei PTCC 1608 could induced the apoptosis in HT29 cell line and it had low toxicity on HEC293 cell line. Therefore, it seems that L. casei has potential uses as probiotic for pharmaceutical applications including prevention and treatment of colon cancer.

Lactobacillus casei Exerts Anti-Proliferative Effects Accompanied by Apoptotic Cell Death and Up-Regulation of TRAIL in Colon Carcinoma Cells

Science.gov (United States)

Santarmaki, Valentina; Aindelis, Georgios; Tompoulidou, Evgenia; Lamprianidou, Eleftheria E.; Saxami, Georgia; Ypsilantis, Petros; Lampri, Evangeli S.; Simopoulos, Constantinos; Kotsianidis, Ioannis; Galanis, Alex; Kourkoutas, Yiannis; Dimitrellou, Dimitra; Chlichlia, Katerina

2016-01-01

Probiotic microorganisms such as lactic acid bacteria (LAB) exert a number of strain-specific health-promoting activities attributed to their immunomodulatory, anti-inflammatory and anti-carcinogenic properties. Despite recent attention, our understanding of the biological processes involved in the beneficial effects of LAB strains is still limited. To this end, the present study investigated the growth-inhibitory effects of Lactobacillus casei ATCC 393 against experimental colon cancer. Administration of live Lactobacillus casei (as well as bacterial components thereof) on murine (CT26) and human (HT29) colon carcinoma cell lines raised a significant concentration- and time-dependent anti-proliferative effect, determined by cell viability assays. Specifically, a dramatic decrease in viability of colon cancer cells co-incubated with 109 CFU/mL L. casei for 24 hours was detected (78% for HT29 and 52% for CT26 cells). In addition, live L. casei induced apoptotic cell death in both cell lines as revealed by annexin V and propidium iodide staining. The significance of the in vitro anti-proliferative effects was further confirmed in an experimental tumor model. Oral daily administration of 109 CFU live L. casei for 13 days significantly inhibited in vivo growth of colon carcinoma cells, resulting in approximately 80% reduction in tumor volume of treated mice. Tumor growth inhibition was accompanied by L. casei-driven up-regulation of the TNF-related apoptosis-inducing ligand TRAIL and down-regulation of Survivin. Taken together, these findings provide evidence for beneficial tumor-inhibitory, anti-proliferative and pro-apoptotic effects driven by this probiotic LAB strain. PMID:26849051

Lactobacillus casei Exerts Anti-Proliferative Effects Accompanied by Apoptotic Cell Death and Up-Regulation of TRAIL in Colon Carcinoma Cells.

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Angeliki Tiptiri-Kourpeti

Full Text Available Probiotic microorganisms such as lactic acid bacteria (LAB exert a number of strain-specific health-promoting activities attributed to their immunomodulatory, anti-inflammatory and anti-carcinogenic properties. Despite recent attention, our understanding of the biological processes involved in the beneficial effects of LAB strains is still limited. To this end, the present study investigated the growth-inhibitory effects of Lactobacillus casei ATCC 393 against experimental colon cancer. Administration of live Lactobacillus casei (as well as bacterial components thereof on murine (CT26 and human (HT29 colon carcinoma cell lines raised a significant concentration- and time-dependent anti-proliferative effect, determined by cell viability assays. Specifically, a dramatic decrease in viability of colon cancer cells co-incubated with 10(9 CFU/mL L. casei for 24 hours was detected (78% for HT29 and 52% for CT26 cells. In addition, live L. casei induced apoptotic cell death in both cell lines as revealed by annexin V and propidium iodide staining. The significance of the in vitro anti-proliferative effects was further confirmed in an experimental tumor model. Oral daily administration of 10(9 CFU live L. casei for 13 days significantly inhibited in vivo growth of colon carcinoma cells, resulting in approximately 80% reduction in tumor volume of treated mice. Tumor growth inhibition was accompanied by L. casei-driven up-regulation of the TNF-related apoptosis-inducing ligand TRAIL and down-regulation of Survivin. Taken together, these findings provide evidence for beneficial tumor-inhibitory, anti-proliferative and pro-apoptotic effects driven by this probiotic LAB strain.

Lactobacillus casei Exerts Anti-Proliferative Effects Accompanied by Apoptotic Cell Death and Up-Regulation of TRAIL in Colon Carcinoma Cells.

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Tiptiri-Kourpeti, Angeliki; Spyridopoulou, Katerina; Santarmaki, Valentina; Aindelis, Georgios; Tompoulidou, Evgenia; Lamprianidou, Eleftheria E; Saxami, Georgia; Ypsilantis, Petros; Lampri, Evangeli S; Simopoulos, Constantinos; Kotsianidis, Ioannis; Galanis, Alex; Kourkoutas, Yiannis; Dimitrellou, Dimitra; Chlichlia, Katerina

2016-01-01

Probiotic microorganisms such as lactic acid bacteria (LAB) exert a number of strain-specific health-promoting activities attributed to their immunomodulatory, anti-inflammatory and anti-carcinogenic properties. Despite recent attention, our understanding of the biological processes involved in the beneficial effects of LAB strains is still limited. To this end, the present study investigated the growth-inhibitory effects of Lactobacillus casei ATCC 393 against experimental colon cancer. Administration of live Lactobacillus casei (as well as bacterial components thereof) on murine (CT26) and human (HT29) colon carcinoma cell lines raised a significant concentration- and time-dependent anti-proliferative effect, determined by cell viability assays. Specifically, a dramatic decrease in viability of colon cancer cells co-incubated with 10(9) CFU/mL L. casei for 24 hours was detected (78% for HT29 and 52% for CT26 cells). In addition, live L. casei induced apoptotic cell death in both cell lines as revealed by annexin V and propidium iodide staining. The significance of the in vitro anti-proliferative effects was further confirmed in an experimental tumor model. Oral daily administration of 10(9) CFU live L. casei for 13 days significantly inhibited in vivo growth of colon carcinoma cells, resulting in approximately 80% reduction in tumor volume of treated mice. Tumor growth inhibition was accompanied by L. casei-driven up-regulation of the TNF-related apoptosis-inducing ligand TRAIL and down-regulation of Survivin. Taken together, these findings provide evidence for beneficial tumor-inhibitory, anti-proliferative and pro-apoptotic effects driven by this probiotic LAB strain.

Green synthesis of NiO nanoparticles using Moringa oleifera extract and their biomedical applications: Cytotoxicity effect of nanoparticles against HT-29 cancer cells.

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Ezhilarasi, A Angel; Vijaya, J Judith; Kaviyarasu, K; Maaza, M; Ayeshamariam, A; Kennedy, L John

2016-11-01

Green protocols for the synthesis of nickel oxide nanoparticles using Moringa oleifera plant extract has been reported in the present study as they are cost effective and ecofriendly, moreover this paper records that the nickel oxide (NiO) nanoparticles prepared from green method shows better cytotoxicity and antibacterial activity. The NiO nanoparticles were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), High resolution transmission electron microscopy (HRTEM), Energy dispersive X-ray analysis (EDX), and Photoluminescence spectroscopy (PL). The formation of a pure nickel oxide phase was confirmed by XRD and FTIR. The synthesized NiO nanoparticles was single crystalline having face centered cubic phase and has two intense photoluminescence emissions at 305.46nm and 410nm. The formation of nano- and micro-structures was confirmed by HRTEM. The in-vitro cytotoxicity and cell viability of human cancer cell HT-29 (Colon Carcinoma cell lines) and antibacterial studies against various bacterial strains were studied with various concentrations of nickel oxide nanoparticles prepared from Moringa oleifera plant extract. MTT assay measurements on cell viability and morphological studies proved that the synthesized NiO nanoparticles posses cytotoxic activity against human cancer cells and the various zones of inhibition (mm), obtained revealed the effective antibacterial activity of NiO nanoparticles against various Gram positive and Gram negative bacterial pathogens. Copyright © 2016 Elsevier B.V. All rights reserved.

Pharmacological blockade of aquaporin-1 water channel by AqB013 restricts migration and invasiveness of colon cancer cells and prevents endothelial tube formation in vitro.

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Dorward, Hilary S; Du, Alice; Bruhn, Maressa A; Wrin, Joseph; Pei, Jinxin V; Evdokiou, Andreas; Price, Timothy J; Yool, Andrea J; Hardingham, Jennifer E

2016-02-24

Aquaporins (AQP) are water channel proteins that enable fluid fluxes across cell membranes, important for homeostasis of the tissue environment and for cell migration. AQP1 knockout mouse models of human cancers showed marked inhibition of tumor-induced angiogenesis, and in pre-clinical studies of colon adenocarcinomas, forced over-expression of AQP1 was shown to increase angiogenesis, invasion and metastasis. We have synthesized small molecule antagonists of AQP1. Our hypothesis is that inhibition of AQP1 will reduce migration and invasiveness of colon cancer cells, and the migration and tube-forming capacity of endothelial cells in vitro. Expression of AQP1 in cell lines was assessed by quantitative (q) PCR, western blot and immunofluorescence, while expression of AQP1 in human colon tumour tissue was assessed by immunohistochemistry. The effect of varying concentrations of the AQP1 inhibitor AqB013 was tested on human colon cancer cell lines expressing high versus low levels of AQP1, using wound closure (migration) assays, matrigel invasion assays, and proliferation assays. The effect of AqB013 on angiogenesis was tested using an endothelial cell tube-formation assay. HT29 colon cancer cells with high AQP1 levels showed significant inhibition of migration compared to vehicle control of 27.9% ± 2.6% (p migration of HCT-116 cells with low AQP1 expression. In an invasion assay, HT29 cells treated with 160 μM of AqB013, showed a 60.3% ± 8.5% decrease in invasion at 144 hours (p < 0.0001) and significantly decreased rate of invasion compared with the vehicle control (F-test, p = 0.001). Almost complete inhibition of endothelial tube formation (angiogenesis assay) was achieved at 80 μM AqB013 compared to vehicle control (p < 0.0001). These data provide good evidence for further testing of the inhibitor as a therapeutic agent in colon cancer.

Development of an Anti-HER2 Monoclonal Antibody H2Mab-139 Against Colon Cancer.

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Kaneko, Mika K; Yamada, Shinji; Itai, Shunsuke; Kato, Yukinari

2018-02-01

Human epidermal growth factor receptor 2 (HER2) expression has been reported in several cancers, such as breast, gastric, lung, pancreatic, and colorectal cancers. HER2 is overexpressed in those cancers and is associated with poor clinical outcomes. Trastuzumab, a humanized anti-HER2 antibody, provides significant survival benefits for patients with HER2-overexpressing breast cancers and gastric cancers. In this study, we developed a novel anti-HER2 monoclonal antibody (mAb), H 2 Mab-139 (IgG 1 , kappa) and investigated it against colon cancers using flow cytometry, western blot, and immunohistochemical analyses. Flow cytometry analysis revealed that H 2 Mab-139 reacted with colon cancer cell lines, such as Caco-2, HCT-116, HCT-15, HT-29, LS 174T, COLO 201, COLO 205, HCT-8, SW1116, and DLD-1. Although H 2 Mab-139 strongly reacted with LN229/HER2 cells on the western blot, we did not observe a specific signal for HER2 in colon cancer cell lines. Immunohistochemical analyses revealed sensitive and specific reactions of H 2 Mab-139 against colon cancers, indicating that H 2 Mab-139 is useful in detecting HER2 overexpression in colon cancers using flow cytometry and immunohistochemical analyses.

Formation of hydrogen peroxide in cell culture media by apple polyphenols and its effect on antioxidant biomarkers in the colon cell line HT-29.

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Bellion, Phillip; Olk, Melanie; Will, Frank; Dietrich, Helmut; Baum, Matthias; Eisenbrand, Gerhard; Janzowski, Christine

2009-10-01

Beneficial health effects of diets containing fruits have partly been attributed to polyphenols which display a spectrum of bioactive effects, including antioxidant activity. However, polyphenols can also exert prooxidative effects in vitro. In this study, polyphenol-mediated hydrogen peroxide (H(2)O(2)) formation was determined after incubation of apple juice extracts (AEs) and polyphenols in cell culture media. Effects of extracellular H(2)O(2 )on total glutathione (tGSH; =GSH + GSSG) and cellular reactive oxygen species (ROS) level of HT-29 cells were studied by coincubation +/- catalase (CAT). AEs ( > or =30 microg/mL) significantly generated H(2)O(2) in DMEM, depending on their composition. Similarly, H(2)O(2) was measured for individual apple polyphenols/degradation products (phenolic acids > epicatechin, flavonols > dihydrochalcones). Highest concentrations were generated by compounds bearing the o-catechol moiety. H(2)O(2) formation was found to be pH dependent; addition of CAT caused a complete decomposition of H(2)O(2) whereas superoxide dismutase was less/not effective. At incubation of HT-29 cells with quercetin (1-100 microM), generated H(2)O(2) slightly contributed to antioxidant cell protection by modulation of tGSH- and ROS-level. In conclusion, H(2)O(2) generation in vitro by polyphenols has to be taken into consideration when interpreting results of such cell culture experiments. Unphysiologically high polyphenol concentrations, favoring substantial H(2)O(2 )formation, are not expected to be met in vivo, even under conditions of high end nutritional uptake.

The apoptotic effects of Brucea javanica fruit extract against HT29 cells associated with p53 upregulation and inhibition of NF-κB translocation

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Bagheri E

2018-03-01

Full Text Available Elham Bagheri,1 Fatemeh Hajiaghaalipour,2 Shaik Nyamathulla,1 Nur’Ain Salehen3 1Department of Pharmacy, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia; 2Institute of Biological Science, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia; 3Department of Biomedical Science, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia Background: Brucea javanica (L. Merr. is a plant from the genus Brucea, which is used in local traditional medicine to treat various diseases. Recent studies revealed an impressive anticancer efficiency of B. javanica extract in different types of cancer cells.Purpose: In this study, we have investigated the cytotoxic effects of the B. javanica hexane, ethanolic extracts against colon cancer cells. HT29 colon cells were selected as an in vitro cancer model to evaluate the anticancer activity of B. javanica ethanolic extract (BJEE and the possible mechanisms of action that induced apoptosis.Methods: 3-(4,5-dimethylthiazol-2-yl-2, 5,-diphenyltetrazolium bromide (MTT, lactate dehydrogenase, acridine orange/propidium iodide, and annexin-V-fluorescein isothiocyanate assays were performed to determine the antiproliferative and apoptosis validation of BJEE on cancer cells. Measurement of reactive oxygen species (ROS production, caspase activities, nucleus factor-κB activity, and gene expression experiments was done to investigate the potential mechanisms of action in the apoptotic process.Results: The results obtained from this study illustrated the significant antiproliferative effect of BJEE on colorectal cancer cells, with a concentration value that inhibits 50% of the cell growth of 25±3.1 µg/mL after 72 h of treatment. MTT assay demonstrated that the BJEE is selectively toxic to cancer cells, and BJEE induced cell apoptosis via activation of caspase-8 along with modulation of apoptosis-related proteins such as Fas, CD40, tumor necrosis factor-related apoptosis-inducing ligands

Prolonged exposure of colon cancer cells to 5-fluorouracil nanoparticles improves its anticancer activity

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Essam Tawfik

2017-02-01

Full Text Available In this study, we aimed to improve the anticancer effect of 5-FU on human colon cancer cell lines by incorporating in poly(d,l lactic-co-glycolic acid (PLGA nanoparticles (NPs. The 5-FU-PLGA NPs were prepared by nanoprecipitation technique. Prepared NPs were moderately dispersed with an average diameter of 133 ± 25.19 nm. Scanning Electron Microscope (SEM images revealed spherical structures with subtle surface irregularity. Free 5-FU dose–response curves were constructed (12.5–2000 μM using MTT assay on HCT 116 and HT-29 cell lines for 1, 3, and 5 days. The calculated IC50 on HCT 116 were 185 μM after 1 day, 11.3 μM after 3 days, and 1.48 μM after 5 days. On HT-29, IC50 was only reached after 5 days of 5-FU treatment (11.25 μM. The HCT 116 viability following treatment with 100 μM 5-FU in free or NPs forms for 3 days was 38.8% and 18.6%, respectively. Similarly, when 250 μM was applied, HCT 116 viability was 17.03% and 14.6% after treatment with free and NPs forms of 5-FU, respectively. Moreover, HT-29 cell viability after 250 μM 5-FU treatment in free or NPs forms was 55.45% and 34.01%, respectively. We also noticed that HCT 116 cells were more sensitive to 5-FU-PLGA NPs as compared to HT-29 cells. Overall, our data indicate that 5-FU activity is time dependent and the prolonged effects created by PLGA NPs may contribute, at least in part, to the noticed enhancement of the anticancer activity of 5-FU drug.

Down-regulation of 5-HT1B and 5-HT1D receptors inhibits proliferation, clonogenicity and invasion of human pancreatic cancer cells.

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Nilgun Gurbuz

Full Text Available Pancreatic ductal adenocarcinoma is characterized by extensive local tumor invasion, metastasis and early systemic dissemination. The vast majority of pancreatic cancer (PaCa patients already have metastatic complications at the time of diagnosis, and the death rate of this lethal type of cancer has increased over the past decades. Thus, efforts at identifying novel molecularly targeted therapies are priorities. Recent studies have suggested that serotonin (5-HT contributes to the tumor growth in a variety of cancers including prostate, colon, bladder and liver cancer. However, there is lack of evidence about the impact of 5-HT receptors on promoting pancreatic cancer. Having considered the role of 5-HT-1 receptors, especially 5-HT1B and 5-HT1D subtypes in different types of malignancies, the aim of this study was to investigate the role of 5-HT1B and 5-HT1D receptors in PaCa growth and progression and analyze their potential as cytotoxic targets. We found that knockdown of 5-HT1B and 5-HT1D receptors expression, using specific small interfering RNA (siRNA, induced significant inhibition of proliferation and clonogenicity of PaCa cells. Also, it significantly suppressed PaCa cells invasion and reduced the activity of uPAR/MMP-2 signaling and Integrin/Src/Fak-mediated signaling, as integral tumor cell pathways associated with invasion, migration, adhesion, and proliferation. Moreover, targeting 5-HT1B and 5-HT1D receptors down-regulates zinc finger ZEB1 and Snail proteins, the hallmarks transcription factors regulating epithelial-mesenchymal transition (EMT, concomitantly with up-regulating of claudin-1 and E-Cadherin. In conclusion, our data suggests that 5-HT1B- and 5-HT1D-mediated signaling play an important role in the regulation of the proliferative and invasive phenotype of PaCa. It also highlights the therapeutic potential of targeting of 5-HT1B/1D receptors in the treatment of PaCa, and opens a new avenue for biomarkers identification

Down-regulation of 5-HT1B and 5-HT1D receptors inhibits proliferation, clonogenicity and invasion of human pancreatic cancer cells.

Directory of Open Access Journals (Sweden)

Nilgun Gurbuz

Full Text Available Pancreatic ductal adenocarcinoma is characterized by extensive local tumor invasion, metastasis and early systemic dissemination. The vast majority of pancreatic cancer (PaCa patients already have metastatic complications at the time of diagnosis, and the death rate of this lethal type of cancer has increased over the past decades. Thus, efforts at identifying novel molecularly targeted therapies are priorities. Recent studies have suggested that serotonin (5-HT contributes to the tumor growth in a variety of cancers including prostate, colon, bladder and liver cancer. However, there is lack of evidence about the impact of 5-HT receptors on promoting pancreatic cancer. Having considered the role of 5-HT-1 receptors, especially 5-HT1B and 5-HT1D subtypes in different types of malignancies, the aim of this study was to investigate the role of 5-HT1B and 5-HT1D receptors in PaCa growth and progression and analyze their potential as cytotoxic targets. We found that knockdown of 5-HT1B and 5-HT1D receptors expression, using specific small interfering RNA (siRNA, induced significant inhibition of proliferation and clonogenicity of PaCa cells. Also, it significantly suppressed PaCa cells invasion and reduced the activity of uPAR/MMP-2 signaling and Integrin/Src/Fak-mediated signaling, as integral tumor cell pathways associated with invasion, migration, adhesion, and proliferation. Moreover, targeting 5-HT1B and 5-HT1D receptors down-regulates zinc finger ZEB1 and Snail proteins, the hallmarks transcription factors regulating epithelial-mesenchymal transition (EMT, concomitantly with up-regulating of claudin-1 and E-Cadherin. In conclusion, our data suggests that 5-HT1B- and 5-HT1D- mediated signaling play an important role in the regulation of the proliferative and invasive phenotype of PaCa. It also highlights the therapeutic potential of targeting of 5-HT1B/1D receptors in the treatment of PaCa, and opens a new avenue for biomarkers identification

Rheinanthrone, a metabolite of sennoside A, triggers macrophage activation to decrease aquaporin-3 expression in the colon, causing the laxative effect of rhubarb extract.

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Kon, Risako; Ikarashi, Nobutomo; Nagoya, Chika; Takayama, Tomoko; Kusunoki, Yoshiki; Ishii, Makoto; Ueda, Harumi; Ochiai, Wataru; Machida, Yoshiaki; Sugita, Kazuyuki; Sugiyama, Kiyoshi

2014-02-27

Aquaporin-3 (AQP3) is expressed in mucosal epithelial cells in the colon and is important for regulating fecal water content. We examined the role of AQP3 in the laxative effect of rhubarb extract. After orally administering rhubarb extract or its major component (sennoside A) to rats, the fecal water content, AQP3 expression and prostaglandin E2 (PGE2) concentrations in the colon were examined. The mechanism by which sennoside A decreases the expression of AQP3 was examined using the human colon cancer HT-29 cells and macrophage-derived Raw264.7 cells. During diarrhea by rhubarb extract administration, the PGE2 levels in the colon increased while the AQP3 expression significantly decreased. Similar changes were also observed when sennoside A was administered. When sennoside A or its metabolites, rheinanthrone and rhein were added to Raw264.7 cells, a significant increase in the PGE2 concentration was observed only in cells treated with rheinanthrone. Fifteen minutes after adding PGE2 to the HT-29 cells, the AQP3 expression decreased to approximately 40% of the control. When pretreated with indomethacin, sennoside A neither decreased the AQP3 expression nor induced diarrhea. Sennoside A may decrease AQP3 expression in the colon to inhibit water transport from the luminal to the vascular side, leading to a laxative effect. The decreases in the levels of AQP3 are caused by rheinanthrone, which is a metabolite of sennoside A, this metabolite activates the macrophages in the colon and increases the secretion of PGE2; PGE2 acts as a paracrine factor and decreases AQP3 expression in colon mucosal epithelial cells. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

SU-E-T-668: Radiosensitizing Effect of Bosutinib On Prostate and Colon Cancers: A Pilot in Vitro Study

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Wang, B; Cvetkovic, D; Chen, L; Ma, C [Fox Chase Cancer Center, Philadelphia, PA (United States); Wang, C [Fox Chase Cancer Center, Philadelphia, PA (United States); West China Hospital, Sichuan University, Chengdu, Sichuan (China)

2015-06-15

Purpose: Recently it has been reported that Bosutinib, a clinical kinase inhibitor, can enhance the tumor cell chemosensitivity by overriding DNA damage checkpoints. However, to the best of our knowledge, there is no report on its effect on cell radiosensitivity in the literature. The objective of the present study is to determine whether Bosutinib has the potential to be used as a radiosensitizer for various cancer cell lines. Methods: In this study, we tested 4 cell lines derived from human prostate (LNCaP, PC-3, DU-145) and colon (HT-29) cancers. The cells were seeded into 12-well plates 24 hours prior to the radiation treatments. For each cell line, we designed 4 study groups, namely, the control, Bosutinib, radiotherapy, and radiotherapy+Bosutinib groups. We used 6 MV photon beams from a Siemens Artiste accelerator to deliver 2 Gy dose in one fraction to the cells in the radiotherapy and radiotherapy+Bosutinib groups. Immediately after irradiation, the cells in the radiotherapy+Bosutinib group were treated with Bosutinib (1µM) for 3 hours. The cell survival was evaluated through clonogenic assays. Results: The cell survival rates of the LNCaP, PC-3, DU-145, and HT-29 cells were found to be 21%, 92%, 76%, and 93% for the radiotherapy group; 21%, 69%, 67%, and 81% for the radiotherapy+Bosutinib group; and 103%, 107%, 86%, and 102% for the Bosutinib group, respectively. Although synergetic cell killing was not seen for the LNCaP and DU-145 cell lines in this study, the cell survival data from the clonogenic assay indicated that Bosutinib could enhance the sensitivity of PC-3 and HT-29 cells to radiation treatment. Conclusion: Our preliminary results demonstrated the possibility of Bosutinib as a radiosensitizer for certain prostate and colon cancers, which are resistant to radiotherapy. Further studies are warranted to quantify the radiosensitizing effect of Bosutinib.

SU-E-T-668: Radiosensitizing Effect of Bosutinib On Prostate and Colon Cancers: A Pilot in Vitro Study

International Nuclear Information System (INIS)

Wang, B; Cvetkovic, D; Chen, L; Ma, C; Wang, C

2015-01-01

Purpose: Recently it has been reported that Bosutinib, a clinical kinase inhibitor, can enhance the tumor cell chemosensitivity by overriding DNA damage checkpoints. However, to the best of our knowledge, there is no report on its effect on cell radiosensitivity in the literature. The objective of the present study is to determine whether Bosutinib has the potential to be used as a radiosensitizer for various cancer cell lines. Methods: In this study, we tested 4 cell lines derived from human prostate (LNCaP, PC-3, DU-145) and colon (HT-29) cancers. The cells were seeded into 12-well plates 24 hours prior to the radiation treatments. For each cell line, we designed 4 study groups, namely, the control, Bosutinib, radiotherapy, and radiotherapy+Bosutinib groups. We used 6 MV photon beams from a Siemens Artiste accelerator to deliver 2 Gy dose in one fraction to the cells in the radiotherapy and radiotherapy+Bosutinib groups. Immediately after irradiation, the cells in the radiotherapy+Bosutinib group were treated with Bosutinib (1µM) for 3 hours. The cell survival was evaluated through clonogenic assays. Results: The cell survival rates of the LNCaP, PC-3, DU-145, and HT-29 cells were found to be 21%, 92%, 76%, and 93% for the radiotherapy group; 21%, 69%, 67%, and 81% for the radiotherapy+Bosutinib group; and 103%, 107%, 86%, and 102% for the Bosutinib group, respectively. Although synergetic cell killing was not seen for the LNCaP and DU-145 cell lines in this study, the cell survival data from the clonogenic assay indicated that Bosutinib could enhance the sensitivity of PC-3 and HT-29 cells to radiation treatment. Conclusion: Our preliminary results demonstrated the possibility of Bosutinib as a radiosensitizer for certain prostate and colon cancers, which are resistant to radiotherapy. Further studies are warranted to quantify the radiosensitizing effect of Bosutinib

In vitro anti-proliferative activity on colon cancer cell line (HT-29) of Thai medicinal plants selected from Thai/Lanna medicinal plant recipe database "MANOSROI III".

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Manosroi, Aranya; Akazawa, Hiroyuki; Akihisa, Toshihiro; Jantrawut, Pensak; Kitdamrongtham, Worapong; Manosroi, Worapaka; Manosroi, Jiradej

2015-02-23

Thai/Lanna region has its own folklore wisdoms including the traditional medicinal plant recipes. Thai/Lanna medicinal plant recipe database "MANOSROI III" has been developed by Prof. Dr. Jiradej Manosroi. It consists of over 200,000 recipes for all diseases including cancer. To investigate the anti-proliferative and apoptotic activities on human colon cancer cell line (HT-29) as well as the cancer cell selectivity of the methanolic extracts (MEs) and fractions of the 23 selected plants from the "MANOSROI III" database. The 23 selected plants were extracted with methanol under reflux and evaluated for their anti-proliferative activity by sulforhodamine B assay. The 5 plants (Gloriosa superba, Caesalpinia sappan, Fibraurea tinctoria, Ventilago denticulata and Psophocarpus tetragonolobus) with potent anti-proliferative activity were fractionated by liquid-liquid partition to give 4 fractions including each hexane (HF), methanol-water (MF), n-butanol (BF) and water (WF) fractions. They were tested for anti-proliferative activity and cancer cell selectivity. The ME and fractions of G. superba which showed potent anti-proliferative activity were further examined for morphological changes and apoptotic activities by acridine orange (AO)/ethidium bromide (EB) staining. The ME of G. superba root showed active with the highest anti-proliferative activity at 9.17 and 1.58 folds of cisplatin and doxorubicin, respectively. After liquid-liquid partition, HF of V. denticulata, MFs of F. tinctoria, V. denticulata and BF of P. tetragonolobus showed higher anti-proliferative activities than their MEs. The MF of G. superba indicated the highest anti-proliferative activity at 7.73 and 1.34 folds of cisplatin and doxorubicin, respectively, but only 0.86 fold of its ME. The ME and HF, MF and BF of G. superba and MF of F. tinctoria demonstrated high cancer cell selectivity. At 50 µg/ml, ME, HF, MF and BF of G. superba demonstrated higher apoptotic activities than the two standard drugs

Radioimmunotoxin Therapy of Experimental Colon and Ovarian Cancer

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Buchsbaum, Donald J.; Vallera, Daniel A.

2006-02-09

To pursue the development of radiolabeled immunotoxins (RIT) for colon cancer, it was first necessary to identify an immunotoxin (IT) that could selectively kill colon cancer cell lines. Recently, our collaborators in the Vallera laboratory have observed that potent recombinant IT can be synthesized using recombinant single chain antibodies (sFv) spliced to truncated diphtheria toxin (DT) consisting of the first 390 amino acids of native DT. DT was chosen as a toxin because it is a catalytic bacterial toxin that is easily manipulated in genetic engineering studies. Also, the Vallera lab has developed new procedures for preparing the sFv fusion toxins from bacterial inclusion bodies such as DT and another good genetic engineering toxin pseudomonas exotoxin (PE) based on detergent refolding. This allows for enhanced yields and higher purity that is essential for generating the protein that will be needed for preparation of larger amounts of RIT for therapy. Many potential sFvs were considered for targeting colon cancer. The best results have been obtained with an sFv recognizing EpCam. EpCam, also known as ESA or EGP40, is a 40 kDa epithelial transmembrane glycoprotein found on the basolateral surface of simple, pseudostratified, and transitional epithelia. It has been found overexpressed on 81% of adenocarcinomas of the colon (Went et al. Human pathology 35:122, 2004). EpCam sliced to DT (DTEpCam) was highly potent in studies in which we measured its ability to inhibit the proliferation of the HT-29 and COLO 205 colon cancer cell lines since we measured its IC50 at 1-2 x 10-2 nM. Potency is important, but is also critical that DTEpCam is selective in its cytotoxicity against EpCam-expressing target colon cancer cells. The activity of DTEpCam was highly selective since irrelevant control IT that did not recognize any markers on cancer cells, did not show any activity against the same colon cancer cell lines. Also, blocking studies were performed in which DTEpCam was

Radioimmunotoxin Therapy of Experimental Colon and Ovarian Cancer

International Nuclear Information System (INIS)

Buchsbaum, Donald J.; Vallera, Daniel A.

2006-01-01

To pursue the development of radiolabeled immunotoxins (RIT) for colon cancer, it was first necessary to identify an immunotoxin (IT) that could selectively kill colon cancer cell lines. Recently, our collaborators in the Vallera laboratory have observed that potent recombinant IT can be synthesized using recombinant single chain antibodies (sFv) spliced to truncated diphtheria toxin (DT) consisting of the first 390 amino acids of native DT. DT was chosen as a toxin because it is a catalytic bacterial toxin that is easily manipulated in genetic engineering studies. Also, the Vallera lab has developed new procedures for preparing the sFv fusion toxins from bacterial inclusion bodies such as DT and another good genetic engineering toxin pseudomonas exotoxin (PE) based on detergent refolding. This allows for enhanced yields and higher purity that is essential for generating the protein that will be needed for preparation of larger amounts of RIT for therapy. Many potential sFvs were considered for targeting colon cancer. The best results have been obtained with an sFv recognizing EpCam. EpCam, also known as ESA or EGP40, is a 40 kDa epithelial transmembrane glycoprotein found on the basolateral surface of simple, pseudostratified, and transitional epithelia. It has been found overexpressed on 81% of adenocarcinomas of the colon (Went et al. Human pathology 35:122, 2004). EpCam sliced to DT (DTEpCam) was highly potent in studies in which we measured its ability to inhibit the proliferation of the HT-29 and COLO 205 colon cancer cell lines since we measured its IC50 at 1-2 x 10-2 nM. Potency is important, but is also critical that DTEpCam is selective in its cytotoxicity against EpCam-expressing target colon cancer cells. The activity of DTEpCam was highly selective since irrelevant control IT that did not recognize any markers on cancer cells, did not show any activity against the same colon cancer cell lines. Also, blocking studies were performed in which DTEpCam was

Effects of maple (Acer) plant part extracts on proliferation, apoptosis and cell cycle arrest of human tumorigenic and non-tumorigenic colon cells.

Science.gov (United States)

González-Sarrías, Antonio; Li, Liya; Seeram, Navindra P

2012-07-01

Phenolic-enriched extracts of maple sap and syrup, obtained from the sugar and red maple species (Acer saccharum Marsh, A. rubrum L., respectively), are reported to show anticancer effects. Despite traditional medicinal uses of various other parts of these plants by Native Americans, they have not been investigated for anticancer activity. Here leaves, stems/twigs, barks and sapwoods of both maple species were evaluated for antiproliferative effects against human colon tumorigenic (HCT-116, HT-29, Caco-2) and non-tumorigenic (CCD-18Co) cells. Extracts were standardized to total phenolic and ginnalin-A (isolated in our laboratory) levels. Overall, the extracts inhibited the growth of the colon cancer more than normal cells (over two-fold), their activities increased with their ginnalin-A levels, with red > sugar maple extracts. The red maple leaf extract, which contained the highest ginnalin-A content, was the most active extract (IC₅₀  = 35 and 16 µg/mL for extract and ginnalin-A, respectively). The extracts were not cytotoxic nor did they induce apoptosis of the colon cancer cells. However, cell cycle analyses revealed that the antiproliferative effects of the extracts were mediated through cell cycle arrest in the S-phase. The results from the current study suggest that these maple plant part extracts may have potential anticolon cancer effects. Copyright © 2011 John Wiley & Sons, Ltd.

Selected chemo-ecological studies of marine opisthobranchs from Indian coasts

Digital Repository Service at National Institute of Oceanography (India)

Fontana, A.; Ciavatta, M.L.; DeSouza, L.; Mollo, E.; Naik, C.G.; Parameswaran, P.S.; Wahidullah, S.; Cimino, G.

),27?29 tunicates (ecteinascidins, e.g. 12)30, 31 and sponges (renieramycins, e.g. 13).32?34 Jorumycin (10) was indeed cytotoxic against several tumor cell lines [HT29 (human colon carcinoma), A549 (human lung carcinoma), Mel28 (human melanoma), P388 (mouse lym...

Lebein, a snake venom disintegrin, suppresses human colon cancer cells proliferation and tumor-induced angiogenesis through cell cycle arrest, apoptosis induction and inhibition of VEGF expression.

Science.gov (United States)

Zakraoui, Ons; Marcinkiewicz, Cezary; Aloui, Zohra; Othman, Houcemeddine; Grépin, Renaud; Haoues, Meriam; Essafi, Makram; Srairi-Abid, Najet; Gasmi, Ammar; Karoui, Habib; Pagès, Gilles; Essafi-Benkhadir, Khadija

2017-01-01

Lebein, is an heterodimeric disintegrin isolated from Macrovipera lebetina snake venom that was previously characterized as an inhibitor of ADP-induced platelet aggregation. In this study, we investigated the effect of Lebein on the p53-dependent growth of human colon adenocarcinoma cell lines. We found that Lebein significantly inhibited LS174 (p53wt), HCT116 (p53wt), and HT29 (p53mut) colon cancer cell viability by inducing cell cycle arrest through the modulation of expression levels of the tumor suppression factor p53, cell cycle regulating proteins cyclin D1, CDK2, CDK4, retinoblastoma (Rb), CDK1, and cyclin-dependent kinase inhibitors p21 and p27. Interestingly, Lebein-induced apoptosis of colon cancer cells was dependent on their p53 status. Thus, in LS174 cells, cell death was associated with PARP cleavage and the activation of caspases 3 and 8 while in HCT116 cells, Lebein induced caspase-independent apoptosis through increased expression of apoptosis inducing factor (AIF). In LS174 cells, Lebein triggers the activation of the MAPK ERK1/2 pathway through induction of reactive oxygen species (ROS). It also decreased cell adhesion and migration to fibronectin through down regulation of α5β1 integrin. Moreover, Lebein significantly reduced the expression of two angiogenesis stimulators, Vascular Endothelial Growth Factor (VEGF) and Neuropilin 1 (NRP1). It inhibited the VEGF-induced neovascularization process in the quail embryonic CAM system and blocked the development of human colon adenocarcinoma in nude mice. Overall, our work indicates that Lebein may be useful to design a new therapy against colon cancer. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

(2Alpha,3beta)-2,3-dihydroxyolean-12-en-28-oic acid, a new natural triterpene from Olea europea, induces caspase dependent apoptosis selectively in colon adenocarcinoma cells.

Science.gov (United States)

Reyes, Fernando J; Centelles, Josep J; Lupiáñez, José A; Cascante, Marta

2006-11-27

Triterpenoids are known to induce apoptosis and to be anti-tumoural. Maslinic acid, a pentacyclic triterpene, is present in high concentrations in olive pomace. This study examines the response of HT29 and Caco-2 colon-cancer cell lines to maslinic-acid treatment. At concentrations inhibiting cell growth by 50-80% (IC50HT29=61+/-1 microM, IC80HT29=76+/-1 microM and IC50Caco-2=85+/-5 microM, IC80Caco-2=116+/-5 microM), maslinic acid induced strong G0/G1 cell-cycle arrest and DNA fragmentation, and increased caspase-3 activity. However, maslinic acid did not alter the cell cycle or induce apoptosis in the non-tumoural intestine cell lines IEC-6 and IEC-18. Moreover, maslinic acid induced cell differentiation in colon adenocarcinoma cells. These findings support a role for maslinic acid as a tumour suppressant and as a possible new therapeutic tool for aberrant cell proliferation in the colon. In this report, we demonstrate for the first time that, in tumoural cancer cells, maslinic acid exerts a significant anti-proliferation effect by inducing an apoptotic process characterized by caspase-3 activation by a p53-independent mechanism, which occurs via mitochondrial disturbances and cytochrome c release.

The pharmacology of TD-8954, a potent and selective 5-HT4 receptor agonist with gastrointestinal prokinetic properties

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David T Beattie

2011-05-01

Full Text Available This study evaluated the in vitro and in vivo pharmacological properties of TD-8954, a potent and selective 5-HT4 receptor agonist. TD-8954 had high affinity (pKi = 9.4 for human recombinant 5-HT4(c (h5-HT4(c receptors, and selectivity (> 2,000-fold over all other 5-HT receptors and non-5-HT receptors, ion channels, enzymes and transporters tested (n = 78. TD-8954 produced an elevation of cAMP in HEK-293 cells expressing the h5-HT4(c receptor (pEC50 = 9.3, and contracted the guinea pig colonic longitudinal muscle/myenteric plexus (LMMP preparation (pEC50 = 8.6. TD-8954 had moderate intrinsic activity (IA in the in vitro assays. In conscious guinea pigs, subcutaneous (s.c. administration of TD 8954 (0.03 - 3 mg/kg increased the colonic transit of carmine red dye, reducing the time taken for its excretion. Following intraduodenal (i.d. dosing to anesthetized rats, TD 8954 (0.03 - 10 mg/kg evoked a dose-dependent relaxation of the esophagus. Following oral administration to conscious dogs, TD 8954 (10 and 30 µg/kg produced an increase in contractility of the antrum, duodenum and jejunum. In a single ascending oral dose study in healthy human subjects, TD-8954 (0.1 - 20 mg increased bowel movement frequency and reduced the time to first stool. It is concluded that TD-8954 is a potent and selective 5-HT4 receptor agonist in vitro, with robust in vivo stimulatory activity in the gastrointestinal (GI tract of guinea pigs, rats, dogs and humans. TD-8954 may have clinical utility in patients with disorders of reduced GI motility.

HT-29 and Caco-2 Reporter Cell Lines for Functional Studies of Nuclear Factor Kappa B Activation

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Giuliana Mastropietro

2015-01-01

Full Text Available The NF-κB is a transcription factor which plays a key role in regulating biological processes. In response to signals, NF-κB activation occurs via phosphorylation of its inhibitor, which dissociates from the NF-κB dimer allowing the translocation to the nucleus, inducing gene expression. NF-κB activation has direct screening applications for drug discovery for several therapeutic indications. Thus, pathway-specific reporter cell systems appear as useful tools to screen and unravel the mode of action of probiotics and natural and synthetic compounds. Here, we describe the generation, characterization, and validation of human epithelial reporter cell lines for functional studies of NF-κB activation by different pro- and anti-inflammatory agents. Caco-2 and HT-29 cells were transfected with a pNF-κB-hrGFP plasmid which contains the GFP gene under the control of NF-κB binding elements. Three proinflammatory cytokines (TNF-α, IL-1β, and LPS were able to activate the reporter systems in a dose-response manner, which corresponds to the activation of the NF-κB signaling pathway. Finally, the reporter cell lines were validated using lactic acid bacteria and a natural compound. We have established robust Caco-2-NF-κB-hrGFP and HT-29-NF-κB-hrGFP reporter cell lines which represent a valuable tool for primary screening and identification of bacterial strains and compounds with a potential therapeutic interest.

Characterisation of 5-HT3C, 5-HT3D and 5-HT3E receptor subunits: evolution, distribution and function.

Science.gov (United States)

Holbrook, Joanna D; Gill, Catherine H; Zebda, Noureddine; Spencer, Jon P; Leyland, Rebecca; Rance, Kim H; Trinh, Han; Balmer, Gemma; Kelly, Fiona M; Yusaf, Shahnaz P; Courtenay, Nicola; Luck, Jane; Rhodes, Andrew; Modha, Sundip; Moore, Stephen E; Sanger, Gareth J; Gunthorpe, Martin J

2009-01-01

The 5-HT(3) receptor is a member of the 'Cys-loop' family of ligand-gated ion channels that mediate fast excitatory and inhibitory transmission in the nervous system. Current evidence points towards native 5-HT(3) receptors originating from homomeric assemblies of 5-HT(3A) or heteromeric assembly of 5-HT(3A) and 5-HT(3B). Novel genes encoding 5-HT(3C), 5-HT(3D), and 5-HT(3E) have recently been described but the functional importance of these proteins is unknown. In the present study, in silico analysis (confirmed by partial cloning) indicated that 5-HT(3C), 5-HT(3D), and 5-HT(3E) are not human-specific as previously reported: they are conserved in multiple mammalian species but are absent in rodents. Expression profiles of the novel human genes indicated high levels in the gastrointestinal tract but also in the brain, Dorsal Root Ganglion (DRG) and other tissues. Following the demonstration that these subunits are expressed at the cell membrane, the functional properties of the recombinant human subunits were investigated using patch clamp electrophysiology. 5-HT(3C), 5-HT(3D), and 5-HT(3E) were all non-functional when expressed alone. Co-transfection studies to determine potential novel heteromeric receptor interactions with 5-HT(3A) demonstrated that the expression or function of the receptor was modified by 5-HT(3C) and 5-HT(3E), but not 5-HT(3D). The lack of distinct effects on current rectification, kinetics or pharmacology of 5-HT(3A) receptors does not however provide unequivocal evidence to support a direct contribution of 5-HT(3C) or 5-HT(3E) to the lining of the ion channel pore of novel heteromeric receptors. The functional and pharmacological contributions of these novel subunits to human biology and diseases such as irritable bowel syndrome for which 5-HT(3) receptor antagonists have major clinical usage, therefore remains to be fully determined.

The novel anthraquinone derivative IMP1338 induces death of human cancer cells by p53-independent S and G2/M cell cycle arrest.

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Choi, Hyun Kyung; Ryu, Hwani; Son, A-Rang; Seo, Bitna; Hwang, Sang-Gu; Song, Jie-Young; Ahn, Jiyeon

2016-04-01

To identify novel small molecules that induce selective cancer cell death, we screened a chemical library containing 1040 compounds in HT29 colon cancer and CCD18-Co normal colon cells, using a phenotypic cell-based viability assay system with the Cell Counting Kit-8 (CCK-8). We discovered a novel anthraquinone derivative, N-(4-[{(9,10-dioxo-9,10-dihydro-1-anthracenyl)sulfonyl}amino]phenyl)-N-methylacetamide (IMP1338), which was cytotoxic against the human colon cancer cells tested. The MTT cell viability assay showed that treatment with IMP1338 selectively inhibited HCT116, HCT116 p53(-/-), HT29, and A549 cancer cell proliferation compared to that of Beas2B normal epithelial cells. To elucidate the cellular mechanism underlying the cytotoxicity of IMP1338, we examined the effect of IMP1338 on the cell cycle distribution and death of cancer cells. IMP1338 treatment significantly arrested the cell cycle at S and G2/M phases by DNA damage and led to apoptotic cell death, which was determined using FACS analysis with Annexin V/PI double staining. Furthermore, IMP1338 increased caspase-3 cleavage in wild-type p53, p53 knockout HCT116, and HT29 cells as determined using immunoblotting. In addition, IMP1338 markedly induced the phosphorylation of histone H2AX and Chk1 in both cell lines while the combination of 5-fluorouracil (5-FU) and radiation inhibited the viability of HCT116, HCT116 p53(-/-), and HT29 cells compared to 5-FU or radiation alone. Our findings indicated that IMP1338 induced p53-independent cell death through S and G2/M phase arrest as well as DNA damage. These results provide a basis for future investigations assessing the promising anticancer properties of IMP1338. Copyright © 2016. Published by Elsevier Masson SAS.

Prehistoric human colonization of India

Indian Academy of Sciences (India)

Unknown

2. Earliest human colonization of south Asia. The early human colonization of south Asia is represented largely by an abundance of stone tool assemblages. The oldest known tools ..... component among finished tools is conspicuous in the hinterland riverine ...... sativum), green gram (Vigna radiata), gram/chicken pea.

Downregulation of CD147 expression by RNA interference inhibits HT29 cell proliferation, invasion and tumorigenicity in vitro and in vivo.

Science.gov (United States)

Li, Rui; Pan, Yuqin; He, Bangshun; Xu, Yeqiong; Gao, Tianyi; Song, Guoqi; Sun, Huiling; Deng, Qiwen; Wang, Shukui

2013-12-01

We investigated the effect of CD147 silencing on HT29 cell proliferation and invasion. We constructed a novel short hairpin RNA (shRNA) expression vector pYr-mir30-shRNA. The plasmid was transferred to HT29 cells. The expression of CD147, MCT1 (lactate transporters monocarboxylate transporter 1) and MCT4 (lactate transporters monocarboxylate transporter 4) were monitored by quantitative PCR and western blotting, respectively. The MMP-2 (matrix metalloproteinase-2) and MMP-9 (matrix metalloproteinase-9) activities were determined by gelatin zymography assay, while the intracellular lactate concentration was determined by the lactic acid assay kit. WST-8 assay was used to determine the HT29 cell proliferation and the chemosensitivity. Invasion assay was used to determine the invasion of HT29 cells. In addition, we established a colorectal cancer model, and detected CD147 expression in vivo. The results showed that the expression of CD147 and MCT1 was significantly reduced at both mRNA and protein levels, and also the activity of MMP-2 and MMP-9 was reduced. The proliferation and invasion were decreased, but chemosensitivity to cisplatin was increased. In vivo, the CD147 expression was also significantly decreased, and reduced the tumor growth after CD147 gene silencing. The results demonstrated that silencing of CD147 expression inhibited the proliferation and invasion, suggesting CD147 silencing might be an adjuvant gene therapy strategy to chemotherapy.

Frondoside A Enhances the Anti-Cancer Effects of Oxaliplatin and 5-Fluorouracil on Colon Cancer Cells

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Samir Attoub

2018-05-01

Full Text Available Over recent years, we have demonstrated that Frondoside A, a triterpenoid glycoside isolated from an Atlantic sea cucumber, has potent in vitro and in vivo anti-cancer effects against human pancreatic, breast, and lung cancer. We have also demonstrated that Frondoside A is able to potentiate and/or synergize the anti-cancer effects of major classical cytotoxic agents, namely, gemcitabine, paclitaxel, and cisplatin, in the treatment of pancreatic, breast, and lung cancer, respectively. This study evaluates the impact of Frondoside A alone and in combination with the standard cytotoxic drugs oxaliplatin and 5-fluorouracil (5-FU in the treatment of colon cancer using three human colon cancer cell lines, namely, HT-29, HCT-116, and HCT8/S11. We demonstrate that Frondoside A, oxaliplatin, and 5-FU cause a concentration- and time-dependent reduction in the number of HT-29 colon cancer cells. A concentration of 2.5 µM of Frondoside A led to almost 100% inhibition of cell numbers at 72 h. A similar effect was only observed with a much higher concentration (100 µM of oxaliplatin or 5-FU. The reduction in cell numbers by Frondoside A, oxaliplatin, and 5-FU was also confirmed in two other colon cancer cell lines, namely, HCT8/S11 and HCT-116, treated for 48 h. The combinations of low concentrations of these drugs for 48 h in vitro clearly demonstrated that Frondoside A enhances the inhibition of cell numbers induced by oxaliplatin or 5-FU. Similarly, such a combination also efficiently inhibited colony growth in vitro. Interestingly, we found that the inhibition of ERK1/2 phosphorylation was significantly enhanced when Frondoside A was used in combination treatments. Moreover, we show that Frondoside A and 5-FU, when used alone, induce a concentration-dependent induction of apoptosis and that their pro-apoptotic effect is dramatically enhanced when used in combination. We further demonstrate that apoptosis induction upon the treatment of colon cancer

Genistein inhibits proliferation of colon cancer cells by attenuating a negative effect of epidermal growth factor on tumor suppressor FOXO3 activity

International Nuclear Information System (INIS)

Qi, Wentao; Weber, Christopher R; Wasland, Kaarin; Savkovic, Suzana D

2011-01-01

Soy consumption is associated with a lower incidence of colon cancer which is believed to be mediated by one of its of components, genistein. Genistein may inhibit cancer progression by inducing apoptosis or inhibiting proliferation, but mechanisms are not well understood. Epidermal growth factor (EGF)-induced proliferation of colon cancer cells plays an important role in colon cancer progression and is mediated by loss of tumor suppressor FOXO3 activity. The aim of this study was to assess if genistein exerts anti-proliferative properties by attenuating the negative effect of EGF on FOXO3 activity. The effect of genistein on proliferation stimulated by EGF-mediated loss of FOXO3 was examined in human colonic cancer HT-29 cells. EGF-induced FOXO3 phosphorylation and translocation were assessed in the presence of genistein. EGF-mediated loss of FOXO3 interactions with p53 (co-immunoprecipitation) and promoter of p27kip1 (ChIP assay) were examined in presence of genistein in cells with mutated p53 (HT-29) and wild type p53 (HCT116). Silencing of p53 determined activity of FOXO3 when it is bound to p53. Genistein inhibited EGF-induced proliferation, while favoring dephosphorylation and nuclear retention of FOXO3 (active state) in colon cancer cells. Upstream of FOXO3, genistein acts via the PI3K/Akt pathway to inhibit EGF-stimulated FOXO3 phosphorylation (i.e. favors active state). Downstream, EGF-induced disassociation of FOXO3 from mutated tumor suppressor p53, but not wild type p53, is inhibited by genistein favoring FOXO3-p53(mut) interactions with the promoter of the cell cycle inhibitor p27kip1 in colon cancer cells. Thus, the FOXO3-p53(mut) complex leads to elevated p27kip1 expression and promotes cell cycle arrest. These novel anti-proliferative mechanisms of genistein suggest a possible role of combining genistein with other chemoreceptive agents for the treatment of colon cancer

Mechanisms underlying 3-bromopyruvate-induced cell death in colon cancer.

Science.gov (United States)

Sun, Yiming; Liu, Zhe; Zou, Xue; Lan, Yadong; Sun, Xiaojin; Wang, Xiu; Zhao, Surong; Jiang, Chenchen; Liu, Hao

2015-08-01

3-Bromopyruvate (3BP) is an energy-depleting drug that inhibits Hexokinase II activity by alkylation during glycolysis, thereby suppressing the production of ATP and inducing cell death. As such, 3BP can potentially serve as an anti-tumorigenic agent. Our previous research showed that 3BP can induce apoptosis via AKT /protein Kinase B signaling in breast cancer cells. Here we found that 3BP can also induce colon cancer cell death by necroptosis and apoptosis at the same time and concentration in the SW480 and HT29 cell lines; in the latter, autophagy was also found to be a mechanism of cell death. In HT29 cells, combined treatment with 3BP and the autophagy inhibitor 3-methyladenine (3-MA) exacerbated cell death, while viability in 3BP-treated cells was enhanced by concomitant treatment with the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone (z-VAD-fmk) and the necroptosis inhibitor necrostatin (Nec)-1. Moreover, 3BP inhibited tumor growth in a SW480 xenograft mouse model. These results indicate that 3BP can suppress tumor growth and induce cell death by multiple mechanisms at the same time and concentration in different types of colon cancer cell by depleting cellular energy stores.

Pet imaging of human pituitary 5-HT2 receptors with F-18 setoperone

Energy Technology Data Exchange (ETDEWEB)

Fischman, A.J.; Bonab, A.A.; Babich, J.W. [Massachusetts General Hospital, Boston, MA (United States)] [and others

1995-05-01

Serotonin (5-HT) receptors play an important role in the regulation of pituitary function. In particular, 5HT agonists stimulate ACTH, {beta}-endorphin, prolactin and growth hormone secretion but inhibit TSH release. 5-HT binding sites have been identified by autoradiographic studies of rat and human pituitary. In the present investigation, we used PET with F-18 setoperone to image 5-HT2 receptors in normal humans. Setoperone, a piperidine derivative with potent 5-HT2 receptor blocking properties was labelled with F-18 by nucleophilic substitution on the nitro derivative. After HPLC purification, specific activity was between 10,000 and 15,000 mCi/{mu} mole and radiochemical purity was >98%. Six healthy male volunteers were injected with 5-7 mCi of F-18. Setoperone and serial PET images and arterial blood samples were collected over 2 hrs. Specific binding to 5-HT2 receptors in the frontal cortex (FC), striatum (ST) and pituitary (P) was quantitated using the cerebellum (C) as reference. The tracer showed clear retention in FC, ST and P (known to contain a high density of 5-HT2 receptors) relative to C (known to be devoid of 5-HT2 receptors). In all subjects, FC/C, ST/C and P/C ratios increased during the first hr. and remained stable thereafter. For FC and ST, the ratios reached similar values; 3.92{plus_minus}0.73 and 3.53{plus_minus}0.32. For pituitary, a significantly higher ratio, was measured at all times; 6.53{plus_minus}1.82 (p<0.01). These results indicate that F-18 setoperone is an effective PET radiopharmaceutical for imaging 5-HT2 receptors in the human pituitary. Future applications of this agent could provide important new insights into neuroendocrine function.

The effects of TNF-alpha and inhibitors of arachidonic acid metabolism on human colon HT-29 cells depend on differentiation status

Czech Academy of Sciences Publication Activity Database

Kovaříková, Martina; Hofmanová, Jiřina; Souček, Karel; Kozubík, Alois

2004-01-01

Roč. 72, č. 1 (2004), s. 23-31 ISSN 0301-4681 R&D Projects: GA ČR GA525/01/0419; GA ČR GP524/02/P051; GA AV ČR IBS5004009 Institutional research plan: CEZ:AV0Z5004920 Keywords : colon cancer * cell differentiation * TNF-alpha Subject RIV: BO - Biophysics Impact factor: 4.481, year: 2004

Interaction of cruciferin-based nanoparticles with Caco-2 cells and Caco-2/HT29-MTX co-cultures.

Science.gov (United States)

Akbari, Ali; Lavasanifar, Afsaneh; Wu, Jianping

2017-12-01

The objective of this work was to assess the potential of Cruciferin/Calcium (Cru/Ca) and Cruciferin/Chitosan (Cru/Cs) nanoparticles for oral drug delivery. For this purpose, Cru/Ca and Cru/Cs nanoparticles were developed through cold gelation of Cruciferin, a major canola protein, and in interaction with calcium and chitosan, respectively. The extent and rate of particle uptake in Caco-2 cells and Caco-2/HT29 co-culture was then evaluated by fluorescence spectroscopy as well as flow cytometry. Through pre-incubation of Caco-2 cell monolayer with specific endocytosis inhibitors, the mechanism of cell uptake was investigated. Our results showed that the uptake of negatively-charged Cru/Ca particles to be ∼3 times higher than positively-charged Cru/Cs ones by Caco-2 cells. Presence of mucus secreted by HT29 cells in their co-culture with Caco-2 had negligible influence on the uptake and transport of both particles. In contrast to Cru/Ca particles which were dissociated in the simulated gastrointestinal conditions, digestion of Cru/Cs particles resulted in 6- and 2-fold increase in the cellular uptake and transport of encapsulated coumarin in the latter particles, respectively. While the presence of mucus in Caco-2/HT29 co-culture caused 40-50% decrease of cellular uptake and transport for coumarin encapsulated in digested Cru/Cs particles, it had no significant effect on the cell uptake and transport of coumarin associated with Cru/Ca particles after digestion. Energy-dependent mechanisms were the dominant mechanism for uptake of both undigested and digested particles. Therefore, in Caco-2/HT29 co-culture which closely simulated intestinal epithelial cells, undigested Cru/Ca and Cru/Cs particles had the ability to penetrate mucus layers, while digested Cru/Cs particles showed mucoadhesive property, and digested Cru/Ca particles were dissociated. Our results points to a potential for cruciferin based nanoparticles for oral drug delivery. The long-term objective of

Chloroquine potentiates the anti-cancer effect of 5-fluorouracil on colon cancer cells

International Nuclear Information System (INIS)

Sasaki, Kazuhito; Hiyoshi, Masaya; Kaneko, Manabu; Kitayama, Joji; Takahashi, Koki; Nagawa, Hirokazu; Tsuno, Nelson H; Sunami, Eiji; Tsurita, Giichiro; Kawai, Kazushige; Okaji, Yurai; Nishikawa, Takeshi; Shuno, Yasutaka; Hongo, Kumiko

2010-01-01

Chloroquine (CQ), the worldwide used anti-malarial drug, has recently being focused as a potential anti-cancer agent as well as a chemosensitizer when used in combination with anti-cancer drugs. It has been shown to inhibit cell growth and/or to induce cell death in various types of cancer. 5-Fluorouracil (5-FU) is the chemotherapeutic agent of first choice in colorectal cancer, but in most cases, resistance to 5-FU develops through various mechanisms. Here, we focused on the combination of CQ as a mechanism to potentiate the inhibitory effect of 5-FU on human colon cancer cells. HT-29 cells were treated with CQ and/or 5-FU, and their proliferative ability, apoptosis and autophagy induction effects, and the affection of the cell cycle were evaluated. The proliferative ability of HT-29 was analyzed by the MTS assay. Apoptosis was quantified by flow-cytometry after double-staining of the cells with AnnexinV/PI. The cell cycle was evaluated by flow-cytometry after staining of cells with PI. Autophagy was quantified by flow-cytometry and Western blot analysis. Finally, to evaluate the fate of the cells treated with CQ and/or 5-FU, the colony formation assay was performed. 5-FU inhibited the proliferative activity of HT-29 cells, which was mostly dependent on the arrest of the cells to the G0/G1-phase but also partially on apoptosis induction, and the effect was potentiated by CQ pre-treatment. The potentiation of the inhibitory effect of 5-FU by CQ was dependent on the increase of p21 Cip1 and p27 Kip1 and the decrease of CDK2. Since CQ is reported to inhibit autophagy, the catabolic process necessary for cell survival under conditions of cell starvation or stress, which is induced by cancer cells as a protective mechanism against chemotherapeutic agents, we also analyzed the induction of autophagy in HT-29. HT-29 induced autophagy in response to 5-FU, and CQ inhibited this induction, a possible mechanism of the potentiation of the anti-cancer effect of 5-FU. Our

A key role of microRNA-29b for the suppression of colon cancer cell migration by American ginseng.

Directory of Open Access Journals (Sweden)

Deepak Poudyal

Full Text Available Metastasis of colon cancer cells increases the risk of colon cancer mortality. We have recently shown that American ginseng prevents colon cancer, and a Hexane extract of American Ginseng (HAG has particularly potent anti-inflammatory and anti-cancer properties. Dysregulated microRNA (miR expression has been observed in several disease conditions including colon cancer. Using global miR expression profiling, we observed increased miR-29b in colon cancer cells following exposure to HAG. Since miR-29b plays a role in regulating the migration of cancer cells, we hypothesized that HAG induces miR-29b expression to target matrix metalloproteinase-2 (MMP-2 thereby suppressing the migration of colon cancer cells. Results are consistent with this hypothesis. Our study supports the understanding that targeting MMP-2 by miR-29b is a mechanism by which HAG suppresses the migration of colon cancer cells.

Extravirgin olive oil up-regulates CB₁ tumor suppressor gene in human colon cancer cells and in rat colon via epigenetic mechanisms.

Science.gov (United States)

Di Francesco, Andrea; Falconi, Anastasia; Di Germanio, Clara; Micioni Di Bonaventura, Maria Vittoria; Costa, Antonio; Caramuta, Stefano; Del Carlo, Michele; Compagnone, Dario; Dainese, Enrico; Cifani, Carlo; Maccarrone, Mauro; D'Addario, Claudio

2015-03-01

Extravirgin olive oil (EVOO) represents the typical lipid source of the Mediterranean diet, an eating habit pattern that has been associated with a significant reduction of cancer risk. Diet is the more studied environmental factor in epigenetics, and many evidences suggest dysregulation of epigenetic pathways in cancer. The aim of our study was to investigate the effects of EVOO and its phenolic compounds on endocannabinoid system (ECS) gene expression via epigenetic regulation in both human colon cancer cells (Caco-2) and rats exposed to short- and long-term dietary EVOO. We observed a selective and transient up-regulation of CNR1 gene - encoding for type 1 cannabinoid receptor (CB₁) - that was evoked by exposure of Caco-2 cells to EVOO (100 ppm), its phenolic extracts (OPE, 50 μM) or authentic hydroxytyrosol (HT, 50 μM) for 24 h. None of the other major elements of the ECS (i.e., CB₂; GPR55 and TRPV1 receptors; and NAPE-PLD, DAGL, FAAH and MAGL enzymes) was affected at any time point. The stimulatory effect of OPE and HT on CB₁ expression was inversely correlated to DNA methylation at CNR1 promoter and was associated with reduced proliferation of Caco-2 cells. Interestingly, CNR1 gene was less expressed in Caco-2 cells when compared to normal colon mucosa cells, and again this effect was associated with higher level of DNA methylation at CNR1. Moreover, in agreement with the in vitro studies, we also observed a remarkable (~4-fold) and selective increase in CB₁ expression in the colon of rats receiving dietary EVOO supplementation for 10 days. Consistently, CpG methylation of rat Cnr1 promoter, miR23a and miR-301a, previously shown to be involved in the pathogenesis of colorectal cancer and predicted to target CB₁ mRNA, was reduced after EVOO administration down to ~50% of controls. Taken together, our findings demonstrating CB₁ gene expression modulation by EVOO or its phenolic compounds via epigenetic mechanism, both in vitro and in vivo, may

Role of protein kinase C and epidermal growth factor receptor signalling in growth stimulation by neurotensin in colon carcinoma cells

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Dajani Olav

2011-10-01

Full Text Available Abstract Background Neurotensin has been found to promote colon carcinogenesis in rats and mice, and proliferation of human colon carcinoma cell lines, but the mechanisms involved are not clear. We have examined signalling pathways activated by neurotensin in colorectal and pancreatic carcinoma cells. Methods Colon carcinoma cell lines HCT116 and HT29 and pancreatic adenocarcinoma cell line Panc-1 were cultured and stimulated with neurotensin or epidermal growth factor (EGF. DNA synthesis was determined by incorporation of radiolabelled thymidine into DNA. Levels and phosphorylation of proteins in signalling pathways were assessed by Western blotting. Results Neurotensin stimulated the phosphorylation of both extracellular signal-regulated kinase (ERK and Akt in all three cell lines, but apparently did so through different pathways. In Panc-1 cells, neurotensin-induced phosphorylation of ERK, but not Akt, was dependent on protein kinase C (PKC, whereas an inhibitor of the β-isoform of phosphoinositide 3-kinase (PI3K, TGX221, abolished neurotensin-induced Akt phosphorylation in these cells, and there was no evidence of EGF receptor (EGFR transactivation. In HT29 cells, in contrast, the EGFR tyrosine kinase inhibitor gefitinib blocked neurotensin-stimulated phosphorylation of both ERK and Akt, indicating transactivation of EGFR, independently of PKC. In HCT116 cells, neurotensin induced both a PKC-dependent phosphorylation of ERK and a metalloproteinase-mediated transactivation of EGFR that was associated with a gefitinib-sensitive phosphorylation of the downstream adaptor protein Shc. The activation of Akt was also inhibited by gefitinib, but only partly, suggesting a mechanism in addition to EGFR transactivation. Inhibition of PKC blocked neurotensin-induced DNA synthesis in HCT116 cells. Conclusions While acting predominantly through PKC in Panc-1 cells and via EGFR transactivation in HT29 cells, neurotensin used both these pathways in HCT116

Role of protein kinase C and epidermal growth factor receptor signalling in growth stimulation by neurotensin in colon carcinoma cells

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Müller, Kristin M; Tveteraas, Ingun H; Aasrum, Monica; Ødegård, John; Dawood, Mona; Dajani, Olav; Christoffersen, Thoralf; Sandnes, Dagny L

2011-01-01

Neurotensin has been found to promote colon carcinogenesis in rats and mice, and proliferation of human colon carcinoma cell lines, but the mechanisms involved are not clear. We have examined signalling pathways activated by neurotensin in colorectal and pancreatic carcinoma cells. Colon carcinoma cell lines HCT116 and HT29 and pancreatic adenocarcinoma cell line Panc-1 were cultured and stimulated with neurotensin or epidermal growth factor (EGF). DNA synthesis was determined by incorporation of radiolabelled thymidine into DNA. Levels and phosphorylation of proteins in signalling pathways were assessed by Western blotting. Neurotensin stimulated the phosphorylation of both extracellular signal-regulated kinase (ERK) and Akt in all three cell lines, but apparently did so through different pathways. In Panc-1 cells, neurotensin-induced phosphorylation of ERK, but not Akt, was dependent on protein kinase C (PKC), whereas an inhibitor of the β-isoform of phosphoinositide 3-kinase (PI3K), TGX221, abolished neurotensin-induced Akt phosphorylation in these cells, and there was no evidence of EGF receptor (EGFR) transactivation. In HT29 cells, in contrast, the EGFR tyrosine kinase inhibitor gefitinib blocked neurotensin-stimulated phosphorylation of both ERK and Akt, indicating transactivation of EGFR, independently of PKC. In HCT116 cells, neurotensin induced both a PKC-dependent phosphorylation of ERK and a metalloproteinase-mediated transactivation of EGFR that was associated with a gefitinib-sensitive phosphorylation of the downstream adaptor protein Shc. The activation of Akt was also inhibited by gefitinib, but only partly, suggesting a mechanism in addition to EGFR transactivation. Inhibition of PKC blocked neurotensin-induced DNA synthesis in HCT116 cells. While acting predominantly through PKC in Panc-1 cells and via EGFR transactivation in HT29 cells, neurotensin used both these pathways in HCT116 cells. In these cells, neurotensin-induced activation of ERK

The Adhesion of Lactobacillus salivarius REN to a Human Intestinal Epithelial Cell Line Requires S-layer Proteins.

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Wang, Ran; Jiang, Lun; Zhang, Ming; Zhao, Liang; Hao, Yanling; Guo, Huiyuan; Sang, Yue; Zhang, Hao; Ren, Fazheng

2017-03-10

Lactobacillus salivarius REN, a novel probiotic isolated from Chinese centenarians, can adhere to intestinal epithelial cells and subsequently colonize the host. We show here that the surface-layer protein choline-binding protein A (CbpA) of L. salivarius REN was involved in adherence to the human colorectal adenocarcinoma cell line HT-29. Adhesion of a cbpA deletion mutant was significantly reduced compared with that of wild-type, suggesting that CbpA acts as an adhesin that mediates the interaction between the bacterium and its host. To identify the molecular mechanism of adhesion, we determined the crystal structure of a truncated form of CbpA that is likely involved in binding to its cell-surface receptor. The crystal structure identified CbpA as a peptidase of the M23 family whose members harbor a zinc-dependent catalytic site. Therefore, we propose that CbpA acts as a multifunctional surface protein that cleaves the host extracellular matrix and participates in adherence. Moreover, we identified enolase as the CbpA receptor on the surface of HT-29 cells. The present study reveals a new class of surface-layer proteins as well as the molecular mechanism that may contribute to the ability of L. salivarius REN to colonize the human gut.

Hydrogen sulfide lowers proliferation and induces protective autophagy in colon epithelial cells.

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Ya C Wu

Full Text Available Hydrogen sulfide (H(2S is a gaseous bacterial metabolite that reaches high levels in the large intestine. In the present study, the effect of H(2S on the proliferation of normal and cancerous colon epithelial cells was investigated. An immortalized colon epithelial cell line (YAMC and a panel of colon cancer cell lines (HT-29, SW1116, HCT116 were exposed to H(2S at concentrations similar to those found in the human colon. H(2S inhibited normal and cancerous colon epithelial cell proliferation as measured by MTT assay. The anti-mitogenic effect of H(2S was accompanied by G(1-phase cell cycle arrest and the induction of the cyclin-dependent kinase inhibitor p21(Cip. Moreover, exposure to H(2S led to features characteristic of autophagy, including increased formation of LC3B(+ autophagic vacuoles and acidic vesicular organelles as determined by immunofluorescence and acridine orange staining, respectively. Abolition of autophagy by RNA interference targeting Vps34 or Atg7 enhanced the anti-proliferative effect of H(2S. Further mechanistic investigation revealed that H(2S stimulated the phosphorylation of AMP-activated protein kinase (AMPK and inhibited the phosphorylation of mammalian target of rapamycin (mTOR and S6 kinase. Inhibition of AMPK significantly reversed H(2S-induced autophagy and inhibition of cell proliferation. Collectively, we demonstrate that H(2S inhibits colon epithelial cell proliferation and induces protective autophagy via the AMPK pathway.

HMG-CoA reductase regulates CCL17-induced colon cancer cell migration via geranylgeranylation and RhoA activation

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Al-Haidari, Amr A.; Syk, Ingvar; Thorlacius, Henrik

2014-01-01

Highlights: • Simvastatin blocked CCL17-induced and CCR4-dependent RhoA activation in HT29 cells. • CCL17/CCR4-mediated migration of colon cancer cells was antagonised by simvastatin. • Cell migration recovered by adding Mevalonate and geranylgeranyl pyrophosphate. • Targeting HMG-CoA reductase might be useful to inhibit colon cancer metastasis. - Abstract: Background: Simvastatin is widely used to lower cholesterol levels in patients with cardiovascular diseases, although accumulating evidence suggests that statins, such as simvastatin, also exert numerous anti-tumoral effects. Aim: The aim of this study was to examine the effect of simvastatin on colon cancer cell migration. Methods: Migration assays were performed to evaluate CCL17-induced colon cancer cell (HT-29) chemotaxis. In vitro tumor growth and apoptosis were assessed using a proliferation assay and annexin V assay, respectively. Active RhoA protein levels in CCL17-stimulated colon cancer cells were quantified using a G-LISA assay. Results: We found that simvastatin dose-dependently decreased CCL17-induced colon cancer cell migration. Simvastatin had no effect on colon cancer cell proliferation or apoptosis. Inhibition of beta chemokine receptor 4, CCR4, reduced CCL17-evoked activation of RhoA in colon cancer cells. Moreover, administration of mevalonate reversed the inhibitory effect of simvastatin on CCL17-induced colon cancer cell migration. Interestingly, co-incubation with geranylgeranyl pyrophosphate (GGPP) antagonized the inhibitory impact of simvastatin on colon cancer cell migration triggered by CCL17. Moreover, we observed that simvastatin decreased CCL17-induced activation of RhoA in colon cancer cells. Administration of mevalonate and GGPP reversed the inhibitory effect of simvastatin on CCL17-provoked RhoA activation in colon cancer cells. Conclusions: Taken together, our findings show for the first time that HMG-CoA reductase regulates CCL17-induced colon cancer cell migration via

Glycosylation-related gene expression in HT29-MTX-E12 cells upon infection by Helicobacter pylori.

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Cairns, Michael T; Gupta, Ananya; Naughton, Julie A; Kane, Marian; Clyne, Marguerite; Joshi, Lokesh

2017-10-07

To identify glycosylation-related genes in the HT29 derivative cell line, HT29-MTX-E12, showing differential expression on infection with Helicobacter pylori ( H. pylori ). Polarised HT29-MTX-E12 cells were infected for 24 h with H. pylori strain 26695. After infection RNA was isolated from both infected and non-infected host cells. Sufficient infections were carried out to provide triplicate samples for microarray analysis and for qRT-PCR analysis. RNA was isolated and hybridised to Affymetrix arrays. Analysis of microarray data identified genes significantly differentially expressed upon infection. Genes were grouped into gene ontology functional categories. Selected genes associated with host glycan structure (glycosyltransferases, hydrolases, lectins, mucins) were validated by real-time qRT-PCR analysis. Infection of host cells was confirmed by the isolation of live bacteria after 24 h incubation and by PCR amplification of bacteria-specific genes from the host cell RNA. H. pylori do not survive incubation under the adopted culture conditions unless they associate with the adherent mucus layer of the host cell. Microarray analysis identified a total of 276 genes that were significantly differentially expressed ( P < 0.05) upon H. pylori infection and where the fold change in expression was greater than 2. Six of these genes are involved in glycosylation-related processes. Real-time qRT-PCR demonstrated significant downregulation (1.8-fold, P < 0.05) of the mucin MUC20. REG4 was heavily expressed and significantly downregulated (3.1-fold, P < 0.05) upon infection. Gene ontology analysis was consistent with previous studies on H. pylori infection. Gene expression data suggest that infection with H. pylori causes a decrease in glycan synthesis, resulting in shorter and simpler glycan structures.

Characteristics of [18F] fluorodeoxyglucose uptake in human colon cancer cells

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Kim, Chae Kyun; Chung, June Key; Jeong, Jae Min; Lee, Myung Chul; Koh, Chang Soon

1997-01-01

Cancer tissues are characterized by increased glucose uptake. 18 F-fluorodeoxyglucose(FDG), a glucose analogue is used for the diagnosis of cancer in PET studies. This study was aimed to compare the glucose uptake and glucose transporter 1(GLUT1) expression in various human colon cancer cells. We measured FDG uptake by cell retention study and expression of GLUT1 using Western blotting. Human colon cancer cells, SNU-C2A, SNU-C4 and SNU-C5, were used. The cells were incubated with 1μ Ci/ml of FDG in HEPES- buffered saline for one hour. The FDG uptake of SNU-C2A, SNU-C4 and SNU-C5 were 16.8±1.36, 12.3±5.55 and 61.0±2.17 cpm/μg of protein, respectively. Dose-response and time-course studies represent that FDG uptake of cancer cells were dose dependent and time dependent. The rate of FDG uptake of SNU-C2A, SNU-C4 and SNU-C5 were 0.29±0.03, 0.21±0.09 and 1.07±0.07 cpm/min/μg of protein, respectively. Western blot analysis showed that the GLUT1 expression of SNU-C5 was significantly higher than those of SNU-C2A and SNU-C4. These results represent that FDG uptake into human colon cancer cells are different from each other. In addition, FDG uptake and expression of GLUT1 are closely related in human colon cancer cells

Arsenic downregulates tight junction claudin proteins through p38 and NF-κB in intestinal epithelial cell line, HT-29

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Jeong, Chang Hee; Seok, Jin Sil; Petriello, Michael C.; Han, Sung Gu

2017-01-01

Arsenic is a naturally occurring metalloid that often is found in foods and drinking water. Human exposure to arsenic is associated with the development of gastrointestinal problems such as fluid loss, diarrhea and gastritis. Arsenic is also known to induce toxic responses including oxidative stress in cells of the gastrointestinal track. Tight junctions (TJs) regulate paracellular permeability and play a barrier role by inhibiting the movement of water, solutes and microorganisms in the paracellular space. Since oxidative stress and TJ damage are known to be associated, we examined whether arsenic produces TJ damage such as downregulation of claudins in the human colorectal cell line, HT-29. To confirm the importance of oxidative stress in arsenic-induced TJ damage, effects of the antioxidant compound (e.g., N-acetylcysteine (NAC)) were also determined in cells. HT-29 cells were treated with arsenic trioxide (40 μM, 12 h) to observe the modified expression of TJ proteins. Arsenic decreased expression of TJ proteins (i.e., claudin-1 and claudin-5) and transepithelial electrical resistance (TEER) whereas pretreatment of NAC (5–10 mM, 1 h) attenuated the observed claudins downregulation and TEER. Arsenic treatment produced cellular oxidative stress via superoxide generation and lowering glutathione (GSH) levels, while NAC restored cellular GSH levels and decreased oxidative stress. Arsenic increased phosphorylation of p38 and nuclear translocation of nuclear factor-kappa B (NF-κB) p65, while NAC attenuated these intracellular events. Results demonstrated that arsenic can damage intestinal epithelial cells by proinflammatory process (oxidative stress, p38 and NF-κB) which resulted in the downregulation of claudins and NAC can protect intestinal TJs from arsenic toxicity.

Crataegus azarolus Leaves Induce Antiproliferative Activity, Cell Cycle Arrest, and Apoptosis in Human HT-29 and HCT-116 Colorectal Cancer Cells.

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Mustapha, Nadia; Pinon, Aline; Limami, Youness; Simon, Alain; Ghedira, Kamel; Hennebelle, Thierry; Chekir-Ghedira, Leila

2016-05-01

Limited success has been achieved in extending the survival of patients with metastatic colorectal cancer (CRC). There is a strong need for novel agents in the treatment and prevention of CRC. Therefore, in the present study we evaluated the antiproliferative and pro-apoptotic potential of Crataegus azarolus ethyl acetate extract in HCT-116 and HT-29 human colorectal cancer cell lines. Moreover, we attempted to investigate the signaling pathways that should be involved in its cytotoxic effect. The Crataegus azarolus ethyl acetate extract-induced growth inhibitory effect was associated with DNA fragmentation, sub-G1 peak, loss of mitochondrial potential, and poly (ADP-ribose) polymerase (PARP) cleavage. In addition, ethyl acetate extract of Crataegus azarolus induced the cleavage of caspase-8. It has no effect on steady-state levels of total Bcl-2 protein. Whereas Bax levels decreased significantly in a dose-dependent manner in both tested cell lines. Taken together, these findings confirm the involvement of the extrinsic pathway of apoptosis. The apoptotic cell death induced by ethyl acetate extract of Crataegus azarolus was accompanied by an enhancement of the p21 expression but not through p53 activation in human colorectal cancer cells. The above-mentioned data provide insight into the molecular mechanisms of Crataegus azarolus ethyl acetate extract-induced apoptosis in CRC. Therefore, this compound should be a potential anticancer agent for the treatment of CRC. © 2015 Wiley Periodicals, Inc.

Induction of apoptosis in HT-29 cells by extracts from isothiocyanates-rich varieties of Brassica oleracea.

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Mas, Sergi; Crescenti, Anna; Gassó, Patricia; Deulofeu, Ramon; Molina, Rafael; Ballesta, Antonio; Kensler, Thomas W; Lafuente, Amalia

2007-01-01

Among the vegetables with anti-carcinogenic properties, members of the genus Brassica are the most effective at reducing the risk of cancer. This property may be explained by their principle bioactive compounds, isothiocyanates (ITCs). The aim of this study was to measure the amounts of ITCs in extracts from vegetables of the Brasssica genus and assay them for potency of induction of apoptosis in a colorectal cancer cell line (HT-29). ITCs were determined by the cyclocondensation assay with 1,2-benzenedithiol and induction of apoptosis by assessment of cell viability, caspase-3 activity and DNA fragmentation. Purple cabbage extract showed the highest ITC concentration per gram, fresh weight, followed by black cabbage and Romanesco cauliflower. At ITC concentrations of 7.08 microg/mL these extracts decreased cell viability and induced caspase-3 and DNA fragmentation at 48h. Brussels sprouts showed the strongest effects on cell viability and caspase-3 activity. Varieties of Brassica Oleracea are rich sources of ITCs that potently inhibit the growth of colon cancer cells by inducting apoptosis. All the extracts showed anticancer activity at ITC concentrations of between 3.54 to 7.08 mug/mL, which are achievable in vivo. Our results showed that ITC concentration and the chemopreventive responses of plant extracts vary among the varieties of Brassica Oleracea studied and among their cultivars.

A Schiff base-derived copper (II) complex is a potent inducer of apoptosis in colon cancer cells by activating the intrinsic pathway.

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Hajrezaie, Maryam; Paydar, Mohammadjavad; Moghadamtousi, Soheil Zorofchian; Hassandarvish, Pouya; Gwaram, Nura Suleiman; Zahedifard, Maryam; Rouhollahi, Elham; Karimian, Hamed; Looi, Chung Yeng; Ali, Hapipah Mohd; Abdul Majid, Nazia; Abdulla, Mahmood Ameen

2014-01-01

Metal-based drugs with extensive clinical applications hold great promise for the development of cancer chemotherapeutic agents. In the last few decades, Schiff bases and their complexes have become well known for their extensive biological potential. In the present study, we examined the antiproliferative effect of a copper (II) complex on HT-29 colon cancer cells. The Cu(BrHAP)2 Schiff base compound demonstrated a potent antiproliferative effect in HT-29 cells, with an IC50 value of 2.87  μg/ml after 72 h of treatment. HT-29 cells treated with Cu (II) complexes underwent apoptosis death, as exhibited by a progressive elevation in the proportion of the G1 cell population. At a concentration of 6.25  μg/ml, the Cu(BrHAP)2 compound caused significant elevation in ROS production following perturbation of mitochondrial membrane potential and cytochrome c release, as assessed by the measurement of fluorescence intensity in stained cells. Furthermore, the activation of caspases 3/7 and 9 was part of the Cu (II) complex-induced apoptosis, which confirmed the involvement of mitochondrial-mediated apoptosis. Meanwhile, there was no significant activation of caspase-8. Taken together, these results imply that the Cu(BrHAP)2 compound is a potential candidate for further in vivo and clinical colon cancer studies to develop novel chemotherapeutic agents derived from metal-based agents.

Nutrient Availability Alters the Effect of Autophagy on Sulindac Sulfide-Induced Colon Cancer Cell Apoptosis

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Shiun-Kwei Chiou

2012-01-01

Full Text Available Autophagy is a catabolic process by which a cell degrades its intracellular materials to replenish itself. Induction of autophagy under various cellular stress stimuli can lead to either cell survival or cell death via apoptotic and/or autophagic (nonapoptotic pathways. The NSAID sulindac sulfide induces apoptosis in colon cancer cells. Here, we show that inhibition of autophagy under serum-deprived conditions resulted in significant reductions of sulindac sulfide-induced apoptosis in HT-29 colon cancer cells. In contrast, inhibition of autophagy under conditions where serum is available significantly increased sulindac sulfide-induced apoptosis in HT-29 cells. We previously showed that the apoptosis inhibitor, survivin, plays a role in regulating NSAID-induced apoptosis and autophagic cell death. Here, we show that survivin protein half-life is increased in the presence of autophagy inhibitors under serum-deprived conditions, but not under conditions when serum is available. Thus, the increased levels of survivin may be a factor contributing to inhibition of sulindac sulfide-induced apoptosis under serum-deprived conditions. These results suggest that whether a cell lives or dies due to autophagy induction depends on the balance of factors that regulate both autophagic and apoptotic processes.

Chemopreventive properties of raisins originating from Greece in colon cancer cells.

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Kountouri, Aggeliki M; Gioxari, Aristea; Karvela, Evangelia; Kaliora, Andriana C; Karvelas, Michalis; Karathanos, Vaios T

2013-02-26

Colorectal cancer is one of the major causes of cancer-related mortality in humans in both developed and developing countries. Dietary patterns influence the risk of colon cancer development, while plant-derived foods have gained great interest, due to the high content of antioxidants. Corinthian raisins (Currants, CR) and Sultanas (S) (Vitis vinifera L., Vitaceae) are dried vine fruits produced in Greece with many culinary uses in both the Mediterranean and the Western nutrition. In the present study, we investigated the effects of CR and S on human colon cancer cells. Methanol extracts of CR and S were used at different concentrations. The total polyphenol content and anti-radical activity were measured by Folin-Ciocalteu and DPPH, respectively. Antioxidant, anti-inflammatory and anti-proliferative effects on HT29 cell culture were evaluated. All extracts exhibited DPPH˙ scavenging activity in a dose-dependent manner. Both products suppressed cell proliferation, while the levels of glutathione and cyclooxygenase 2 were significantly decreased. A significant reduction in IL-8 levels and NF-kappaB p65 activation was also observed. Both antioxidant and anti-inflammatory effects were dependent on the duration of exposure. Results indicate that the methanol extracts of CR and S exhibit anti-radical activity in vitro, as well as cancer preventive efficacy on colon cancer cells, with S having slightly higher activity. The beneficial properties of these unique dried grapes are attributed to their high content of phenolic compounds.

Cellular Homeostasis and Antioxidant Response in Epithelial HT29 Cells on Titania Nanotube Arrays Surface

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Rabiatul Basria SMN Mydin

2017-01-01

Full Text Available Cell growth and proliferative activities on titania nanotube arrays (TNA have raised alerts on genotoxicity risk. Present toxicogenomic approach focused on epithelial HT29 cells with TNA surface. Fledgling cell-TNA interaction has triggered G0/G1 cell cycle arrests and initiates DNA damage surveillance checkpoint, which possibly indicated the cellular stress stimuli. A profound gene regulation was observed to be involved in cellular growth and survival signals such as p53 and AKT expressions. Interestingly, the activation of redox regulator pathways (antioxidant defense was observed through the cascade interactions of GADD45, MYC, CHECK1, and ATR genes. These mechanisms furnish to protect DNA during cellular division from an oxidative challenge, set in motion with XRRC5 and RAD50 genes for DNA damage and repair activities. The cell fate decision on TNA-nanoenvironment has been reported to possibly regulate proliferative activities via expression of p27 and BCL2 tumor suppressor proteins, cogent with SKP2 and BCL2 oncogenic proteins suppression. Findings suggested that epithelial HT29 cells on the surface of TNA may have a positive regulation via cell-homeostasis mechanisms: a careful circadian orchestration between cell proliferation, survival, and death. This nanomolecular knowledge could be beneficial for advanced medical applications such as in nanomedicine and nanotherapeutics.

Inhibition of Lipopolysaccharide-Induced Interleukin 8 in Human Adenocarcinoma Cell Line HT-29 by Spore Probiotics: B. coagulans and B. subtilis (natto).

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Azimirad, Masoumeh; Alebouyeh, Masoud; Naji, Tahereh

2017-03-01

Probiotics are used as a treatment for different intestinal disorders. They confer health benefits by different ways. This study was aimed to investigate immunomodulatory effect of Bacillus probiotic spores on the production of lipopolysaccharide (LPS)-induced interleukin 8 (IL-8) in HT-29 intestinal epithelial cells. Differentiated intestinal epithelial cell line was used as a model for the study of colonization of purified spores (Bacillus subtilis (natto) and B. coagulans) and their anti-inflammatory effects. MTT assay and trypan blue staining were used for the detection of optimal concentration of the purified spores and LPS. Pre-treatment assay was done by treatment of the cells with the purified spores for 2 h, followed by challenges with LPS for 3 and 18 h. Post-treatment assay was done by initial treatment of the cells with LPS for 18 h, followed by the spores for 3 and 6 h. Levels of IL-8 secretion and its mRNA expression were measured by ELISA and relative Q real-time PCR. Our results showed similar rates of adherence to intestinal epithelial cells by the spore probiotics, while displaying no cytotoxic effect. In the pre-treatment assay, a significant decrease in IL-8, at both protein and mRNA levels, was measured for B. coagulans spores after the addition of LPS, which was higher than those observed for Bacillus subtilis (natto) spores. In the post-treatment assay, while Bacillus subtilis (but not B. coagulans) diminished the LPS-stimulated IL-8 levels after 3 h of incubation, the inhibitory effect was not constant. In conclusion, ability of Bacillus spore probiotics for adherence to intestinal epithelial cell and their anti-inflammatory effects, through interference with LPS/IL-8 signaling, was shown in this study. Further studies are needed to characterize responsible bacterial compounds associated with these effects.

Human wound colonization by Lucilia eximia and Chrysomya rufifacies (Diptera: Calliphoridae): myiasis, perimortem, or postmortem colonization?

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Sanford, Michelle R; Whitworth, Terry L; Phatak, Darshan R

2014-05-01

The infestation of human or animal tissues by fly larvae has been given distinctive terminology depending on the timing and location of colonization. Wounds and orifices colonized by Diptera in a living human or animal are typically referred to as myiasis. When the colonization occurs after death, it is referred to as postmortem colonization and can be used to estimate the minimum postmortem interval. What happens when the human, as in the case presented here, has a necrotic limb while the human remains alive, at least for a short period of time? The case presented here documents perimortem wound colonization by Lucilia eximia (Wiedemann) and Chrysomya rufifacies (Macquart) and the considerations for approximating development temperatures and estimating the time of colonization (TOC). This represents the first record of L. eximia in human myiasis in the United States and the first record of the co-occurrence of L. eximia and C. rufifacies in human myiasis in the United States. The TOC was estimated using both ambient and body temperature. Insect colonization before death complicates the estimation of TOC and minimum postmortem interval and illustrates the problem of temperature approximation in forensic entomology casework.

The effect of Saccharomyces boulardii on human colon cells and inflammation in rats with trinitrobenzene sulfonic acid-induced colitis.

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Lee, Sang Kil; Kim, Youn Wha; Chi, Sung-Gil; Joo, Yeong-Shil; Kim, Hyo Jong

2009-02-01

Saccharomyces boulardii (S. boulardii) has beneficial effects in the treatment of intestinal inflammation; however, little is known about the mechanisms by which these effects occur. We investigated the effects of S. boulardii on the expression of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and interleukin-8 (IL-8), using human HT-29 colonocytes and a rat model of trinitrobenzene sulfonic acid (TNBS)-induced colitis. The effect of S. boulardii on gene expression was assessed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), and Northern blot and Western blot assays. Pharmacological inhibitors for various signaling pathways were used to determine the signaling pathways implicated in the S. boulardii regulation of PPAR-gamma and IL-8. We found that S. boulardii up-regulated and down-regulated PPAR-gamma and IL-8 expression at the transcription level, both in vitro and in vivo (P Saccharomyces boulardii blocked tumor necrosis factor-alpha (TNF-alpha) regulation of PPAR-gamma and IL-8 through disruption of TNF-alpha-mediated nuclear factor kappa B (NF-kappaB) activation. Furthermore, S. boulardii suppressed colitis and expression of pro-inflammatory cytokine genes in vivo (P boulardii reduces colonic inflammation and regulates inflammatory gene expression.

[Effect of piperine on 5-HT and synaptophysin expression of rats with irritable bowel syndrome].

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Wu, Shu-Juan; Wang, Ren-Ye; Xue, Ji-Xiong; Pan, Jian-Chun

2013-12-01

This study is to explore the amelioration of piperine on chronic acute combining stress rat with depression-like behavior, visceral sensitivity, and its effect on the expression of serotonin (5-HT) and synaptophysin. Forty two SD rats were divided into seven groups: blank group, model group, piperine (12.5, 25, 50 and 100 mgkg-1, ig) and imipramine (10 mgkg-1, ip) groups. The rat model of irritable bowel syndrome was established by chronic acute combining stress, and then to evaluate depression-like behavior and visceral sensitivity. The expressions of 5-HT and synaptophysin in the hippocampus and colon were determined by high performance liquid chromatography (HPLC) and Western blotting, respectively. The duration of immobility of IBS rat in the forced swimming test had been significantly increased, the sucrose consumption of IBS rat had been reduced and visceral sensitivity was obviously elevated in the IBS model group as compared with those in the normal control group (P<0.05, P<0.01). As compared with those in the normal control group, the expression of 5-HT significantly decreased, 5-HIAA/5-HT ratio significantly increased in the hippocampus of IBS model group (P<0.05), but opposite presentations were noted in the colon (P<0.05). As compared with that in the normal control group, the synaptophysin expression in the hippocampus decreased significantly but obviously increased in the colon (P<0.05). Piperine improved the behavior of IBS rats, and reversed the levels of 5-HT and 5-HIAA, and 5-HIAA/5-HT proportion in the hippocampus and colon (P<0.05); besides, they significantly reverse the synaptophysin level in the hippocampus and colon (P<0.05). The presence of depression and visceral sensitivity had been changed in IBS rats, with abnormal expression of 5-HT and synaptophysin in the brain-gut system. Piperine can ameliorate the changes of the behavior and regulation of serotonin and synaptophysin expression in IBS rat model.

Pharmacology of the hypothermic response to 5-HT1A receptor activation in humans.

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Lesch, K P; Poten, B; Söhnle, K; Schulte, H M

1990-01-01

The selective 5-HT1A receptor ligand ipsapirone (IPS) caused dose-related hypothermia in humans. The response was attenuated by the nonselective 5-HT1/2 receptor antagonist metergoline and was completely antagonized by the nonselective beta-adrenoceptor antagonist pindolol, which interacts stereoselectively with the 5-HT1A receptor. The selective beta 1-adrenergic antagonist betaxolol had no effect. The findings indicate that IPS-induced hypothermia specifically involves activation of (presynaptic) 5-HT1A receptors. Therefore, the hypothermic response to IPS may provide a convenient in vivo paradigma to assess the function of the presynaptic 5-HT receptor in affective disorders and its involvement in the effects of psychotropic drugs.

Impact of Mesenchymal Stem Cell secreted PAI-1 on colon cancer cell migration and proliferation

Energy Technology Data Exchange (ETDEWEB)

Hogan, Niamh M. [Discipline of Surgery, School of Medicine, National University of Ireland, Galway (Ireland); Joyce, Myles R. [Department of Colorectal Surgery, University College Hospital, Galway (Ireland); Murphy, J. Mary; Barry, Frank P.; O’Brien, Timothy [Regenerative Medicine Institute, National University of Ireland, Galway (Ireland); Kerin, Michael J. [Discipline of Surgery, School of Medicine, National University of Ireland, Galway (Ireland); Dwyer, Roisin M., E-mail: roisin.dwyer@nuigalway.ie [Discipline of Surgery, School of Medicine, National University of Ireland, Galway (Ireland)

2013-06-14

Highlights: •MSCs were directly co-cultured with colorectal cancer (CRC) cells on 3D scaffolds. •MSCs influence CRC protein/gene expression, proliferation and migration. •We report a significant functional role of MSC-secreted PAI-1 in colon cancer. -- Abstract: Mesenchymal Stem Cells are known to engraft and integrate into the architecture of colorectal tumours, with little known regarding their fate following engraftment. This study aimed to investigate mediators of Mesenchymal Stem Cell (MSC) and colon cancer cell (CCC) interactions. Mesenchymal Stem Cells and colon cancer cells (HT29 and HCT-116) were cultured individually or in co-culture on 3-dimensional scaffolds. Conditioned media containing all secreted factors was harvested at day 1, 3 and 7. Chemokine secretion and expression were analyzed by Chemi-array, ELISA (Macrophage migration inhibitory factor (MIF), plasminogen activator inhibitor type 1 (PAI-1)) and RQ-PCR. Colon cancer cell migration and proliferation in response to recombinant PAI-1, MSCs and MSCs + antibody to PAI-1 was analyzed using Transwell inserts and an MTS proliferation assay respectively. Chemi-array revealed secretion of a wide range of factors by each cell population, including PAI-1and MIF. ELISA analysis revealed Mesenchymal Stem Cells to secrete the highest levels of PAI-1 (MSC mean 10.6 ng/mL, CCC mean 1.01 ng/mL), while colon cancer cells were the principal source of MIF. MSC-secreted PAI-1 stimulated significant migration of both CCC lines, with an antibody to the chemokine shown to block this effect (67–88% blocking,). A cell-line dependant effect on CCC proliferation was shown for Mesenchymal Stem Cell-secreted PAI-1 with HCT-116 cells showing decreased proliferation at all concentrations, and HT29 cells showing increased proliferation in the presence of higher PAI-1 levels. This is the first study to identify PAI-1 as an important mediator of Mesenchymal Stem Cell/colon cancer cell interactions and highlights the

Impact of Mesenchymal Stem Cell secreted PAI-1 on colon cancer cell migration and proliferation

International Nuclear Information System (INIS)

Hogan, Niamh M.; Joyce, Myles R.; Murphy, J. Mary; Barry, Frank P.; O’Brien, Timothy; Kerin, Michael J.; Dwyer, Roisin M.

2013-01-01

Highlights: •MSCs were directly co-cultured with colorectal cancer (CRC) cells on 3D scaffolds. •MSCs influence CRC protein/gene expression, proliferation and migration. •We report a significant functional role of MSC-secreted PAI-1 in colon cancer. -- Abstract: Mesenchymal Stem Cells are known to engraft and integrate into the architecture of colorectal tumours, with little known regarding their fate following engraftment. This study aimed to investigate mediators of Mesenchymal Stem Cell (MSC) and colon cancer cell (CCC) interactions. Mesenchymal Stem Cells and colon cancer cells (HT29 and HCT-116) were cultured individually or in co-culture on 3-dimensional scaffolds. Conditioned media containing all secreted factors was harvested at day 1, 3 and 7. Chemokine secretion and expression were analyzed by Chemi-array, ELISA (Macrophage migration inhibitory factor (MIF), plasminogen activator inhibitor type 1 (PAI-1)) and RQ-PCR. Colon cancer cell migration and proliferation in response to recombinant PAI-1, MSCs and MSCs + antibody to PAI-1 was analyzed using Transwell inserts and an MTS proliferation assay respectively. Chemi-array revealed secretion of a wide range of factors by each cell population, including PAI-1and MIF. ELISA analysis revealed Mesenchymal Stem Cells to secrete the highest levels of PAI-1 (MSC mean 10.6 ng/mL, CCC mean 1.01 ng/mL), while colon cancer cells were the principal source of MIF. MSC-secreted PAI-1 stimulated significant migration of both CCC lines, with an antibody to the chemokine shown to block this effect (67–88% blocking,). A cell-line dependant effect on CCC proliferation was shown for Mesenchymal Stem Cell-secreted PAI-1 with HCT-116 cells showing decreased proliferation at all concentrations, and HT29 cells showing increased proliferation in the presence of higher PAI-1 levels. This is the first study to identify PAI-1 as an important mediator of Mesenchymal Stem Cell/colon cancer cell interactions and highlights the

Oral and Fecal Campylobacter concisus Strains induce Barrier dysfunction by Apoptosis in HT-29/B6 Intestinal Epithelial Cells

DEFF Research Database (Denmark)

Nielsen, Hans Linde; Nielsen, Henrik Ib; Ejlertsen, Tove

in Ussing chambers. Tight junction (TJ) protein expression was determined by Western blotting, and subcellular TJ distribution was analyzed by confocal laser-scanning microscopy. Apoptosis induction was examined by TUNEL-staining and Western blot of caspase-3 activation. All strains invaded confluent HT-29...

Decreased expression of 17β-hydroxysteroid dehydrogenase type 1 is associated with DNA hypermethylation in colorectal cancer located in the proximal colon

International Nuclear Information System (INIS)

Rawłuszko, Agnieszka Anna; Horbacka, Karolina; Krokowicz, Piotr; Jagodziński, Paweł Piotr

2011-01-01

The importance of 17β-estradiol (E2) in the prevention of large bowel tumorigenesis has been shown in many epidemiological studies. Extragonadal E2 may form by the aromatase pathway from androstenedione or the sulfatase pathway from estrone (E1) sulfate followed by E1 reduction to E2 by 17-β-hydroxysteroid dehydrogenase (HSD17B1), so HSD17B1 gene expression may play an important role in the production of E2 in peripheral tissue, including the colon. HSD17B1 expression was analyzed in colorectal cancer cell lines (HT29, SW707) and primary colonic adenocarcinoma tissues collected from fifty two patients who underwent radical colon surgical resection. Histopathologically unchanged colonic mucosa located at least 10-20 cm away from the cancerous lesions was obtained from the same patients. Expression level of HSD17B1 using quantitative PCR and western blot were evaluated. DNA methylation level in the 5' flanking region of HSD17B1 CpG rich region was assessed using bisulfite DNA sequencing and HRM analysis. The influence of DNA methylation on HSD17B1 expression was further evaluated by ChIP analysis in HT29 and SW707 cell lines. The conversion of estrone (E1) in to E2 was determined by electrochemiluminescence method. We found a significant decrease in HSD17B1 transcript (p = 0.0016) and protein (p = 0.0028) levels in colorectal cancer (CRC) from the proximal but not distal colon and rectum. This reduced HSD17B1 expression was associated with significantly increased DNA methylation (p = 0.003) in the CpG rich region located in the 5' flanking sequence of the HSD17B1 gene in CRC in the proximal but not distal colon and rectum. We also showed that 5-dAzaC induced demethylation of the 5' flanking region of HSD17B1, leading to increased occupation of the promoter by Polymerase II, and increased transcript and protein levels in HT29 and SW707 CRC cells, which contributed to the increase in E2 formation. Our results showed that reduced HSD17B1 expression can

Chemoprevention of colon carcinogenesis by polyethylene glycol: suppression of epithelial proliferation via modulation of SNAIL/beta-catenin signaling.

Science.gov (United States)

Roy, Hemant K; Kunte, Dhananjay P; Koetsier, Jennifer L; Hart, John; Kim, Young L; Liu, Yang; Bissonnette, Marc; Goldberg, Michael; Backman, Vadim; Wali, Ramesh K

2006-08-01

Polyethylene glycol (PEG) is one of the most potent chemopreventive agents against colorectal cancer; however, the mechanisms remain largely unexplored. In this study, we assessed the ability of PEG to target cyclin D1-beta-catenin-mediated hyperproliferation in the azoxymethane-treated rat model and the human colorectal cancer cell line, HT-29. Azoxymethane-treated rats were randomized to AIN-76A diet alone or supplemented with 5% PEG-8000. After 30 weeks, animals were euthanized and biopsies of aberrant crypt foci and uninvolved crypts were subjected to immunohistochemical and immunoblot analyses. PEG markedly suppressed both early and late markers of azoxymethane-induced colon carcinogenesis (fractal dimension by 80%, aberrant crypt foci by 64%, and tumors by 74%). In both azoxymethane-treated rats and HT-29 cells treated with 5% PEG-3350 for 24 hours, PEG decreased proliferation (45% and 52%, respectively) and cyclin D1 (78% and 56%, respectively). Because beta-catenin is the major regulator of cyclin D1 in colorectal cancer, we used the T-cell factor (Tcf)-TOPFLASH reporter assay to show that PEG markedly inhibited beta-catenin transcriptional activity. PEG did not alter total beta-catenin expression but rather its nuclear localization, leading us to assess E-cadherin expression (a major determinant of beta-catenin subcellular localization), which was increased by 73% and 71% in the azoxymethane-rat and HT-29 cells, respectively. We therefore investigated the effect of PEG treatment on levels of the negative regulator of E-cadherin, SNAIL, and observed a 50% and 75% decrease, respectively. In conclusion, we show, for the first time, a molecular mechanism through which PEG imparts its antiproliferative and hence profound chemopreventive effect.

Tumour-Derived Interleukin-1 Beta Induces Pro-inflammatory Response in Human Mesenchymal Stem Cells

DEFF Research Database (Denmark)

Alajez, Nehad M; Al-toub, Mashael; Almusa, Abdulaziz

’ secreted factors as represented by a panel of human cancer cell lines (breast (MCF7 and MDA-MB-231); prostate (PC-3); lung (NCI-H522); colon (HT-29) and head & neck (FaDu)) on the biological characteristics of MSCs. Background Over the past several years, significant amount of research has emerged......, the goal of this study was to assess the cellular and molecular changes in MSCs in response to secreted factors present in conditioned media (CM) from a panel of human tumor cell lines covering a spectrum of human cancers (Breast, Prostate, Lung, colon, and head and neck). Research Morphological changes...... with bipolar processes. In association with phenotypic changes, genome-wide gene expression and bioinformatics analysis revealed an enhanced pro-inflammatory response of those MSCs. Pharmacological inhibitions of FAK and MAPKK severely impaired the pro-inflammatory response of MSCs to tumor CM (~80-99%, and 55...

Colonic spirochetosis in animals and humans.

Science.gov (United States)

Smith, James L

2005-07-01

Colonic spirochetosis is a disease caused by the gram-negative bacteria Brachyspira aalborgi and Brachyspira pilosicoli. B. pilosicoli induces disease in both humans and animals, whereas B. aalborgi affects only humans and higher primates. Symptoms in humans include diarrhea, rectal bleeding, and abdominal cramps. Colonic spirochetosis is common in third world countries; however, in developed countries, the disease is observed mainly in homosexual males. Terminally ill patients infected with Brachyspira are particularly at risk for developing spirochetemia. Diarrhea, poor growth performance, and decreased feed-to-gain efficiency is seen in pigs with colonic spirochetosis. The disease in chickens is characterized by delayed and/or reduced egg production, diarrhea, poor feed conversion, and retarded growth. Thus, colonic spirochetosis can represent a serious economic loss in the swine and poultry industries. The organisms are transmitted by the fecal-oral route, and several studies have demonstrated that human, primate, pig, dog, or bird strains of B. pilosicoli can be transmitted to pigs, chickens, and mice. B. pilosicoli may be a zoonotic pathogen, and although it has not been demonstrated, there is a possibility that both B. pilosicoli and B. aalborgi can be transferred to humans via contact with the feces of infected animals, meat from infected animals, or food contaminated by food handlers. Neither B. pilosicoli nor B. aalborgi has been well characterized in terms of basic cellular functions, pathogenicity, or genetics. Studies are needed to more thoroughly understand these Brachyspira species and their disease mechanisms.

Expression and role of 5-HT7 receptor in brain and intestine in rats with irritable bowel syndrome.

Science.gov (United States)

Zou, Bai-cang; Dong, Lei; Wang, Yan; Wang, Sheng-hao; Cao, Ming-bo

2007-12-05

The 5-hydroxytryptamine7 receptor (5-HT(7) receptor, 5-HT(7)R) plays an important role in the regulation of smooth muscle relaxation and visceral sensation and might be involved in the pathogenesis of the gastrointestinal dyskinesia, abdominal pain and visceral paresthesia in irritable bowel syndrome (IBS). The aim of this study was to investigate the role of the 5-HT(7) receptor in the pathogenesis of IBS. A rat model of irritable bowel syndrome with diarrhea (IBS-D) was established by colonic instillation of acetic acid and restraint stress. A rat model with irritable bowel syndrome with constipation (IBS-C) was established by stomach irrigated with 0 - 4 degrees C cool water daily for 14 days. The content and distribution of 5-HT in the brain and gut were examined by immunohistochemistry and the mRNA expression of the 5-HT(7) receptor was determined by fluorescent quantitative reverse transcription polymerase chain reaction. The accumulation of cyclic adenosine monophosphate (cAMP) in all the same tissues was measured by radioimmunity. The models of IBS were reliable by identification. The immunohistochemistry results showed that there were significantly more 5-HT positive cells in the IBS-D group than in the control group in the hippocampus, hypothalamus, jejunum, ileum, proximate colon and distal colon (P intestine is related to the IBS pathogenesis. The up-regulated expression of the 5-HT(7) receptor in the brain and colon might play an important role in the pathogenesis of IBS-C.

Regulatory effect of evodiamine on the malignant biological behaviors and Wnt/β-catenin signaling pathway of colorectal cancer cell lines HT29

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Yuan-Hui Wang

2016-04-01

Full Text Available Objective: To study the regulatory effect of evodiamine on the malignant biological behaviors and Wnt/β-catenin signaling pathway of colorectal cancer cell lines HT29. Methods: Colorectal cancer cell lines HT29 were cultured and divided into blank control group and evodiamine group, and after different treatment, cell viability, proportion of different cell cycle as well as the contents of VEGFA, VEGFB, VEGFC, MMP3, MMP14, Wnt and β-catenin were detected. Results: (1 Cell viability: MTT value of evodiamine group was significantly lower than that of blank control group; (2 Cell cycle: proportion of both S phase and G2/M phase of evodiamine group were lower than those of blank control group, and proportion of G0/ G1 phase was higher than that of blank control group; (3 VEGF and MMP contents: VEGFA, VEGFB, VEGFC, MMP3 and MMP14 contents of evodiamine group were lower than those of blank control group; (4 Wnt/β-catenin signaling pathway: Wnt and β-catenin contents of evodiamine group were lower than those of blank control group. Conclusion: Evodiamine can inhibit the proliferation of colorectal cancer cell lines HT29 and down-regulate the expression of VEGF and MMP, and the effect may be achieved by inhibiting the activation of Wnt/β-catenin signaling pathway.

Biosynthesis of human colonic mucin: Muc2 is the prominent secretory mucin

NARCIS (Netherlands)

Tytgat, K. M.; Büller, H. A.; Opdam, F. J.; Kim, Y. S.; Einerhand, A. W.; Dekker, J.

1994-01-01

Human colonic epithelium produces large amounts of mucin. The aim of this study was to examine mucin biosynthesis in the human colon. Human colonic mucin was isolated using CsCl density gradients, and polyclonal antiserum was raised. Biosynthesis of colonic mucins was studied by labeling colonic

Vitamin E analogue, D-alpha tocopherol succinate, enhances x-ray induced growth delay of human adenocarcinoma cancer cell line

International Nuclear Information System (INIS)

Jaworska, A.; Ottesen, T.E.

2003-01-01

The purpose of this study was to assess the effects of d-alpha Tocopherol succinate (alpha-TS) in modifying radiation-induced viability reduction and apoptosis occurrence in the model for normal and cancer cells. Our hypothesis was that alpha-TS enhances the growth-inhibitory effect of x-irradiation in cancer cells and that the effect is more pronounced in these cells than in normal cells. Murine NIH 3T3 Swiss albino embryonic cells and HT29 human Caucasian colon adenocarcinoma cells were used in the experiments. Alpha-TS was added to the cultures 1 h prior to irradiation with doses of 2 or 5Gy of x-ray. After irradiation cells were incubated for 73 h. Trypan blue exclusion viability test and estimation of apoptosis and necrosis were made. Apoptotic and necrotic cells were counted in fluorescence microscope using fluorescence dyes: propidium iodide and Hoechst 33342. For experiments with the dose of 5 Gy at least five series of experiments were performed. At lower doses (up to approximately 25μM/ml) treatment with alpha-TS alone enhanced growth of both cell lines. At higher doses treatment with alpha-TS alone delayed the growth of the cell cultures, accompanied by 20-25% necrosis. At the concentrations higher than 25μM/mL alpha-TS alone caused growth delay of both cell cultures, being much more pronounced for the cancer cell line HT29. At the concentrations of 50 μM/mL, responsible for about 30-60% of growth delay, there was observed a synergy effect for x-rays and alpha-TS for both cell lines. The effect was more pronounced for HT29 cells (DMF=0.48 for HT29 versus DMF=0.73 for NIH 3T3). These results may confirm the views of the literature reports suggesting that use of vitamin E together with radiation could be favorable for colon cancer treatment; however, more experiments using more advanced techniques are needed

SN38 conjugated hyaluronic acid gold nanoparticles as a novel system against metastatic colon cancer cells.

Science.gov (United States)

Hosseinzadeh, Hosniyeh; Atyabi, Fatemeh; Varnamkhasti, Behrang Shiri; Hosseinzadeh, Reza; Ostad, Seyed Nasser; Ghahremani, Mohammad Hossein; Dinarvand, Rassoul

2017-06-30

Combination of chemotherapy and photothermal therapy has been proposed for better treatment of metastatic colon cancer. In this study SN38, a highly potent cytotoxic agent, was conjugated to negatively charged hyaluronic acid (HA), which was deposited on the surface of the positively charged gold nanoparticles via electrostatic interaction. The drug conjugation and its interaction with gold nanoparticles were verified by 1 H NMR and UV-vis spectroscopies, respectively. The prepared SN38-HA gold NPs are negatively charged spherical nanoparticles with an average size of 75±10nm. In vitro release study revealed that drug release in acidic conditions (pH 5.2) was faster than that in physiological pH. Red light emitting diode (LED, 630nm, 30mW) was used as a light source for photothermal experiments. The drug release in acidic conditions was increased up to 30% using red LED illumination (6min) in comparison with experiment carried out indark. The cytotoxicity study on MUC1 positive HT29, SW480 colon cancer cells and MUC1 negative CHO cells, showed higher toxicity of the nanoparticles on HT29 and SW480 cell lines compared to CHO cells. Confocal microscopy images along with flow cytometry analysis confirm the cytotoxicity results. The incubation time for reaching IC50 decreases from 48h to 24h by LED illumination after nanoparticle treatment. Migratory potential of the HT29 and SW480 cell lines was reduced by co-application of SN38-HA gold NPs and LED radiation. Also anti-proliferative study indicates that LED radiation has increased the cytotoxicity of the nanoparticles and this effect is remained up to 8days. Copyright © 2017 Elsevier B.V. All rights reserved.

Phenolic composition of selected herbal infusions and their anti-inflammatory effect on a colonic model in vitro in HT-29 cells

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Elda Herrera-Carrera

2015-12-01

Full Text Available Some herbal infusions used in folk medicine in Mexico to treat gastrointestinal disorders were evaluated. Antioxidant activity and phenolic compounds were analyzed on the lyophilized aqueous crude extracts (LACE of arnica (Aster gymnocephalus, chamomile (Chamaemelum nobile, cumin (Cominum cyminum, desert resurrection plant (DRP (Selaginella lepidophylla, laurel (Listea glaucescens, marjoram (Origanum majorana, mint (Mentha spicata, salvilla (Buddleia scordioides and yerbaniz (Tagetes lucida. Total phenolic content ranged from 8.0 to 70.7 μg GAE/mg for DRP and laurel respectively. Major phenolic compounds were identified by gas chromatography–mass spectrometry and high-performance liquid chromatography. The IC50 determined by the degradation of the deoxy-d-ribose ranged from 2,452.53 to 5,097.11 μg/mL. The cytoprotective effect of the LACE alone and on indomethacin-induced oxidative stress in HT-29 cells was tested. The tetrazolium dye MTT assay was performed in concentrations of 0.125–10 mg/mL allowing choosing the lowest concentration for this experimentation. Inflammation markers were measured by Western blotting. None of the extracts inhibited COX-1 by themselves; however, it was observed that extracts have a modulation effect over COX-2, TNFα, NFκB, and IL-8. By the decrease in the expression of pro-inflammatory cytokines, it follows that salvilla, chamomile, and laurel show promising anti-inflammatory effects.

Serotonin disturbs colon epithelial tolerance of commensal E. coli by increasing NOX2-derived superoxide.

Science.gov (United States)

Banskota, Suhrid; Regmi, Sushil Chandra; Gautam, Jaya; Gurung, Pallavi; Lee, Yu-Jeong; Ku, Sae Kwang; Lee, Jin-Hyung; Lee, Jintae; Chang, Hyeun Wook; Park, Sang Joon; Kim, Jung-Ae

2017-05-01

Adherent-invasive E. coli colonization and Toll-like receptor (TLR) expression are increased in the gut of inflammatory bowel disease (IBD) patients. However, the underlying mechanism of such changes has not been determined. In the current study, it was examined whether gut serotonin (5-hydroxytryptamine, 5-HT) can induce adherent-invasive E. coli colonization and increase TLR expression. In a co-culture system, commensal E. coli strain (BW25113, BW) adhered minimally to colon epithelial cells, but this was significantly enhanced by 5-HT to the level of a pathogenic strain (EDL933). Without inducing bacterial virulence, such as, biofilm formation, 5-HT enhanced BW-induced signaling in colon epithelial cells, that is, NADPH oxidase (NOX)-dependent superoxide production, the up-regulations of IL-8, TLR2, TLR4, and ICAM-1, and the down-regulations of E-cadherin and claudin-2. In a manner commensurate with these gene modulations, BW induced an increase in NF-κB and a decrease in GATA reporter signals in colon epithelial cells. However, 5-HT-enhanced BW adhesion and colon epithelial responses were blocked by knock-down of NOX2, TLR2, or TLR4. In normal mice, 5-HT induced the invasion of BW into gut submucosa, and the observed molecular changes were similar to those observed in vitro, except for significant increases in TNFα and IL-1β, and resulted in death. In dextran sulfate sodium-induced colitis mice (an IBD disease model), in which colonic 5-HT levels were markedly elevated, BW administration induced death in along with large amount of BW invasion into colon submucosa, and time to death was negatively related to the amount of BW injected. Taken together, our results demonstrate that 5-HT induces the invasion of commensal E. coli into gut submucosa by amplifying commensal bacteria-induced epithelial signaling (superoxide production and the inductions of NOX2 and TLR2/TLR4). The authors suggest that these changes may constitute the molecular basis for the

Protective effect of surface layer proteins isolated from four Lactobacillus strains on hydrogen-peroxide-induced HT-29 cells oxidative stress.

Science.gov (United States)

Zhao, Bin-Bin; Meng, Jun; Zhang, Qiu-Xiang; Kang, Ting-Ting; Lu, Rong-Rong

2017-09-01

The objective of this study was to explore the antioxidant effect of the surface layer proteins (SLPs) and their mechanism. We investigated four SLPs which were extracted from L. casei zhang, L. rhamnosus, L. gasseri and L. acidophilus NCFM respectively using LiCl. The protective effect of SLPs on H 2 O 2 -induced HT-29 cells oxidative injury was investigated. As results, SLPs (100μg/mL) could significantly mitigate HT-29 cells cytotoxicity, improve the activities of total antioxidant capacity (T-AOC), catalase (CAT) and superoxide dismutase (SOD), decrease the contents of malondialdehyde (MDA) and lactate dehydrogenase (LDH), compared with H 2 O 2 -induced group (Pproteins of caspase-3 and caspase-9 (Pcells induced by H 2 O 2 , and the mechanism could be attributed to SLPs' ability to enhance the activity of the intracellular antioxidant enzyme system, reduce ROS accumulation and to inhibit apoptosis by regulating mitochondrial pathway. Copyright © 2017 Elsevier B.V. All rights reserved.

Zebrafish Axenic Larvae Colonization with Human Intestinal Microbiota.

Science.gov (United States)

Arias-Jayo, Nerea; Alonso-Saez, Laura; Ramirez-Garcia, Andoni; Pardo, Miguel A

2018-04-01

The human intestine hosts a vast and complex microbial community that is vital for maintaining several functions related with host health. The processes that determine the gut microbiome composition are poorly understood, being the interaction between species, the external environment, and the relationship with the host the most feasible. Animal models offer the opportunity to understand the interactions between the host and the microbiota. There are different gnotobiotic mice or rat models colonized with the human microbiota, however, to our knowledge, there are no reports on the colonization of germ-free zebrafish with a complex human intestinal microbiota. In the present study, we have successfully colonized 5 days postfertilization germ-free zebrafish larvae with the human intestinal microbiota previously extracted from a donor and analyzed by high-throughput sequencing the composition of the transferred microbial communities that established inside the zebrafish gut. Thus, we describe for first time which human bacteria phylotypes are able to colonize the zebrafish digestive tract. Species with relevant interest because of their linkage to dysbiosis in different human diseases, such as Akkermansia muciniphila, Eubacterium rectale, Faecalibacterium prausnitzii, Prevotella spp., or Roseburia spp. have been successfully transferred inside the zebrafish digestive tract.

Morphological modulation of human fibrosarcoma HT-1080 cells by hydroxybenzoate compounds during apoptosis

Directory of Open Access Journals (Sweden)

Jassem G Mahdi

2015-10-01

Full Text Available Hydroxybenzoate (HB compounds have shown to modulate the morphology in human fibrosarcoma HT-1080 cells. The changes in HT-1080 cells showed marker signs of apoptosis, which included the condensation of nucleus, cell round, blebbing and the formation of apoptotic bodies. The different stages of apoptosis were assessed microscopically using different staining and immunohistochemical techniques, as well as scanning electron microscopy. In addition, HB compounds increased the expression of caspase-3, which is closely associated with the development of the modulation in HT-1080 cells that are undergoing the programmed cell death. Both acetyl salicylic acid (ASA and HBZn compounds were dose and treatment duration dependent.

Prostaglandin E2 stimulates Fas ligand expression via the EP1 receptor in colon cancer cells.

LENUS (Irish Health Repository)

O'Callaghan, G

2012-02-03

Fas ligand (FasL\\/CD95L) is a member of the tumour necrosis factor superfamily that triggers apoptosis following crosslinking of the Fas receptor. Despite studies strongly implicating tumour-expressed FasL as a major inhibitor of the anti-tumour immune response, little is known about the mechanisms that regulate FasL expression in tumours. In this study, we show that the cyclooxygenase (COX) signalling pathway, and in particular prostaglandin E(2) (PGE(2)), plays a role in the upregulation of FasL expression in colon cancer. Suppression of either COX-2 or COX-1 by RNA interference in HCA-7 and HT29 colon tumour cells reduced FasL expression at both the mRNA and protein level. Conversely, stimulation with PGE(2) increased FasL expression and these cells showed increased cytotoxicity against Fas-sensitive Jurkat T cells. Prostaglandin E(2)-induced FasL expression was mediated by signalling via the EP1 receptor. Moreover, immunohistochemical analysis using serial sections of human colon adenocarcinomas revealed a strong positive correlation between COX-2 and FasL (r=0.722; P<0.0001) expression, and between EP1 receptor and FasL (r=0.740; P<0.0001) expression, in the tumour cells. Thus, these findings indicate that PGE(2) positively regulates FasL expression in colon tumour cells, adding another pro-neoplastic activity to PGE(2).

Cytotoxic activity of isolated constituents from leaves of Premna serratifolia on MCF-7 and HT-29 cell lines

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Mahesh Biradi

2015-03-01

Full Text Available Premna serratifolia (Syn: Premna integrifolia is an important medicinal herb known as “Agnimantha” in Ayurveda and traditionally used for anticancer activity. The objective of present study was to isolate the cytotoxic phytoconstituents from the n-hexane soluble fraction of P. serratifolia leaf extract. Unsaponifiable portion of n-hexane soluble fraction was subjected to silica based column chromatography. The major constituents present in all the sub-fractions were identified by TLC and phytochemical tests. Two constituents were isolated and they were purified. Sub-fractions with isolates were tested for cytotoxic effect by BSL bioassay. Two isolates were found to be active and which were tested on cancer cell lines MCF-7 and HT-29 for their cytotoxicity. Among two isolates, one compound has shown significant cytotoxicity. From the results we conclude that the plant isolates showed cytotoxicity against selected human cancer cell lines.

Potential anticancer properties of bioactive compounds of Gymnema sylvestre and its biofunctionalized silver nanoparticles.

Science.gov (United States)

Arunachalam, Kantha Deivi; Arun, Lilly Baptista; Annamalai, Sathesh Kumar; Arunachalam, Aarrthy M

2015-01-01

Gymnema sylvestre is an ethno-pharmacologically important medicinal plant used in many polyherbal formulations for its potential health benefits. Silver nanoparticles (SNPs) were biofunctionalized using aqueous leaf extracts of G. sylvestre. The anticancer properties of the bioactive compounds and the biofunctionalized SNPs were compared using the HT29 human adenoma colon cancer cell line. The preliminary phytochemical screening for bioactive compounds from aqueous extracts revealed the presence of alkaloids, triterpenes, flavonoids, steroids, and saponins. Biofunctionalized SNPs were synthesized using silver nitrate and characterized by ultraviolet-visible spectroscopy, scanning electron microscopy, energy-dispersive X-ray analysis, Fourier transform infrared spectroscopy, and X-ray diffraction for size and shape. The characterized biofunctionalized G. sylvestre were tested for its in vitro anticancer activity against HT29 human colon adenocarcinoma cells. The biofunctionlized G. sylvestre SNPs showed the surface plasmon resonance band at 430 nm. The scanning electron microscopy images showed the presence of spherical nanoparticles of various sizes, which were further determined using the Scherrer equation. In vitro cytotoxic activity of the biofunctionalized green-synthesized SNPs (GSNPs) indicated that the sensitivity of HT29 human colon adenocarcinoma cells for cytotoxic drugs is higher than that of Vero cell line for the same cytotoxic agents and also higher than the bioactive compound of the aqueous extract. Our results show that the anticancer properties of the bioactive compounds of G. sylvestre can be enhanced through biofunctionalizing the SNPs using the bioactive compounds present in the plant extract without compromising their medicinal properties.

Radiation-induced dysfunction of colonic transport: role of enteric nervous system and of serotonine

International Nuclear Information System (INIS)

Francois, Agnes

1998-01-01

One of the most commonly observed features of radiation-induced injury of the gastrointestinal tract is the appearance of severe diarrhea. One difficulty in understanding the origin of radiation-induced diarrhea is the multiplicity of factors implicated, depending on the type of radiation, the dose received and the irradiated field. Colonic transport is regulated for a great part by the enteric nervous system (ENS), in close association with immunocompetent cells, especially mast cells. The aim of this study was to investigate whether the neuro-immune regulation of colonic transport could be implicated in radiation-induced attenuation and recovery of colonic functions. Male Wistar rats were whole-body irradiated at 3.8 Gy neutron or 5 and 10 Gy gamma. At 1 and 3 days after exposure, the colonic epithelium was hypo-responsive to neural stimulation (submucosal plexus). Mechanistic studies were performed after 10 Gy exposure. The decreased colonic transport was associated with the disappearance of both submucosal mast cells and histamine-mediated pathway, together with decreased responses to exogenous histamine. Similarly, the response to exogenous 5-HT was decreased, without any modification of either the neural (5-HT 3 ) or non-neural (5-HT 4 ) pathways. Seven days after exposure, colonic transport capacity returned to normal in spite of the absence of mast cells. However these observations were associated with the reappearance of a histaminergic pathway, the origin of which is still unknown. The part played by 5-HT 3 receptors was increased, together with the appearance of a neurally-associated 5-HT4 receptor-pathway. These results suggest that the decreased influence of the ENS on colonic transport observed 1 and 3 days after exposure may be due to both the disappearance of neuro-immune links and the hypo-responsiveness of colonic epithelium to the mediators released by ENS. The functional recovery at seven days may be related on one hand to the return of altered

5-Fluorouracil modulation of radiosensitivity in cultured human carcinoma cells

International Nuclear Information System (INIS)

Smalley, S.R.; Kimler, B.F.; Evans, R.G.

1991-01-01

We evaluated conventional pulse exposure versus continuous exposure models of 5-fluorouracil (5-FU) radiosensitization in HT-29 (human colon adenocarcinoma) and DU-145 (human prostate cancer adenocarcinoma) cell lines. Cell survival following treatment with drug and/or radiation was determined by colony formation assays. Radiation was delivered either by itself, approximately midway through a 1-hr exposure to 5-FU (10 micrograms/ml), or at various times following initiation of exposure to 5-FU (0.5 microgram/ml) present throughout the entire period of incubation. Drug concentrations were selected to approximate those achieved in vivo in humans. HT-29 cells showed a plating efficiency of 87% and similar cytotoxicity (survival reduced to 0.57-0.71) for all 5-FU conditions. The Do's of the radiation survival curves were not different for 1 hr of 5-FU exposure versus radiation alone. However, continuous exposure conditions demonstrated statistically significantly different Do's from radiation alone and pulse 5-FU exposure. DU-145 cells displayed a plating efficiency of 17% and cytotoxicities of 0.10-0.91 for the 5-FU conditions. DU-145 cells showed different radiation 5-FU interactions: 5-FU produced statistically significant changes in Do well as the differences between cell lines insofar as their radiosensitization by 5-FU underscore the caution required in extrapolating these radiobiologic models to the clinical setting

Isolation and in vitro expansion of human colonic stem cells

NARCIS (Netherlands)

Jung, P.; Sato, T.; Merlos-Suarez, A.; Barriga, F.M.; Iglesias, M.; Rossell, D.; Auer, H.; Gallardo, M.; Blasco, M.A.; Sancho, E.; Clevers, H.; Batlle, E.

2011-01-01

Here we describe the isolation of stem cells of the human colonic epithelium. Differential cell surface abundance of ephrin type-B receptor 2 (EPHB2) allows the purification of different cell types from human colon mucosa biopsies. The highest EPHB2 surface levels correspond to epithelial colonic

Prehistoric human colonization of India

Indian Academy of Sciences (India)

Unknown

J. Biosci. | Vol. 26 | No. 4 | Suppl. | November 2001. V N Misra. 492 ... humans differ from the other apes in their upright posture, ... characterized by Levallois flakes and blades and by the ... and the coastal region running parallel to them, northeast ..... November 2001. Prehistoric human colonization of India. 497. Figure 1.

TFF3-dependent resistance of human colorectal adenocarcinoma cells HT-29/B6 to apoptosis is mediated by miR-491-5p regulation of lncRNA PRINS

OpenAIRE

Hanisch, Carlos; Sharbati, Jutta; Kutz-Lohroff, Barbara; Huber, Otmar; Einspanier, Ralf; Sharbati, Soroush

2017-01-01

Tumour necrosis factor-? (TNF-?) is a double-edged cytokine associated with pathogenesis of inflammatory-related cancers being also able to induce cancer cell death. In the process of tumour development or metastasis, cancer cells can become resistant to TNF-?. In trefoil factor 3 (TFF3) overexpressing colorectal adenocarcinoma cells (HT-29/B6), we observed enhanced resistance against TNF-?/interferon gamma-induced apoptosis. TFF3 is a secreted small peptide that supports intestinal tissue re...

Evaluation of chemical composition, antioxidant, antibacterial, cytotoxic and apoptotic effects of Aloysia citrodora extract on colon cancer cell line

Directory of Open Access Journals (Sweden)

Amir Mirzaie

2016-06-01

Full Text Available Background: Aloysia citrodora belongs to the Verbenaceae family of plants, a well-known herbal medicine in Iran. The aim of the present study was to investigate the chemical composition, antioxidant, antibacterial, cytotoxic and apoptotic effect of A. citrodora extract against human colon cancer (HT29 cells by using real-time polymerase chain reaction and flow-cytometry methods. Methods: This experimental study was carried out in Islamic Azad University, East Tehran Branch, from March to September of 2014. At first, the A. citrodora chemical constituents were analyzed by gas chromatography-mass spectrometry (GC-MS technique. In addition, antioxidant assay, antibacterial and anti-cancer effect was performed using 1,1-diphenyl-2-picrylhydrazyl (DPPH, disk diffusion and 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT methods, respectively. The half maximal inhibitory concentration (IC50 value was calculated. We extracted total RNA molecules by using RNX solution, after which cDNA was synthesized. Finally, the pro-apoptotic (Bax and anti-apoptotic (Bcl2 gene expression was performed by real-time polymerase chain reaction and apoptotic effects were analyzed using Flow-cytometry method. Results: GC-MS analysis of Aloysia citrodora extract was shown 37 major components and the most frequent component was belonged to Spathulenol (17.57% and Caryophyllene oxide (15.15% The antioxidant activity of the extract was IC50= 0.6±0.03 mg/ml. The maximum and minimum antibacterial effects of extract were belonged to gram-negative and gram-positive bacteria, respectively. Cytotoxic results revealed that the A.citrodora extract have IC50= 20.1±0.78 mg/ml against colon cancer (HT29 cell line and real-time polymerase chain reaction results showed the expression level of Bax and Bcl2 was increased and decreased respectively in colon cancer cell line (3.470±0.72 (P< 0.05, 0.43±0.35 (P< 0.05. In addition, the flow-cytometry results indicated the 38

Inflammasome-independent NLRP3 is required for epithelial-mesenchymal transition in colon cancer cells.

Science.gov (United States)

Wang, Hong; Wang, Yajing; Du, Qianming; Lu, Ping; Fan, Huimin; Lu, Jinrong; Hu, Rong

2016-03-15

Inflammasome NLRP3 plays a crucial role in the process of colitis and colitis--associated colon cancer. Even though much is known regarding the NLRP3 inflammasome that regulates pro-inflammatory cytokine release in innate immune cells, the role of NLRP3 in non-immune cells is still unclear. In this study, we showed that NLRP3 was highly expressed in mesenchymal-like colon cancer cells (SW620), and was upregulated by tumor necrosis factors-α (TNF-α) and transforming growth factor-β1 (TGF-β1) respectively, during EMT in colon cancer epithelial cells HCT116 and HT29. Knockdown of NLRP3 retained epithelial spindle-like morphology of HCT116 and HT29 cells and reversed the mesenchymal characteristic of SW620 cells, indicated by the decreased expression of vimentin and MMP9 and increased expression of E-cadherin. In addition, knockdown of NLRP3 in colorectal carcinoma cells displayed diminished cell migration and invasion. Interestingly, during the EMT process induced by TNF-α or TGF-β1, the cleaved caspase-1 and ASC speck were not detected, indicating that NLRP3 functions in an inflammasome-independent way. Further studies demonstrated that NLRP3 protein expression was regulated by NF-κB signaling in TNF-α or TGF-β1-induced EMT, as verified by the NF-κB inhibitor Bay 11-7082. Moreover, NLRP3 knockdown reduced the expression of Snail1, indicating that NLRP3 may promote EMT through regulating Snail1. In summary, our results showed that the NLRP3 expression, not the inflammasome activation, was required for EMT in colorectal cancer cells. Copyright © 2016 Elsevier Inc. All rights reserved.

Cetuximab improves AZD6244 antitumor activity in colorectal cancer HT29 cells in vitro and in nude mice by attenuating HER3/Akt pathway activation.

Science.gov (United States)

Zhang, Qin; Xiao, He; Jin, Feng; Li, Mengxia; Luo, Jia; Wang, Ge

2018-07-01

The present study investigated the molecular mechanism by which the epidermal growth factor receptor (EGFR) inhibitor cetuximab enhances the antitumor activity of the mitogen-activated protein kinase kinase (MEK) inhibitor AZD6244 in colorectal cancer HT29 cells. HT29 cells were treated with AZD6244 plus cetuximab and then subjected to the following assays: Cell Counting kit-8, BrdU-incorporation, flow cytometric cell cycle distribution and apoptosis analysis, western blot analysis, and nude mouse xenografts. The combination of AZD6244 and cetuximab significantly reduced HT29 cell viability and proliferation compared with AZD6244 alone. The combination treatment reduced the IC 50 value from 108.12±10.05 to 28.45±1.92 nM. AZD6244 and cetuximab also induced cell cycle arrest at G1 phase and reduced S phase (88.53% vs. 93.39%, P=0.080; 8.73% vs. 4.24%, P=0.082, respectively). Combination of AZD6244 with cetuximab significantly induced tumor cells apoptosis (14.61% vs. 8.99%, P=0.046). Inhibition of EGFR activity using cetuximab partially abrogated the feedback-activation of phosphorylated receptor tyrosine-protein kinase erB-3 (p-HER3) and p-AKT serine/threonine kinase (AKT), as well as prevented reactivation of p-extracellular regulated kinase (ERK) conferred by AZD6244 treatment. Combination of AZD6244 and cetuximab also inhibited HT29 cell xenograft growth in nude mice and suppressed HER3 and p-AKT levels in xenografts. The EGFR inhibitor cetuximab enhanced the antitumor activity of the MEK inhibitor AZD6244 in colorectal cells in vitro and in vivo . Co-inhibition of MEK and EGFR may be a promising treatment strategy in colorectal cancers.

Ellagitannins from pomegranate ameliorates 5-fluorouracil-induced intestinal mucositis in rats while enhancing its chemotoxicity against HT-29 colorectal cancer cells through intrinsic apoptosis induction.

Science.gov (United States)

Chen, Xiao-Xin; Lam, Kar Ho; Feng, Yibin; Xu, Kai; Sze, Stephen C W; Tang, Chi Wai; Leung, George P H; Lee, Calvin Kai-Fai; Shi, Jun; Yang, Zhijun; Li, Sheng-Tao; Zhang, Zhang-Jin; Zhang, Yanbo

2018-06-19

Worldwide, colorectal cancer (CRC) is a deleterious disease causing millions of death annually. 5-Fluorouracil (5-FU) is a first-line chemotherapy for CRC, but chemoresistance and gastrointestinal mucositis limit its efficacy. Polyphenol-rich foods are increasingly popular due to their potential beneficial role in cancer. Ellagitannins is a group of phenolic compounds commonly found in pomegranate, strawberries, raspberries, etc. The objective of this study was to explore whether ellagitannins from pomegranate (PETs) could ameliorate 5-FU-induced intestinal mucositis and enhance its efficacy against CRC. The results showed that PETs (100 mg/kg) counteracted 5-FU-induced intestinal mucositis in rats. The number of apoptotic cells per crypt was reduced from 1.50±0.21 to 0.85±0.18 (P<0.05). Moreover, PETs induced HT-29 CRC cell death through intrinsic apoptosis as demonstrated by dissipation of mitochondrial membrane potential, increased Bax to Bcl-2 ratio, and cleavage of caspase 9 and caspase 3. PETs and 5-FU combination treatments exhibited synergistic cytotoxicity against HT-29 cells with a weighted combination index of 0.3494. PETs (80 µg/mL) and 5-FU (40 µg/mL) treatments for 48 h induced 14.03±0.76% and 16.42±1.15% of HT-29 cells to undergo apoptosis while the combination treatment further increased apoptosis cells to 34.00±1.54% (P<0.05). Combination treatment of the cells also enhanced S phase cell cycle arrest as compared with PETs or 5-FU monotherapy (P<0.05). These results suggest that dietary ellagitannins from pomegranate could alleviate intestinal mucositis in rats induced by 5-FU while enhancing its toxicity against HT-29 cells through potentiation of apoptosis and cell cycle arrest.

The 5-HT(4) receptor levels in hippocampus correlates inversely with memory test performance in humans

DEFF Research Database (Denmark)

Haahr, Mette Ewers; Fisher, Patrick; Holst, Klaus Kähler

2013-01-01

The cerebral serotonin (5-HT) system is involved in cognitive functions such as memory and learning and animal studies have repeatedly shown that stimulation of the 5-HT type 4 receptor (5-HT(4) R) facilitates memory and learning and further that the 5-HT(4) R modulates cellular memory processes...... in hippocampus. However, any associations between memory functions and the expression of the 5-HT(4) R in the human hippocampus have not been investigated. Using positron emission tomography with the tracer [(11) C]SB207145 and Reys Auditory Verbal Learning Test we aimed to examine the individual variation...... of the 5-HT4R binding in hippocampus in relation to memory acquisition and consolidation in healthy young volunteers. We found significant, negative associations between the immediate recall scores and left and right hippocampal BP(ND) , (p = 0.009 and p = 0.010 respectively) and between the right...

Humans Can Taste Glucose Oligomers Independent of the hT1R2/hT1R3 Sweet Taste Receptor.

Science.gov (United States)

Lapis, Trina J; Penner, Michael H; Lim, Juyun

2016-08-23

It is widely accepted that humans can taste mono- and disaccharides as sweet substances, but they cannot taste longer chain oligo- and polysaccharides. From the evolutionary standpoint, the ability to taste starch or its oligomeric hydrolysis products would be highly adaptive, given their nutritional value. Here, we report that humans can taste glucose oligomer preparations (average degree of polymerization 7 and 14) without any other sensorial cues. The same human subjects could not taste the corresponding glucose polymer preparation (average degree of polymerization 44). When the sweet taste receptor was blocked by lactisole, a known sweet inhibitor, subjects could not detect sweet substances (glucose, maltose, and sucralose), but they could still detect the glucose oligomers. This suggests that glucose oligomer detection is independent of the hT1R2/hT1R3 sweet taste receptor. Human subjects described the taste of glucose oligomers as "starchy," while they describe sugars as "sweet." The dose-response function of glucose oligomer was also found to be indistinguishable from that of glucose on a molar basis. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

CysLT(1)R antagonists inhibit tumor growth in a xenograft model of colon cancer.

Science.gov (United States)

Savari, Sayeh; Liu, Minghui; Zhang, Yuan; Sime, Wondossen; Sjölander, Anita

2013-01-01

The expression of the inflammatory G-protein coupled receptor CysLT1R has been shown to be upregulated in colon cancer patients and associated with poor prognosis. The present study investigated the correlation between CysLT1R and colon cancer development in vivo using CysLT1R antagonists (ZM198,615 or Montelukast) and the nude mouse xenograft model. Two drug administration regimens were established. The first regimen was established to investigate the importance of CysLT1R in tumor initiation. Nude mice were inoculated with 50 µM CysLT1R antagonist-pretreated HCT-116 colon cancer cells and received continued treatment (5 mg/kg/day, intraperitoneally). The second regimen aimed to address the role of CysLT1R in tumor progression. Nude mice were inoculated with non-pretreated HCT-116 cells and did not receive CysLT1R antagonist treatment until recordable tumor appearance. Both regimens resulted in significantly reduced tumor size, attributed to changes in proliferation and apoptosis as determined by reduced Ki-67 levels and increased levels of p21(WAF/Cip1) (Pcolon cancer cell line HCT-116 and CysLT1R antagonists. In addition to significant reductions in cell proliferation, adhesion and colony formation, we observed induction of cell cycle arrest and apoptosis in a dose-dependent manner. The ability of Montelukast to inhibit growth of human colon cancer xenograft was further validated by using two additional colon cancer cell lines, SW-480 and HT-29. Our results demonstrate that CysLT1R antagonists inhibit growth of colon cancer xenografts primarily by reducing proliferation and inducing apoptosis of the tumor cells.

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African Journals Online (AJOL)

DR TONUKARI NYEROVWO

2011-02-18

Feb 18, 2011 ... provide novel genetic data involved in the pathogenesis of disease. In this study ... participate in cell-cell interactions by binding to integrins .... 4˚C with primary rabbit polyclonal anti-human ADAM9 antibody .... network-based analysis of systemic inflammation in humans. ... colon cancer cell line HT29 cells.

Regorafenib effects on human colon carcinoma xenografts monitored by dynamic contrast-enhanced computed tomography with immunohistochemical validation.

Directory of Open Access Journals (Sweden)

Clemens C Cyran

Full Text Available To investigate dynamic contrast-enhanced computed tomography for monitoring the effects of regorafenib on experimental colon carcinomas in rats by quantitative assessments of tumor microcirculation parameters with immunohistochemical validation.Colon carcinoma xenografts (HT-29 implanted subcutaneously in female athymic rats (n = 15 were imaged at baseline and after a one-week treatment with regorafenib by dynamic contrast-enhanced computed tomography (128-slice dual-source computed tomography. The therapy group (n = 7 received regorafenib daily (10 mg/kg bodyweight. Quantitative parameters of tumor microcirculation (plasma flow, mL/100 mL/min, endothelial permeability (PS, mL/100 mL/min, and tumor vascularity (plasma volume, % were calculated using a 2-compartment uptake model. Dynamic contrast-enhanced computed tomography parameters were validated with immunohistochemical assessments of tumor microvascular density (CD-31, tumor cell apoptosis (TUNEL, and proliferation (Ki-67.Regorafenib suppressed tumor vascularity (15.7±5.3 to 5.5±3.5%; p<0.05 and tumor perfusion (12.8±2.3 to 8.8±2.9 mL/100 mL/min; p = 0.063. Significantly lower microvascular density was observed in the therapy group (CD-31; 48±10 vs. 113±25, p<0.05. In regorafenib-treated tumors, significantly more apoptotic cells (TUNEL; 11844±2927 vs. 5097±3463, p<0.05 were observed. Dynamic contrast-enhanced computed tomography tumor perfusion and tumor vascularity correlated significantly (p<0.05 with microvascular density (CD-31; r = 0.84 and 0.66 and inversely with apoptosis (TUNEL; r = -0.66 and -0.71.Regorafenib significantly suppressed tumor vascularity (plasma volume quantified by dynamic contrast-enhanced computed tomography in experimental colon carcinomas in rats with good-to-moderate correlations to an immunohistochemical gold standard. Tumor response biomarkers assessed by dynamic contrast-enhanced computed tomography may be a promising future

A crosstalk between muscarinic and CRF2 receptors regulates cellular adhesion properties of human colon cancer cells.

Science.gov (United States)

Pelissier-Rota, M; Chartier, N T; Bonaz, B; Jacquier-Sarlin, M R

2017-07-01

Patients with inflammatory bowel disease often suffer from chronic and relapsing intestinal inflammation that favor the development of colitis associated cancer. An alteration of the epithelial intestinal barrier function observed in IBD is supposed to be a consequence of stress. It has been proposed that corticotrophin-releasing factor receptor (CRF2), one of the two receptors of CRF, the principal neuromediator of stress, acts on cholinergic nerves to induce stress-mediated epithelial barrier dysfunction. Non-neuronal acetylcholine (Ach) and muscarinic receptors (mAchR) also contribute to alterations of epithelial cell functions. In this study, we investigated the mechanisms through which stress and Ach modulate epithelial cell adhesive properties. We show that Ach-induced activation of mAchR in HT-29 cells results in cell dissociation together with changes in cell-matrix contacts, which correlates with the acquisition of invasive potential consistent with a matrix metalloproteinase (MMP) mode of invasion. These processes result from mAchR subsequent stimulation of the cascade of src/Erk and FAK activation. Ach-induced secretion of laminin 332 leads to α3β1 integrin activation and RhoA-dependent reorganization of the actin cytoskeleton. We show that Ach-mediated effects on cell adhesion are blocked by astressin 2b, a CRF2 antagonist, suggesting that Ach action depends partly on CRF2 signaling. This is reinforced by the fact that Ach-mediated activation of mAchR stimulates both the synthesis and the release of CRF2 ligands in HT-29 cells (effects blocked by atropine). In summary, our data provides evidence for a novel intracellular circuit involving mAchR acting on CRF2-signaling that could mediate colonic mucosal barrier dysfunction and exacerbate mucosal inflammation. Copyright © 2017. Published by Elsevier B.V.

The effect of milk fat globules on adherence and internalization of Salmonella Enteritidis to HT-29 cells.

Science.gov (United States)

Guri, A; Griffiths, M; Khursigara, C M; Corredig, M

2012-12-01

Milk fat globules were extracted from bovine and goat milk and incubated with HT-29 human adenocarcinoma cells to assess the attachment and internalization of Salmonella Enteritidis. Because the expression of bacterial adhesins is highly affected by the presence of antibiotic, the attachment was studied with and without antibiotic in the cell growth medium. Although no inhibitory effect of the fat globules was observed in the presence of the antibiotic, milk fat globules significantly inhibited the binding and internalization of Salmonella in medium free of antibiotic. The fat globules from both bovine and goat milk markedly reduced bacterial binding and invasion compared with controls, and the cells treated with goat milk-derived fat globules demonstrated greater protective properties than those derived from bovine milk. The effect of heat treatment on bovine fat globules was also investigated, and it was shown that the fat globules from heated milk had a higher degree of inhibition than those from unheated milk. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Hyaluronic acid–nimesulide conjugates as anticancer drugs against CD44-overexpressing HT-29 colorectal cancer in vitro and in vivo

Directory of Open Access Journals (Sweden)

Jian YS

2017-03-01

Full Text Available You-Sin Jian,1 Ching-Wen Chen,1 Chih-An Lin,2 Hsiu-Ping Yu,1 Hua-Yang Lin,3 Ming-Yuan Liao,1 Shu-Huan Wu,1 Yan-Fu Lin,1 Ping-Shan Lai1,2,4,5 1Department of Chemistry, 2PhD Program in Tissue Engineering and Regenerative Medicine, National Chung Hsing University, Taichung, 3Preclinical Development Research Department, Holy Stone Healthcare Co., Ltd., Taipei, 4Research Center for Sustainable Energy and Nanotechnology, 5Rong Hsing Research Center for Translational Medicine, National Chung Hsing University, Taichung, Taiwan Abstract: Carrier-mediated drug delivery systems are promising therapeutics for targeted delivery and improved efficacy and safety of potent cytotoxic drugs. Nimesulide is a multifactorial cyclooxygenase 2 nonsteroidal anti-inflammatory drug with analgesic, antipyretic and potent anticancer properties; however, the low solubility of nimesulide limits its applications. Drugs conjugated with hyaluronic acid (HA are innovative carrier-mediated drug delivery systems characterized by CD44-mediated endocytosis of HA and intracellular drug release. In this study, hydrophobic nimesulide was conjugated to HA of two different molecular weights (360 kDa as HA with high molecular weight [HAH] and 43kDa as HA with low molecular weight [HAL] to improve its tumor-targeting ability and hydrophilicity. Our results showed that hydrogenated nimesulide (N-[4-amino-2-phenoxyphenyl]methanesulfonamide was successfully conjugated with both HA types by carbodiimide coupling and the degree of substitution of nimesulide was 1%, which was characterized by 1H nuclear magnetic resonance 400 MHz and total correlation spectroscopy. Both Alexa Fluor® 647 labeled HAH and HAL could selectively accumulate in CD44-overexpressing HT-29 colorectal tumor area in vivo, as observed by in vivo imaging system. In the in vitro cytotoxic test, HA–nimesulide conjugate displayed >46% cell killing ability at a nimesulide concentration of 400 µM in HT-29 cells, whereas

An Apta-Biosensor for Colon Cancer Diagnostics

Directory of Open Access Journals (Sweden)

Mojgan Ahmadzadeh Raji

2015-09-01

Full Text Available This paper reports the design and implementation of an aptasensor using a modified KCHA10a aptamer. This aptasensor consists of a functionalized electrodes using various materials including 11-mercaptoandecanoic acid (11-MUA and modified KCHA10a aptamer. The HCT 116, HT 29 and HEp-2 cell lines are used in this study to demonstrate the functionality of aptasensor for colon cancer detection purposes. Flow cytometry, fluorescence microscopy and electrochemical cyclic voltammetry are used to verify the binding between the target cells and aptamer. The limit of detection (LOD of this aptasensor is equal to seven cancer cells. Based on the experimental results, the proposed sensor can be employed for point-of-care cancer disease diagnostics.

Celecoxib and tauro-ursodeoxycholic acid co-treatment inhibits cell growth in familial adenomatous polyposis derived LT97 colon adenoma cells

Energy Technology Data Exchange (ETDEWEB)

Heumen, Bjorn W.H. van, E-mail: b.vanheumen@mdl.umcn.nl [Department of Gastroenterology and Hepatology, Radboud University Nijmegen Medical Centre, Nijmegen (Netherlands); Roelofs, Hennie M.J.; Morsche, Rene H.M. te [Department of Gastroenterology and Hepatology, Radboud University Nijmegen Medical Centre, Nijmegen (Netherlands); Marian, Brigitte [Institute of Cancer Research, Wien University, Vienna (Austria); Nagengast, Fokko M.; Peters, Wilbert H.M. [Department of Gastroenterology and Hepatology, Radboud University Nijmegen Medical Centre, Nijmegen (Netherlands)

2012-04-15

Chemoprevention would be a desirable strategy to avoid duodenectomy in patients with familial adenomatous polyposis (FAP) suffering from duodenal adenomatosis. We investigated the in vitro effects on cell proliferation, apoptosis, and COX-2 expression of the potential chemopreventives celecoxib and tauro-ursodeoxycholic acid (UDCA). HT-29 colon cancer cells and LT97 colorectal micro-adenoma cells derived from a patient with FAP, were exposed to low dose celecoxib and UDCA alone or in combination with tauro-cholic acid (CA) and tauro-chenodeoxycholic acid (CDCA), mimicking bile of FAP patients treated with UDCA. In HT-29 cells, co-treatment with low dose celecoxib and UDCA resulted in a decreased cell growth (14-17%, p < 0.01). A more pronounced decrease (23-27%, p < 0.01) was observed in LT97 cells. Cell growth of HT-29 cells exposed to 'artificial bile' enriched with UDCA, was decreased (p < 0.001), either in the absence or presence of celecoxib. In LT97 cells incubated with 'artificial bile' enriched with UDCA, cell growth was decreased only in the presence of celecoxib (p < 0.05). No clear evidence was found for involvement of proliferating cell nuclear antigen, caspase-3, or COX-2 in the cellular processes leading to the observed changes in cell growth. In conclusion, co-treatment with low dose celecoxib and UDCA has growth inhibitory effects on colorectal adenoma cells derived from a patient with FAP, and further research on this combination as promising chemopreventive strategy is desired. -- Highlights: Black-Right-Pointing-Pointer Celecoxib and UDCA acid co-treatment decreases cell growth in colon tumor cells. Black-Right-Pointing-Pointer UDCA enriched 'artificial bile' decreases LT-97 cell growth only in presence of celecoxib. Black-Right-Pointing-Pointer PCNA, caspase-3, nor COX-2 seem to be involved in the observed changes in cell growth.

Celecoxib and tauro-ursodeoxycholic acid co-treatment inhibits cell growth in familial adenomatous polyposis derived LT97 colon adenoma cells

International Nuclear Information System (INIS)

Heumen, Bjorn W.H. van; Roelofs, Hennie M.J.; Morsche, René H.M. te; Marian, Brigitte; Nagengast, Fokko M.; Peters, Wilbert H.M.

2012-01-01

Chemoprevention would be a desirable strategy to avoid duodenectomy in patients with familial adenomatous polyposis (FAP) suffering from duodenal adenomatosis. We investigated the in vitro effects on cell proliferation, apoptosis, and COX-2 expression of the potential chemopreventives celecoxib and tauro-ursodeoxycholic acid (UDCA). HT-29 colon cancer cells and LT97 colorectal micro-adenoma cells derived from a patient with FAP, were exposed to low dose celecoxib and UDCA alone or in combination with tauro-cholic acid (CA) and tauro-chenodeoxycholic acid (CDCA), mimicking bile of FAP patients treated with UDCA. In HT-29 cells, co-treatment with low dose celecoxib and UDCA resulted in a decreased cell growth (14–17%, p < 0.01). A more pronounced decrease (23–27%, p < 0.01) was observed in LT97 cells. Cell growth of HT-29 cells exposed to ‘artificial bile’ enriched with UDCA, was decreased (p < 0.001), either in the absence or presence of celecoxib. In LT97 cells incubated with ‘artificial bile’ enriched with UDCA, cell growth was decreased only in the presence of celecoxib (p < 0.05). No clear evidence was found for involvement of proliferating cell nuclear antigen, caspase-3, or COX-2 in the cellular processes leading to the observed changes in cell growth. In conclusion, co-treatment with low dose celecoxib and UDCA has growth inhibitory effects on colorectal adenoma cells derived from a patient with FAP, and further research on this combination as promising chemopreventive strategy is desired. -- Highlights: ► Celecoxib and UDCA acid co-treatment decreases cell growth in colon tumor cells. ► UDCA enriched ‘artificial bile’ decreases LT-97 cell growth only in presence of celecoxib. ► PCNA, caspase-3, nor COX-2 seem to be involved in the observed changes in cell growth.

Emigrating Beyond Earth Human Adaptation and Space Colonization

CERN Document Server

Smith, Cameron M

2012-01-01

For four million years humankind has been actively expanding geographically and in doing so has adapted to a wide variety of hostile environments. Now we are looking towards the ultimate adaptation - the colonization of space. Emigrating Beyond Earth illustrates that this is not a technocratic endeavor, but a natural continuation of human evolution; a journey not just for the engineer and rocket scientist, but for everyman. Based on the most current understanding of our universe, human adaptation and evolution, the authors explain why space colonization must be planned as an adaptation to, rather than the conquest of, space. Emigrating Beyond Earth argues that space colonization is an insurance policy for our species, and that it isn't about rockets and robots, it's about humans doing what we've been doing for four million years: finding new places and new ways to live. Applying a unique anthropological approach, the authors outline a framework for continued human space exploration and offer a glimpse of a po...

Changes of Serotonin (5-HT), 5-HT2A Receptor, and 5-HT Transporter in the Sprague-Dawley Rats of Depression,Myocardial Infarction and Myocardial Infarction Co-exist with Depression

Institute of Scientific and Technical Information of China (English)

Mei-Yan Liu; Yah-Ping Ren; Wan-Lin Wei; Guo-Xiang Tian; Guo Li

2015-01-01

Background:To evaluate whether serotonin (5-HT),5-HT2A receptor (5-HT2AR),and 5-HT transporter (serotonin transporter [SERT]) are associated with different disease states of depression,myocardial infarction (MI) and MI co-exist with depression in Sprague-Dawley rats.Methods:After established the animal model of four groups include control,depression,MI and MI with depression,we measured 5-HT,5-HT2AR and SERT from serum and platelet lysate.Results:The serum concentration of 5-HT in depression rats decreased significantly compared with the control group (303.25 ± 9.99 vs.352.98 ± 13.73;P =0.000),while that in MI group increased (381.78 ± 14.17 vs.352.98 ± 13.73;P =0.000).However,the depression + MI group had no change compared with control group (360.62 ± 11.40 vs.352.98 ± 13.73;P =0.036).The changes of the platelet concentration of 5-HT in the depression,MI,and depression + MI group were different from that of serum.The levels of 5-HT in above three groups were lower than that in the control group (380.40 ± 17.90,387.75 ± 22.28,246.40 ± 18.99 vs.500.29 ± 20.91;P =0.000).The platelet lysate concentration of 5-HT2AR increased in depression group,MI group,and depression + MI group compared with the control group (370.75 ± 14.75,393.47 ± 15.73,446.66 ± 18.86 vs.273.66 ± 16.90;P =0.000).The serum and platelet concentration of SERT in the depression group,MI group and depression + MI group were all increased compared with the control group (527.51 ± 28.32,602.02 ± 23.32,734.76 ± 29.59 vs.490.56 ± 16.90;P =0.047,P =0.000,P =0.000 in each and 906.38 ± 51.84,897.33 ± 60.34,1030.17 ± 58.73 vs.708.62 ± 51.15;P =0.000 in each).Conclusions:The concentration of 5-HT2AR in platelet lysate and SERT in serum and platelet may be involved in the pathway of MI with depression.Further studies should examine whether elevated 5-HT2AR and SERT may contribute to the biomarker in MI patients with depression.

Reparative properties of the traditional Chinese medicine Cordyceps sinensis (Chinese caterpillar mushroom) using HT29 cell culture and rat gastric damage models of injury.

Science.gov (United States)

Marchbank, Tania; Ojobo, Ehighale; Playford, Christopher J; Playford, Raymond J

2011-05-01

Cordyceps sinensis (CS) is a traditional Chinese medicine and health food used to support many organ systems. It is commercially produced by cultivation in a liquid medium or on a solid (grain/potato) phase. We tested the effects of hot water extracts of liquid-phase and solid-phase commercially grown CS on its ability to influence proliferation (using Alamar blue, an oxidation/reduction indicator), migration (serial-wounded monolayer photomicroscopy), invasion through collagen gel (fluorometric assay) and indomethacin-induced apoptosis (active caspase-3 colorimetric assay) of human colon cancer HT29 cells. An in vivo study used a rat gastric damage model (indomethacin 20 mg/kg and 4 h restraint with oral administration). The CS extract stimulated cell proliferation threefold when added at 10 μg/ml (P < 0·01). Cell migration increased by 69 % and invasion by 17 % when CS was added at 5 mg/ml (P < 0·01). The results also showed that 93 % of the pro-proliferative activity was soluble in ethanol, whereas pro-migratory activity was divided (61:49) into both ethanol-soluble and ethanol-insoluble sub-fractions. Indomethacin-induced apoptosis was not affected by the presence of CS. CS reduced the amount of gastric injury by 63 % when administered orally at 20 mg/ml (P < 0·01), the results being similar to using the potent cytoprotective agent epidermal growth factor at 25 μg/ml (83 % reduction). We conclude that both methods of cultivated CS possess biological activity when analysed using a variety of gut models of injury and repair. Functional foods, such as CS, could provide a novel approach for the prevention and treatment of injury to the bowel.

Effect of calcium electroporation in combination with metformin in vivo and correlation between viability and intracellular ATP level after calcium electroporation in vitro

DEFF Research Database (Denmark)

Frandsen, Stine Krog; Gehl, Julie

2017-01-01

cancer cell lines: Breast (MDA-MB231) and colon (HT29), and in normal human fibroblasts (HDF-n), as well as investigating viability in human bladder cancer cells (SW780) and human small cell lung cancer cells (H69) where we have previously published intracellular ATP levels. RESULTS: Calcium...... with calcium alone (pHDF-n, and MDA-MB231; p

HT-COMET: a novel automated approach for high throughput assessment of human sperm chromatin quality

Science.gov (United States)

Albert, Océane; Reintsch, Wolfgang E.; Chan, Peter; Robaire, Bernard

2016-01-01

STUDY QUESTION Can we make the comet assay (single-cell gel electrophoresis) for human sperm a more accurate and informative high throughput assay? SUMMARY ANSWER We developed a standardized automated high throughput comet (HT-COMET) assay for human sperm that improves its accuracy and efficiency, and could be of prognostic value to patients in the fertility clinic. WHAT IS KNOWN ALREADY The comet assay involves the collection of data on sperm DNA damage at the level of the single cell, allowing the use of samples from severe oligozoospermic patients. However, this makes comet scoring a low throughput procedure that renders large cohort analyses tedious. Furthermore, the comet assay comes with an inherent vulnerability to variability. Our objective is to develop an automated high throughput comet assay for human sperm that will increase both its accuracy and efficiency. STUDY DESIGN, SIZE, DURATION The study comprised two distinct components: a HT-COMET technical optimization section based on control versus DNAse treatment analyses (n = 3–5), and a cross-sectional study on 123 men presenting to a reproductive center with sperm concentrations categorized as severe oligozoospermia, oligozoospermia or normozoospermia. PARTICIPANTS/MATERIALS, SETTING, METHODS Sperm chromatin quality was measured using the comet assay: on classic 2-well slides for software comparison; on 96-well slides for HT-COMET optimization; after exposure to various concentrations of a damage-inducing agent, DNAse, using HT-COMET; on 123 subjects with different sperm concentrations using HT-COMET. Data from the 123 subjects were correlated to classic semen quality parameters and plotted as single-cell data in individual DNA damage profiles. MAIN RESULTS AND THE ROLE OF CHANCE We have developed a standard automated HT-COMET procedure for human sperm. It includes automated scoring of comets by a fully integrated high content screening setup that compares well with the most commonly used semi

Colon cancer associated transcripts in human cancers.

Science.gov (United States)

Chen, Yincong; Xie, Haibiao; Gao, Qunjun; Zhan, Hengji; Xiao, Huizhong; Zou, Yifan; Zhang, Fuyou; Liu, Yuchen; Li, Jianfa

2017-10-01

Long non-coding RNAs serve as important regulators in complicated cellular activities, including cell differentiation, proliferation and death. Dysregulation of long non-coding RNAs occurs in the formation and progression of cancers. The family of colon cancer associated transcripts, long non-coding RNAs colon cancer associated transcript-1 and colon cancer associated transcript-2 are known as oncogenes involved in various cancers. Colon cancer associated transcript-1 is a novel lncRNA located in 8q24.2, and colon cancer associated transcript-2 maps to the 8q24.21 region encompassing rs6983267. Colon cancer associated transcripts have close associations with clinical characteristics, such as lymph node metastasis, high TNM stage and short overall survival. Knockdown of them can reverse the malignant phenotypes of cancer cells, including proliferation, migration, invasion and apoptosis. Moreover, they can increase the expression level of c-MYC and oncogenic microRNAs via activating a series of complex mechanisms. In brief, the family of colon cancer associated transcripts may serve as potential biomarkers or therapeutic targets for human cancers. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Promoter hypermethylation mediated downregulation of FBP1 in human hepatocellular carcinoma and colon cancer.

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Mingquan Chen

Full Text Available FBP1, fructose-1,6-bisphosphatase-1, a gluconeogenesis regulatory enzyme, catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and inorganic phosphate. The mechanism that it functions to antagonize glycolysis and was epigenetically inactivated through NF-kappaB pathway in gastric cancer has been reported. However, its role in the liver carcinogenesis still remains unknown. Here, we investigated the expression and DNA methylation of FBP1 in primary HCC and colon tumor. FBP1 was lowly expressed in 80% (8/10 human hepatocellular carcinoma, 66.7% (6/9 liver cancer cell lines and 100% (6/6 colon cancer cell lines, but was higher in paired adjacent non-tumor tissues and immortalized normal cell lines, which was well correlated with its promoter methylation status. Methylation was further detected in primary HCCs, gastric and colon tumor tissues, but none or occasionally in paired adjacent non-tumor tissues. Detailed methylation analysis of 29 CpG sites at a 327-bp promoter region by bisulfite genomic sequencing confirmed its methylation. FBP1 silencing could be reversed by chemical demethylation treatment with 5-aza-2'-deoxycytidine (Aza, indicating direct epigenetic silencing. Restoring FBP1 expression in low expressed cells significantly inhibited cell growth and colony formation ability through the induction of G2-M phase cell cycle arrest. Moreover, the observed effects coincided with an increase in reactive oxygen species (ROS generation. In summary, epigenetic inactivation of FBP1 is also common in human liver and colon cancer. FBP1 appears to be a functional tumor suppressor involved in the liver and colon carcinogenesis.

Effect of Proton Beam on Cancer Progressive and Metastatic Enzymes

International Nuclear Information System (INIS)

Sohn, Y. H.; Nam, K. S.; Oh, Y. H.; Kim, M. K.; Kim, M. Y.; Jang, J. S.

2008-04-01

The purpose of this study was to investigate the effect of proton beam on enzymes for promotion/progression of carcinogenesis and metastasis of malignant tumor cells to clarify proton beam-specific biological effects. The changes of cancer chemopreventive enzymes in human colorectal adenocarcinoma HT-29 cells irradiated with proton beams were tested by measuring the activities of quinine reductase (QR), glutathione S-transferase (GST), and ornithine decarboxylase (ODC), glutathione (GSH) levels, and expression of cyclooxygenase-2 (COX-2). We also examined the effect of proton beam on the ODC activity and expression of COX-2 in human breast cancer cell. We then assessed the metastatic capabilities of HT-29 and MDA-MB-231 cells irradiated with proton beam by measuring the invasiveness of cells through Matrigel-coated membrane and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MMP activity in MDA-MB-231 and HT-29 cells. QR activity of irradiated HT-29 cells was slightly increased. Proton irradiation at dose of 32 Gy in HT-29 cells increased GST activity by 1.23-fold. In addition GSH levels in HT-29 cells was significantly increased 1.23- (p<0.05), 1.32- (p<0.01) and 1.34-fold (p<0.01) with the proton irradiation at doses of 8, 16 and 32 Gy, respectively. These results suggest that colon cancer chemopreventive activity was increased with the proton irradiation by increasing QR and GST activities and GSH levels and inhibiting ODC activity. Proton ion irradiation decreased the invasiveness of TPA-treated HT-29 cells and MDA-MB-231 cells through Matrigel-coated membrane. Proton ion irradiation pretreatment decreased TPA-induced MMP activity in MDA-MB-231 and HT-29 cells. Further studies are necessary to investigate if these findings could be translated to in vivo situations

Detection of human cancer in an animal model using radio-labelled tumour-associated monoclonal antibodies

International Nuclear Information System (INIS)

Epenetos, A.A.; Arklie, J.; Knowles, R.W.; Bodmer, W.F.

1982-01-01

Monoclonal antibodies to epithelial-cell antigenic determinants, labelled with 123 I and 125 I, were administered parenterally to immunodeficient mice bearing human tumours derived from a human cancer cell line. Anterior, posterior and lateral radioscans of the body were taken with a gamma scintillation camera at various times from immediately to 65 days after injection. Visual displays of the images were processed by standard computer techniques. The model used a human colon-cancer cell line, HT29, and the monoclonal antibody, AUAl, which is specific to an epithelial proliferating antigen. Tumour detection was achieved in all the mice. The smallest tumour detectable appeared to be about 1 mm in diameter. The degree of antibody uptake in a tumour depended on its size and the blood supply of its surrounding tissues. (author)

Lower sensitivity of FHC fetal colon epithelial cells to photodynamic therapy compared to HT-29 colon adenocarcinoma cells despite higher intracellular accumulation of hypericin

Czech Academy of Sciences Publication Activity Database

Mikeš, J.; Hýžďalová, Martina; Kočí, Lenka; Jendželovský, R.; Koval, J.; Vaculová, Alena; Hofmanová, Jiřina; Kozubík, Alois; Fedoročko, P.

2011-01-01

Roč. 10, č. 4 (2011), s. 626-632 ISSN 1474-905X R&D Projects: GA ČR(CZ) GA301/07/1557 Grant - others:GA ČR(CZ) GA305/09/1526 Program:GA Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : photodynamic therapy * hypericin * colon cancer Subject RIV: BO - Biophysics Impact factor: 2.584, year: 2011

Neurotensin Phosphorylates GSK-3α/β through the Activation of PKC in Human Colon Cancer Cells

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Qingding Wang

2006-09-01

Full Text Available Neurotensin (NT, a gastrointestinal hormone, binds its receptor [neurotensin receptor (NTR] to regulate the growth of normal and neoplastic intestinal cells; molecular mechanisms remain largely undefined. Glycogen synthase kinase-3 (GSK-3 regulates diverse cellular processes, including cell growth and apoptosis. Here, we show that NT induces the phosphorylation of GSK-3α/β in the human colon cancer cell line HT29, HCT116, or SW480, which possesses high-affinity NTR. The effect of NT was blocked by inhibitors of protein kinase C (PKC, but not by inhibitors of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK1 or phosphatidylinositol-3 kinase, suggesting a predominant role for PKC in GSK-3β phosphorylation by NT. Pretreatment with Gö6976 (which inhibits PKCα and PKCβ1 or downregulation of endogenous PKCα or PKCβ1 blocked NT-mediated GSK-3β (but not GSK-3α phosphorylation. Moreover, a selective PKCβ inhibitor, LY379196, reduced NT-mediated GSK-3β (but not GSK-3α phosphorylation, suggesting a role for PKCbβ in the NT-mediated phosphorylation of GSK-3β and an undefined kinase in the NT-mediated phosphorylation of GSK-3α. Treatment with NT or the GSK-3 inhibitor SB216763 increased the expression of cyclin D1, a downstream effector protein of GSK-3 and a critical protein for the proliferation of various cells. Our results indicate that NT uses PKC-dependent pathways to modulate GSK-3, which may play a role in the NT regulation of intestinal cell growth.

Expression of adrenomedullin in human colorectal tumors and its role in cell growth and invasion in vitro and in xenograft growth in vivo

International Nuclear Information System (INIS)

Nouguerède, Emilie; Berenguer, Caroline; Garcia, Stéphane; Bennani, Bahia; Delfino, Christine; Nanni, Isabelle; Dahan, Laetitia; Gasmi, Mohamed; Seitz, Jean-François; Martin, Pierre-Marie; Ouafik, L'Houcine

2013-01-01

Adrenomedullin (AM) is a multifunctional peptide vasodilator that transduces its effects through calcitonin receptor-like receptor/receptor activity-modifying protein-2 and -3 (CLR/RAMP2 and CLR/RAMP3). In this study, real-time quantitative reverse transcription demonstrated a significant expression of AM mRNA in tumor samples from colorectal cancer (CRC) patients in clinical stage II, III, and IV when compared with normal colorectal tissue. AM, CLR, RAMP2, and RAMP3 proteins were immunohistochemically localized in the carcinomatous epithelial compartment of CRC tissue. Tissue microarray analysis revealed a clear increase of AM, CLR, RAMP2, and RAMP3 staining in lymph node and distant metastasis when compared with primary tumors. The human colon carcinoma cells HT-29 expressed and secreted AM into the culture medium with a significant increase under hypoxia. Treatment of HT-29 cells with synthetic AM stimulated cell proliferation and invasion in vitro. Incubation with anti-AM antibody (αAM), anti-AM receptors antibodies (αAMR), or AM antagonist AM 22–52 inhibited significantly basal levels of proliferation of HT-29 cells, suggesting that AM may function as an autocrine growth factor for CRC cells. Treatment with αAM significantly suppressed the growth of HT-29 tumor xenografts in vivo. Histological examination of αAM-treated tumors showed evidence of disruption of tumor vascularity with decreased microvessel density, depletion of endothelial cells and pericytes, and increased tumor cell apoptosis. These findings highlight the potential importance of AM and its receptors in the progression of CRC and support the conclusion that αAM treatment inhibits tumor growth by suppression of angiogenesis and tumor growth, suggesting that AM may be a useful therapeutic target

Enhancement of P53-Mutant Human Colorectal Cancer Cells Radiosensitivity by Flavonoid Fisetin

International Nuclear Information System (INIS)

Chen Wenshu; Lee Yijang; Yu Yichu; Hsaio Chinghui

2010-01-01

Purpose: The aim of this study was to investigate whether fisetin is a potential radiosensitizer for human colorectal cancer cells, which are relatively resistant to radiotherapy. Methods and Materials: Cell survival was examined by clonogenic survival assay, and DNA fragmentation was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. The effects of treatments on cell cycle distribution and apoptosis were examined by flow cytometry. Western blot analysis was performed to ascertain the protein levels of γ-H2AX, phospho-Chk2, active caspase-3, PARP cleavage, phospho-p38, phospho-AKT, and phospho-ERK1/2. Results: Fisetin pretreatment enhanced the radiosensitivity of p53-mutant HT-29 human colorectal cancer cells but not human keratocyte HaCaT cells; it also prolonged radiation-induced G 2 /M arrest, enhanced radiation-induced cell growth arrest in HT-29 cells, and suppressed radiation-induced phospho-H2AX (Ser-139) and phospho-Chk2 (Thr-68) in p53-mutant HT-29 cells. Pretreatment with fisetin enhanced radiation-induced caspase-dependent apoptosis in HT-29 cells. Fisetin pretreatment augmented radiation-induced phosphorylation of p38 mitogen-activated protein kinase, which is involved in caspase-mediated apoptosis, and SB202190 significantly reduced apoptosis and radiosensitivity in fisetin-pretreated HT-29 cells. By contrast, both phospho-AKT and phospho-ERK1/2, which are involved in cell proliferation and antiapoptotic pathways, were suppressed after irradiation combined with fisetin pretreatment. Conclusions: To our knowledge, this study is the first to provide evidence that fisetin exerts a radiosensitizing effect in p53-mutant HT-29 cells. Fisetin could potentially be developed as a novel radiosensitizer against radioresistant human cancer cells.

Comparative effects of RRR-alpha- and RRR-gamma-tocopherol on proliferation and apoptosis in human colon cancer cell lines.

Science.gov (United States)

Campbell, Sharon E; Stone, William L; Lee, Steven; Whaley, Sarah; Yang, Hongsong; Qui, Min; Goforth, Paige; Sherman, Devin; McHaffie, Derek; Krishnan, Koyamangalath

2006-01-17

Mediterranean societies, with diets rich in vitamin E isoforms, have a lower risk for colon cancer than those of northern Europe and the Americas. Vitamin E rich diets may neutralize free radicals generated by fecal bacteria in the gut and prevent DNA damage, but signal transduction activities can occur independent of the antioxidant function. The term vitamin E represents eight structurally related compounds, each differing in their potency and mechanisms of chemoprevention. The RRR-gamma-tocopherol isoform is found primarily in the US diet, while RRR-alpha-tocopherol is highest in the plasma. The effectiveness of RRR-alpha- and RRR-gamma-tocopherol at inhibiting cell growth and inducing apoptosis in colon cancer cell lines with varying molecular characteristics (SW480, HCT-15, HCT-116 and HT-29) and primary colon cells (CCD-112CoN, nontransformed normal phenotype) was studied. Colon cells were treated with and without RRR-alpha- or RRR-gamma-tocopherol using varying tocopherol concentrations and time intervals. Cell proliferation and apoptosis were measured using the trypan blue assay, annexin V staining, DNA laddering and caspase activation. Treatment with RRR-gamma-tocopherol resulted in significant cell death for all cancer cell lines tested, while RRR-alpha-tocopherol did not. Further, RRR-gamma-tocopherol treatment showed no cytotoxicity to normal colon cells CCD-112CoN at the highest concentration and time point tested. RRR-gamma-tocopherol treatment resulted in cleavage of PARP, caspase 3, 7, and 8, but not caspase 9. Differences in the percentage cell death and apoptosis were observed in different cell lines suggesting that molecular differences in these cell lines may influence the ability of RRR-gamma-tocopherol to induce cell death. This is the first study to demonstrate that multiple colon cancer cell lines containing varying genetic alterations will under go growth reduction and apoptosis in the presence of RRR-gamma-tocopherol without damage to

Comparative effects of RRR-alpha- and RRR-gamma-tocopherol on proliferation and apoptosis in human colon cancer cell lines

International Nuclear Information System (INIS)

Campbell, Sharon E; Krishnan, Koyamangalath; Stone, William L; Lee, Steven; Whaley, Sarah; Yang, Hongsong; Qui, Min; Goforth, Paige; Sherman, Devin; McHaffie, Derek

2006-01-01

Mediterranean societies, with diets rich in vitamin E isoforms, have a lower risk for colon cancer than those of northern Europe and the Americas. Vitamin E rich diets may neutralize free radicals generated by fecal bacteria in the gut and prevent DNA damage, but signal transduction activities can occur independent of the antioxidant function. The term vitamin E represents eight structurally related compounds, each differing in their potency and mechanisms of chemoprevention. The RRR-γ-tocopherol isoform is found primarily in the US diet, while RRR-α-tocopherol is highest in the plasma. The effectiveness of RRR-α- and RRR-γ-tocopherol at inhibiting cell growth and inducing apoptosis in colon cancer cell lines with varying molecular characteristics (SW480, HCT-15, HCT-116 and HT-29) and primary colon cells (CCD-112CoN, nontransformed normal phenotype) was studied. Colon cells were treated with and without RRR-α- or RRR-γ-tocopherol using varying tocopherol concentrations and time intervals. Cell proliferation and apoptosis were measured using the trypan blue assay, annexin V staining, DNA laddering and caspase activation. Treatment with RRR-γ-tocopherol resulted in significant cell death for all cancer cell lines tested, while RRR-α-tocopherol did not. Further, RRR-γ-tocopherol treatment showed no cytotoxicity to normal colon cells CCD-112CoN at the highest concentration and time point tested. RRR-γ-tocopherol treatment resulted in cleavage of PARP, caspase 3, 7, and 8, but not caspase 9. Differences in the percentage cell death and apoptosis were observed in different cell lines suggesting that molecular differences in these cell lines may influence the ability of RRR-γ-tocopherol to induce cell death. This is the first study to demonstrate that multiple colon cancer cell lines containing varying genetic alterations will under go growth reduction and apoptosis in the presence of RRR-γ-tocopherol without damage to normal colon cells. The amount growth

Correlating the Metabolic Stability of Psychedelic 5-HT2A Agonists with Anecdotal Reports of Human Oral Bioavailability

DEFF Research Database (Denmark)

Leth-Petersen, Sebastian; Bundgaard, Christoffer; Hansen, Martin

2014-01-01

2,5-Dimethoxyphenethylamines and their N-benzylated derivatives are potent 5-HT2A agonists with psychedelic effects in humans. The N-benzylated derivatives are among the most selective 5-HT2A agonists currently available and their usage as biochemical and brain imaging tools is increasing, yet ve...

Urotensin-II receptor is over-expressed in colon cancer cell lines and in colon carcinoma in humans.

Science.gov (United States)

Federico, Alessandro; Zappavigna, Silvia; Romano, Marco; Grieco, Paolo; Luce, Amalia; Marra, Monica; Gravina, Antonietta Gerarda; Stiuso, Paola; D'Armiento, Francesco Paolo; Vitale, Giovanni; Tuccillo, Concetta; Novellino, Ettore; Loguercio, Carmela; Caraglia, Michele

2014-01-01

Urotensin (U)-II receptor (UTR) has been previously reported to be over-expressed in a number of tumours. Whether UTR-related pathway plays a role in colon carcinogenesis is unknown. We evaluated UTR protein and mRNA expression in human epithelial colon cancer cell lines and in normal colon tissue, adenomatous polyps and colon cancer. U-II protein expression was assessed in cancer cell lines. Moreover, we evaluated the effects of U-II(4-11) (an UTR agonist), antagonists and knockdown of UTR protein expression through a specific shRNA, on proliferation, invasion and motility of human colon cancer cells. Cancer cell lines expressed U-II protein and UTR protein and mRNA. By immunohistochemistry, UTR was expressed in 5-30% of epithelial cells in 45 normal controls, in 30-48% in 21 adenomatous polyps and in 65-90% in 48 colon adenocarcinomas. UTR mRNA expression was increased by threefold in adenomatous polyps and eightfold in colon cancer, compared with normal colon. U-II(4-11) induced a 20-40% increase in cell growth while the blockade of the receptor with specific antagonists caused growth inhibition of 20-40%. Moreover, the knock down of UTR with a shRNA or the inhibition of UTR with the antagonist urantide induced an approximately 50% inhibition of both motility and invasion. UTR appears to be involved in the regulation of colon cancer cell invasion and motility. These data suggest that UTR-related pathway may play a role in colon carcinogenesis and that UTR may function as a target for therapeutic intervention in colon cancer. © 2013 Stichting European Society for Clinical Investigation Journal Foundation.

A method for high purity intestinal epithelial cell culture from adult human and murine tissues for the investigation of innate immune function.

Science.gov (United States)

Graves, Christina L; Harden, Scott W; LaPato, Melissa; Nelson, Michael; Amador, Byron; Sorenson, Heather; Frazier, Charles J; Wallet, Shannon M

2014-12-01

Intestinal epithelial cells (IECs) serve as an important physiologic barrier between environmental antigens and the host intestinal immune system. Thus, IECs serve as a first line of defense and may act as sentinel cells during inflammatory insults. Despite recent renewed interest in IEC contributions to host immune function, the study of primary IEC has been hindered by lack of a robust culture technique, particularly for small intestinal and adult tissues. Here, a novel adaptation for culture of primary IEC is described for human duodenal organ donor tissue as well as duodenum and colon of adult mice. These epithelial cell cultures display characteristic phenotypes and are of high purity. In addition, the innate immune function of human primary IEC, specifically with regard to Toll-like receptor (TLR) expression and microbial ligand responsiveness, is contrasted with a commonly used intestinal epithelial cell line (HT-29). Specifically, TLR expression at the mRNA level and production of cytokine (IFNγ and TNFα) in response to TLR agonist stimulation is assessed. Differential expression of TLRs as well as innate immune responses to ligand stimulation is observed in human-derived cultures compared to that of HT-29. Thus, use of this adapted method to culture primary epithelial cells from adult human donors and from adult mice will allow for more appropriate studies of IECs as innate immune effectors. Published by Elsevier B.V.

Quantification of 5-HT{sub 1A} receptors in human brain using p-MPPF kinetic modelling and PET

Energy Technology Data Exchange (ETDEWEB)

Sanabria-Bohorquez, S.M.; Veraart, C. [Neural Rehabilitation Engineering Lab., Univ. Catholique de Louvain, Brussels (Belgium); Biver, F.; Damhaut, P.; Wikler, D.; Goldman, S. [PET/Biomedical Cyclotron Unit, Univ. Libre de Bruxelles (Belgium)

2002-01-01

Serotonin-1A (5-HT{sub 1A}) receptors are implicated in neurochemical mechanisms underlying anxiety and depression and their treatment. Animal studies have suggested that 4-(2'-methoxyphenyl)-1-[2'-[N-(2''-pyridinyl)-p-[{sup 18}F]fluorobenzamido] ethyl] piperazine (p-MPPF) may be a suitable positron emission tomography (PET) tracer of 5-HT{sub 1A} receptors. To test p-MPPF in humans, we performed 60-min dynamic PET scans in 13 healthy volunteers after single bolus injection. Metabolite quantification revealed a fast decrease in tracer plasma concentration, such that at 5 min post injection about 25% of the total radioactivity in plasma corresponded to p-MPPF. Radioactivity concentration was highest in hippocampus, intermediate in neocortex and lowest in basal ganglia and cerebellum. The interactions between p-MPPF and 5-HT{sub 1A} receptors were described using linear compartmental models with plasma input and reference tissue approaches. The two quantification methods provided similar results which are in agreement with previous reports on 5-HT{sub 1A} receptor brain distribution. In conclusion, our results show that p-MPPF is a suitable PET radioligand for 5-HT{sub 1A} receptor human studies. (orig.)

Inhibition of PCAF histone acetyltransferase, cytotoxicity and cell permeability of 2-acylamino-1-(3- or 4-carboxy-phenyl)benzamides.

Science.gov (United States)

Park, Woong Jae; Ma, Eunsook

2012-11-05

Small molecule HAT inhibitors are useful tools to unravel the role of histone acetyltransferases (HATs) in the cell and they also have relevance in oncology. We synthesized a series of 2-acylamino-1-(3- or 4-carboxyphenyl)benzamides 8–19 bearing C6, C8, C10, C12, C14, and C16 acyl chains at the 2-amino position of 2-aminobenzoic acid. Enzyme inhibition of these compounds was investigated using in vitro PCAF HAT assays. The inhibitory activities of compounds 8–10, 16, and 19 were similar to that of anacardic acid, and 17 was found to be more active than anacardic acid at 100 μM. Compounds 11–15 showed the low inhibitory activity on PCAF HAT. The cytotoxicity of the synthesized compounds was evaluated by SRB (sulforhodamine B) assay against seven human cancer cell lines: HT-29 (colon), HCT-116 (colon), MDA-231 (breast), A549 (lung), Hep3B (hepatoma), HeLa (cervical) and Caki (kidney) and one normal cell line (HSF). Compound 17 was more active than anacardic acid against human colon cancer (HCT 116, IC(50): 29.17 μM), human lung cancer (A549, IC₅₀: 32.09 μM) cell lines. 18 was more active than anacardic acid against human colon cancer (HT-29, IC₅₀: 35.49 μM and HCT 116, IC₅₀: 27.56 μM), human lung cancer (A549, IC₅₀: 30.69 μM), and human cervical cancer (HeLa, IC₅₀: 34.41 μM) cell lines. The apparent permeability coefficient (P(app), cm/s) values of two compounds (16 and 17) were evaluated as 68.21 and 71.48 × 10⁻⁶ cm/s by Caco-2 cell permeability assay.

Inhibition of PCAF Histone Acetyltransferase, Cytotoxicity and Cell Permeability of 2-Acylamino-1-(3- or 4-Carboxy-phenylbenzamides

Directory of Open Access Journals (Sweden)

Eunsook Ma

2012-11-01

Full Text Available Small molecule HAT inhibitors are useful tools to unravel the role of histone acetyltransferases (HATs in the cell and they also have relevance in oncology. We synthesized a series of 2-acylamino-1-(3- or 4-carboxyphenylbenzamides 8–19 bearing C6, C8, C10, C12, C14, and C16 acyl chains at the 2-amino position of 2-aminobenzoic acid. Enzyme inhibition of these compounds was investigated using in vitro PCAF HAT assays. The inhibitory activities of compounds 8–10, 16, and 19 were similar to that of anacardic acid, and 17 was found to be more active than anacardic acid at 100 μM. Compounds 11–15 showed the low inhibitory activity on PCAF HAT. The cytotoxicity of the synthesized compounds was evaluated by SRB (sulforhodamine B assay against seven human cancer cell lines: HT-29 (colon, HCT-116 (colon, MDA-231 (breast, A549 (lung, Hep3B (hepatoma, HeLa (cervical and Caki (kidney and one normal cell line (HSF. Compound 17 was more active than anacardic acid against human colon cancer (HCT 116, IC50: 29.17 μM, human lung cancer (A549, IC50: 32.09 μM cell lines. 18 was more active than anacardic acid against human colon cancer (HT-29, IC50: 35.49 μM and HCT 116, IC50: 27.56 μM, human lung cancer (A549, IC50: 30.69 μM, and human cervical cancer (HeLa, IC50: 34.41 μM cell lines. The apparent permeability coefficient (Papp, cm/s values of two compounds (16 and 17 were evaluated as 68.21 and 71.48 × 10−6 cm/s by Caco-2 cell permeability assay.

Antitumor Activity of Total Flavonoids from Daphne genkwa in Colorectal Cancer.

Science.gov (United States)

Du, Wen-Juan; Yang, Xiao-Lin; Song, Zi-Jing; Wang, Jiao-Ying; Zhang, Wen-Jun; He, Xin; Zhang, Run-Qi; Zhang, Chun-Feng; Li, Fei; Yu, Chun-Hao; Wang, Chong-Zhi; Yuan, Chun-Su

2016-02-01

Daphne genkwa Sieb.et Zucc. is a well-known medicinal plant. This study was designed to investigate the anticancer effects of total flavonoids in D. genkwa (TFDG) in vitro and in vivo. HT-29 and SW-480 human colorectal cancer cells were cultured to investigate the anticancer activity of TFDG. In addition, the Apc(Min/+) mouse model was applied in the in vivo experiment. Results of the cell experiment revealed that TFDG possessed significant inhibitory effects on HT-29 and SW-480 human colorectal cancer cells (both p colon (both p cancer therapeutics, and TFDG's action is likely linked to its ability to regulate immune function and inhibit the production of inflammatory cytokines. Copyright © 2015 John Wiley & Sons, Ltd.

1,25(OH)2D3 attenuates TGF-β1/β2-induced increased migration and invasion via inhibiting epithelial-mesenchymal transition in colon cancer cells.

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Chen, Shanwen; Zhu, Jing; Zuo, Shuai; Ma, Ju; Zhang, Junling; Chen, Guowei; Wang, Xin; Pan, Yisheng; Liu, Yucun; Wang, Pengyuan

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) has been reported to inhibit proliferation and migration of multiple types of cancer cells. However, the mechanism underlying its anti-metastasis effect is not fully illustrated. In this study, the effect of 1,25(OH)2D3 on TGF-β1/β2-induced epithelial-mesenchymal transition (EMT) is tested in colon cancer cells. The results suggest that 1,25(OH)2D3 inhibited TGF-β1/β2-induced increased invasion and migration of in SW-480 and HT-29 cells. 1,25(OH)2D3 also inhibited the cadherin switch in SW-480 and HT-29 cells. TGF-β1/β2-induced increased expression of EMT-related transcription factors was also inhibited by 1,25(OH)2D3. 1,25(OH)2D3 also inhibited the secretion of MMP-2 and MMP-9 and increased expression of F-actin induced by TGF-β1/β2 in SW-480 cells. Taken together, this study suggests that the suppression of EMT might be one of the mechanisms underlying the anti-metastasis effect of 1,25(OH)2D3 in colon cancer cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Psychopharmacology of 5-HT1A receptors

International Nuclear Information System (INIS)

Cowen, Philip J.

2000-01-01

Serotonin 1A (5-HT 1A ) receptors are located on both 5-HT cell bodies where they act as inhibitory autoreceptors and at postsynaptic sites where they mediate the effects of 5-HT released from nerve terminals. The sensitivity of 5-HT 1A receptors in humans can be measured using the technique of pharmacological challenge. For example, acute administration of a selective 5-HT 1A receptor agonist, such as ipsapirone, decreases body temperature and increases plasma cortisol through activation of pre- and postsynaptic 5-HT 1A receptors, respectively. Use of this technique has demonstrated that unmedicated patients with major depression have decreased sensitivity of both pre- and postsynaptic 5-HT 1A receptors. Treatment with selective serotonin reuptake inhibitors further down-regulates 5-HT 1A receptor activity. Due to the hypotheses linking decreased sensitivity of 5-HT 1A autoreceptors with the onset of antidepressant activity, there is current interest in the therapeutic efficacy of combined treatment with selective serotonin reuptake inhibitors and 5-HT 1A receptor antagonists

Colonic Fermentation: A Neglected Topic in Human Physiology Education

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Valeur, Jorgen; Berstad, Arnold

2010-01-01

Human physiology textbooks tend to limit their discussion of colonic functions to those of absorbing water and electrolytes and storing waste material. However, the colon is a highly active metabolic organ, containing an exceedingly complex society of microbes. By means of fermentation, gastrointestinal microbes break down nutrients that cannot be…

Oncogenic KRAS activates an embryonic stem cell-like program in human colon cancer initiation.

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Le Rolle, Anne-France; Chiu, Thang K; Zeng, Zhaoshi; Shia, Jinru; Weiser, Martin R; Paty, Philip B; Chiu, Vi K

2016-01-19

Colorectal cancer is the third most frequently diagnosed cancer worldwide. Prevention of colorectal cancer initiation represents the most effective overall strategy to reduce its associated morbidity and mortality. Activating KRAS mutation (KRASmut) is the most prevalent oncogenic driver in colorectal cancer development, and KRASmut inhibition represents an unmet clinical need. We apply a systems-level approach to study the impact of KRASmut on stem cell signaling during human colon cancer initiation by performing gene set enrichment analysis on gene expression from human colon tissues. We find that KRASmut imposes the embryonic stem cell-like program during human colon cancer initiation from colon adenoma to stage I carcinoma. Expression of miR145, an embryonic SC program inhibitor, promotes cell lineage differentiation marker expression in KRASmut colon cancer cells and significantly suppresses their tumorigenicity. Our data support an in vivo plasticity model of human colon cancer initiation that merges the intrinsic stem cell properties of aberrant colon stem cells with the embryonic stem cell-like program induced by KRASmut to optimize malignant transformation. Inhibition of the embryonic SC-like program in KRASmut colon cancer cells reveals a novel therapeutic strategy to programmatically inhibit KRASmut tumors and prevent colon cancer.

Involvement of CRF2 signaling in enterocyte differentiation.

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Ducarouge, Benjamin; Pelissier-Rota, Marjolaine; Powell, Rebecca; Buisson, Alain; Bonaz, Bruno; Jacquier-Sarlin, Muriel

2017-07-28

the crypts of colon and in human colon carcinoma cell lines. Furthermore, CRF2 expression is down regulated according to the kinetic of HT-29 cell differentiation. By performing functional assays, we found that Ucn3-induced CRF2 signaling alters both para- and trans-cellular permeability of differentiated HT-29 and Caco-2 cells. These effects are partly mediated by Ucn3-induced morphological changes associated with the disruption of mature AJ in HT-29 cells and tight junctions (TJ) in Caco-2 cells. Ucn3-mediated activation of CRF2 decreases mRNA and protein expression levels of KLF4 a transcription factor involved in IEC differentiation. This signaling is correlated to a down-regulation of key IEC markers such as DPPIV and AP, at both transcriptional and post-transcriptional levels. Our findings suggest that CRF2 signaling could modulate IEC differentiation. These mechanisms could be relevant to the stress induced epithelial alterations found in inflammatory bowel diseases.

Comparative genome-based identification of a cell wall-anchored protein from Lactobacillus plantarum increases adhesion of Lactococcus lactis to human epithelial cells.

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Zhang, Bo; Zuo, Fanglei; Yu, Rui; Zeng, Zhu; Ma, Huiqin; Chen, Shangwu

2015-09-15

Adhesion to host cells is considered important for Lactobacillus plantarum as well as other lactic acid bacteria (LAB) to persist in human gut and thus exert probiotic effects. Here, we sequenced the genome of Lt. plantarum strain NL42 originating from a traditional Chinese dairy product, performed comparative genomic analysis and characterized a novel adhesion factor. The genome of NL42 was highly divergent from its closest neighbors, especially in six large genomic regions. NL42 harbors a total of 42 genes encoding adhesion-associated proteins; among them, cwaA encodes a protein containing multiple domains, including five cell wall surface anchor repeat domains and an LPxTG-like cell wall anchor motif. Expression of cwaA in Lactococcus lactis significantly increased its autoaggregation and hydrophobicity, and conferred the new ability to adhere to human colonic epithelial HT-29 cells by targeting cellular surface proteins, and not carbohydrate moieties, for CwaA adhesion. In addition, the recombinant Lc. lactis inhibited adhesion of Staphylococcus aureus and Escherichia coli to HT-29 cells, mainly by exclusion. We conclude that CwaA is a novel adhesion factor in Lt. plantarum and a potential candidate for improving the adhesion ability of probiotics or other bacteria of interest.

Expression of DIAPH1 is up-regulated in colorectal cancer and its down-regulation strongly reduces the metastatic capacity of colon carcinoma cells.

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Lin, Yuan-Na; Izbicki, Jakob R; König, Alexandra; Habermann, Jens K; Blechner, Christine; Lange, Tobias; Schumacher, Udo; Windhorst, Sabine

2014-04-01

In most cases, metastatic colorectal cancer is not curable, thus new approaches are necessary to identify novel targets for colorectal cancer therapy. Actin-binding-proteins (ABPs) directly regulate motility of metastasising tumor cells, and for cortactin an association with colon cancer metastasis has been already shown. However, as its depletion only incompletely inhibits metastasis, additional, more suitable cellular targets have to be identified. Here we analyzed expression of the ABPs, DIAPH1, VASP, N-WASP, and fascin in comparison with cortactin and found that, besides cortactin, DIAPH1 was expressed with the highest frequency (63%) in colorectal cancer. As well as cortactin, DIAPH1 was not detectable in normal colon tissue and expression of both proteins was positively correlated with metastasis of colorectal cancer. To analyse the mechanistic role of DIAPH1 for metastasis of colon carcinoma cells in comparison with cortactin, expression of the proteins was stably down-regulated in the human colon carcinoma cell lines HT-29, HROC-24 and HCT-116. Analysis of metastasis of colon carcinoma cells in SCID mice revealed that depletion of DIAPH1 reduced metastasis 60-fold and depletion of cortactin 16-fold as compared with control cells. Most likely the stronger effect of DIAPH1 depletion on colon cancer metastasis is due to the fact that in vitro knock down of DIAPH1 impaired all steps of metastasis; adhesion, invasion and migration while down-regulation of cortactin only reduced adhesion and invasion. This very strong reducing effect of DIAPH1 depletion on colon carcinoma cell metastasis makes the protein a promising therapeutic target for individualized colorectal cancer therapy. © 2013 UICC.

Resveratrol Modulates the Topoisomerase Inhibitory Potential of Doxorubicin in Human Colon Carcinoma Cells

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Anika Schroeter

2014-12-01

Full Text Available Resveratrol (RSV is currently being widely discussed as potentially useful for anticancer therapy in combination with classical chemotherapeutics, e.g., the topoisomerase II (TOP II poison doxorubicin (DOX. However, there is still a lack of knowledge of possible interference at the target enzyme, especially since RSV itself has recently been described to act as a TOP poison. We therefore sought to address the question whether RSV affects DOX-induced genotoxic and cytotoxic effects with special emphasis on TOP II in HT-29 colon carcinoma cells. RSV was found to counteract DOX-induced formation of DNA-TOP-intermediates at ≥100 µM for TOP IIα and at 250 µM for TOP IIβ. As a consequence, RSV modulated the DNA-strand breaking potential of DOX by mediating protective effects with an apparent maximum at 100 µM. At higher concentration ranges (≥200 µM RSV diminished the intracellular concentrations of DOX. Nevertheless, the presence of RSV slightly enhanced the cytotoxic effects of DOX after 1.5 h and 24 h of incubation. Taken together, at least in cell culture RSV was found to affect the TOP-poisoning potential of DOX and to modulate its cytotoxic effectiveness. Thus, further studies are needed to clarify the impact of RSV on the therapeutic effectiveness of DOX under in vivo conditions.

Identification of a novel human deoxynivalenol metabolite enhancing proliferation of intestinal and urinary bladder cells

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Warth, Benedikt; Del Favero, Giorgia; Wiesenberger, Gerlinde; Puntscher, Hannes; Woelflingseder, Lydia; Fruhmann, Philipp; Sarkanj, Bojan; Krska, Rudolf; Schuhmacher, Rainer; Adam, Gerhard; Marko, Doris

2016-09-01

The mycotoxin deoxynivalenol (DON) is an abundant contaminant of cereal based food and a severe issue for global food safety. We report the discovery of DON-3-sulfate as a novel human metabolite and potential new biomarker of DON exposure. The conjugate was detectable in 70% of urine samples obtained from pregnant women in Croatia. For the measurement of urinary metabolites, a highly sensitive and selective LC-MS/MS method was developed and validated. The method was also used to investigate samples from a duplicate diet survey for studying the toxicokinetics of DON-3-sulfate. To get a preliminary insight into the biological relevance of the newly discovered DON-sulfates, in vitroexperiments were performed. In contrast to DON, sulfate conjugates lacked potency to suppress protein translation. However, surprisingly we found that DON-sulfates enhanced proliferation of human HT-29 colon carcinoma cells, primary human colon epithelial cells (HCEC-1CT) and, to some extent, also T24 bladder cancer cells. A proliferative stimulus, especially in tumorigenic cells raises concern on the potential impact of DON-sulfates on consumer health. Thus, a further characterization of their toxicological relevance should be of high priority.

TAp63 suppress metastasis via miR-133b in colon cancer cells.

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Lin, C W; Li, X R; Zhang, Y; Hu, G; Guo, Y H; Zhou, J Y; Du, J; Lv, L; Gao, K; Zhang, Y; Deng, H

2014-04-29

TAp63 is a tumour-suppressor protein that is often underexpressed in various types of cancer. It has been shown to activate gene transcription depending on the transcription domain and to be closely related with metastasis. In this study, we demonstrate that TAp63 suppresses metastasis in colon cancer cells through microRNA-133b. We evaluated the correlation of TAp63 and miR-133b with HT-29 and SW-620 cells and investigated the roles of TAp63 in the expression of RhoA, E-cadherin and vimentin. We further investigated the roles of TAp63-mediated invasion and migration of colon cancer cells. TAp63 expression is downregulated in colon cancer, and microRNA-133b is a transcriptional target of TAp63. Furthermore, microRNA-133b is essential for the inhibitory effects of TAp63 on RhoA, E-cadherin and vimentin. Moreover, TAp63 inhibits cell migration and invasion through microRNA-133b. Correspondingly, the inhibitory effect of TAp63 on RhoA, E-cadherin, vimentin, migration and invasion can be blocked by the microRNA-133b inhibitor. TAp63 and microRNA-133b were able to suppress the metastasis of colon cancer. Both TAp63 and microRNA-133b may be potential biomarkers for diagnosis in colon cancer metastasis and may provide unique therapeutic targets for this common malignancy.

Kinetic modeling of 11C-SB207145 binding to 5-HT4 receptors in the human brain in vivo

DEFF Research Database (Denmark)

Marner, Lisbeth; Gillings, Nic; Comley, Robert A

2009-01-01

The serotonin 4 receptor (5-HT(4) receptor) is known to be involved in learning and memory. We evaluated for the first time the quantification of a novel 5-HT(4) receptor radioligand, (11)C-SB207145, for in vivo brain imaging with PET in humans. METHODS: For evaluation of reproducibility, 6...

Evaluation of an in vitro faecal degradation method for early assessment of the impact of colonic degradation on colonic absorption in humans.

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Tannergren, Christer; Borde, Anders; Boreström, Cecilia; Abrahamsson, Bertil; Lindahl, Anders

2014-06-16

The objective of this study was to develop and evaluate an in vitro method to investigate bacterial-mediated luminal degradation of drugs in colon in humans. This would be a valuable tool for the assessment of drug candidates during early drug development, especially for compounds intended to be developed as oral extended release formulations. Freshly prepared faecal homogenate from healthy human volunteers (n=3-18), dog (n=6) and rat (colon and caecal content, n=3) was homogenised with 3.8 parts (w/w) physiological saline under anaerobical conditions. Four model compounds (almokalant, budesonide, ximelagatran and metoprolol) were then incubated (n=3-18) separately in the human faecal homogenate for up to 120min at 37°C. In addition, ximelagatran was also incubated in the faecal or colonic content from dog and rat. The mean (±SD) in vitro half-life for almokalant, budesonide and ximelagatran was 39±1, 68±21 and 26±12min, respectively, in the human faecal homogenate. Metoprolol was found to be stable in the in vitro model. The in vitro degradation data was then compared to literature data on fraction absorbed after direct colon administration in humans. The percentage of drug remaining after 60min of in vitro incubation correlated (R(2)=0.90) with the fraction absorbed from colon in humans. The mean in vitro half-life of ximelagatran was similar in human faeces (26±12min) and rat colon content (34±31min), but significantly (pdegradation in vivo was rapidly degraded in the faecal homogenates as well as quantitatively since a correlation was established between percentage degraded in vitro at 60min and fraction absorbed in the colon for the model drugs, which have no other absorption limiting properties. Also, the method is easy to use from a technical point of view, which suggests that the method is suitable for use in early assessment of colonic absorption of extended release formulation candidates. Further improvement of the confidence in the use of the

Specific oncogenic activity of the Src-family tyrosine kinase c-Yes in colon carcinoma cells.

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Florence Sancier

Full Text Available c-Yes, a member of the Src tyrosine kinase family, is found highly activated in colon carcinoma but its importance relative to c-Src has remained unclear. Here we show that, in HT29 colon carcinoma cells, silencing of c-Yes, but not of c-Src, selectively leads to an increase of cell clustering associated with a localisation of β-catenin at cell membranes and a reduction of expression of β-catenin target genes. c-Yes silencing induced an increase in apoptosis, inhibition of growth in soft-agar and in mouse xenografts, inhibition of cell migration and loss of the capacity to generate liver metastases in mice. Re-introduction of c-Yes, but not c -Src, restores transforming properties of c-Yes depleted cells. Moreover, we found that c-Yes kinase activity is required for its role in β-catenin localisation and growth in soft agar, whereas kinase activity is dispensable for its role in cell migration. We conclude that c-Yes regulates specific oncogenic signalling pathways important for colon cancer progression that is not shared with c-Src.

Calreticulin is a fine tuning molecule in epibrassinolide-induced apoptosis through activating endoplasmic reticulum stress in colon cancer cells.

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Obakan-Yerlikaya, Pinar; Arisan, Elif Damla; Coker-Gurkan, Ajda; Adacan, Kaan; Ozbey, Utku; Somuncu, Berna; Baran, Didem; Palavan-Unsal, Narcin

2017-06-01

Epibrassinolide (EBR), a member of brassinostreoids plant hormones with cell proliferation promoting role in plants, is a natural polyhydroxysteroid with structural similarity to steroid hormones of vertebrates. EBR has antiproliferative and apoptosis-inducing effect in various cancer cells. Although EBR has been shown to affect survival and mitochondria-mediated apoptosis pathways in a p53-independent manner, the exact molecular targets of EBR are still under investigation. Our recent SILAC (Stable Isotope Labeling by Amino Acids in Cell Culture) data showed that the most significantly altered protein after EBR treatment was calreticulin (CALR). CALR, a chaperone localized in endoplasmic reticulum (ER) lumen, plays role in protein folding and buffering Ca 2+ ions. The alteration of CALR may cause ER stress and unfolded protein response correspondingly the induction of apoptosis. Unfolded proteins are conducted to 26S proteasomal degradation following ubiquitination. Our study revealed that EBR treatment caused ER stress and UPR by altering CALR expression causing caspase-dependent apoptosis in HCT 116, HT29, DLD-1, and SW480 colon cancer cells. Furthermore, 48 h EBR treatment did not caused UPR in Fetal Human Colon cells (FHC) and Mouse Embryonic Fibroblast cells (MEF). In addition our findings showed that HCT 116 colon cancer cells lacking Bax and Puma expression still undergo UPR and related apoptosis. CALR silencing and rapamycin co-treatment prevented EBR-induced UPR and apoptosis, whereas 26S proteasome inhibition further increased the effect of EBR in colon cancer cells. All these findings showed that EBR is an ER stress and apoptotic inducer in colon cancer cells without affecting non-malignant cells. © 2017 Wiley Periodicals, Inc.

Histochemical and radioautographic studies of normal human fetal colon

International Nuclear Information System (INIS)

Lev, R.; Orlic, D.; New York Medical Coll., N.Y.

1974-01-01

Twenty fetal and infant colons ranging from 10 weeks in utero to 20 months postpartum, and 12 adult human colons were examined using histochemical techniques in conjunction with in vitro radioautography using Na 2 35 SO 4 as a sulfomucin precursor. Only the sulfated components of mucus in fetal goblet cells was found to differ significantly from adult colonic mucins. In the fetus sulfomucin staining was much weaker than in the adult, and was more intense in the left colon which is the reverse of the adult pattern. Sulfomucin was concentrated in the crypts throughout the fetal colon whereas in the adult right colon it predominated in the surface cells. As in the adult, saponification liberated carboxyl groups, possibly belonging to sialic acid, and vicinal hydroxyl groups from fetal mucins suggesting that this procedure hydrolyses an ester linkage between these 2 reactive groups. During the middle trimester of fetal life the colon possesses villi whose constituent cells display alkaline phosphatase in their surface coat. These and other morphological and histochemical similarities to fetal small intestine suggest that the fetal colon may have a limited capacity to absorb materials contained within swallowed amniotic fluid during this period. (orig.) [de

Colonic lymphoid follicles associated with colonic neoplasms

International Nuclear Information System (INIS)

Glick, S.N.; Teplick, S.K.; Ross, W.M.

1986-01-01

The authors prospectively evaluated 62 patients over 40 years old in whom lymphoid follicles were demonstrated on double-contrast enema examinations. Eighteen patients (29%) had no current radiographic evidence of, or history of, colonic neoplasms. Forty-four patients (71%) had an associated neoplasm. Fourteen patients had associated colonic carcinoma, and ten patients had a history of a previously resected colon cancer. One patient had previously undergone resection for ''polyps.'' Twenty-two patients had an associated ''polyp.'' There were no clinical or radiographic features that could reliably distinguish the neoplastic from the nonneoplastic groups. However, lymphoid follicles in the left colon or diffusely involving the colon were more likely to be associated with a colonic neoplasm. Lymphoid follicles were almost always identified near a malignant lesion

Comparative effects of RRR-alpha- and RRR-gamma-tocopherol on proliferation and apoptosis in human colon cancer cell lines

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Sherman Devin

2006-01-01

Full Text Available Abstract Background Mediterranean societies, with diets rich in vitamin E isoforms, have a lower risk for colon cancer than those of northern Europe and the Americas. Vitamin E rich diets may neutralize free radicals generated by fecal bacteria in the gut and prevent DNA damage, but signal transduction activities can occur independent of the antioxidant function. The term vitamin E represents eight structurally related compounds, each differing in their potency and mechanisms of chemoprevention. The RRR-γ-tocopherol isoform is found primarily in the US diet, while RRR-α-tocopherol is highest in the plasma. Methods The effectiveness of RRR-α- and RRR-γ-tocopherol at inhibiting cell growth and inducing apoptosis in colon cancer cell lines with varying molecular characteristics (SW480, HCT-15, HCT-116 and HT-29 and primary colon cells (CCD-112CoN, nontransformed normal phenotype was studied. Colon cells were treated with and without RRR-α- or RRR-γ-tocopherol using varying tocopherol concentrations and time intervals. Cell proliferation and apoptosis were measured using the trypan blue assay, annexin V staining, DNA laddering and caspase activation. Results Treatment with RRR-γ-tocopherol resulted in significant cell death for all cancer cell lines tested, while RRR-α-tocopherol did not. Further, RRR-γ-tocopherol treatment showed no cytotoxicity to normal colon cells CCD-112CoN at the highest concentration and time point tested. RRR-γ-tocopherol treatment resulted in cleavage of PARP, caspase 3, 7, and 8, but not caspase 9. Differences in the percentage cell death and apoptosis were observed in different cell lines suggesting that molecular differences in these cell lines may influence the ability of RRR-γ-tocopherol to induce cell death. Conclusion This is the first study to demonstrate that multiple colon cancer cell lines containing varying genetic alterations will under go growth reduction and apoptosis in the presence of RRR

Transcription factor Runx2 knockdown regulates colon cancer transplantation tumor growth in vitro: an experimental study

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Bin Xu1

2017-05-01

Full Text Available Objective: To study the effect of transcription factor Runx2 knockdown on colon cancer transplantation tumor growth in vitro. Methods: Colon cancer cell lines HT29 were cultured and transfected with negative control (NC - shRNA plasmids and Runx2-shRNA plasmids respectively, the colon cancer cells transfected with shRNA were subcutaneously injected into C57 nude mice, and they were included in NC group and Runx2 knockdown group respectively. 1 week, 2 weeks and 3 weeks after model establishment, serum was collected to determine the contents of tumor markers, and tumor lesions were collected to determine proliferation and apoptosis gene expression. Results: CCSA-2, CEA and CA19-9 levels in serum as well as Rac1, Wnt3a, PLD2 and FAM96B protein expression in transplantation tumor lesions of Runx2 knockdown group were significantly lower than those of NC group while MS4A12, ASPP2 and Fas protein expression in transplantation tumor lesions of Runx2 knockdown group were significantly higher than those of NC group. Conclusion: Transcription factor Runx2 knockdown could inhibit the colon cancer transplantation tumor growth in vitro.

Continued colonization of the human genome by mitochondrial DNA.

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Miria Ricchetti

2004-09-01

Full Text Available Integration of mitochondrial DNA fragments into nuclear chromosomes (giving rise to nuclear DNA sequences of mitochondrial origin, or NUMTs is an ongoing process that shapes nuclear genomes. In yeast this process depends on double-strand-break repair. Since NUMTs lack amplification and specific integration mechanisms, they represent the prototype of exogenous insertions in the nucleus. From sequence analysis of the genome of Homo sapiens, followed by sampling humans from different ethnic backgrounds, and chimpanzees, we have identified 27 NUMTs that are specific to humans and must have colonized human chromosomes in the last 4-6 million years. Thus, we measured the fixation rate of NUMTs in the human genome. Six such NUMTs show insertion polymorphism and provide a useful set of DNA markers for human population genetics. We also found that during recent human evolution, Chromosomes 18 and Y have been more susceptible to colonization by NUMTs. Surprisingly, 23 out of 27 human-specific NUMTs are inserted in known or predicted genes, mainly in introns. Some individuals carry a NUMT insertion in a tumor-suppressor gene and in a putative angiogenesis inhibitor. Therefore in humans, but not in yeast, NUMT integrations preferentially target coding or regulatory sequences. This is indeed the case for novel insertions associated with human diseases and those driven by environmental insults. We thus propose a mutagenic phenomenon that may be responsible for a variety of genetic diseases in humans and suggest that genetic or environmental factors that increase the frequency of chromosome breaks provide the impetus for the continued colonization of the human genome by mitochondrial DNA.

In vitro study of alpha 2-adrenoceptor turnover and metabolism using the adenocarcinoma cell line HT29

International Nuclear Information System (INIS)

Paris, H.; Taouis, M.; Galitzky, J.

1987-01-01

The biosynthesis rate of the receptor was studied in postconfluent HT29 cells, when its density expressed as fmol/mg of cell membrane protein is constant, by following the recovery of the receptor binding capacity after blockade with the non-reversible alpha-adrenergic antagonist benextramine. Study of the inhibition of [ 3 H]yohimbine and [ 3 H]UK-14,304 binding showed that benextramine was a more potent antagonist at alpha 2-adrenoceptor than phenoxybenzamine. The incubation of intact HT29 cells for 30 min in the presence of 10(-5) M benextramine irreversibly blocked more than 95% of the alpha 2-adrenoceptors and totally suppressed the inhibitory effect of UK-14,304 on cyclic AMP production. The blockade appeared specific, since benextramine effects were prevented by alpha 2-adrenergic agents. Moreover, neither vasoactive intestinal polypeptide responsiveness nor other tested aspects of the regulation of the adenylate cyclase was altered by the treatment. Study of the time course of receptor recovery after irreversible blockade indicated that alpha 2-adrenoceptors reappeared in the cells with a monoexponential kinetic. The linearization of the repopulation curve obtained with the labeled antagonist [ 3 H]yohimbine allowed the determination of the rate constant for receptor degradation (k = 0.0268 +/- 0.0025 hr-1) and the rate of receptor synthesis (6.91 +/- 0.64 fmol/mg of cell membrane protein/hr) corresponding to the synthesis of about 500 receptors/cell/hr. The alpha 2-adrenoceptor half-life was 26 +/- 3 hr. Measurement of the biological effects associated to the alpha-adrenoceptor stimulation during the course of receptor recovery indicated a relationship between the number of cell receptors and the percentage of inhibition of the cyclic AMP accumulation induced by forskolin

Inhibitory effects of different forms of tocopherols, tocopherol phosphates, and tocopherol quinones on growth of colon cancer cells.

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Dolfi, Sonia C; Yang, Zhihong; Lee, Mao-Jung; Guan, Fei; Hong, Jungil; Yang, Chung S

2013-09-11

Tocopherols are the major source of dietary vitamin E. In this study, the growth inhibitory effects of different forms of tocopherols (T), tocopheryl phosphates (TP), and tocopherol quinones (TQ) on human colon cancer HCT116 and HT29 cells were investigated. δ-T was more active than γ-T in inhibiting colon cancer cell growth, decreasing cancer cell colony formation, and inducing apoptosis; however, α-T was rather ineffective. Similarly, the rate of cellular uptake also followed the ranking order δ-T > γ-T ≫ α-T. TP and TQ generally had higher inhibitory activities than their parent compounds. Interestingly, the γ forms of TP and TQ were more active than the δ forms in inhibiting cancer cell growth, whereas the α forms were the least effective. The potencies of γ-TQ and δ-TQ (showing IC50 values of ∼0.8 and ∼2 μM on HCT116 cells after a 72 h incubation, respectively) were greater than 100-fold and greater than 20-fold higher, respectively, than those of their parent tocopherols. Induction of cancer cell apoptosis by δ-T, γ-TP, and γ-TQ was characterized by the cleavage of caspase 3 and PARP1 and DNA fragmentation. These studies demonstrated the higher growth inhibitory activity of δ-T than γ-T, the even higher activities of the γ forms of TP and TQ, and the ineffectiveness of the α forms of tocopherol and their metabolites against colon cancer cells.

Rab27A mediated by NF-κB promotes the stemness of colon cancer cells via up-regulation of cytokine secretion.

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Feng, Feixue; Jiang, Yinghao; Lu, Huanyu; Lu, Xiaozhao; Wang, Shan; Wang, Lifeng; Wei, Mengying; Lu, Wei; Du, Zhichao; Ye, Zichen; Yang, Guodong; Yuan, Fang; Ma, Yanxia; Lei, Xiaoying; Lu, Zifan

2016-09-27

Recent evidences have unveiled critical roles of cancer stem cells (CSCs) in tumorigenicity, but how interactions between CSC and tumor environments help maintain CSC initiation remains obscure. The small GTPases Rab27A regulates autocrine and paracrine cytokines by monitoring exocytosis of extracellular vesicles, and is reported to promote certain tumor progression. We observe that overexpression of Rab27A increased sphere formation efficiency (SFE) by increasing the proportion of CD44+ and PKH26high cells in HT29 cell lines, and accelerating the growth of colosphere with higher percentage of cells at S phase. Mechanism study revealed that the supernatant derived from HT29 sphere after Rab27A overexpression was able to expand sphere numbers with elevated secretion of VEGF and TGF-β. In tumor implanting nude mice model, tumor initiation rates and tumor sizes were enhanced by Rab27A with obvious angiogenesis. As a contrast, knocking down Rab27A impaired the above effects. More importantly, the correlation between higher p65 level and Rab27A in colon sphere was detected, p65 was sufficient to induce up-regulation of Rab27A and a functional NF-κB binding site in the Rab27A promoter was demonstrated. Altogether, our findings reveal a unique mechanism that tumor environment related NF-κB signaling promotes various colon cancer stem cells (cCSCs) properties via an amplified paracrine mechanism regulated by higher Rab27A level.

Effect of the 5-HT4 receptor and serotonin transporter on visceral hypersensitivity in rats

International Nuclear Information System (INIS)

Chi, Yan; Liu, Xin-Guang; Wang, Hua-Hong; Li, Jun-Xia; Li, Yi-Xuan

2012-01-01

Visceral hypersensitivity plays an important role in motor and sensory abnormalities associated with irritable bowel syndrome, but the underlying mechanisms are not fully understood. The present study was designed to evaluate the expression of the 5-HT 4 receptor and the serotonin transporter (SERT) as well as their roles in chronic visceral hypersensitivity using a rat model. Neonatal male Sprague-Dawley rats received intracolonic injections of 0.5% acetic acid (0.3-0.5 mL at different times) between postnatal days 8 and 21 to establish an animal model of visceral hypersensitivity. On day 43, the threshold intensity for a visually identifiable contraction of the abdominal wall and body arching were recorded during rectal distention. Histological evaluation and the myeloperoxidase activity assay were performed to determine the severity of inflammation. The 5-HT 4 receptor and SERT expression of the ascending colon were monitored using immunohistochemistry and Western blot analyses; the plasma 5-HT levels were measured using an ELISA method. As expected, transient colonic irritation at the neonatal stage led to visceral hypersensitivity, but no mucosal inflammation was later detected during adulthood. Using this model, we found reduced SERT expression (0.298 ± 0.038 vs 0.634 ± 0.200, P < 0.05) and increased 5-HT 4 receptor expression (0.308 ± 0.017 vs 0.298 ± 0.021, P < 0.05). Treatment with fluoxetine (10 mg·kg −1 ·day −1 , days 36-42), tegaserod (1 mg·kg −1 ·day −1 , day 43), or the combination of both, reduced visceral hypersensitivity and plasma 5-HT levels. Fluoxetine treatment increased 5-HT 4 receptor expression (0.322 ± 0.020 vs 0.308 ± 0.017, P < 0.01) but not SERT expression (0.219 ± 0.039 vs 0.298 ± 0.038, P = 0.654). These results indicate that both the 5-HT 4 receptor and SERT play a role in the pathogenesis of visceral hypersensitivity, and its mechanism may be involved in the local 5-HT level

Effect of the 5-HT4 receptor and serotonin transporter on visceral hypersensitivity in rats

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Chi Yan

2012-10-01

Full Text Available Visceral hypersensitivity plays an important role in motor and sensory abnormalities associated with irritable bowel syndrome, but the underlying mechanisms are not fully understood. The present study was designed to evaluate the expression of the 5-HT4 receptor and the serotonin transporter (SERT as well as their roles in chronic visceral hypersensitivity using a rat model. Neonatal male Sprague-Dawley rats received intracolonic injections of 0.5% acetic acid (0.3-0.5 mL at different times between postnatal days 8 and 21 to establish an animal model of visceral hypersensitivity. On day 43, the threshold intensity for a visually identifiable contraction of the abdominal wall and body arching were recorded during rectal distention. Histological evaluation and the myeloperoxidase activity assay were performed to determine the severity of inflammation. The 5-HT4 receptor and SERT expression of the ascending colon were monitored using immunohistochemistry and Western blot analyses; the plasma 5-HT levels were measured using an ELISA method. As expected, transient colonic irritation at the neonatal stage led to visceral hypersensitivity, but no mucosal inflammation was later detected during adulthood. Using this model, we found reduced SERT expression (0.298 ± 0.038 vs 0.634 ± 0.200, P < 0.05 and increased 5-HT4 receptor expression (0.308 ± 0.017 vs 0.298 ± 0.021, P < 0.05. Treatment with fluoxetine (10 mg·kg-1·day-1, days 36-42, tegaserod (1 mg·kg-1·day-1, day 43, or the combination of both, reduced visceral hypersensitivity and plasma 5-HT levels. Fluoxetine treatment increased 5-HT4 receptor expression (0.322 ± 0.020 vs 0.308 ± 0.017, P < 0.01 but not SERT expression (0.219 ± 0.039 vs 0.298 ± 0.038, P = 0.654. These results indicate that both the 5-HT4 receptor and SERT play a role in the pathogenesis of visceral hypersensitivity, and its mechanism may be involved in the local 5-HT level.

Psychopharmacology of 5-HT{sub 1A} receptors

Energy Technology Data Exchange (ETDEWEB)

Cowen, Philip J

2000-07-01

Serotonin{sub 1A} (5-HT{sub 1A}) receptors are located on both 5-HT cell bodies where they act as inhibitory autoreceptors and at postsynaptic sites where they mediate the effects of 5-HT released from nerve terminals. The sensitivity of 5-HT{sub 1A} receptors in humans can be measured using the technique of pharmacological challenge. For example, acute administration of a selective 5-HT{sub 1A} receptor agonist, such as ipsapirone, decreases body temperature and increases plasma cortisol through activation of pre- and postsynaptic 5-HT{sub 1A} receptors, respectively. Use of this technique has demonstrated that unmedicated patients with major depression have decreased sensitivity of both pre- and postsynaptic 5-HT{sub 1A} receptors. Treatment with selective serotonin reuptake inhibitors further down-regulates 5-HT{sub 1A} receptor activity. Due to the hypotheses linking decreased sensitivity of 5-HT{sub 1A} autoreceptors with the onset of antidepressant activity, there is current interest in the therapeutic efficacy of combined treatment with selective serotonin reuptake inhibitors and 5-HT{sub 1A} receptor antagonists.

Aspirin acetylates multiple cellular proteins in HCT-116 colon cancer cells: Identification of novel targets.

Science.gov (United States)

Marimuthu, Srinivasan; Chivukula, Raghavender S V; Alfonso, Lloyd F; Moridani, Majid; Hagen, Fred K; Bhat, G Jayarama

2011-11-01

Epidemiological and clinical observations provide consistent evidence that regular intake of aspirin may effectively inhibit the occurrence of epithelial tumors; however, the molecular mechanisms are not completely understood. In the present study, we determined the ability of aspirin to acetylate and post-translationally modify cellular proteins in HCT-116 human colon cancer cells to understand the potential mechanisms by which it may exerts anti-cancer effects. Using anti-acetyl lysine antibodies, here we demonstrate that aspirin causes the acetylation of multiple proteins whose molecular weight ranged from 20 to 200 kDa. The identity of these proteins was determined, using immuno-affinity purification, mass spectrometry and immuno-blotting. A total of 33 cellular proteins were potential targets of aspirin-mediated acetylation, while 16 were identified as common to both the control and aspirin-treated samples. These include enzymes of glycolytic pathway, cytoskeleton proteins, histones, ribosomal and mitochondrial proteins. The glycolytic enzymes which were identified include aldolase, glyceraldehyde-3-phosphate dehydrogenase, enolase, pyruvate kinase M2, and lactate dehydrogenase A and B chains. Immunoblotting experiment showed that aspirin also acetylated glucose-6-phosphate dehydrogenase and transketolase, both enzymes of pentose phosphate pathway involved in ribonucleotide biosynthesis. In vitro assays of these enzymes revealed that aspirin did not affect pyruvate kinase and lactate dehydrogenase activity; however, it decreased glucose 6 phosphate dehydrogenase activity. Similar results were also observed in HT-29 human colon cancer cells. Selective inhibition of glucose-6-phosphate dehydrogenase may represent an important mechanism by which aspirin may exert its anti-cancer effects through inhibition of ribonucleotide synthesis.

Tuft (caveolated) cells in two human colon carcinoma cell lines.

OpenAIRE

Barkla, D. H.; Whitehead, R. H.; Foster, H.; Tutton, P. J.

1988-01-01

The presence of an unusual cell type in two human colon carcinoma cell lines is reported. The cells show the same morphology as "tuft" (caveolated) cells present in normal gastrointestinal epithelium. Tuft cells were seen in cell line LIM 1863 growing in vitro and in human colon carcinoma cell line LIM 2210 growing as subcutaneous solid tumour xenografts in nude mice. Characteristic morphologic features of tuft cells included a wide base, narrow apex and a tuft of long microvilli projecting f...

Preclinical In Vitro and In Vivo Evaluation of [18F]FE@SUPPY for Cancer PET Imaging: Limitations of a Xenograft Model for Colorectal Cancer

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T. Balber

2018-01-01

Full Text Available Molecular imaging probes such as PET-tracers have the potential to improve the accuracy of tumor characterization by directly visualizing the biochemical situation. Thus, molecular changes can be detected early before morphological manifestation. The A3 adenosine receptor (A3AR is described to be highly expressed in colon cancer cell lines and human colorectal cancer (CRC, suggesting this receptor as a tumor marker. The aim of this preclinical study was the evaluation of F18FE@SUPPY as a PET-tracer for CRC using in vitro imaging and in vivo PET imaging. First, affinity and selectivity of FE@SUPPY and its metabolites were determined, proving the favorable binding profile of FE@SUPPY. The human adenocarcinoma cell line HT-29 was characterized regarding its hA3AR expression and was subsequently chosen as tumor graft. Promising results regarding the potential of F18FE@SUPPY as a PET-tracer for CRC imaging were obtained by autoradiography as ≥2.3-fold higher accumulation of F18FE@SUPPY was found in CRC tissue compared to adjacent healthy colon tissue from the same patient. Nevertheless, first in vivo studies using HT-29 xenografts showed insufficient tumor uptake due to (1 poor conservation of target expression in xenografts and (2 unfavorable pharmacokinetics of F18FE@SUPPY in mice. We therefore conclude that HT-29 xenografts are not adequate to visualize hA3ARs using F18FE@SUPPY.

Measuring endogenous 5-HT release by emission tomography: promises and pitfalls

DEFF Research Database (Denmark)

Paterson, Louise M; Tyacke, Robin J; Nutt, David J

2010-01-01

Molecular in vivo neuroimaging techniques can be used to measure regional changes in endogenous neurotransmitters, evoked by challenges that alter synaptic neurotransmitter concentration. This technique has most successfully been applied to the study of endogenous dopamine release using positron......, with reference to the dopaminergic system. Studies that aim to image acute, endogenous 5-HT release or depletion at 5-HT receptor targets are summarised, with particular attention to studies in humans. Radiotracers targeting the 5-HT(1A), 5-HT(2A), and 5-HT(4) receptors and the serotonin reuptake transporter...... have been explored for their sensitivity to 5-HT fluctuations, but with mixed outcomes; tracers for these targets cannot reliably image endogenous 5-HT in humans. Shortcomings in our basic knowledge of the mechanisms underlying changes in binding potential are addressed, and suggestions are made...

Identification of downstream metastasis-associated target genes regulated by LSD1 in colon cancer cells.

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Chen, Jiang; Ding, Jie; Wang, Ziwei; Zhu, Jian; Wang, Xuejian; Du, Jiyi

2017-03-21

This study aims to identify downstream target genes regulated by lysine-specific demethylase 1 (LSD1) in colon cancer cells and investigate the molecular mechanisms of LSD1 influencing invasion and metastasis of colon cancer. We obtained the expression changes of downstream target genes regulated by small-interfering RNA-LSD1 and LSD1-overexpression via gene expression profiling in two human colon cancer cell lines. An Affymetrix Human Transcriptome Array 2.0 was used to identify differentially expressed genes (DEGs). We screened out LSD1-target gene associated with proliferation, metastasis, and invasion from DEGs via Gene Ontology and Pathway Studio. Subsequently, four key genes (CABYR, FOXF2, TLE4, and CDH1) were computationally predicted as metastasis-related LSD1-target genes. ChIp-PCR was applied after RT-PCR and Western blot validations to detect the occupancy of LSD1-target gene promoter-bound LSD1. A total of 3633 DEGs were significantly upregulated, and 4642 DEGs were downregulated in LSD1-silenced SW620 cells. A total of 4047 DEGs and 4240 DEGs were upregulated and downregulated in LSD1-overexpressed HT-29 cells, respectively. RT-PCR and Western blot validated the microarray analysis results. ChIP assay results demonstrated that LSD1 might be negative regulators for target genes CABYR and CDH1. The expression level of LSD1 is negatively correlated with mono- and dimethylation of histone H3 lysine4(H3K4) at LSD1- target gene promoter region. No significant mono-methylation and dimethylation of H3 lysine9 methylation was detected at the promoter region of CABYR and CDH1. LSD1- depletion contributed to the upregulation of CABYR and CDH1 through enhancing the dimethylation of H3K4 at the LSD1-target genes promoter. LSD1- overexpression mediated the downregulation of CABYR and CDH1expression through decreasing the mono- and dimethylation of H3K4 at LSD1-target gene promoter in colon cancer cells. CABYR and CDH1 might be potential LSD1-target genes in colon

EMT is the dominant program in human colon cancer

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Tollenaar Rob AEM

2011-01-01

Full Text Available Abstract Background Colon cancer has been classically described by clinicopathologic features that permit the prediction of outcome only after surgical resection and staging. Methods We performed an unsupervised analysis of microarray data from 326 colon cancers to identify the first principal component (PC1 of the most variable set of genes. PC1 deciphered two primary, intrinsic molecular subtypes of colon cancer that predicted disease progression and recurrence. Results Here we report that the most dominant pattern of intrinsic gene expression in colon cancer (PC1 was tightly correlated (Pearson R = 0.92, P -135 with the EMT signature-- both in gene identity and directionality. In a global micro-RNA screen, we further identified the most anti-correlated microRNA with PC1 as MiR200, known to regulate EMT. Conclusions These data demonstrate that the biology underpinning the native, molecular classification of human colon cancer--previously thought to be highly heterogeneous-- was clarified through the lens of comprehensive transcriptome analysis.

Strategies For Human Exploration Leading To Human Colonization of Space

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Smitherman, David; Everett, Harmon

2009-01-01

Enabling the commercial development of space is key to the future colonization of space and key to a viable space exploration program. Without commercial development following in the footsteps of exploration it is difficult to justify and maintain public interest in the efforts. NASA's exploration program has suffered from the lack of a good commercial economic strategy for decades. Only small advances in commercial space have moved forward, and only up to Earth orbit with the commercial satellite industry. A way to move beyond this phase is to begin the establishment of human commercial activities in space in partnership with the human exploration program. In 2007 and 2008, the authors researched scenarios to make space exploration and commercial space development more feasible as part of their graduate work in the Space Architecture Program at the Sasakawa International Center for Space Architecture at the University of Houston, Houston, Texas. Through this research it became apparent that the problems facing future colonization are much larger than the technology being developed or the international missions that our space agencies are pursuing. These issues are addressed in this paper with recommendations for space exploration, commercial development, and space policy that are needed to form a strategic plan for human expansion into space. In conclusion, the authors found that the current direction in space as carried out by our space agencies around the world is definitely needed, but is inadequate and incapable of resolving all of the issues that inhibit commercial space development. A bolder vision with strategic planning designed to grow infrastructures and set up a legal framework for commercial markets will go a long way toward enabling the future colonization of space.

Characterization and significance of ACE2 and Mas receptor in human colon adenocarcinoma.

Science.gov (United States)

Bernardi, Stella; Zennaro, Cristina; Palmisano, Silvia; Velkoska, Elena; Sabato, Nicoletta; Toffoli, Barbara; Giacomel, Greta; Buri, Luigi; Zanconati, Fabrizio; Bellini, Giuseppe; Burrell, Louise M; De Manzini, Nicolò; Fabris, Bruno

2012-03-01

A new arm of the renin-angiotensin system (RAS) has been recently characterized; this includes angiotensin converting enzyme (ACE)2 and angiotensin (Ang)1-7, a heptapeptide acting through the Mas receptor (MasR). Recent studies show that Ang1-7 has an antiproliferative action on lung adenocarcinoma cells. The aim of this study was to characterize RAS expression in human colon adenocarcinoma and to investigate whether Ang1-7 exerts an antiproliferative effect on human colon adenocarcinoma cells. Gene, protein expression and enzymatic activity of the main components of the RAS were determined on non-neoplastic colon mucosa as well as on the tumor mass and the mucosa taken 5 cm distant from it, both collected from patients with colon adenocarcinoma. Two different human colon cancer cell lines were treated with AngII and Ang1-7. The novel finding of this study was that MasR was significantly upregulated in colon adenocarcinoma compared with non-neoplastic colon mucosa, which showed little or no expression of it. ACE gene expression and enzymatic activity were also increased in the tumors. However, AngII and Ang1-7 did not have any pro-/antiproliferative effects in the cell lines studied. The data suggest that upregulation of the MasR could be used as a diagnostic marker of colon adenocarcinoma.

Colonic complications following human bone marrow transplantation

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Paulino Martínez Hernández-Magro

2015-01-01

Full Text Available Background: Human bone marrow transplantation (BMT becomes an accepted treatment of leukemia, aplastic anemia, immunodeficiency syndromes, and hematologic malignancies. Colorectal surgeons must know how to determine and manage the main colonic complications. Objective: To review the clinical features, clinical and pathological staging of graft vs host disease (GVHD, and treatment of patients suffering with colonic complications of human bone marrow transplantation. Patients and methods: We have reviewed the records of all patients that received an allogeneic bone marrow transplant and were evaluated at our Colon and Rectal Surgery department due to gastrointestinal symptoms, between January 2007 and January 2012. The study was carried out in patients who developed colonic complications, all of them with clinical, histopathological or laboratory diagnosis. Results: The study group was constituted by 77 patients, 43 male and 34 female patients. We identified colonic complications in 30 patients (38.9%; five patients developed intestinal toxicity due to pretransplant chemotherapy (6.4%; graft vs. host disease was present in 16 patients (20%; 13 patients (16.8% developed acute colonic GVHD, and 3 (3.8% chronic GVHD. Infection was identified in 9 patients (11.6%. Conclusions: The three principal colonic complications are the chemotherapy toxicity, GVHD, and superinfection; the onset of symptoms could help to suspect the type of complication (0–20 day chemotherapy toxicity, 20 and more GVHD, and infection could appear in any time of transplantation. Resumo: Experiência: O transplante de medula óssea humana (MOH passou a ser um tratamento adotado para leucemia, anemia aplástica, síndromes de imunodeficiência e neoplasias hematológicas. Cirurgiões colorretais devem saber como determinar e tratar as principais complicações do cólon. Objetivo: Revisar as características clínicas, estadiamentos clínico e patológico da doença do enxerto

PHP-HT (VitaResc Biotech).

Science.gov (United States)

Baldwin, A; Wiley, E

2001-04-01

VitaResc (formerly Apex) is developing PHP-HT, pyridoxalated hemoglobin polyoxyethylene conjugate, for the potential treatment of nitric oxide-induced shock (characterized by hypotension), associated with various etiologies, initially in septic shock. A phase I safety study and an initial phase I/II patient trial for NO-induced shock have been completed, and VitaResc has enrolled patients in three of five planned cohorts in a continuation of these trials to include a protocol of continuous infusion and dose escalation [330680,349187,390918]. The results from the dose escalation trials are expected to provide the basis for a randomized, controlled phase II/III pivotal trial of PHP-HT [390918]. VitaResc has licensed PHP-HT exclusively from Ajinomoto for all indications, worldwide, except Japan [275263]. Ajinomoto originally developed the human derived and chemically modified hemoglobin preparation as a blood substitute, but no development has been reported by the company since 1997 [275277,303577]. The other potential indications of PHP-HT include shock associated with burns, pancreatitis, hemodialysis and cytokine therapies [275277]. VitaResc expects the annual market potential of PHP-HT to exceed 1 billion dollars [330680].

The G-protein coupled chemoattractant receptor FPR2 promotes malignant phenotype of human colon cancer cells

Science.gov (United States)

Xiang, Yi; Yao, Xiaohong; Chen, Keqiang; Wang, Xiafei; Zhou, Jiamin; Gong, Wanghua; Yoshimura, Teizo; Huang, Jiaqiang; Wang, Rongquan; Wu, Yuzhang; Shi, Guochao; Bian, Xiuwu; Wang, Jiming

2016-01-01

The G-protein coupled chemoattractant receptor formylpeptide receptor-2 (FPR2 in human, Fpr2 in mice) is expressed by mouse colon epithelial cells and plays a critical role in mediating mucosal homeostasis and inflammatory responses. However, the biological role of FPR2 in human colon is unclear. Our investigation revealed that a considerable number of human colon cancer cell lines expressed FPR2 and its ligands promoted cell migration and proliferation. Human colon cancer cell lines expressing high levels of FPR2 also formed more rapidly growing tumors in immunocompromised mice as compared with cell lines expressing lower levels of FPR2. Knocking down of FPR2 from colon cancer cell lines highly expressing FPR2 reduced their tumorigenicity. Clinically, FPR2 is more highly expressed in progressive colon cancer, associated with poorer patient prognosis. These results suggest that FPR2 can be high-jacked by colon cancer cells for their growth advantage, thus becoming a potential target for therapeutic development. PMID:27904774

Acetylcholine release by human colon cancer cells mediates autocrine stimulation of cell proliferation.

Science.gov (United States)

Cheng, Kunrong; Samimi, Roxana; Xie, Guofeng; Shant, Jasleen; Drachenberg, Cinthia; Wade, Mark; Davis, Richard J; Nomikos, George; Raufman, Jean-Pierre

2008-09-01

Most colon cancers overexpress M3 muscarinic receptors (M3R), and post-M3R signaling stimulates human colon cancer cell proliferation. Acetylcholine (ACh), a muscarinic receptor ligand traditionally regarded as a neurotransmitter, may be produced by nonneuronal cells. We hypothesized that ACh release by human colon cancer cells results in autocrine stimulation of proliferation. H508 human colon cancer cells, which have robust M3R expression, were used to examine effects of muscarinic receptor antagonists, acetylcholinesterase inhibitors, and choline transport inhibitors on cell proliferation. A nonselective muscarinic receptor antagonist (atropine), a selective M3R antagonist (p-fluorohexahydro-sila-difenidol hydrochloride), and a choline transport inhibitor (hemicholinum-3) all inhibited unstimulated H508 colon cancer cell proliferation by approximately 40% (P<0.005). In contrast, two acetylcholinesterase inhibitors (eserine-hemisulfate and bis-9-amino-1,2,3,4-tetrahydroacridine) increased proliferation by 2.5- and 2-fold, respectively (P<0.005). By using quantitative real-time PCR, expression of choline acetyltransferase (ChAT), a critical enzyme for ACh synthesis, was identified in H508, WiDr, and Caco-2 colon cancer cells. By using high-performance liquid chromatography-electrochemical detection, released ACh was detected in H508 and Caco-2 cell culture media. Immunohistochemistry in surgical specimens revealed weak or no cytoplasmic staining for ChAT in normal colon enterocytes (n=25) whereas half of colon cancer specimens (n=24) exhibited moderate to strong staining (P<0.005). We conclude that ACh is an autocrine growth factor in colon cancer. Mechanisms that regulate colon epithelial cell production and release of ACh warrant further investigation.

Dispersal time for ancient human migrations: Americas and Europe colonization

Science.gov (United States)

Flores, J. C.

2007-07-01

I apply the recently proposed intermittence strategy to investigate the ancient human migrations in the world. That is, the Americas colonization (Bering-bridge and Pacific-coast theories) and Neanderthal replacement in Europe around 45000 years before the present. Using a mathematical equation related to diffusion and ballistic motion, I calculate the colonization time in all these cases in good agreement with archeological data (including Neolithic transition in Europe). Moreover, to support these calculations, I obtain analytically the effective speed of colonization in Europe veff=0.62 [km/yr] and related to the Aurignacian culture propagation.

Human Milk Hyaluronan Enhances Innate Defense of the Intestinal Epithelium*

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Hill, David R.; Rho, Hyunjin K.; Kessler, Sean P.; Amin, Ripal; Homer, Craig R.; McDonald, Christine; Cowman, Mary K.; de la Motte, Carol A.

2013-01-01

Breast-feeding is associated with enhanced protection from gastrointestinal disease in infants, mediated in part by an array of bioactive glycan components in milk that act through molecular mechanisms to inhibit enteric pathogen infection. Human milk contains hyaluronan (HA), a glycosaminoglycan polymer found in virtually all mammalian tissues. We have shown that synthetic HA of a specific size range promotes expression of antimicrobial peptides in intestinal epithelium. We hypothesize that hyaluronan from human milk also enhances innate antimicrobial defense. Here we define the concentration of HA in human milk during the first 6 months postpartum. Importantly, HA isolated from milk has a biological function. Treatment of HT-29 colonic epithelial cells with human milk HA at physiologic concentrations results in time- and dose-dependent induction of the antimicrobial peptide human β-defensin 2 and is abrogated by digestion of milk HA with a specific hyaluronidase. Milk HA induction of human β-defensin 2 expression is also reduced in the presence of a CD44-blocking antibody and is associated with a specific increase in ERK1/2 phosphorylation, suggesting a role for the HA receptor CD44. Furthermore, oral administration of human milk-derived HA to adult, wild-type mice results in induction of the murine Hβ D2 ortholog in intestinal mucosa and is dependent upon both TLR4 and CD44 in vivo. Finally, treatment of cultured colonic epithelial cells with human milk HA enhances resistance to infection by the enteric pathogen Salmonella typhimurium. Together, our observations suggest that maternally provided HA stimulates protective antimicrobial defense in the newborn. PMID:23950179

Agonist/antagonist interactions with cloned human 5-HT(1A) receptors: Variations in intrinsic activity studied in transfected HeLa cells

NARCIS (Netherlands)

Boddeke, H.W.G.M.; Fargin, A.; Raymond, J.R.; Schoeffter, P.; Hoyer, D.

1992-01-01

The characteristics of 5-HT(1A)-recognition sites and receptor-mediated release of intracellular calcium were established in two transfected HeLa cell lines (HA 6 and HA 7) expressing different levels of human 5-HT(1A) receptors (about 3000 and 500 fmol/mg protein, Fargin et al. 1989; 1991; Raymond

Comparison of photocytotoxicyty of PDT with hypericin by model of healthy versus malignant colon epithelium cells

International Nuclear Information System (INIS)

Mikes, J.; Kleban, J.; Jendzelovsky, R.; Solar, P.; Fedorocko, P.; Hyzdalova, M.

2006-01-01

Photodynamic therapy (PDT) is becoming a rapidly developing method in cancer therapy, recently. PDT is based on administration of nontoxic/weakly toxic photosensitive compound and its activation with light. The phototoxicity of PDT depends on generation of superoxide radicals (Type-I reaction), which in turn might form peroxide and hydroxyl radicals, and production of singlet oxygen ( 1 O 2 ) (Type-II reaction) after irradiation with light of appropriate wavelength which properly overlaps the photosensitizer's absorbing spectra. Oxidative damage in the cell induced by reactive oxygen species depends on the intracellular localisation and affects different cell organelles. Although PDT is of use in clinical practise, new promising photosensitive compounds with advantageous attributes are discovered continuously. Hypericin, one of these compounds, is known to affect cell cycle and proliferation, to alter gene expression and to induce cell death. Due to its spectral characteristics, hypericin is applicable for treatment of superficial malignancies and therefore also for treatment of colon adenocarcinomas. We compared two cell lines of identical histological origin, one as a model of colon adenocarcinoma (HT29) and second as a model of healthy colon epithelium, to evaluate photo-cytotoxicity of PDT with hypericin to healthy tissue and determine applicability of this therapy in treatment of colon malignancies. (authors)

Colonic transit time is related to bacterial metabolism and mucosal turnover in the human gut

DEFF Research Database (Denmark)

Roager, Henrik Munch; Hansen, Lea Benedicte Skov; Bahl, Martin Iain

Little is known about how colonic transit time relates to human colonic metabolism, and its importance for host health, although stool consistency, a proxy for colonic transit time, has recently been negatively associated with gut microbial richness. To address the relationships between colonic t...... imply a healthy gut microbial ecosystem and points at colonic transit time as a highly important factor to consider in microbiome and metabolomics studies.......Little is known about how colonic transit time relates to human colonic metabolism, and its importance for host health, although stool consistency, a proxy for colonic transit time, has recently been negatively associated with gut microbial richness. To address the relationships between colonic...... transit time and the gut microbial composition and metabolism, we assessed the colonic transit time of 98 subjects using radiopaque markers, and profiled their gut microbiota by16S rRNA gene sequencing and their urine metabolome by ultra performance liquid chromatography mass spectrometry. Based...

A silyl andrographolide analogue suppresses Wnt/β-catenin signaling pathway in colon cancer.

Science.gov (United States)

Reabroi, Somrudee; Chairoungdua, Arthit; Saeeng, Rungnapha; Kasemsuk, Teerapich; Saengsawang, Witchuda; Zhu, Weiming; Piyachaturawat, Pawinee

2018-05-01

Hyperactivation of Wnt/β-catenin signaling implicated in oncogenesis of colorectal cancer (CRC) is a potential molecular target for chemotherapy. An andrographolide analogue, 3A.1 (19-tert-butyldiphenylsilyl-8, 17-epoxy andrographolide) has previously been reported to be potently cytotoxic toward cancer cells by unknown molecular mechanisms. The present study explored the anti-cancer activity of analogue 3A.1 on Wnt/β-catenin signaling in colon cancer cells (HT29 cells) which were more sensitive to the others (HCT116 and SW480 cells). Analogue 3A.1 inhibited viability of HT29 cells with IC 50 value of 11.1 ± 1.4 μM at 24 h, which was more potent than that of the parent andrographolide. Analogue 3A.1 also suppressed the proliferation of HT29 cells and induced cell apoptosis in a dose-dependent manner. Its apoptotic activity was accompanied with increased expressions of proteins related to DNA damages; PARP-1 and γ-H2AX. In addition, analogue 3A.1 significantly inhibited T-cell factor and lymphoid enhancer factor (TCF/LEF) promoter activity of Wnt/β-catenin signaling. Accordingly, the expressions of Wnt target genes and β-catenin protein were suppressed. Moreover, analogue 3A.1 increased the activity of GSK-3β kinase, which is a negative regulator responsible for degradation of intracellular β-catenin. This mode of action was further supported by the absence of the effects after treatment with a GSK-3β inhibitor, and over-expression of a mutant β-catenin (S33Y). Our findings reveal, for the first time, an insight into the molecular mechanism of the anti-cancer activity of analogue 3A.1 through the inhibition of Wnt/β-catenin/GSK-3β pathway and provide a therapeutic potential of the andrographolide analogue 3A.1 in CRC treatment. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Effect of Uncaria tomentosa Extract on Apoptosis Triggered by Oxaliplatin Exposure on HT29 Cells

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Liliane Z. de Oliveira

2014-01-01

Full Text Available Background/Aim. The use of herbal products as a supplement to minimize the effects of chemotherapy for cancer treatment requires further attention with respect to the activity and toxicity of chemotherapy. Uncaria tomentosa extract, which contains oxindole alkaloids, is one of these herbal products. The objective of this study was to evaluate whether Uncaria tomentosa extract modulates apoptosis induced by chemotherapy exposure. Materials and Methods. Colorectal adenocarcinoma cells (HT29 cells were grown in the presence of oxaliplatin and/or Uncaria tomentosa extract. Results. The hydroalcoholic extract of Uncaria tomentosa enhanced chemotherapy-induced apoptosis, with an increase in the percentage of Annexin positive cells, an increase in caspase activities, and an increase of DNA fragments in culture of the neoplastic cells. Moreover, antioxidant activity may be related to apoptosis. Conclusion. Uncaria tomentosa extract has a role for cancer patients as a complementary therapy. Further studies evaluating these beneficial effects with other chemotherapy drugs are recommended.

Induction of farnesoid X receptor signaling in germ-free mice colonized with a human microbiota

DEFF Research Database (Denmark)

Wahlström, Annika; Kovatcheva-Datchary, Petia; Ståhlman, Marcus

2017-01-01

The gut microbiota influences the development and progression of metabolic diseases partly by metabolism of bile acids (BAs) and modified signaling through the farnesoid X receptor (FXR). In this study, we aimed to determine how the human gut microbiota metabolizes murine BAs and affects FXR...... signaling in colonized mice. We colonized germ-free mice with cecal content from a mouse donor or feces from a human donor and euthanized the mice after short-term (2 weeks) or long-term (15 weeks) colonization. We analyzed the gut microbiota and BA composition and expression of FXR target genes in ileum...... and liver. We found that cecal microbiota composition differed between mice colonized with mouse and human microbiota and was stable over time. Human and mouse microbiota reduced total BA levels similarly, but the humanized mice produced less secondary BAs. The human microbiota was able to reduce the levels...

Human colon tissue in organ culture: calcium and multi-mineral-induced mucosal differentiation.

Science.gov (United States)

Dame, Michael K; Veerapaneni, Indiradevi; Bhagavathula, Narasimharao; Naik, Madhav; Varani, James

2011-01-01

We have recently shown that a multi-mineral extract from the marine red algae, Lithothamnion calcareum, suppresses colon polyp formation and inflammation in mice. In the present study, we used intact human colon tissue in organ culture to compare responses initiated by Ca(2+) supplementation versus the multi-mineral extract. Normal human colon tissue was treated for 2 d in culture with various concentrations of calcium or the mineral-rich extract. The tissue was then prepared for histology/immunohistochemistry, and the culture supernatants were assayed for levels of type I procollagen and type I collagen. At higher Ca(2+) concentrations or with the mineral-rich extract, proliferation of epithelial cells at the base and walls of the mucosal crypts was suppressed, as visualized by reduced Ki67 staining. E-cadherin, a marker of differentiation, was more strongly expressed at the upper third of the crypt and at the luminal surface. Treatment with Ca(2+) or with the multi-mineral extract influenced collagen turnover, with decreased procollagen and increased type I collagen. These data suggest that calcium or mineral-rich extract has the capacity to (1) promote differentiation in human colon tissue in organ culture and (2) modulate stromal function as assessed by increased levels of type I collagen. Taken together, these data suggest that human colon tissue in organ culture (supporting in vivo finding in mice) will provide a valuable model for the preclinical assessment of agents that regulate growth and differentiation in the colonic mucosa.

Synthesis and biological evaluation of 6H-1-benzopyrano[4,3-b]quinolin-6-one derivatives as inhibitors of colon cancer cell growth

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Tie-Ling Li

2015-08-01

Full Text Available A convenient synthesis of 6H-1-benzopyrano[4,3-b]quinolin-6-one derivatives was reported using 4-chloro-2-oxo-2H-chromene-3-carbaldehyde with different aromatic amines using silica sulfuric acid. The compounds were tested for their anticancer activity against colon (HCT-116 and S1-MI-80, prostate (PC3 and DU-145, breast (MCF-7 and MDAMB-231 cancer cells. These com-pounds showed more selectivity and potent cytotoxic activity against colon cancer cells. 3c was tested against five other colon cancer cell lines (HT-29, HCT-15, LS-180, LS-174, and LoVo, which had similar cytotoxicity and selectivity. 3c did not induce PXR-regulated ABCB1 or ABCG2 transporters. In fact, 3c induced cytotoxicity in HEK293 cells over expressing ABCB1 or ABCG2 to the same extent as in normal HEK293 cells. It was cytotoxic approximately 3- and 5-fold to resistant colon carcinoma S1-MI-80 cells. 3c also produced concentration-dependent changes in HCT-116 colon cancer cells, in mitochondrial membrane potential, leading to apoptosis, and sub-micromolar concentrations caused chromosomal DNA fragmentation.

Inferring human colonization history using a copying model.

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Garrett Hellenthal

2008-05-01

Full Text Available Genome-wide scans of genetic variation can potentially provide detailed information on how modern humans colonized the world but require new methods of analysis. We introduce a statistical approach that uses Single Nucleotide Polymorphism (SNP data to identify sharing of chromosomal segments between populations and uses the pattern of sharing to reconstruct a detailed colonization scenario. We apply our model to the SNP data for the 53 populations of the Human Genome Diversity Project described in Conrad et al. (Nature Genetics 38,1251-60, 2006. Our results are consistent with the consensus view of a single "Out-of-Africa" bottleneck and serial dilution of diversity during global colonization, including a prominent East Asian bottleneck. They also suggest novel details including: (1 the most northerly East Asian population in the sample (Yakut has received a significant genetic contribution from the ancestors of the most northerly European one (Orcadian. (2 Native North [corrected] Americans have received ancestry from a source closely related to modern North-East Asians (Mongolians and Oroquen that is distinct from the sources for native South [corrected] Americans, implying multiple waves of migration into the Americas. A detailed depiction of the peopling of the world is available in animated form.

Human Colon Tumors Express a Dominant-Negative Form of SIGIRR That Promotes Inflammation and Colitis-Associated Colon Cancer in Mice.

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Zhao, Junjie; Bulek, Katarzyna; Gulen, Muhammet F; Zepp, Jarod A; Karagkounis, Georgio; Martin, Bradley N; Zhou, Hao; Yu, Minjia; Liu, Xiuli; Huang, Emina; Fox, Paul L; Kalady, Matthew F; Markowitz, Sanford D; Li, Xiaoxia

2015-12-01

Single immunoglobulin and toll-interleukin 1 receptor (SIGIRR), a negative regulator of the Toll-like and interleukin-1 receptor (IL-1R) signaling pathways, controls intestinal inflammation and suppresses colon tumorigenesis in mice. However, the importance of SIGIRR in human colorectal cancer development has not been determined. We investigated the role of SIGIRR in development of human colorectal cancer. We performed RNA sequence analyses of pairs of colon tumor and nontumor tissues, each collected from 68 patients. Immunoblot and immunofluorescence analyses were used to determine levels of SIGIRR protein in primary human colonic epithelial cells, tumor tissues, and colon cancer cell lines. We expressed SIGIRR and mutant forms of the protein in Vaco cell lines. We created and analyzed mice that expressed full-length (control) or a mutant form of Sigirr (encoding SIGIRR(N86/102S), which is not glycosylated) specifically in the intestinal epithelium. Some mice were given azoxymethane (AOM) and dextran sulfate sodium to induce colitis-associated cancer. Intestinal tissues were collected and analyzed by immunohistochemical and gene expression profile analyses. RNA sequence analyses revealed increased expression of a SIGIRR mRNA isoform, SIGIRR(ΔE8), in colorectal cancer tissues compared to paired nontumor tissues. SIGIRR(ΔE8) is not modified by complex glycans and is therefore retained in the cytoplasm-it cannot localize to the cell membrane or reduce IL1R signaling. SIGIRR(ΔE8) interacts with and has a dominant-negative effect on SIGIRR, reducing its glycosylation, localization to the cell surface, and function. Most SIGIRR detected in human colon cancer tissues was cytoplasmic, whereas in nontumor tissues it was found at the cell membrane. Mice that expressed SIGIRR(N86/102S) developed more inflammation and formed larger tumors after administration of azoxymethane and dextran sulfate sodium than control mice; colon tissues from these mutant mice expressed

The first microbial colonizers of the human gut

NARCIS (Netherlands)

Milani, Christian; Duranti, Sabrina; Bottacini, Francesca; Casey, Eoghan; Turroni, Francesca; Mahony, Jennifer; Belzer, Clara; Palacio, Susana Delgado; Montes, Silvia Arboleya; Mancabelli, Leonardo; Lugli, Gabriele Andrea; Rodriguez, Juan Miguel; Bode, Lars; Vos, De Willem; Gueimonde, Miguel; Margolles, Abelardo; Sinderen, Van Douwe; Ventura, Marco

2017-01-01

The human gut microbiota is engaged in multiple interactions affecting host health during the host's entire life span. Microbes colonize the neonatal gut immediately following birth. The establishment and interactive development of this early gut microbiota are believed to be (at least partially)

Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.

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Sato, Toshiro; Stange, Daniel E; Ferrante, Marc; Vries, Robert G J; Van Es, Johan H; Van den Brink, Stieneke; Van Houdt, Winan J; Pronk, Apollo; Van Gorp, Joost; Siersema, Peter D; Clevers, Hans

2011-11-01

We previously established long-term culture conditions under which single crypts or stem cells derived from mouse small intestine expand over long periods. The expanding crypts undergo multiple crypt fission events, simultaneously generating villus-like epithelial domains that contain all differentiated types of cells. We have adapted the culture conditions to grow similar epithelial organoids from mouse colon and human small intestine and colon. Based on the mouse small intestinal culture system, we optimized the mouse and human colon culture systems. Addition of Wnt3A to the combination of growth factors applied to mouse colon crypts allowed them to expand indefinitely. Addition of nicotinamide, along with a small molecule inhibitor of Alk and an inhibitor of p38, were required for long-term culture of human small intestine and colon tissues. The culture system also allowed growth of mouse Apc-deficient adenomas, human colorectal cancer cells, and human metaplastic epithelia from regions of Barrett's esophagus. We developed a technology that can be used to study infected, inflammatory, or neoplastic tissues from the human gastrointestinal tract. These tools might have applications in regenerative biology through ex vivo expansion of the intestinal epithelia. Studies of these cultures indicate that there is no inherent restriction in the replicative potential of adult stem cells (or a Hayflick limit) ex vivo. Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

Magnetic Resonance Imaging Quantification of Fasted State Colonic Liquid Pockets in Healthy Humans.

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Murray, Kathryn; Hoad, Caroline L; Mudie, Deanna M; Wright, Jeff; Heissam, Khaled; Abrehart, Nichola; Pritchard, Susan E; Al Atwah, Salem; Gowland, Penny A; Garnett, Martin C; Amidon, Gregory E; Spiller, Robin C; Amidon, Gordon L; Marciani, Luca

2017-08-07

The rate and extent of drug dissolution and absorption from solid oral dosage forms is highly dependent on the volume of liquid in the gastrointestinal tract (GIT). However, little is known about the time course of GIT liquid volumes after drinking a glass of water (8 oz), particularly in the colon, which is a targeted site for both locally and systemically acting drug products. Previous magnetic resonance imaging (MRI) studies offered novel insights on GIT liquid distribution in fasted humans in the stomach and small intestine, and showed that freely mobile liquid in the intestine collects in fairly distinct regions or "pockets". Based on this previous pilot data, we hypothesized that (1) it is possible to quantify the time course of the volume and number of liquid pockets in the undisturbed colon of fasted healthy humans following ingestion of 240 mL, using noninvasive MRI methods; (2) the amount of freely mobile water in the fasted human colon is of the order of only a few milliliters. Twelve healthy volunteers fasted overnight and underwent fasted abdominal MRI scans before drinking 240 mL (∼8 fluid ounces) of water. After ingesting the water they were scanned at frequent intervals for 2 h. The images were processed to quantify freely mobile water in the total and regional colon: ascending, transverse, and descending. The fasted colon contained (mean ± SEM) 11 ± 5 pockets of resting liquid with a total volume of 2 ± 1 mL (average). The colonic fluid peaked at 7 ± 4 mL 30 min after the water drink. This peak fluid was distributed in 17 ± 7 separate liquid pockets in the colon. The regional analysis showed that pockets of free fluid were found primarily in the ascending colon. The interindividual variability was very high; the subjects showed a range of number of colonic fluid pockets from 0 to 89 and total colonic freely mobile fluid volume from 0 to 49 mL. This is the first study measuring the time course of the number, regional location, and volume of

Oral Candida spp. colonization in human immunodeficiency virus-infected individuals

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D. V. Moris

2008-01-01

Full Text Available Several yeast species of Candida genus can colonize the skin as well as the mucous membrane of the vagina and the digestive tract for short or long periods. Depending on the host's immunological state and the yeast's virulence, colonization can become an infection, invading the colonized tissues and also disseminating. AIDS is characterized by the host's intensive and progressive immunodepression which manifests as diverse symptoms, mainly lesions in the mouth. Oral candidiasis is the most prevalent opportunistic infection in individuals infected with human immunodeficiency virus (HIV and is an important indicator of the disease progress and the immunosuppression increase. The factors involved in the equilibrium between Candida spp. and HIV-infected subjects are sometimes contradictory and were evaluated in the present study specially for colonization.

Effects of cyclooxygenase and lipoxygenase inhibition on basal- and serotonin-induced ion transport in rat colon

DEFF Research Database (Denmark)

Engelmann, Bodil Elisabeth; Bindslev, Niels; Poulsen, Steen Seier

2002-01-01

basal conditions. Furthermore, data suggest neither the COX-1 nor the COX-2 enzyme to be of major importance for 5-HT-induced ion transport in rat colon in vitro. In conclusion, this study supports 5-HT as a mediator of chloride secretion by activating several receptor subtypes and the LOX enzyme...

Attività chemioprotettiva di estratti di spinacio (Spinacia oleracea L.)

OpenAIRE

Milano, Francesco

2013-01-01

It is well known that Spinach (Spinacia oleracea L.) has a variety of biological functions, such as antitumor and chemoprotective activity, but little is known about the modulation of secondary metabolites induction by abiotic stress (i.e. hypoxia) and it’s correlation to human health. In this study I demonstrate that 24 h hypoxia alter the production of secondary metabolites in spinach. In vitro studies on human colon adenocarcinoma HT-29 cell line of different spinach aqueous extracts,show ...

Effect of the 5-HT{sub 4} receptor and serotonin transporter on visceral hypersensitivity in rats

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Chi, Yan; Liu, Xin-Guang; Wang, Hua-Hong; Li, Jun-Xia; Li, Yi-Xuan [Department of Gastroenterology, Peking University First Hospital, Beijing (China)

2012-07-27

Visceral hypersensitivity plays an important role in motor and sensory abnormalities associated with irritable bowel syndrome, but the underlying mechanisms are not fully understood. The present study was designed to evaluate the expression of the 5-HT{sub 4} receptor and the serotonin transporter (SERT) as well as their roles in chronic visceral hypersensitivity using a rat model. Neonatal male Sprague-Dawley rats received intracolonic injections of 0.5% acetic acid (0.3-0.5 mL at different times) between postnatal days 8 and 21 to establish an animal model of visceral hypersensitivity. On day 43, the threshold intensity for a visually identifiable contraction of the abdominal wall and body arching were recorded during rectal distention. Histological evaluation and the myeloperoxidase activity assay were performed to determine the severity of inflammation. The 5-HT{sub 4} receptor and SERT expression of the ascending colon were monitored using immunohistochemistry and Western blot analyses; the plasma 5-HT levels were measured using an ELISA method. As expected, transient colonic irritation at the neonatal stage led to visceral hypersensitivity, but no mucosal inflammation was later detected during adulthood. Using this model, we found reduced SERT expression (0.298 ± 0.038 vs 0.634 ± 0.200, P < 0.05) and increased 5-HT{sub 4} receptor expression (0.308 ± 0.017 vs 0.298 ± 0.021, P < 0.05). Treatment with fluoxetine (10 mg·kg{sup −1}·day{sup −1}, days 36-42), tegaserod (1 mg·kg{sup −1}·day{sup −1}, day 43), or the combination of both, reduced visceral hypersensitivity and plasma 5-HT levels. Fluoxetine treatment increased 5-HT{sub 4} receptor expression (0.322 ± 0.020 vs 0.308 ± 0.017, P < 0.01) but not SERT expression (0.219 ± 0.039 vs 0.298 ± 0.038, P = 0.654). These results indicate that both the 5-HT{sub 4} receptor and SERT play a role in the pathogenesis of visceral hypersensitivity, and its mechanism may be involved in the local 5-HT

Electrochemical fecal pellet sensor for simultaneous real-time ex vivo detection of colonic serotonin signalling and motility

Science.gov (United States)

Morris, Rachel; Fagan-Murphy, Aidan; MacEachern, Sarah J.; Covill, Derek; Patel, Bhavik Anil

2016-03-01

Various investigations have focused on understanding the relationship between mucosal serotonin (5-HT) and colonic motility, however contradictory studies have questioned the importance of this intestinal transmitter. Here we described the fabrication and use of a fecal pellet electrochemical sensor that can be used to simultaneously detect the release of luminal 5-HT and colonic motility. Fecal pellet sensor devices were fabricated using carbon nanotube composite electrodes that were housed in 3D printed components in order to generate a device that had shape and size that mimicked a natural fecal pellet. Devices were fabricated where varying regions of the pellet contained the electrode. Devices showed that they were stable and sensitive for ex vivo detection of 5-HT, and no differences in the fecal pellet velocity was observed when compared to natural fecal pellets. The onset of mucosal 5-HT was observed prior to the movement of the fecal pellet. The release of mucosal 5-HT occurred oral to the fecal pellet and was linked to the contraction of the bowel wall that drove pellet propulsion. Taken, together these findings provide new insights into the role of mucosal 5-HT and suggest that the transmitter acts as a key initiator of fecal pellet propulsion.

Implication of 5-HT(2B) receptors in the serotonin syndrome.

Science.gov (United States)

Diaz, Silvina Laura; Maroteaux, Luc

2011-09-01

The serotonin (5-HT) syndrome occurs in humans after antidepressant overdose or combination of drugs inducing a massive increase in extracellular 5-HT. Several 5-HT receptors are known to participate in this syndrome in humans and animal models. The 5-HT(2B) receptor has been proposed as a positive modulator of serotonergic activity, but whether it is involved in 5-HT syndrome has not yet been studied. We analyzed here, a putative role of 5-HT(2B) receptors in this disorder by forced swimming test (FST) and behavioral assessment in the open field. In FST, genetic (5-HT(2B)(-/-) mice) or pharmacological (antagonist RS127445 at 0.5 mg/kg) ablation of 5-HT(2B) receptors facilitated selective 5-HT reuptake inhibitors (SSRI)-induced increase of immobility time as well as expression of other symptoms related to 5-HT syndrome like hind limb abduction and Straub tail. Increase in immobility was also developed in FST by both wild type (WT) and 5-HT(2B)(-/-) mice after the administration of 5-HT(1A), 5-HT(2A) or 5-HT(2C) receptor agonists, 8-OH-DPAT (5 mg/kg), DOI (1 mg/kg), or WAY161503 (5 mg/kg), respectively. In contrast, the 5-HT(2B) receptor agonist BW723C86 (3 mg/kg) or 5-HT(1B) receptor agonist CGS12066A (2 mg/kg) decreased immobility time in both genotypes. The 5-HT syndrome induced by fluoxetine at high doses was blocked in WT and 5-HT(2B)(-/-) mice by administration of 5-HT(1A) and 5-HT(2C) receptor antagonists (WAY100635 0.5 mg/kg and SB242084 0.5 mg/kg) but not by the 5-HT(2A) receptor antagonist MDL100907 (1 mg/kg). By behavioral assessment, we confirmed that 5-HT(2B)(-/-) mice were more prone to develop 5-HT syndrome symptoms after administration of high dose of SSRIs or the 5-HT precursor 5-Hydroxytryptophan, 5-HTP, even if increases in 5-HT plasma levels were similar in both genotypes. This evidence suggests that the presence of 5-HT(2B) receptors hinders acute 5-HT toxicity once high levels of 5-HT are attained. Therefore, differential agonism

Expanding the Tissue Toolbox : Deriving Colon Tissue from Human Pluripotent Stem Cells

NARCIS (Netherlands)

Bruens, Lotte; Snippert, Hugo J.G.

2017-01-01

Organoid technology holds great potential for disease modeling and regenerative medicine. In this issue of Cell Stem Cell, Múnera et al. (2017) establish the generation of pluripotent stem cell-derived colon organoids that upon transplantation in mice, resembling human colon to a large extent,

Lactobacillus reuteri Inhibition of Enteropathogenic Escherichia coli Adherence to Human Intestinal Epithelium

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Alistair eWalsham

2016-03-01

Full Text Available Enteropathogenic E. coli (EPEC is a major cause of diarrheal infant death in developing countries, and probiotic bacteria have been shown to provide health benefits in gastrointestinal infections. In this study, we have investigated the influence of the gut symbiont Lactobacillus reuteri on EPEC adherence to the human intestinal epithelium. Different host cell model systems including non-mucus-producing HT-29 and mucus-producing LS174T intestinal epithelial cell lines as well as human small intestinal biopsies were used. Adherence of L. reuteri to HT-29 cells was strain-specific, and the mucus-binding proteins CmbA and MUB increased binding to both HT-29 and LS174T cells. L. reuteri ATCC PTA 6475 and ATCC 53608 significantly inhibited EPEC binding to HT-29 but not LS174T cells. While pre-incubation of LS174T cells with ATCC PTA 6475 did not affect EPEC A/E lesion formation, it increased the size of EPEC microcolonies. ATCC PTA 6475 and ATCC 53608 binding to the mucus layer resulted in decreased EPEC adherence to small intestinal biopsy epithelium. Our findings show that L. reuteri reduction of EPEC adhesion is strain-specific and has the potential to target either the epithelium or the mucus layer, providing further rationale for the selection of probiotic strains.

Lactobacillus reuteri Inhibition of Enteropathogenic Escherichia coli Adherence to Human Intestinal Epithelium.

Science.gov (United States)

Walsham, Alistair D S; MacKenzie, Donald A; Cook, Vivienne; Wemyss-Holden, Simon; Hews, Claire L; Juge, Nathalie; Schüller, Stephanie

2016-01-01

Enteropathogenic Escherichia coli (EPEC) is a major cause of diarrheal infant death in developing countries, and probiotic bacteria have been shown to provide health benefits in gastrointestinal infections. In this study, we have investigated the influence of the gut symbiont Lactobacillus reuteri on EPEC adherence to the human intestinal epithelium. Different host cell model systems including non-mucus-producing HT-29 and mucus-producing LS174T intestinal epithelial cell lines as well as human small intestinal biopsies were used. Adherence of L. reuteri to HT-29 cells was strain-specific, and the mucus-binding proteins CmbA and MUB increased binding to both HT-29 and LS174T cells. L. reuteri ATCC PTA 6475 and ATCC 53608 significantly inhibited EPEC binding to HT-29 but not LS174T cells. While pre-incubation of LS174T cells with ATCC PTA 6475 did not affect EPEC attaching/effacing (A/E) lesion formation, it increased the size of EPEC microcolonies. ATCC PTA 6475 and ATCC 53608 binding to the mucus layer resulted in decreased EPEC adherence to small intestinal biopsy epithelium. Our findings show that L. reuteri reduction of EPEC adhesion is strain-specific and has the potential to target either the epithelium or the mucus layer, providing further rationale for the selection of probiotic strains.

FXR silencing in human colon cancer by DNA methylation and KRAS signaling.

Science.gov (United States)

Bailey, Ann M; Zhan, Le; Maru, Dipen; Shureiqi, Imad; Pickering, Curtis R; Kiriakova, Galina; Izzo, Julie; He, Nan; Wei, Caimiao; Baladandayuthapani, Veerabhadran; Liang, Han; Kopetz, Scott; Powis, Garth; Guo, Grace L

2014-01-01

Farnesoid X receptor (FXR) is a bile acid nuclear receptor described through mouse knockout studies as a tumor suppressor for the development of colon adenocarcinomas. This study investigates the regulation of FXR in the development of human colon cancer. We used immunohistochemistry of FXR in normal tissue (n = 238), polyps (n = 32), and adenocarcinomas, staged I-IV (n = 43, 39, 68, and 9), of the colon; RT-quantitative PCR, reverse-phase protein array, and Western blot analysis in 15 colon cancer cell lines; NR1H4 promoter methylation and mRNA expression in colon cancer samples from The Cancer Genome Atlas; DNA methyltransferase inhibition; methyl-DNA immunoprecipitation (MeDIP); bisulfite sequencing; and V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) knockdown assessment to investigate FXR regulation in colon cancer development. Immunohistochemistry and quantitative RT-PCR revealed that expression and function of FXR was reduced in precancerous lesions and silenced in a majority of stage I-IV tumors. FXR expression negatively correlated with phosphatidylinositol-4, 5-bisphosphate 3 kinase signaling and the epithelial-to-mesenchymal transition. The NR1H4 promoter is methylated in ~12% colon cancer The Cancer Genome Atlas samples, and methylation patterns segregate with tumor subtypes. Inhibition of DNA methylation and KRAS silencing both increased FXR expression. FXR expression is decreased early in human colon cancer progression, and both DNA methylation and KRAS signaling may be contributing factors to FXR silencing. FXR potentially suppresses epithelial-to-mesenchymal transition and other oncogenic signaling cascades, and restoration of FXR activity, by blocking silencing mechanisms or increasing residual FXR activity, represents promising therapeutic options for the treatment of colon cancer.

Comparative Study of Green Sub- and Supercritical Processes to Obtain Carnosic Acid and Carnosol-Enriched Rosemary Extracts with in Vitro Anti-Proliferative Activity on Colon Cancer Cells

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Andrea del Pilar Sánchez-Camargo

2016-12-01

Full Text Available In the present work, four green processes have been compared to evaluate their potential to obtain rosemary extracts with in vitro anti-proliferative activity against two colon cancer cell lines (HT-29 and HCT116. The processes, carried out under optimal conditions, were: (1 pressurized liquid extraction (PLE, using an hydroalcoholic mixture as solvent at lab-scale; (2 Single-step supercritical fluid extraction (SFE at pilot scale; (3 Intensified two-step sequential SFE at pilot scale; (4 Integrated PLE plus supercritical antisolvent fractionation (SAF at pilot scale. Although higher extraction yields were achieved by using PLE (38.46% dry weight, this extract provided the lowest anti-proliferative activity with no observed cytotoxic effects at the assayed concentrations. On the other hand, extracts obtained using the PLE + SAF process provided the most active rosemary extracts against both colon cancer cell lines, with LC50 ranging from 11.2 to 12.4 µg/mL and from 21.8 to 31.9 µg/mL for HCT116 and HT-29, respectively. In general, active rosemary extracts were characterized by containing carnosic acid (CA and carnosol (CS at concentrations above 263.7 and 33.9 mg/g extract, respectively. Some distinct compounds have been identified in the SAF extracts (rosmaridiphenol and safficinolide, suggesting their possible role as additional contributors to the observed strong anti-proliferative activity of CA and CS in SAF extracts.

Haliotis tuberculata hemocyanin (HtH): analysis of oligomeric stability of HtH1 and HtH2, and comparison with keyhole limpet hemocyanin KLH1 and KLH2.

Science.gov (United States)

Harris, J R; Scheffler, D; Gebauer, W; Lehnert, R; Markl, J

2000-12-01

The multimeric/higher oligomeric states of the two isoforms of Haliotis tuberculata hemocyanin (HtH1 and HtH2) have been assessed by transmission electron microscopy (TEM) of negatively stained specimens, for comparison with previously published structural data from keyhole limpet hemocyanin (KLH1 and KLH2) [see Harris, J.R., Gebauer, W., Guderian, F.U., Markl, J., 1997a. Keyhole limpet hemocyanin (KLH), I: Reassociation from Immucothel followed by separation of KLH1 and KLH2. Micron, 28, 31-41; Harris, J.R., Gebauer, W., Söhngen, S.M., Nermut, M.V., Markl, J., 1997b. Keyhole limpet hemocyanin (KLH). II: Characteristic reassociation properties of purified KLH1 and KLH2. Micron, 28, 43-56; Harris, J.R., Gebauer, W., Adrian, M., Markl, J., 1998. Keyhole limpet hemocyanin (KLH): Slow in vitro reassociation of KLH1 and KLH2 from Immucothel. Micron, 29, 329-339]. In purified samples of both HtH isoforms, the hollow cylindrical ca 8MDa didecamer predominates together with a small number of decamers, but tri- and longer multidecamers are detectable only in the HtH2. The stability of the two HtH isoforms under varying ionic conditions have been monitored, thereby enabling conditions for the production of stable decamers to be established. The ability of these decamers to reform multimers in the presence of 10 and 100mM concentrations of calcium and magnesium ions in Tris-HCl buffer (pH 7.4), and also of individual HtH1 and HtH2 subunits (produced by pH 9.6 dissociation in glycine-NaOH buffer), to reassociate in the presence of calcium and magnesium ions, has been assessed. For the HtH1 decamers, the predominant multimeric product is the didecamer at 10 and 100mM calcium and magnesium concentrations, whereas for the HtH2 decamers, large numbers of multidecamers are produced, with the reaction proceeding more completely at the higher calcium and magnesium concentration. With the HtH1 subunit, reassociation in the presence of 10 and 100mM calcium and magnesium ions yielded

Survival advantages conferred to colon cancer cells by E-selectin-induced activation of the PI3K-NFκB survival axis downstream of Death receptor-3

International Nuclear Information System (INIS)

Porquet, Nicolas; Huot, Jacques; Poirier, Andrée; Houle, François; Pin, Anne-Laure; Gout, Stéphanie; Tremblay, Pierre-Luc; Paquet, Éric R; Klinck, Roscoe; Auger, François A

2011-01-01

Extravasation of circulating cancer cells is a key event of metastatic dissemination that is initiated by the adhesion of cancer cells to endothelial cells. It requires interactions between adhesion receptors on endothelial cells and their counter-receptors on cancer cells. Notably, E-selectin, a major endothelial adhesion receptor, interacts with Death receptor-3 present on metastatic colon carcinoma cells. This interaction confers metastatic properties to colon cancer cells by promoting the adhesion of cancer cells to endothelial cells and triggering the activation of the pro-migratory p38 and pro-survival ERK pathways in the cancer cells. In the present study, we investigated further the mechanisms by which the E-selectin-activated pathways downstream of DR3 confer a survival advantage to colon cancer cells. Cell survival has been ascertained by using the WST-1 assay and by evaluating the activation of the PI3 kinase/NFκB survival axis. Apoptosis has been assayed by determining DNA fragmentation by Hoechst staining and by measuring cleavage of caspases-8 and -3. DR3 isoforms have been identified by PCR. For more precise quantification, targeted PCR reactions were carried out, and the amplified products were analyzed by automated chip-based microcapillary electrophoresis on an Agilent 2100 Bioanalyzer instrument. Interaction between DR3-expressing HT29 colon carcinoma cells and E-selectin induces the activation of the PI3K/Akt pathway. Moreover, p65/RelA, the anti-apoptotic subunit of NFκB, is rapidly translocated to the nucleus in response to E-selectin. This translocation is impaired by the PI3K inhibitor LY294002. Furthermore, inhibition of the PI3K/Akt pathway increases the cleavage of caspase 8 in colon cancer cells treated with E-selectin and this effect is still further increased when both ERK and PI3K pathways are concomitantly inhibited. Intriguingly, metastatic colon cancer cell lines such as HT29 and SW620 express higher levels of a splice variant of

Survival advantages conferred to colon cancer cells by E-selectin-induced activation of the PI3K-NFκB survival axis downstream of Death receptor-3

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Paquet Éric R

2011-07-01

Full Text Available Abstract Background Extravasation of circulating cancer cells is a key event of metastatic dissemination that is initiated by the adhesion of cancer cells to endothelial cells. It requires interactions between adhesion receptors on endothelial cells and their counter-receptors on cancer cells. Notably, E-selectin, a major endothelial adhesion receptor, interacts with Death receptor-3 present on metastatic colon carcinoma cells. This interaction confers metastatic properties to colon cancer cells by promoting the adhesion of cancer cells to endothelial cells and triggering the activation of the pro-migratory p38 and pro-survival ERK pathways in the cancer cells. In the present study, we investigated further the mechanisms by which the E-selectin-activated pathways downstream of DR3 confer a survival advantage to colon cancer cells. Methods Cell survival has been ascertained by using the WST-1 assay and by evaluating the activation of the PI3 kinase/NFκB survival axis. Apoptosis has been assayed by determining DNA fragmentation by Hoechst staining and by measuring cleavage of caspases-8 and -3. DR3 isoforms have been identified by PCR. For more precise quantification, targeted PCR reactions were carried out, and the amplified products were analyzed by automated chip-based microcapillary electrophoresis on an Agilent 2100 Bioanalyzer instrument. Results Interaction between DR3-expressing HT29 colon carcinoma cells and E-selectin induces the activation of the PI3K/Akt pathway. Moreover, p65/RelA, the anti-apoptotic subunit of NFκB, is rapidly translocated to the nucleus in response to E-selectin. This translocation is impaired by the PI3K inhibitor LY294002. Furthermore, inhibition of the PI3K/Akt pathway increases the cleavage of caspase 8 in colon cancer cells treated with E-selectin and this effect is still further increased when both ERK and PI3K pathways are concomitantly inhibited. Intriguingly, metastatic colon cancer cell lines such as HT

Measuring endogenous 5-HT release by emission tomography: promises and pitfalls

Science.gov (United States)

Paterson, Louise M; Tyacke, Robin J; Nutt, David J; Knudsen, Gitte M

2010-01-01

Molecular in vivo neuroimaging techniques can be used to measure regional changes in endogenous neurotransmitters, evoked by challenges that alter synaptic neurotransmitter concentration. This technique has most successfully been applied to the study of endogenous dopamine release using positron emission tomography, but has not yet been adequately extended to other neurotransmitter systems. This review focuses on how the technique has been applied to the study of the 5-hydroxytryptamine (5-HT) system. The principles behind visualising fluctuations in neurotransmitters are introduced, with reference to the dopaminergic system. Studies that aim to image acute, endogenous 5-HT release or depletion at 5-HT receptor targets are summarised, with particular attention to studies in humans. Radiotracers targeting the 5-HT1A, 5-HT2A, and 5-HT4 receptors and the serotonin reuptake transporter have been explored for their sensitivity to 5-HT fluctuations, but with mixed outcomes; tracers for these targets cannot reliably image endogenous 5-HT in humans. Shortcomings in our basic knowledge of the mechanisms underlying changes in binding potential are addressed, and suggestions are made as to how the selection of targets, radiotracers, challenge paradigms, and experimental design might be optimised to improve our chances of successfully imaging endogenous neurotransmitters in the future. PMID:20664611

1,25(OH)2D3 attenuates TGF-β1/β2-induced increased migration and invasion via inhibiting epithelial–mesenchymal transition in colon cancer cells

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Chen, Shanwen; Zhu, Jing; Zuo, Shuai; Ma, Ju; Zhang, Junling; Chen, Guowei; Wang, Xin; Pan, Yisheng; Liu, Yucun; Wang, Pengyuan, E-mail: wangpengyuan2014@126.com

2015-12-04

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) has been reported to inhibit proliferation and migration of multiple types of cancer cells. However, the mechanism underlying its anti-metastasis effect is not fully illustrated. In this study, the effect of 1,25(OH)2D3 on TGF-β1/β2-induced epithelial–mesenchymal transition (EMT) is tested in colon cancer cells. The results suggest that 1,25(OH)2D3 inhibited TGF-β1/β2-induced increased invasion and migration of in SW-480 and HT-29 cells. 1,25(OH)2D3 also inhibited the cadherin switch in SW-480 and HT-29 cells. TGF-β1/β2-induced increased expression of EMT-related transcription factors was also inhibited by 1,25(OH)2D3. 1,25(OH)2D3 also inhibited the secretion of MMP-2 and MMP-9 and increased expression of F-actin induced by TGF-β1/β2 in SW-480 cells. Taken together, this study suggests that the suppression of EMT might be one of the mechanisms underlying the anti-metastasis effect of 1,25(OH)2D3 in colon cancer cells. - Highlights: • TGF-β1/β2-induced model of EMT was used in this study to test the effect of 1,25(OH)2D3 on EMT in colon cancer cells. • 1,25(OH)2D3 inhibited TGF-β1/β2-induced increased migration and invasion. • 1,25(OH)2D3 inhibited TGF-β1/β2-induced increased level of EMT-related transcription factors. • 1,25(OH)2D3 inhibited TGF-β1/β2-induced increased expression of F-actin in SW-480 cells.

Calorimetric signatures of human cancer cells and their nuclei

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Todinova, S. [Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 21, Sofia 1113 (Bulgaria); Stoyanova, E. [Department of Molecular Immunology, Institute of Biology and Immunology of Reproduction, Bulgarian Academy of Sciences, Tzarigradsko shose Blvd. 73, Sofia 1113 (Bulgaria); Krumova, S., E-mail: sakrumo@gmail.com [Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 21, Sofia 1113 (Bulgaria); Iliev, I. [Institute of Experimental Morphology, Pathology and Anthropology with Museum, Acad. G. Bonchev Str., Bl. 25, Sofia 1113 (Bulgaria); Taneva, S.G. [Institute of Biophysics and Biomedical Engineering, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 21, Sofia 1113 (Bulgaria)

2016-01-10

Graphical abstract: - Highlights: • Two temperature ranges are distinguished in the thermograms of cells/nuclei. • Different thermodynamic properties of cancer and normal human cells/nuclei. • Dramatic reduction of the enthalpy of the low-temperature range in cancer cells. • Oxaliplatin and 5-FU affect the nuclear matrix proteins and the DNA stability. - Abstract: The human cancer cell lines HeLa, JEG-3, Hep G2, SSC-9, PC-3, HT-29, MCF7 and their isolated nuclei were characterized by differential scanning calorimetry. The calorimetric profiles differed from normal human fibroblast (BJ) cells in the two well distinguished temperature ranges—the high-temperature range (H{sub T}, due to DNA-containing structures) and the low-temperature range (L{sub T}, assigned to the nuclear matrix and cellular proteins). The enthalpy of the L{sub T} range, and, respectively the ratio of the enthalpies of the L{sub T}- vs. H{sub T}-range, ΔH{sub L}/ΔH{sub H}, is strongly reduced for all cancer cells compared to normal fibroblasts. On the contrary, for most of the cancer nuclei this ratio is higher compared to normal nuclei. The HT-29 human colorectal cancer cells/nuclei differed most drastically from normal human fibroblast cells/nuclei. Our data also reveal that the treatment of HT-29 cancer cells with cytostatic drugs affects not only the DNA replication but also the cellular proteome.

Composition and antiproliferative effect of essential oil of Origanum vulgare against tumor cell lines.

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Begnini, Karine Rech; Nedel, Fernanda; Lund, Rafael Guerra; Carvalho, Pedro Henrique de Azambuja; Rodrigues, Maria Regina Alves; Beira, Fátima Tereza Alves; Del-Pino, Francisco Augusto Burkert

2014-10-01

Cancer is a leading cause of death and is responsible for one in eight deaths worldwide. The use of herbs as complementary medicine for cancer, especially advanced cancer, has recently increased. The aim of this study was to evaluate in vitro, the antiproliferative effect of Origanum vulgare against human breast adenocarcinoma (MCF-7), and human colon adenocarcinoma (HT-29). The essential oil (EO) was extracted from a bought amount of O. vulgare dried leaves and analyzed in a gas chromatograph interfaced with a mass selective detector. The cytotoxicity test was performed by sulforhodamine B assay. The results show that the EO is composed mostly of 4-terpineol and induces a high cytotoxicity effect in HT-29. In the MCF-7 cell line the EO was less effective. In conclusion, this study showed that O. vulgare main component is 4-terpineol and was effective in inducing cancer cell growth inhibition.

Inhibition of Various Cancer Cells Proliferation of Bornyl Acetate and Essential Oil from Inula graveolens (Linnaeus Desf

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Tunay Karan

2018-05-01

Full Text Available Inula species are medicinal and aromatic plants used for folk medicine extensively. In this work, hydrodistilled essential oil of Inula graveolens (Linnaeus Desf. analyzed by GC-MS which revealed that bornyl acetate was the major product (69.15%. Camphene was the second major compound (11.11%. Antiproliferative activity of the essential oil and bornyl acetate was investigated on HeLa (human cervix carcinoma, HT29 (human colon carcinoma , A549 (human lung carcinoma, MCF7 ( human breast adenocarcinoma cancer cells and FL (human amnion cells normal cells. The cytotoxicity was executed by a Lactate Dehydrogenase (LDH Cytotoxicity Detection Kit. Essential oil and bornyl acetate displayed the outstanding activities on HeLa (IC 50, 64.1, 72.0 µg/mL, HT29 (IC 50, 24.6, 60.5 µg/mL, A549 (IC 50, 28.3, 44.1 µg/mL, MCF-7 (IC 50, 66.5, 85.6 µg/mL, and FL (IC 50, 42.1, 50.6 µg/mL cell lines respectively.

Isolation of Human Colon Stem Cells Using Surface Expression of PTK7.

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Jung, Peter; Sommer, Christian; Barriga, Francisco M; Buczacki, Simon J; Hernando-Momblona, Xavier; Sevillano, Marta; Duran-Frigola, Miquel; Aloy, Patrick; Selbach, Matthias; Winton, Douglas J; Batlle, Eduard

2015-12-08

Insertion of reporter cassettes into the Lgr5 locus has enabled the characterization of mouse intestinal stem cells (ISCs). However, low cell surface abundance of LGR5 protein and lack of high-affinity anti-LGR5 antibodies represent a roadblock to efficiently isolate human colonic stem cells (hCoSCs). We set out to identify stem cell markers that would allow for purification of hCoSCs. In an unbiased approach, membrane-enriched protein fractions derived from in vitro human colonic organoids were analyzed by quantitative mass spectrometry. Protein tyrosine pseudokinase PTK7 specified a cell population within human colonic organoids characterized by highest self-renewal and re-seeding capacity. Antibodies recognizing the extracellular domain of PTK7 allowed us to isolate and expand hCoSCs directly from patient-derived mucosa samples. Human PTK7+ cells display features of canonical Lgr5+ ISCs and include a fraction of cells that undergo differentiation toward enteroendocrine lineage that resemble crypt label retaining cells (LRCs). Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Improved Anticancer Effect of Magnetite Nanocomposite Formulation of GALLIC Acid (Fe₃O₄-PEG-GA) Against Lung, Breast and Colon Cancer Cells.

Science.gov (United States)

Rosman, Raihana; Saifullah, Bullo; Maniam, Sandra; Dorniani, Dena; Hussein, Mohd Zobir; Fakurazi, Sharida

2018-02-02

Lung cancer, breast cancer and colorectal cancer are the most prevalent fatal types of cancers globally. Gallic acid (3,4,5-trihydroxybenzoic acid) is a bioactive compound found in plants and foods, such as white tea, witch hazel and it has been reported to possess anticancer, antioxidant and anti-inflammatory properties. In this study we have redesigned our previously reported anticancer nanocomposite formulation with improved drug loading based on iron oxide magnetite nanoparticles coated with polyethylene glycol and loaded with anticancer drug gallic acid (Fe₃O₄-PEG-GA). The in vitro release profile and percentage drug loading were found to be better than our previously reported formulation. The anticancer activity of pure gallic acid (GA), empty carrier (Fe₃O₄-PEG) nanocarrier and of anticancer nanocomposite (Fe₃O₄-PEG-GA) were screened against human lung cancer cells (A549), human breast cancer cells (MCF-7), human colon cancer cells (HT-29) and normal fibroblast cells (3T3) after incubation of 24, 48 and 72 h using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) MTT assay. The designed formulation (Fe₃O₄-PEG-GA) showed better anticancer activity than free gallic acid (GA). The results of the in vitro studies are highly encouraging to conduct the in vivo studies.

Separation of water-soluble metabolites of benzo[a]pyrene formed by cultured human colon

DEFF Research Database (Denmark)

Autrup, Herman

1979-01-01

A method has been developed to separate conjugated metabolites of benzo[a]pyrene into three major fractions: sulfate esters, glucuronides and glutathione conjugates. In cultured human colon, formation of sulfate esters and glutathione conjugates is the major conjugation pathway, while formation......-hydroxybenzo[a]pyrene were the major substrates for sulfotransferase in cultured human colon....

Oropharyngeal perinatal colonization by human papillomavirus.

Science.gov (United States)

Sánchez-Torices, María Soledad; Corrales-Millan, Rocío; Hijona-Elosegui, Jesús J

2016-01-01

Human papillomavirus (HPV) infection is the most common human sexually transmitted disease. It is clinically relevant because this condition is necessary for the development of epithelial cervical cancer, and it is also a factor closely associated with the occurrence of diverse tumours and various benign and malignant lesions of the head and neck area. The infective mechanism in most of these cases is associated with sexual intercourse, but there is recent scientific evidence suggesting that HPV infection may also be acquired by other routes of infection not necessarily linked to sexual contact. One of them is vertical transmission from mother to child, either during pregnancy or at the time of delivery. The aim of our research was to study maternal-foetal HPV transmission during childbirth in detail, establishing the rate of oropharyngeal neonatal HPV in vaginal deliveries. The presence and type of HPV viral DNA at the time of delivery in samples of maternal cervical secretions, amniotic fluid, venous cord blood samples and neonatal oropharynx in pregnant women (and their babies) were determined. The rate of oropharyngeal neonatal HPV colonization in vaginal deliveries was 58.24%. The maternal and neonatal HPV colonization mechanism is essentially, but not exclusively, transvaginal. Copyright © 2014 Elsevier España, S.L.U. y Sociedad Española de Otorrinolaringología y Cirugía de Cabeza y Cuello. All rights reserved.

Dyospiros kaki phenolics inhibit colitis and colon cancer cell proliferation, but not gelatinase activities.

Science.gov (United States)

Direito, Rosa; Lima, Ana; Rocha, João; Ferreira, Ricardo Boavida; Mota, Joana; Rebelo, Patrícia; Fernandes, Adelaide; Pinto, Rui; Alves, Paula; Bronze, Rosário; Sepodes, Bruno; Figueira, Maria-Eduardo

2017-08-01

Polyphenols from persimmon (Diospyros kaki) have demonstrated radical-scavenging and antiinflammatory activities; however, little is known about the effects of persimmon phenolics on inflammatory bowel diseases (IBD) and colorectal cancer (CRC). Therefore, we aimed in this work to characterize the antiinflammatory and antiproliferative effects of a persimmon phenolic extract (80% acetone in water), using an in vivo model of experimental colitis and a model of cancer cell invasion. Our results show, for the first time, a beneficial effect of a persimmon phenolic extract in the attenuation of experimental colitis and a potential antiproliferative effect on cultured colon cancer cells. Administration of persimmon phenolic extract to mice with TNBS-induced colitis led to a reduction in several functional and histological markers of colon inflammation, namely: attenuation of colon length decrease, reduction of the extent of visible injury (ulcer formation), decrease in diarrhea severity, reduced mortality rate, reduction of mucosal hemorrhage and reduction of general histological features of colon inflammation. In vitro studies also showed that persimmon phenolic extract successfully impaired cell proliferation and invasion in HT-29 cells. Further investigation showed a decreased expression of COX-2 and iNOS in the colonic tissue of colitis mice, two important mediators of intestinal inflammation, but there was no inhibition of the gelatinase MMP-9 and MMP-2 activities. Given the role of inflammatory processes in the progression of CRC and the important link between inflammation and cancer, our results highlight the potential of persimmon polyphenols as a pharmacological tool in the treatment of patients with IBD. Copyright © 2017 Elsevier Inc. All rights reserved.

The influence of hormone therapies on colon and rectal cancer

DEFF Research Database (Denmark)

Mørch, Lina Steinrud; Lidegaard, Øjvind; Keiding, Niels

2016-01-01

followed 1995–2009. Information on HT exposures was from the National Prescription Register and updated daily, while information on colon (n = 8377) and rectal cancers (n = 4742) were from the National Cancer Registry. Potential confounders were obtained from other national registers. Poisson regression...

Use of a temperature-sensitive p53 mutant to evaluate mechanisms of 5-fluorodeoxyuridine-mediated radiosensitization

International Nuclear Information System (INIS)

Naida, J.D.; Davis, M.A.; Lawrence, T.S.

1996-01-01

Purpose/Objective: Evidence exists that fluorodeoxyuridine (FdUrd)-mediated radiosensitization occurs in HT29 human colon carcinoma cells (which are p53 mutant) when these cells progress past the G 1 /S boundary in the presence of the drug. It has been demonstrated that wild type p53 levels increase following fluoropyrimidine treatment and that G 1 arrest is associated with increased p53 levels. We hypothesized that the restoration of wild type p53 function might restore G 1 /S arrest after FdUrd treatment, and that this would prevent FdUrd-mediated radiosensitization. Similarly, we hypothesized that cells containing wild type p53 would not be radiosensitized by FdUrd. Materials and Methods: Two clones of HT29 human colon cancer cells (ts29-A and ts29-G) containing murine temperature-sensitive p53 were constructed using electroporation and Geneticin selection. Incubation of these cells at the permissive temperature of 32 deg. C produces wild type p53 function and at the non permissive temperature of 38 deg. C causes mutant p53 function. A G418 resistant control cell line was also constructed (HT29neo). Cells were incubated at either 32 deg. C or 38 deg. C for 24 hours prior to irradiation and with FdUrd (100 nM) or medium only during the last 14 hours of the temperature shift. To assess progression into S phase, single-parameter (propidium iodide (PI)) and two-parameter (PI and bromodeoxyuridine) flow cytometry were performed at the end of drug exposure. A standard clonogenic assay was used. Results: We found that when ts29-A and ts29-G cells were incubated at the non-permissive (inactive p53 conformation) temperature, they progressed into S phase following exposure to FdUrd and were radiosensitized (enhancement ratio 1.5) to a degree similar to that seen in parental HT29 cells. Cells incubated at the permissive (wild-type p53 conformation) temperature demonstrated G 1 arrest, S phase depletion, and G2 arrest. In addition, FdUrd-mediated radiosensitization was

Central 5-HT4 receptor binding as biomarker of serotonergic tonus in humans

DEFF Research Database (Denmark)

Haahr, M E; Fisher, P M; Jensen, Christian Gaden

2014-01-01

levels, is associated with a decline in brain 5-HT4R binding. A total of 35 healthy men were studied in a placebo-controlled, randomized, double-blind study. Participants were assigned to receive 3 weeks of oral dosing with placebo or fluoxetine, 40 mg per day. Brain 5-HT4R binding was quantified...... at baseline and at follow-up with [(11)C]SB207145 positron emission tomography (PET). Three weeks of intervention with fluoxetine was associated with a 5.2% reduction in brain 5-HT4R binding (P=0.017), whereas placebo intervention did not change 5-HT4R binding (P=0.52). Our findings are consistent...

Serotonin receptors influencing cell proliferation in the jejunal crypt epithelium and in colonic adenocarcinomas.

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Tutton, P J; Barkla, D H

1986-01-01

Serotonin has previously been shown to stimulate cell proliferation in the jejunal crypt epithelium and in colonic tumours. The original classification of serotonin receptors into D and M groups was not conductive to the understanding of these observations. The more recent classification of serotonin receptors into 5HT1 and 5HT2 groups is considered in this report. On the balance of evidence it appears that similar receptors mediate the response to serotonin in the two tissues under consideration and that these receptors resemble those of the 5HT1 group. Such receptors are usually positively linked to adenylate cyclase.

Hydrogen sulfide-releasing naproxen suppresses colon cancer cell growth and inhibits NF-κB signaling

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Kodela R

2015-08-01

Full Text Available Ravinder Kodela,1 Niharika Nath,2 Mitali Chattopadhyay,1 Diandra E Nesbitt,1 Carlos A Velázquez-Martínez,3 Khosrow Kashfi11Department of Physiology, Pharmacology and Neuroscience, Sophie Davis School of Biomedical Education, City University of New York Medical School, 2Department of Life Sciences, New York Institute of Technology, New York, NY, USA; 3Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada Abstract: Colorectal cancer (CRC is the second leading cause of death due to cancer and the third most common cancer in men and women in the USA. Nuclear factor kappa B (NF-κB is known to be activated in CRC and is strongly implicated in its development and progression. Therefore, activated NF-κB constitutes a bona fide target for drug development in this type of malignancy. Many epidemiological and interventional studies have established nonsteroidal anti-inflammatory drugs (NSAIDs as a viable chemopreventive strategy against CRC. Our previous studies have shown that several novel hydrogen sulfide-releasing NSAIDs are promising anticancer agents and are safer derivatives of NSAIDs. In this study, we examined the growth inhibitory effect of a novel H2S-releasing naproxen (HS-NAP, which has a repertoire as a cardiovascular-safe NSAID, for its effects on cell proliferation, cell cycle phase transitions, and apoptosis using HT-29 human colon cancer cells. We also investigated its effect as a chemopreventive agent in a xenograft mouse model. HS-NAP suppressed the growth of HT-29 cells by induction of G0/G1 arrest and apoptosis and downregulated NF-κB. Tumor xenografts in mice were significantly reduced in volume. The decrease in tumor mass was associated with a reduction of cell proliferation, induction of apoptosis, and decreases in NF-κB levels in vivo. Therefore, HS-NAP demonstrates strong anticancer potential in CRC. Keywords: nonsteroidal anti-inflammatory drugs, cell cycle, apoptosis, xenograft, NF

Postirradiation examination report of TRISO and BISO coated ThO2 particles irradiated in capsules HT-31 and HT-33

International Nuclear Information System (INIS)

Sedlak, B.J.

1980-01-01

Capsules HT-31 and HT-33 were uninstrumented capsule experiments irradiated in the target position of the High-Flux Isotope Reactor at Oak Ridge National Laboratory. The experiments were used to evaluate the irradiation performance of (1) fuel fabricated in a 240-mm-diameter coater for production scale-up, (2) TRISO ThO 2 and BISO ThO 2 particles, and (3) fuel with certain OPyC variables. A total of 16 BISO particle samples and 32 TRISO particle samples were irradiated to fast neutron fluences ranging from 4.0 to 11.7 x 10 25 n/m 2 (E > 29 fJ)/sub HTGR/ and heavy metal burnups between 3.5% and 13.2% FIMA at temperatures from 1150 0 to 1530 0 C

The 5-HT2A receptor binding pattern in the human brain is strongly genetically determined

DEFF Research Database (Denmark)

Pinborg, Lars H; Arfan, Haroon; Haugbol, Steven

2007-01-01

With the appropriate radiolabeled tracers, positron emission tomography (PET) enables in vivo human brain imaging of markers for neurotransmission, including neurotransmitter synthesis, receptors, and transporters. Whereas structural imaging studies have provided compelling evidence that the human...... brain anatomy is largely genetically determined, it is currently unknown to what degree neuromodulatory markers are subjected to genetic and environmental influence. Changes in serotonin 2A (5-HT(2A)) receptors have been reported to occur in various neuropsychiatric disorders and an association between...

A cross sectional study of animal and human colonization with Methicillin-Resistant Staphylococcus aureus (MRSA in an Aboriginal community

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Peter Daley

2016-07-01

Full Text Available Abstract Background Methicillin-resistant Staphylococcus aureus (MRSA infections are common among humans in Aboriginal communities in Canada, for unknown reasons. Methods Cross sectional study of humans and dogs in an Aboriginal community of approximately 1200 persons. Our objectives were to measure community-based prevalence of nasal MRSA colonization among humans, use multivariable logistic regression to analyze risk factors for MRSA colonization, and perform molecular typing of Staphylococci isolated to investigate interspecies transmission. Results 461 humans were approached for consent and 442 provided complete data. 109/442 (24.7 %, 95 % C.I. = 20.7–28.7 % of humans were colonized with MRSA. 169/442 (38.2 % of humans had received antibiotics in the last 12 months. Only number of rooms in the house (OR 0.86, p = 0.023 and recreational dog use (OR 7.7, p = 0.002 were significant risk factors for MRSA colonization. 95/109 (87.1 % of MRSA strains from humans were of the same spa type (CMRSA10/USA300. 8/157 (5.1 %, 95 % C.I. = 1.7–8.5 % of dogs were colonized with methicillin-susceptible S. aureus, and no dogs were colonized with MRSA. Conclusions Human MRSA colonization in this community is very common, and a single clone is predominant, suggesting local transmission. Antibiotic use is also very common. Crowding may partially explain high colonization, but most considered risk factors including animal exposure were not predictive. Very few dogs carried human Staphylococcal strains.

Assessing the potential for raw meat to influence human colonization with Staphylococcus aureus

OpenAIRE

Carrel, Margaret; Zhao, Chang; Thapaliya, Dipendra; Bitterman, Patrick; Kates, Ashley E.; Hanson, Blake M.; Smith, Tara C.

2017-01-01

The role of household meat handling and consumption in the transfer of Staphylococcus aureus (S. aureus) from livestock to consumers is not well understood. Examining the similarity of S. aureus colonizing humans and S. aureus in meat from the stores in which those individuals shop can provide insight into the role of meat in human S. aureus colonization. S. aureus isolates were collected from individuals in rural and urban communities in Iowa (n?=?3347) and contemporaneously from meat produc...

Preferential radiosensitization of human prostatic carcinoma cells by mild hyperthermia

International Nuclear Information System (INIS)

Ryu, Samuel; Brown, Stephen L.; Kim, Sang-Hie; Khil, Mark S.; Kim, Jae Ho

1996-01-01

Purpose: Recent cell culture studies by us and others suggest that some human carcinoma cells are more sensitive to heat than are rodent cells following mild hyperthermia. In studying the cellular mechanism of enhanced thermosensitivity of human tumor cells to hyperthermia, prostatic carcinoma cells of human origin were found to be more sensitive to mild hyperthermia than other human cancer cells. The present study was designed to determine the magnitude of radiosensitization of human prostatic carcinoma cells by mild hyperthermia and to examine whether the thermal radiosensitization is related to the intrinsic thermosensitivity of cancer cells. Methods and Materials: Two human prostatic carcinoma cell lines (DU-145 and PC-3) and other carcinoma cells of human origin, in particular, colon (HT-29), breast (MCF-7), lung (A-549), and brain (U-251) were exposed to temperatures of 40-41 deg. C. Single acute dose rate radiation and fractionated radiation were combined with mild hyperthermia to determine thermal radiosensitization. The end point of the study was the colony-forming ability of single-plated cells. Results: DU-145 and PC-3 cells were found to be exceedingly thermosensitive to 41 deg. C for 24 h, relative to other cancer cell lines. Ninety percent of the prostatic cancer cells were killed by a 24 h heat exposure. Prostatic carcinoma cells exposed to a short duration of heating at 41 deg. C for 2 h resulted in a substantial enhancement of radiation-induced cytotoxicity. The thermal enhancement ratios (TERs) of single acute dose radiation following heat treatment 41 deg. C for 2 h were 2.0 in DU-145 cells and 1.4 in PC-3 cells. The TERs of fractionated irradiation combined with continuous heating at 40 deg. C were similarly in the range of 2.1 to 1.4 in prostate carcinoma cells. No significant radiosensitization was observed in MCF-7 and HT-29 cells under the same conditions. Conclusion: The present data suggest that a significant radiosensitization of

Monitoring Cell Death in Regorafenib-Treated Experimental Colon Carcinomas Using Annexin-Based Optical Fluorescence Imaging Validated by Perfusion MRI.

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Philipp M Kazmierczak

Full Text Available To investigate annexin-based optical fluorescence imaging (OI for monitoring regorafenib-induced early cell death in experimental colon carcinomas in rats, validated by perfusion MRI and multiparametric immunohistochemistry.Subcutaneous human colon carcinomas (HT-29 in athymic rats (n = 16 were imaged before and after a one-week therapy with regorafenib (n = 8 or placebo (n = 8 using annexin-based OI and perfusion MRI at 3 Tesla. Optical signal-to-noise ratio (SNR and MRI tumor perfusion parameters (plasma flow PF, mL/100mL/min; plasma volume PV, % were assessed. On day 7, tumors underwent immunohistochemical analysis for tumor cell apoptosis (TUNEL, proliferation (Ki-67, and microvascular density (CD31.Apoptosis-targeted OI demonstrated a tumor-specific probe accumulation with a significant increase of tumor SNR under therapy (mean Δ +7.78±2.95, control: -0.80±2.48, p = 0.021. MRI detected a significant reduction of tumor perfusion in the therapy group (mean ΔPF -8.17±2.32 mL/100 mL/min, control -0.11±3.36 mL/100 mL/min, p = 0.036. Immunohistochemistry showed significantly more apoptosis (TUNEL; 11392±1486 vs. 2921±334, p = 0.001, significantly less proliferation (Ki-67; 1754±184 vs. 2883±323, p = 0.012, and significantly lower microvascular density (CD31; 107±10 vs. 182±22, p = 0.006 in the therapy group.Annexin-based OI allowed for the non-invasive monitoring of regorafenib-induced early cell death in experimental colon carcinomas, validated by perfusion MRI and multiparametric immunohistochemistry.

Autophagy mediates cytotoxicity of human colorectal cancer cells treated with garcinielliptone FC.

Science.gov (United States)

Won, Shen-Jeu; Yen, Cheng-Hsin; Lin, Ting-Yu; Jiang-Shieh, Ya-Fen; Lin, Chun-Nan; Chen, Jyun-Ti; Su, Chun-Li

2018-01-01

The tautomeric pair of garcinielliptone FC (GFC) is a novel tautomeric pair of polyprenyl benzophenonoid isolated from the pericarps of Garcinia subelliptica Merr. (G. subelliptica, Clusiaceae), a tree with abundant sources of polyphenols. Our previous report demonstrated that GFC induced apoptosis on various types of human cancer cell lines including chemoresistant human colorectal cancer HT-29 cells. In the present study, we observed that many autophagy-related genes in GFC-treated HT-29 cells were up- and down-regulated using a cDNA microarray containing oncogenes and kinase genes. GFC-induced autophagy of HT-29 cells was confirmed by observing the formation of acidic vesicular organelles, LC3 puncta, and double-membrane autophagic vesicles using flow cytometry, confocal microscopy, and transmission electron microscopy, respectively. Inhibition of AKT/mTOR/P70S6K signaling as well as formation of Atg5-Atg12 and PI3K/Beclin-1 complexes were observed using Western blot. Administration of autophagy inhibitor (3-methyladenine and shRNA Atg5) and apoptosis inhibitor Z-VAD showed that the GFC-induced autophagy was cytotoxic form and GFC-induced apoptosis enhanced GFC-induced autophagy. Our data suggest the involvement of autophagy and apoptosis in GFC-induced anticancer mechanisms of human colorectal cancer. © 2017 Wiley Periodicals, Inc.

Secreted Human Adipose Leptin Decreases Mitochondrial Respiration in HCT116 Colon Cancer Cells

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Yehuda-Shnaidman, Einav; Nimri, Lili; Tarnovscki, Tanya; Kirshtein, Boris; Rudich, Assaf; Schwartz, Betty

2013-01-01

Obesity is a key risk factor for the development of colon cancer; however, the endocrine/paracrine/metabolic networks mediating this connection are poorly understood. Here we hypothesize that obesity results in secreted products from adipose tissue that induce malignancy-related metabolic alterations in colon cancer cells. Human HCT116 colon cancer cells, were exposed to conditioned media from cultured human adipose tissue fragments of obese vs. non-obese subjects. Oxygen consumption rate (OCR, mostly mitochondrial respiration) and extracellular acidification rate (ECAR, mostly lactate production via glycolysis) were examined vis-à-vis cell viability and expression of related genes and proteins. Our results show that conditioned media from obese (vs. non-obese) subjects decreased basal (40%, prespiration and function in HCT116 colon cancer cells, an effect that is at least partly mediated by leptin. These results highlight a putative novel mechanism for obesity-associated risk of gastrointestinal malignancies, and suggest potential new therapeutic avenues. PMID:24073224

Retinoic acid morpholine amide (RAMA) inhibits expression of Fas ligand through EP1 receptor in colon cancer cells.

Science.gov (United States)

Chen, Shao-Xuan; Du, Shi-Yu; Wang, Yun-Ting; Zhao, Hong-Chuan; Zhang, Yan-Li; Yao, Li

2016-01-01

Among the members of tumour necrosis factor family Fas ligand on binding to its receptor strongly induces apoptosis of tumour-infiltrating lymphocytes (TIL). Thus, FasL acts as an inhibitor of anti-tumour immune response. The present study demonstrates that retinoic acid morpholine amide (RAMA) significantly suppresses FasL expression in colon cancer cells in a dose- and time-dependent manner. The suppression of FasL mRNA and proteins was significant at a concentration of 30 μM after 48 h in CLT85 and HT26 colon cancer cells. There was around 2.6- and 3.2-fold decrease in FasL mRNA after incubation with 30 μM of RAMA in CLT85 cells and HT26 cells, respectively. The results from Western blot showed a decrease in FasL mRNA and protein expression in both CLT85 and HT26 cells after suppression of cyclooxygenase (COX)-2 and COX-1 by RNAi. However, when COX-2-specific silencer RNA (siCOX-2)- and siCOX-1-treated CLT85 and HT26 cells were exposed to RAMA, inhibition of FasL expression was further suppressed. The siCOX-2-treated CLT85 and HT26 cells on exposure to RAMA showed ∼87 and ∼54 % reduction in FasL mRNA, respectively. Co-culture of Jurkat T cells with RAMA-treated HT26 and CLT85 cells decreased the viability of Jurkat T cells by only 2 and 4.3 %, respectively, compared to 19.5 and 37.3 % in control HT26 and CLT85 cells. The results from real-time reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting showed that suppression of EP1 prevented RAMA-induced FasL suppression in CLT85 cells at both the mRNA and protein levels. Thus, RAMA can be a potent therapeutic agent for the treatment of colon tumours.

Opposing effects of low versus high concentrations of water soluble vitamins/dietary ingredients Vitamin C and niacin on colon cancer stem cells (CSCs).

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Sen, Utsav; Shenoy P, Sudheer; Bose, Bipasha

2017-10-01

Colorectal cancer is one of the global causes of cancer deaths. Cancer stem cells (CSCs) inside the tumour niche responsible for metastasis and relapses, and hence need to be targeted for cancer therapeutics. Although dietary fibre and lifestyle changes have been recommended as measures for colorectal cancer prevention, no such recommendations are available for using water soluble vitamins as prophylaxis measure for colorectal cancers. High dose of Vitamin C has been proven to selectively kill colon cancer cells having BRAF and KRAS mutations by inducing oxidative stress. In this study, we show for the first time the opposing effects of the low and high dose of Vitamin C and vitamin B3 on colon CSCs isolated from HT-29 and HCT-15 colorectal carcinoma cell lines. At small doses, both of these vitamins exerted a cell proliferative effect only on CSCs, while there was no change in the proliferation status of non-stem cancer cells and wild-type (WT) populations. On the other hand, the death effects induced by high doses of Vitamin C and B3 were of the order of 50-60% and ∼30% on CSCs from HT-29 and HCT15, respectively. Interestingly, the control fibroblast cell line (NIH3T3) was highly refractory all the tested concentrations of Vitamin C and B3, except for the highest dose - 10,000 μg of Vitamin C that induced only 15% of cell death. Hence, these results indicate the future scope of use of therapeutic doses of Vitamin C and B3 especially in patients with advanced colorectal cancer. © 2017 International Federation for Cell Biology.

Cytotoxic Effects and Anti-Angiogenesis Potential of Pistachio (Pistacia vera L.) Hulls against MCF-7 Human Breast Cancer Cells.

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Seifaddinipour, Maryam; Farghadani, Reyhaneh; Namvar, Farideh; Mohamad, Jamaludin; Abdul Kadir, Habsah

2018-01-05

Pistachio ( Pistacia vera L.) hulls (PVLH) represents a significant by-product of industrial pistachio processing that contains high amounta of phenolic and flavonoid compounds known to act as antioxidants. The current study was designed to evaluate the anti-tumor and anti-angiogenic potentials of PVLH extracts. The cytotoxic effects of hexane, ethyl acetate, methanol, and water PVLH extracts toward human colon cancer (HT-29 and HCT-116), breast adenocarcinoma (MCF-7), lung adenocarcinoma (H23), liver hepatocellular carcinoma (HepG2), cervical cancer (Ca Ski), and normal fibroblast (BJ-5ta) cells were assessed using a MTT cell viability assay. Apoptosis induction was evaluated through the different nuclear staining assays and confirmed by flow cytometry analysis. Anti-angiogenic activities were also determined using chorioallantoic membrane (CAM) assay. PVLH ethyl acetate extracts (PVLH-EAE) demonstrated a suppressive effect with an IC 50 value of 21.20 ± 1.35, 23.00 ± 1.2 and 25.15 ± 1.85 µg/mL against MCF-7, HT-29 and HCT-116, respectively, after 72 h of treatment. Morphological assessment and flow cytometry analysis showed the potential of PVLH-EAE to induce apoptosis. PVLH-EAE at the highest concentration demonstrated significant inhibition of angiogenesis as comparing with control group. Also the expression of Bax increased and the expression of Bcl-2 decreased in treated MCF-7 cells. Thus, the apoptosis induction and angiogenesis potential of PVLH-EAE make it to be the most suitable for further cancer research study to deal with selective antitumor active substances to human cancers especially breast cancer.

Mucus interactions with liposomes encapsulating bioactives: Interfacial tensiometry and cellular uptake on Caco-2 and cocultures of Caco-2/HT29-MTX.

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Li, Yang; Arranz, Elena; Guri, Anilda; Corredig, Milena

2017-02-01

Structuring of delivery matrices in foods aquires careful designing for optimal delivery and subsiquent absorption of the beneficial compounds in the gut. There has been quite improvement in mimicking digestion and absorption in vitro but as of yet little is understood on mucus interference in nutrient absorption Therefore in this study interactions of human intestinal mucus with milk and soy phospholipids liposomes carring hydrophilic (epigallocatechin-3-gallate) or hydrophobic (β-carotene) bioactive molecules were investigated. Liposomes of about 100nm were obtained using microfluidization and their behaviour with the human intestinal mucus were evaluated using drop shape tensiometry. The chemistry of the liposomes (milk or soy) and the encapsulated bioactive structure can affect the viscoelastic behaviour of the complex itself. Empty or loaded liposomes were differently interacting with the mucus at the interface. Mucus-liposomes interactions were also studied using cell cultures, Caco-2 (without mucus) and cocultures Caco-2/HT29-MTX (mucus producing). The interaction of mucus layer with liposomes was at some extent aligned with rheological studies. This work demonstrated that delivery systems may interact with the mucosal surface of intestinal cells, and in vitro approaches allow for screening of such interactions. These highlights could help us in carefully designing the delivery systems and moreover choosing the right carrier and/or bioactive that does not jeopardize the optimal delivery of the bioactive structure. Copyright © 2016 Elsevier Ltd. All rights reserved.

Human milk lactoferrin inactivates two putative colonization factors expressed by Haemophilus influenzae.

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Qiu, J; Hendrixson, D R; Baker, E N; Murphy, T F; St Geme, J W; Plaut, A G

1998-10-13

Haemophilus influenzae is a major cause of otitis media and other respiratory tract disease in children. The pathogenesis of disease begins with colonization of the upper respiratory mucosa, a process that involves evasion of local immune mechanisms and adherence to epithelial cells. Several studies have demonstrated that human milk is protective against H. influenzae colonization and disease. In the present study, we examined the effect of human milk on the H. influenzae IgA1 protease and Hap adhesin, two autotransported proteins that are presumed to facilitate colonization. Our results demonstrated that human milk lactoferrin efficiently extracted the IgA1 protease preprotein from the bacterial outer membrane. In addition, lactoferrin specifically degraded the Hap adhesin and abolished Hap-mediated adherence. Extraction of IgA1 protease and degradation of Hap were localized to the N-lobe of the bilobed lactoferrin molecule and were inhibited by serine protease inhibitors, suggesting that the lactoferrin N-lobe may contain serine protease activity. Additional experiments revealed no effect of lactoferrin on the H. influenzae P2, P5, and P6 outer-membrane proteins, which are distinguished from IgA1 protease and Hap by the lack of an N-terminal passenger domain or an extracellular linker region. These results suggest that human milk lactoferrin may attenuate the pathogenic potential of H. influenzae by selectively inactivating IgA1 protease and Hap, thereby interfering with colonization. Future studies should examine the therapeutic potential of lactoferrin, perhaps as a supplement in infant formulas.

Coumarins and xanthones from the seeds of Mammea siamensis

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Laphookhieo, Surat; Promnart, Phunrawie; Syers, John Keith [Mae Fah Luang University, Tasud, Muang (Thailand). School of Science]. E-mails: mail: surat@mfu.ac.th; laphookhieo@yahoo.com; Kanjana-Opas, Akkharawit [Prince of Songkla University, Hat-Yai (Thailand). Faculty of Agro-Industry. Dept. of Industrial Biotechnology; Ponglimanont, Chanita; Karalai, Chatchanok [Prince of Songkla University, Hat-Yai (Thailand). Faculty of Science. Dept. of Chemistry

2007-07-01

A new coumarin, mammea E/BB cyclo D (1), together with five known compounds, mammea E/BA cyclo D (2), suragin C (3), therapin B (4), 1,7-dihydroxyxanthone (5) and 1-hydroxy-5-methoxyxanthone (6), were isolated from the seeds of Mammea siamensis. Their structures were characterized using 1D and 2D NMR spectral data. Suragin C and therapin B showed cytotoxic activity against breast adenocarcinoma (MCF-7), human cervical cancer (HeLa), colon cancer (HT-29) and human oral cancer (KB). (author)

Discerning apical and basolateral properties of HT-29/B6 and IPEC-J2 cell layers by impedance spectroscopy, mathematical modeling and machine learning.

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Thomas Schmid

Full Text Available Quantifying changes in partial resistances of epithelial barriers in vitro is a challenging and time-consuming task in physiology and pathophysiology. Here, we demonstrate that electrical properties of epithelial barriers can be estimated reliably by combining impedance spectroscopy measurements, mathematical modeling and machine learning algorithms. Conventional impedance spectroscopy is often used to estimate epithelial capacitance as well as epithelial and subepithelial resistance. Based on this, the more refined two-path impedance spectroscopy makes it possible to further distinguish transcellular and paracellular resistances. In a next step, transcellular properties may be further divided into their apical and basolateral components. The accuracy of these derived values, however, strongly depends on the accuracy of the initial estimates. To obtain adequate accuracy in estimating subepithelial and epithelial resistance, artificial neural networks were trained to estimate these parameters from model impedance spectra. Spectra that reflect behavior of either HT-29/B6 or IPEC-J2 cells as well as the data scatter intrinsic to the used experimental setup were created computationally. To prove the proposed approach, reliability of the estimations was assessed with both modeled and measured impedance spectra. Transcellular and paracellular resistances obtained by such neural network-enhanced two-path impedance spectroscopy are shown to be sufficiently reliable to derive the underlying apical and basolateral resistances and capacitances. As an exemplary perturbation of pathophysiological importance, the effect of forskolin on the apical resistance of HT-29/B6 cells was quantified.

Ursodeoxycholic acid inhibits the proliferation of colon cancer cells by regulating oxidative stress and cancer stem-like cell growth

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Kim, EuiJoo

2017-01-01

Introduction The regulation of reactive oxygen species (ROS) exists as a therapeutic target for cancer treatments. Previous studies have shown that ursodeoxycholic acid (UDCA) suppresses the proliferation of colon cancer cells. The aim of this study was to evaluate the effect of UDCA upon the proliferation of colon cancer cells as a direct result of the regulation of ROS. Method Colon cancer cell lines (HT29 and HCT116) were treated with UDCA. The total number of cells and the number of dead cells were determined using cell counters. A fluorescein isothiocyanate-bromodeoxyuridine flow kit was used to analyze cell cycle variations. Upon exposure to UDCA, the protein levels of p27, p21, CDK2, CDK4 and CDK6 were determined using western blotting, and qRT-PCR was used to determine levels of mRNA. We preformed dichlorofluorescindiacetate (DCF-DA) staining to detect alteration of intracellular ROS using fluorescence activated cell sorting (FACS). Colon cancer stem-like cell lines were generated by tumorsphere culture and treated with UDCA for seven days. The total number of tumorspheres was determined using microscopy. Results We found that UDCA reduced the total number of colon cancer cells, but did not increase the number of dead cells. UDCA inhibited the G1/S and G2/M transition phases in colon cancer cells. UDCA induced expression of cell cycle inhibitors such as p27 and p21. However, it was determined that UDCA suppressed levels of CDK2, CDK4, and CDK6. UDCA regulated intracellular ROS generation in colon cancer cells, and induced activation of Erk1/2. Finally, UDCA inhibited formation of colon cancer stem-like cells. Conclusion Our results indicate that UDCA suppresses proliferation through regulation of oxidative stress in colon cancer cells, as well as colon cancer stem-like cells. PMID:28708871

Utilization of solid lipid nanoparticles for enhanced delivery of curcumin in cocultures of HT29-MTX and Caco-2 cells.