Aim:Mitomycin C(MMC)is a commonly used as intravesical treatment for superficial bladder cancer.However,its role in combination with ras inhibition has not been investigated.The aim of this study was to explore the ro...Aim:Mitomycin C(MMC)is a commonly used as intravesical treatment for superficial bladder cancer.However,its role in combination with ras inhibition has not been investigated.The aim of this study was to explore the role of ras in MMC-induced apoptosis in T24 bladder cancer cells and to determine the efficacy of combination therapy in vitro.Methods:We measured the effects of various doses of MMC on apoptosis induction as well as on ras,ERK and Ki-67 protein expression by T24 cell line using immunocytochemistry,flow cytometry and Western blotting.We also tested the effect of siRNA on ras employed singly or in combination with MMC.Results:T24 cells expressed high level of ras protein.MMC treatment increased the level of ras and ERK protein expression after 24 h,and decreased these levels after 72 h.Ras siRNA(100 nmol/L)caused massive apoptosis associated with a marked decrease in ras expression in T24 cells.When combined with low doses of MMC,ras siRNA(50 nmol/L)sensitized T24 cells to apoptosis and decreased their expression of ras.The effect of combined therapy was higher than that of either compound used alone.Expression levels of ERK,a downstream target of ras,declined following combination therapy.Conclusion:Ras siRNA in combination with low dose MMC is a possible treatment strategy for patients with ras-positive bladder tumors.展开更多
Expression of DNA methyltransferase 1(DNMT1),which plays an important role on aberrantly methylated CpG in the promoter regions of tumor sup-pressor genes(TSGs),is higher in bladder cancer cells than in normal bladder...Expression of DNA methyltransferase 1(DNMT1),which plays an important role on aberrantly methylated CpG in the promoter regions of tumor sup-pressor genes(TSGs),is higher in bladder cancer cells than in normal bladder cells.Therefore,its overexpression is closely related to tumor formation.In this study,the eukaryotic vector pshRNA-DNMT1 was constructed and transfected into T24 cells.Levels of DNMT1 mRNA and protein were detected by reverse transcription-polymerase chain reaction(RT-PCR)and western blot.Relative to the blank control at the 24th,48th and 72nd hour after transfection of pshRNA-DNMT1,the inhibitory rates of DNMT1 mRNA levels in T24 cells were 28.44%,52.48%,70.91%,respectively.Those of DNMT1 proteins were 24.27%,57.79%,and 77.74%,respectively.Proliferation and apoptosis were assayed by MTT and flow cytometry with Annexin-V-FITC/PI staining.The growth inhibition rates of pshRNA-DNMT1 at the 24th,48th and 72nd hour after transfection of pshRNA-DNMT1 were(4.34¡0.76)%,(9.87¡1.54)%and(13.78¡1.93)%,respectively.There were statistically significant differ-ences between pshRNA-DNMT1 and the control blank at each time points(P,0.01);24,48 and 72 hours after T24 cells were transfected by pshRNA-DNMT1,the apoptosis rates of pshRNA-DNMT1 were(3.87¡0.81)%,(8.69¡1.23)%and(11.46¡1.24)%,respectively(P,0.01 vs blank control).Based on this case,our conclu-sion is that the recombinant plasmid pshRNA-DNMT1 can silence the expression of gene DNMT1 mRNA and protein effectively,and to some extent,it also can inhibit the proliferation of bladder cancer cell and promote the cellular apoptosis.展开更多
文摘Aim:Mitomycin C(MMC)is a commonly used as intravesical treatment for superficial bladder cancer.However,its role in combination with ras inhibition has not been investigated.The aim of this study was to explore the role of ras in MMC-induced apoptosis in T24 bladder cancer cells and to determine the efficacy of combination therapy in vitro.Methods:We measured the effects of various doses of MMC on apoptosis induction as well as on ras,ERK and Ki-67 protein expression by T24 cell line using immunocytochemistry,flow cytometry and Western blotting.We also tested the effect of siRNA on ras employed singly or in combination with MMC.Results:T24 cells expressed high level of ras protein.MMC treatment increased the level of ras and ERK protein expression after 24 h,and decreased these levels after 72 h.Ras siRNA(100 nmol/L)caused massive apoptosis associated with a marked decrease in ras expression in T24 cells.When combined with low doses of MMC,ras siRNA(50 nmol/L)sensitized T24 cells to apoptosis and decreased their expression of ras.The effect of combined therapy was higher than that of either compound used alone.Expression levels of ERK,a downstream target of ras,declined following combination therapy.Conclusion:Ras siRNA in combination with low dose MMC is a possible treatment strategy for patients with ras-positive bladder tumors.
文摘Expression of DNA methyltransferase 1(DNMT1),which plays an important role on aberrantly methylated CpG in the promoter regions of tumor sup-pressor genes(TSGs),is higher in bladder cancer cells than in normal bladder cells.Therefore,its overexpression is closely related to tumor formation.In this study,the eukaryotic vector pshRNA-DNMT1 was constructed and transfected into T24 cells.Levels of DNMT1 mRNA and protein were detected by reverse transcription-polymerase chain reaction(RT-PCR)and western blot.Relative to the blank control at the 24th,48th and 72nd hour after transfection of pshRNA-DNMT1,the inhibitory rates of DNMT1 mRNA levels in T24 cells were 28.44%,52.48%,70.91%,respectively.Those of DNMT1 proteins were 24.27%,57.79%,and 77.74%,respectively.Proliferation and apoptosis were assayed by MTT and flow cytometry with Annexin-V-FITC/PI staining.The growth inhibition rates of pshRNA-DNMT1 at the 24th,48th and 72nd hour after transfection of pshRNA-DNMT1 were(4.34¡0.76)%,(9.87¡1.54)%and(13.78¡1.93)%,respectively.There were statistically significant differ-ences between pshRNA-DNMT1 and the control blank at each time points(P,0.01);24,48 and 72 hours after T24 cells were transfected by pshRNA-DNMT1,the apoptosis rates of pshRNA-DNMT1 were(3.87¡0.81)%,(8.69¡1.23)%and(11.46¡1.24)%,respectively(P,0.01 vs blank control).Based on this case,our conclu-sion is that the recombinant plasmid pshRNA-DNMT1 can silence the expression of gene DNMT1 mRNA and protein effectively,and to some extent,it also can inhibit the proliferation of bladder cancer cell and promote the cellular apoptosis.
