Objective：To investigate the cellular effects of Pien Tze Huang（片仔癀,PZH） in the HT-29 human colon carcinoma cell line.Methods：The viability of HT-29 cells was determined by MTT assay.A fluorescence-activated ce...Objective：To investigate the cellular effects of Pien Tze Huang（片仔癀,PZH） in the HT-29 human colon carcinoma cell line.Methods：The viability of HT-29 cells was determined by MTT assay.A fluorescence-activated cell sorting（FACS） analysis with annexin-V/propidium iodide（PI） and JC-1 staining were performed to determine cell apoptosis and the loss of mitochondrial membrane potential,respectively.Activation of caspase 3 was evaluated by a colorimetric assay.The mRNA expression levels of Bcl-2 and Bax were measured by reverse transcription polymerase chain reaction（RT-PCR）.Results：PZH,in a dose- and time-dependent manner,reduced viability and induced apoptosis of HT-29 cells.Moreover,PZH treatment resulted in the collapse of the mitochondrial membrane potential,activation of caspase 3,and an increase in the Bax/Bcl-2 ratio.Conclusion：PZH inhibits the growth of HT-29 cells by inducing cancer cell apoptosis via regulation of the Bcl-2 family and activation of caspase 3,which may,in part,explain its anticancer activity.展开更多
Objective: To investigate the anti-colon cancer effects of ethylacetate fraction from Orostachys japonicus(0. japonicus) on HT-29 cancer cells. Methods: The viability of HT-29 cells was assayed by the 3-(4,5-dimethylt...Objective: To investigate the anti-colon cancer effects of ethylacetate fraction from Orostachys japonicus(0. japonicus) on HT-29 cancer cells. Methods: The viability of HT-29 cells was assayed by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium(MTS) method. Apoptosis induction and cell cycle inhibition were confirmed by fluorescein isothiocyanate and propidium iodide staining using flow cytometry.Morphological changes in the nucleus were observed, using a fluorescence microscope with4',6-diamidino-2-phenylindole(DAPI) nuclear staining. The expression levels of the upstream and downstream proteins involved in the anti-cancer mechanism were confirmed by Western blotting. Results: After treating HT-29 cells with different concentrations of ethylacetate fraction from O. japonicus, the viability of cells decreased in a concentration-dependent manner,while apoptosis induction and apoptotic body formation increased. Cell cycle analysis showed that the arrest occurred at the sub-G_1 and S phase. Among the upstream and downstream proteins involved in anti-cancer activity, the level of B cell lymphoma-2 decreased, and the bcl-2-associated x protein increased. The level of pro-caspase-3, pro-caspase-8, and pro-caspase-9 decreased, while the level of cleaved-caspase-3, cleaved-caspase-8, and cleaved-caspase-9 increased. Moreover, the phosphorylation, that is, activation of extracellular signal regulated kinase 1/2, Jun-N-terminal kinase, and p38 increased. Conclusions: Combining the above results, it is thought that the survival of HT-29 cells is suppressed by ethylacetate fraction from0. japonicus through mitochondrial regulation-induced caspase cascade activation, induction of apoptosis and cell cycle arrest.展开更多
Aim:Strong evidence reveals important differences between cancers in the proximal vs.distal colon.Animal models of metastatic colon cancer are available but with varying degrees of reproducibility and several importan...Aim:Strong evidence reveals important differences between cancers in the proximal vs.distal colon.Animal models of metastatic colon cancer are available but with varying degrees of reproducibility and several important limitations.We explored whether there were regional differences in the location of murine colon cancers and assessed the utility of murine models to explore the biological basis for such differences.Methods:We re-analyzed data from our previous studies to assess the regional distribution of murine colon cancer.In survival surgery experiments,we injected HT-29 human colon cancer cells into the wall of the cecum or distal colon of Nu(NCr)-Foxn1 nu or NOD.Cg-Prkdc scid Il2rg Tim1Wji/SzJ mice and compared the development of primary tumors and metastases.Results:Within 7-17 weeks after intramural cecal injection of HT-29 cells,eight mice failed to develop solid primary tumors or metastases.In contrast,within four weeks after cell injection into the distal colon,13 mice developed metastases-12 mice developed subcutaneous metastases;of these,four developed liver metastases and one developed both liver and lung metastases.One mouse developed liver metastases only.Histological examination confirmed these lesions were adenocarcinomas.Conclusion:Our findings reveal the preferential growth of murine colon neoplasia and invasive human orthotopic xenografts in the distal mouse colon.The new approach of injecting cells into the distal colon wall results in a pattern of colon cancer development that closely mimics the progression of metastatic colon cancer in humans.This novel model of colon neoplasia has great potential for exploring anatomical differences in colon cancer and testing novel therapeutics.展开更多
Objective To assess the ability of tetrandrine （Tet） to enhance the sensitivity to irradiation and its mechanism in cell lines of human breast cancer p53-mutant MCF-7/ADR, p53-wild-type MCF-7 and human colon carcino...Objective To assess the ability of tetrandrine （Tet） to enhance the sensitivity to irradiation and its mechanism in cell lines of human breast cancer p53-mutant MCF-7/ADR, p53-wild-type MCF-7 and human colon carcinoma p53-mutant HT-29 as well as in C26 colorectal carcinoma-bearing BALB/c mice. Methods MCF-7/ADR, HT-29 and MCF-7 cells were exposed to irradiation in the absence or presence of tetrandrine. The effect of Tet on the cytotoxicity of X-irradiation in these three cells was determined and the effect of tetrandrine on cell cycle arrest induced by irradiation in its absence or presence was studied by flow cytometry. Moreover, mitotic index measurement determined mitosis of cells to enter mitosis. Western blotting was employed to detect cyclin B1 and Cdc2 proteins in extracts from irradiated or non-irradiated cells of MCF-7/ADR, HT-29 and MCF-7 treated with tetrandrine at various concentrations. Tumor growth delay assay was conducted to determine the radio-sensitization of tetrandrine in vivo. Results Clonogenic assay showed that tetrandrine markedly enhanced the lethal effect of X-rays on p53-mutant MCF-7/ADR and HT-29 cells and the sensitization enhancement ratio （SER） of tetrandrine was 1.51 and 1.63, but its SER was only 1.1 in p53-wt MCF-7 cells. Irradiated p53-mutant MCF-7/ADR and HT-29 cells were only arrested in G2/M phase while MCF-7 cells were arrested in G1 and G2/M phases. Radiation-induced G2 phase arrests were abrogated by tetrandrine in a concentration-dependent manner in MCF-7/ADR and HT-29 cells, whereas redistribution within MCF-7 cell cycle changed slightly. The proportion of cells in M phase increased from 1.3% to 14.7% in MCF-7/ADR cells, and from 1.5% to 13.2% in HT-29 cells, but 2.4% to 7.1% in MCF-7 cells. Furthermore, the levels of cyclin B 1 and Cdc2 expression decreased after X-irradiation in MCF-7/ADR and HT-29 cells, and the mitotic index was also lower. Tet could reverse the decrease and induce the irradiated cells to enter mitosis （M phase）. Endosomatic experiment showed that tetrandrine caused tumor growth delay in irradiated mice. Conclusion Tetrandrine boosts the cell killing activity of irradiation both in vitro and in vivo. Tetrandrine is a potent abrogator for G2 checkpoint control and can sensitize the cells to radiation.展开更多
基金Supported by the National Natural Science Foundation of China(No.81073097)the Developmental Fund of CHEN Ke-ji Integrative Medicine(No.CKJ 2011001)the Natural Science Foundation of Fujian Province of China(No.2010J01195)
文摘Objective：To investigate the cellular effects of Pien Tze Huang（片仔癀,PZH） in the HT-29 human colon carcinoma cell line.Methods：The viability of HT-29 cells was determined by MTT assay.A fluorescence-activated cell sorting（FACS） analysis with annexin-V/propidium iodide（PI） and JC-1 staining were performed to determine cell apoptosis and the loss of mitochondrial membrane potential,respectively.Activation of caspase 3 was evaluated by a colorimetric assay.The mRNA expression levels of Bcl-2 and Bax were measured by reverse transcription polymerase chain reaction（RT-PCR）.Results：PZH,in a dose- and time-dependent manner,reduced viability and induced apoptosis of HT-29 cells.Moreover,PZH treatment resulted in the collapse of the mitochondrial membrane potential,activation of caspase 3,and an increase in the Bax/Bcl-2 ratio.Conclusion：PZH inhibits the growth of HT-29 cells by inducing cancer cell apoptosis via regulation of the Bcl-2 family and activation of caspase 3,which may,in part,explain its anticancer activity.
基金supported by the 2016 Inje University research grant
文摘Objective: To investigate the anti-colon cancer effects of ethylacetate fraction from Orostachys japonicus(0. japonicus) on HT-29 cancer cells. Methods: The viability of HT-29 cells was assayed by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium(MTS) method. Apoptosis induction and cell cycle inhibition were confirmed by fluorescein isothiocyanate and propidium iodide staining using flow cytometry.Morphological changes in the nucleus were observed, using a fluorescence microscope with4',6-diamidino-2-phenylindole(DAPI) nuclear staining. The expression levels of the upstream and downstream proteins involved in the anti-cancer mechanism were confirmed by Western blotting. Results: After treating HT-29 cells with different concentrations of ethylacetate fraction from O. japonicus, the viability of cells decreased in a concentration-dependent manner,while apoptosis induction and apoptotic body formation increased. Cell cycle analysis showed that the arrest occurred at the sub-G_1 and S phase. Among the upstream and downstream proteins involved in anti-cancer activity, the level of B cell lymphoma-2 decreased, and the bcl-2-associated x protein increased. The level of pro-caspase-3, pro-caspase-8, and pro-caspase-9 decreased, while the level of cleaved-caspase-3, cleaved-caspase-8, and cleaved-caspase-9 increased. Moreover, the phosphorylation, that is, activation of extracellular signal regulated kinase 1/2, Jun-N-terminal kinase, and p38 increased. Conclusions: Combining the above results, it is thought that the survival of HT-29 cells is suppressed by ethylacetate fraction from0. japonicus through mitochondrial regulation-induced caspase cascade activation, induction of apoptosis and cell cycle arrest.
文摘Aim:Strong evidence reveals important differences between cancers in the proximal vs.distal colon.Animal models of metastatic colon cancer are available but with varying degrees of reproducibility and several important limitations.We explored whether there were regional differences in the location of murine colon cancers and assessed the utility of murine models to explore the biological basis for such differences.Methods:We re-analyzed data from our previous studies to assess the regional distribution of murine colon cancer.In survival surgery experiments,we injected HT-29 human colon cancer cells into the wall of the cecum or distal colon of Nu(NCr)-Foxn1 nu or NOD.Cg-Prkdc scid Il2rg Tim1Wji/SzJ mice and compared the development of primary tumors and metastases.Results:Within 7-17 weeks after intramural cecal injection of HT-29 cells,eight mice failed to develop solid primary tumors or metastases.In contrast,within four weeks after cell injection into the distal colon,13 mice developed metastases-12 mice developed subcutaneous metastases;of these,four developed liver metastases and one developed both liver and lung metastases.One mouse developed liver metastases only.Histological examination confirmed these lesions were adenocarcinomas.Conclusion:Our findings reveal the preferential growth of murine colon neoplasia and invasive human orthotopic xenografts in the distal mouse colon.The new approach of injecting cells into the distal colon wall results in a pattern of colon cancer development that closely mimics the progression of metastatic colon cancer in humans.This novel model of colon neoplasia has great potential for exploring anatomical differences in colon cancer and testing novel therapeutics.
基金supported by a grant from the Jiangsu Natural Science Foundation (No. BK2005203).
文摘Objective To assess the ability of tetrandrine （Tet） to enhance the sensitivity to irradiation and its mechanism in cell lines of human breast cancer p53-mutant MCF-7/ADR, p53-wild-type MCF-7 and human colon carcinoma p53-mutant HT-29 as well as in C26 colorectal carcinoma-bearing BALB/c mice. Methods MCF-7/ADR, HT-29 and MCF-7 cells were exposed to irradiation in the absence or presence of tetrandrine. The effect of Tet on the cytotoxicity of X-irradiation in these three cells was determined and the effect of tetrandrine on cell cycle arrest induced by irradiation in its absence or presence was studied by flow cytometry. Moreover, mitotic index measurement determined mitosis of cells to enter mitosis. Western blotting was employed to detect cyclin B1 and Cdc2 proteins in extracts from irradiated or non-irradiated cells of MCF-7/ADR, HT-29 and MCF-7 treated with tetrandrine at various concentrations. Tumor growth delay assay was conducted to determine the radio-sensitization of tetrandrine in vivo. Results Clonogenic assay showed that tetrandrine markedly enhanced the lethal effect of X-rays on p53-mutant MCF-7/ADR and HT-29 cells and the sensitization enhancement ratio （SER） of tetrandrine was 1.51 and 1.63, but its SER was only 1.1 in p53-wt MCF-7 cells. Irradiated p53-mutant MCF-7/ADR and HT-29 cells were only arrested in G2/M phase while MCF-7 cells were arrested in G1 and G2/M phases. Radiation-induced G2 phase arrests were abrogated by tetrandrine in a concentration-dependent manner in MCF-7/ADR and HT-29 cells, whereas redistribution within MCF-7 cell cycle changed slightly. The proportion of cells in M phase increased from 1.3% to 14.7% in MCF-7/ADR cells, and from 1.5% to 13.2% in HT-29 cells, but 2.4% to 7.1% in MCF-7 cells. Furthermore, the levels of cyclin B 1 and Cdc2 expression decreased after X-irradiation in MCF-7/ADR and HT-29 cells, and the mitotic index was also lower. Tet could reverse the decrease and induce the irradiated cells to enter mitosis （M phase）. Endosomatic experiment showed that tetrandrine caused tumor growth delay in irradiated mice. Conclusion Tetrandrine boosts the cell killing activity of irradiation both in vitro and in vivo. Tetrandrine is a potent abrogator for G2 checkpoint control and can sensitize the cells to radiation.
