AIM: To investigate the effects of antisense humantelomerase RNA (hTR) on the biologic behavior of humangastric cancer cell line: MKN-45 by gene transfection and itspotential role in the gene therapy of gastric cancer...AIM: To investigate the effects of antisense humantelomerase RNA (hTR) on the biologic behavior of humangastric cancer cell line: MKN-45 by gene transfection and itspotential role in the gene therapy of gastric cancer.METHODS: The hTR cDNA fragment was cloned from MKN-45 through RT-PCR and subcloned into eukaryoticexpression vector (pEF6/V5-His-TOPO) in cis-direction ortrans-direction by DNA recombinant methods. Theconstructed sense, antisense and empty vectors weretransfected into MKN-45 cell lines separately by lipofectin-mediated DNA transfection technology. After drugselection, the expression of antisense hTR gene in stabletransfectants and normal MKN-45 cells was detected by RT-PCR, the telomerase activity by TRAP, the apoptoticfeatures by PI and Hoechst 33258 staining, the cell cycledistribution by flow cytometry and the population doublingtime by cell counting. Comparison among the stabletransfectants and normal MKN-45 cells was made.RESULTS: The sense, antisense hTR eukaryotic expressionvectors and empty vector were successfully constructed andproved to be the same as original design by restrictionendonuclease analysis and sequencing. Then, they weresuccessfully transfected into MKN-45 cell lines separatelywith lipofectin. The expression of antisense hTR gene wasonly detected in MKN-45 cells stably transfected withantisense hTR vector (named as MKN-45-ahTR) but not inthe control cells. In MKN-45-ahTR, the telomerase activitywas inhibited by 75 %, the apoptotic rate was increased to25.3 %, the percentage of cells in the G0/G1 phase wasincreased to 65 %, the proliferation index was decreased to35 % and the population doubling time was prolonged to 35.3hours. However, the telomerase activity, the apoptotic rate,the distribution of cell cycle, the proliferation index and thepopulation doubling time were not different among the controlcells.CONCLUSION: Antisense hTR can significantly inhibittelomerase activity and proliferation of MKN-45 cells andinduce cell apoptosis. Antisense gene therapy based onteiomerase inhibition can be a potential therapeuticapproach to the treatment of gastric cancer.展开更多
AIM: To explore the inducing effect of human mutant p27 gene on the apoptosis of the human gastric cancer cell line MKN-45 and its associated mechanisms. METHODS: The recombinant adenovirus Ad-p27mt was constructed to...AIM: To explore the inducing effect of human mutant p27 gene on the apoptosis of the human gastric cancer cell line MKN-45 and its associated mechanisms. METHODS: The recombinant adenovirus Ad-p27mt was constructed to infect the human gastric cancer cell line MKN-45. Using flow cytometry, TUNEL assay and DNA fragment analysis, we measured the apoptotic effect of Ad-p27mt on the human gastric cancer cells. RESULTS: Ad-p27mt was successfully constructed and the infection efficiency reached 100%. After 18 h of infection, we observed an apoptotic hypodiploid peak on the flow cytometer before G1-S and apoptotic characteristic bands in the DNA electrophoresis. The apoptotic rate detected by TUNEL method was significantly higher in the Ad-p27mt group (89.4±3.12%)compared to the control group (3.12±0.13%, P < 0.01).CONCLUSION: Human mutant p27 can induce apoptosis of the human gastric cancer cells in vitro.展开更多
AIM: To investigate the effects of mifepristone on the invasive and metastatic potential of human gastric adenocarcinoma cell line MKN-45 and its mechanisms. METHODS: After incubation with various concentrations of m...AIM: To investigate the effects of mifepristone on the invasive and metastatic potential of human gastric adenocarcinoma cell line MKN-45 and its mechanisms. METHODS: After incubation with various concentrations of mifepristone (5, 10, 20 umol/L), the adhesion to artificial basement membrane, Matrigel, and the migration of MKN-45 cells were assayed using MTT assay and Transwell cell culture chambers, respectively. Enzyme- linked immunoabsorbent assay (ELISA) and flow cytometry were used to determine the expression of vascular endothelial growth factor (VEGF) and integrin 133 in the cells. After subcutaneous transplantation of MKN-45 cells in nude mice, mifepristone (50 mg/kg.d) was administrated subcutaneously for 8 wk to assess its effects on tumor metastasis. Immunohistochemical analysis was used to detect the expression of VEGF and microvascular density (MVD) in xenografted tumors. RESULTS: Mifepristone dose-dependently inhibited the heterotypic adhesion to Matrigel of MKN-45 cells. The inhibition was accompanied by a significant down-regulation of integrin 133 expression in the cells. After incubation with 5, 10, 20 umol/L mifepristone, the number of migrated MKN-45 cells was 72+8, 50+6, 41+5 in experiment group, and 94+16 in control group (P<0.01). Meanwhile, secreted VEGF protein of MKN-45 cells in mifepristone-treated group (14.2+2.9, 8.9+3.1, 5.4+2.1 ng/g per liter) was significantly lower than that in control group (22.7+4.3 ng/g per liter, P<0.01). In vivo, mifepristone decreased the number of metastatic foci in lungs of nude mice and down-regulated the expression of VEGF and MVD in the xenograted tumors. CONCLUSION: Mifepristone can effectively inhibit the invasive and metastatic potential of human gastric adenocarcinoma cell line MKN-45 in vitro and in vivo through inhibition of heterotypic adhesion to basement membrane, cell migration and angiogenesis.展开更多
Objective To explore the effects of mifepristone on the growth of human gastric cancer cell line MKN-45 and its possible mechanisms. Methods In situ hybridization was used to detect the expression of progesterone rece...Objective To explore the effects of mifepristone on the growth of human gastric cancer cell line MKN-45 and its possible mechanisms. Methods In situ hybridization was used to detect the expression of progesterone receptor (PR) mRNA in MKN-45 cells. Proliferation, cell cycle distribution, and the expression of Bcl-xL and vascular endothelial growth factor (VEGF) of MKN-45 cells incubated with various concentrations of mifepristone (1, 5, 10, and 20 μmol/L) were analyzed using MTT reduction assay, flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR), and enzyme-linked immunoab-sorbent assay (ELISA), respectively. After transplantation of MKN-45 cells underneath the skin of athymic mice, mifepristone was administrated with the dose of 50 mg/(kg·d) for 6 weeks to evaluate the tumor growth. Apoptosis and the expression of proliferating cell nuclear antigen (PCNA) in xenografted tumors were detected using transmission electron microscopy and immunohistochemical staining, respectively. Results PR mRNA was highly expressed in cultured MKN-45 cell. Mifepristone dose-dependently inhibited the pr-oliferation of MKN-45 cells, and the inhibitory rate was dramatically increased from 7.21% to 47.23%. The inhibitory effect was accompanied by a dose-dependent increase in the percentage of cells in G 0 /G 1 phase, and with a concurrent decrease in the proportion of S- and G 2 /M-phase cells and the proliferative index from 57.65% to 24.54%. Meanwhile, mifepristone down-regulated the expression of Bcl-xL and VEGF in a dose-dependent manner. In vivo, mifepristone effectively inhibited the growth of xenografted tumors in nude mice (55.14% for inhibitory rate), induced apoptosis, and down-regulated PCNA expression in gastric cancer. Conclusion Mifepristone exerts significant growth inhibitory effects on PR-positive human MKN-45 gastric cancer cells via multiple mechanisms, and may be a beneficial agent against the tumor.展开更多
基金the National Natural Science Foundation of China,No.39770725
文摘AIM: To investigate the effects of antisense humantelomerase RNA (hTR) on the biologic behavior of humangastric cancer cell line: MKN-45 by gene transfection and itspotential role in the gene therapy of gastric cancer.METHODS: The hTR cDNA fragment was cloned from MKN-45 through RT-PCR and subcloned into eukaryoticexpression vector (pEF6/V5-His-TOPO) in cis-direction ortrans-direction by DNA recombinant methods. Theconstructed sense, antisense and empty vectors weretransfected into MKN-45 cell lines separately by lipofectin-mediated DNA transfection technology. After drugselection, the expression of antisense hTR gene in stabletransfectants and normal MKN-45 cells was detected by RT-PCR, the telomerase activity by TRAP, the apoptoticfeatures by PI and Hoechst 33258 staining, the cell cycledistribution by flow cytometry and the population doublingtime by cell counting. Comparison among the stabletransfectants and normal MKN-45 cells was made.RESULTS: The sense, antisense hTR eukaryotic expressionvectors and empty vector were successfully constructed andproved to be the same as original design by restrictionendonuclease analysis and sequencing. Then, they weresuccessfully transfected into MKN-45 cell lines separatelywith lipofectin. The expression of antisense hTR gene wasonly detected in MKN-45 cells stably transfected withantisense hTR vector (named as MKN-45-ahTR) but not inthe control cells. In MKN-45-ahTR, the telomerase activitywas inhibited by 75 %, the apoptotic rate was increased to25.3 %, the percentage of cells in the G0/G1 phase wasincreased to 65 %, the proliferation index was decreased to35 % and the population doubling time was prolonged to 35.3hours. However, the telomerase activity, the apoptotic rate,the distribution of cell cycle, the proliferation index and thepopulation doubling time were not different among the controlcells.CONCLUSION: Antisense hTR can significantly inhibittelomerase activity and proliferation of MKN-45 cells andinduce cell apoptosis. Antisense gene therapy based onteiomerase inhibition can be a potential therapeuticapproach to the treatment of gastric cancer.
基金Supported by the Natural Science Foundation of Shanghai,No. 04ZB14072
文摘AIM: To explore the inducing effect of human mutant p27 gene on the apoptosis of the human gastric cancer cell line MKN-45 and its associated mechanisms. METHODS: The recombinant adenovirus Ad-p27mt was constructed to infect the human gastric cancer cell line MKN-45. Using flow cytometry, TUNEL assay and DNA fragment analysis, we measured the apoptotic effect of Ad-p27mt on the human gastric cancer cells. RESULTS: Ad-p27mt was successfully constructed and the infection efficiency reached 100%. After 18 h of infection, we observed an apoptotic hypodiploid peak on the flow cytometer before G1-S and apoptotic characteristic bands in the DNA electrophoresis. The apoptotic rate detected by TUNEL method was significantly higher in the Ad-p27mt group (89.4±3.12%)compared to the control group (3.12±0.13%, P < 0.01).CONCLUSION: Human mutant p27 can induce apoptosis of the human gastric cancer cells in vitro.
基金Supported by the National Key Research Project Foundation of China,No.96-905- 02-01,and the National Natural Science Foundation of China,No.39630340
文摘AIM: To investigate the effects of mifepristone on the invasive and metastatic potential of human gastric adenocarcinoma cell line MKN-45 and its mechanisms. METHODS: After incubation with various concentrations of mifepristone (5, 10, 20 umol/L), the adhesion to artificial basement membrane, Matrigel, and the migration of MKN-45 cells were assayed using MTT assay and Transwell cell culture chambers, respectively. Enzyme- linked immunoabsorbent assay (ELISA) and flow cytometry were used to determine the expression of vascular endothelial growth factor (VEGF) and integrin 133 in the cells. After subcutaneous transplantation of MKN-45 cells in nude mice, mifepristone (50 mg/kg.d) was administrated subcutaneously for 8 wk to assess its effects on tumor metastasis. Immunohistochemical analysis was used to detect the expression of VEGF and microvascular density (MVD) in xenografted tumors. RESULTS: Mifepristone dose-dependently inhibited the heterotypic adhesion to Matrigel of MKN-45 cells. The inhibition was accompanied by a significant down-regulation of integrin 133 expression in the cells. After incubation with 5, 10, 20 umol/L mifepristone, the number of migrated MKN-45 cells was 72+8, 50+6, 41+5 in experiment group, and 94+16 in control group (P<0.01). Meanwhile, secreted VEGF protein of MKN-45 cells in mifepristone-treated group (14.2+2.9, 8.9+3.1, 5.4+2.1 ng/g per liter) was significantly lower than that in control group (22.7+4.3 ng/g per liter, P<0.01). In vivo, mifepristone decreased the number of metastatic foci in lungs of nude mice and down-regulated the expression of VEGF and MVD in the xenograted tumors. CONCLUSION: Mifepristone can effectively inhibit the invasive and metastatic potential of human gastric adenocarcinoma cell line MKN-45 in vitro and in vivo through inhibition of heterotypic adhesion to basement membrane, cell migration and angiogenesis.
文摘Objective To explore the effects of mifepristone on the growth of human gastric cancer cell line MKN-45 and its possible mechanisms. Methods In situ hybridization was used to detect the expression of progesterone receptor (PR) mRNA in MKN-45 cells. Proliferation, cell cycle distribution, and the expression of Bcl-xL and vascular endothelial growth factor (VEGF) of MKN-45 cells incubated with various concentrations of mifepristone (1, 5, 10, and 20 μmol/L) were analyzed using MTT reduction assay, flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR), and enzyme-linked immunoab-sorbent assay (ELISA), respectively. After transplantation of MKN-45 cells underneath the skin of athymic mice, mifepristone was administrated with the dose of 50 mg/(kg·d) for 6 weeks to evaluate the tumor growth. Apoptosis and the expression of proliferating cell nuclear antigen (PCNA) in xenografted tumors were detected using transmission electron microscopy and immunohistochemical staining, respectively. Results PR mRNA was highly expressed in cultured MKN-45 cell. Mifepristone dose-dependently inhibited the pr-oliferation of MKN-45 cells, and the inhibitory rate was dramatically increased from 7.21% to 47.23%. The inhibitory effect was accompanied by a dose-dependent increase in the percentage of cells in G 0 /G 1 phase, and with a concurrent decrease in the proportion of S- and G 2 /M-phase cells and the proliferative index from 57.65% to 24.54%. Meanwhile, mifepristone down-regulated the expression of Bcl-xL and VEGF in a dose-dependent manner. In vivo, mifepristone effectively inhibited the growth of xenografted tumors in nude mice (55.14% for inhibitory rate), induced apoptosis, and down-regulated PCNA expression in gastric cancer. Conclusion Mifepristone exerts significant growth inhibitory effects on PR-positive human MKN-45 gastric cancer cells via multiple mechanisms, and may be a beneficial agent against the tumor.