| Part I Effects of metformin combined with lobaplatin on inhibiting proliferation,migration and inducing apoptosis of ovarian cancer cellsObjective:To observe the effect of metformin combined with lobaplatin on the biological behavior of ovarian cancer cells,and to provide a new target and theoretical basis for the treatment of ovarian cancer.Methods:Epithelial ovarian cancer cell line SKOV3 cells were selected for in vitro culture.Logarithmic growth phase cells were selected for experiment,and different concentrations of metformin(0.5mmol/L,2.5mmol/L,5mmol/L)and lobaplatin(0.5μg/mL,1μg/mL,2μg/mL)were co-cultured.After 48 hours of culture in the incubator,the proliferation inhibition rate of ovarian cancer cells was detected by MTT assay,and the semi-inhibitory concentration(IC50)of metformin and lobaplatin was calculated,and the calculated result of 1/2 of IC50 was used as the drug concentration of subsequent experimental studies.Ovarian cancer cell line SKOV3 was co-cultured with metformin,lobaplatin and the combination of the two drugs for 0-72 hours,and the proliferation inhibition rate of ovarian cancer SKOV3 cells was detected again at 24h,48h and 72h.The change of migration ability of cells in each group was detected by the scratch test,and the change of apoptosis level of SKOV3 cells in each group was detected by flow cytometry.Results:1.Inhibitory effects of metformin and lobaplatin at different concentrations on proliferation of ovarian cancer SKOV3 cells:(1)different concentrations of metformin(0.5mmol/L,2.5mmol/L,5mmol/L)inhibited the proliferation of ovarian cancer SKOV3 cells at 24 h,48 h and 72 h in a dose-and time-dependent manner,with statistically significant differences between groups(P<0.05).(2)Lobaplatin concentration of 0.5μg/ml,1μg/ml and 2μg/ml significantly inhibited the survival of ovarian cancer SKOV3 cells at 24 h,48 h and 72 h in a dose-and time-dependent manner,with statistically significant differences between groups(P<0.01).2.The IC50 of metformin against SKOV3 cell proliferation was 6.2 mmol/L,and lobaplatin against SKOV3 cell proliferation was 4.3μg/mL.Therefore,the concentrations of metformin and lobaplatin were 3 mmol/L and 2μg/mL,respectively.3.To compare the inhibitory effects of metformin alone,lobaplatin and combined treatment on the migration of ovarian cancer cell line SKOV3:Scratch test results showed that metformin and lobaplatin alone could inhibit the migration ability of ovarian cancer cell line SKOV3 compared with blank control group,and the inhibition effect was significantly enhanced when the two drugs were combined,with statistical significance(P<0.05).4.To compare the effects of metformin alone,lobaplatin and combined treatment on apoptosis of ovarian cancer cell line SKOV3:Flow cytometry results showed that metformin and lobaplatin alone increased the apoptosis rate of ovarian cancer cell line SKOV3 compared with the blank control group,while the combined effect of metformin and lobaplatin was stronger,and the apoptosis rate of SKOV3 cell was significantly increased,with statistical significance(P<0.05).Conclusion:1.Both metformin and lobaplatin can inhibit the proliferation and migration of ovarian cancer cell line SKOV3,and the effect of the combination of metformin and lobaplatin is significantly stronger than that of single drug.2.Both metformin and lobaplatin can promote the apoptosis of ovarian cancer cell line SKOV3,and the effect of the combination of metformin and lobaplatin on inducing apoptosis is significantly stronger than that of single drug,which has a significant synergistic effect.3.Metformin can enhance the sensitivity of ovarian cancer cells to lobaplatin.Part Ⅱ Effects of metformin combined with lobaplatin on PARP-1/SIRT1/mTOR signaling pathway in ovarian cancer cellsObjective:To observe the effects of metformin combined with lobaplatin on the expression of PARP-1/SIRT1/mTOR signaling pathway protein and the expression of downstream apoptosis-inhibiting protein Survivin in ovarian cancer cells,and to further elucidate the possible mechanism of metformin and lobaplatin on the biological behavior of ovarian cancer cell line SKOV3.Methods:Ovarian cancer cell line SKOV3 was cultured in vitro,Metformin and lobaplatin were used alone or in combination48 h later,the cell protein and mRNA were extracted,and the expression of PARP-1,SIRT1,mTOR and the downstream inhibitor of apoptosis protein Survivin were detected by RT-PCR and Western blot,respectively.Results:1.RT-PCR was used to detect the expression of PARP-1mRNA and survivin mRNA:Compared with the control group,the expression levels of PARP-1 mRNA,SIRT1-mRNA,mTOR-mRNA and Survivin-mRNA were significantly decreased in the metformin alone or lobaplatin group(P<0.05),while the above changes were more obvious in the combination group(P<0.01).2.Western blot was used to detect the expression of PARP-1/SIRT1/mTOR signal pathway protein and the downstream inhibitor of apoptosis protein survivin.Metformin alone or lobaplatin could inhibit the protein expression of PARP-1/SIRT1/mTOR signal pathway protein,but the inhibition effect was more obvious when metformin+lobaplatin were combined,and the difference was statistically significant(P<0.01).at the same time,the expression of survivin,an inhibitor of apoptosis protein,decreased significantly(P<0.01).Conclusion:Metformin can increase the sensitivity of ovarian cancer cells to lobaplatin,which may be related to inhibiting the proliferation of ovarian cancer cells by inhibiting PARP-1/SIRT1/mTOR signal pathway,and promoting the apoptosis of ovarian cancer cells and inhibiting the biological behavior progress of ovarian cancer by affecting the expression of downstream apoptosis-inhibitor protein survivin. |
