Effect Of Metformin On The Biological Behavior Of Bladder Cancer Cells And Increase Their Sensitivity To Cisplatin

PART ONEEFFECT OF METFORMIN ON THE MIGRATION, INVASION AND PROLIFERATION OF T24 BLADDER CANCER CELLSObjective:To observe the influence of metformin on the migration, invasion and proliferation of bladder carcinoma cells.Methods:Cell migration assay were used to observe migrationn change of T24 cell lines, and transwell invasion assay were used to observe invasion change of T24 cell lines. After influenced by different concentrations of metformin for different time, the changes of T24 bladder cancer cell lines proliferation was detected by MTT assay.Results:Cell migration assay showed that metformin could i-nhibit migration of the bladder cancer cell lines T24, and transwell invasion assay showed that metformin could inhibit invasion of the bladder cancer cell lines T24.MTT assay showed that the proliferation of the bladder cancer cell lines T24 were inhibited by metformin in a concentration and time dependent mannerConclusion:Metformin could inhibit proliferation, migration and invasion of bladder cancer cell line T24 in a concentration and-time dependent mannerPART TOWEFFECT OF METFORMIN ON THE PROLIFERATION, APOPTOSIS AND INVASION OF T24 BLADDER CANCER CELLSObjective:To observe the influence of metformin on proliferation, apoptosis and invasion of T24 bladder carcinoma cells.Methods:By different concentrations of metformin for 48h,Annexin V-FITC/PI double staining and flow cytometry (FCM) analysis were used to observe its apoptosis changes after treated. RT-PCR was used to detect proliferation, apoptosis-related genes, cyclinDl, HIF-1, c-Myc, caspase-8, caspase-9, and migration and invasion-related genes MMP-2,, MMP-9 in T24 bladder cancer cell lines. Western blot was used to detect proliferation, apoptosis-related proteins, cyclinD1, HIF-1, c-Myc, caspase-8, caspase-9,and migration and invasion-related genes MMP-2,, MMP-9 in T24 bladder cancer cell lines.Results:After metformin treatment for 48h, Annexin V-FITC/PI double staining and flow cytometry (FCM) analysis showed metformin promoted apoptosis of bladder cancer cell lines T24 in a concentration manner.Downregulated its protein expression of cyclinD1, HIF-l,c-Myc,upregulated its protein expression of caspase8, caspase9,there was obvious changes for protein expression mentioned above during every concentration group(P<0.05), while there was no significant changes for protein expression of MMP-2,MMP-9 during every concentration group(P>0.05).Conclusion:Metformin could promote apoptosis of cell lines T24, inhibit expression of proliferation and apoptosis-related proteins cyclinD 1, HIF-1, c-Myc expression, promote protein caspas-e8 and caspase9 upregulated via a concentration dependent manner, but does not affect their migration and invasion associated regulatory proteins expression of MMP-2 and MMP-9, and these changes may be associated with AMPK or mTOR signaling pathway related changes caused by metformin. PART THREESTUDY OF METFORMIN IMPROVE BLADDER CANCER T24 AND BIU-87 CISPLATIN SENSITIVITY AND SEARCH ITS MECHANISM IN VITROObjective:To study related mechanisms about different concentra-tions of metformin on bladder cancer T24 and BIU-87 cells sensitivity to cisplatin.Methods:After treated by cisplatin (0.5 uM), metformin (1、2、4、 8 uM), alone or in combination, MTT was used to observe the prolif-eration of T24 and BIU-87 cells. Cisplatin (0.5uM), metfor-min (4uM), cisplatin (0.5uM) combinedwith metformin (4uM) wereselected respectively, after T24 and BIU-87 cells were treated for 48h, flow cytometry was used to observe the cell cycle; Western blot was used to detect AMPK, p-AMPK, mTOR, p-mTOR protein expression.Results:MTT results showed that after treated by different concentr-ateionn of metformin (1,2,4 uM) in combination with cisplatin blad-dercancer cell line T24 and BIU-87 for 48h, the metformin could promote the power of cisplatin on proliferation inhibition of T24 and BIU-87 cells in a concentration dependent manner, differencein each concentration gro up was statistically significant (P<0.05), but cisplatin combined with m-etformin (4uM) and metformin (8uM) on cell proliferation group, there was no statistically significant (P>0.05). Flow cytometry showe-d cisplatin in combination with metformin can more significantly promo-te T24 and BIU-87 cell cycle arrest in subGl period, the number of cells 1-ocated in subGl period in combination group was significant (P＜0.05) d-ifferent compared with monotherapy treatment group and control group. Western blot showed that the expression of p-mTOR protein in combined treatment group of T24 andBIU-87 cells was significantly reduced compa-red with monotherapy treatment group and the control group, difference was statistically remarkable (P＜0.05), whereas there was no significant c-hange about.the expression of AMPK, p-AMPK, mTOR protein (P>0.05).Conclusion:Low concentrations of metformin can significantly impro-ve cisplatin capacity for bladder cancer cell proliferation inhibition, whi-ch may be related to the signaling pathway of mTOR inhibition.PART FOURMETFORMIN IMPROVE THE ABILITY OF CISPLATIN ON T24 BLADDER CANCER CELLS DERIVED XENOGRAFT TUMOR GROWTH AND ANGIOGENESIS INHIBITION IN VIVOObjective:In vivo study, to observe a certain concentration of metformin in combination with cisplatin effect on bladder cancer T24-derived tumor growth and angiogenesis ability.Methods:T24 cell xenografts in nude mice, after using a certain concentration of cisplatin (2mg/Kg/4d) and metformin (250mg/Kg/4d) alone or in combination, changes were observed in tumor growth, immunohistochemistry assay was used to observe xenografts Ki-67 proliferation index and microvessel density CD34 index change.Results:The nude mice experiments show cisplatin drug metformin could inhibit the growth of tumor volume and an increase in weight, which was statistically significant (P＜0.05) with the monotherapy treatment group and the control group, the difference; at the same time, immunohistochemistry the results showed that cisplatin plus metformin significantly reduced xenograft can more Ki-67-positive cells and microvessel density, which has a statistically significant (P<0.05) with the monotherapy treatment group and control group differences.Conclusions:In vivo, metformin can significantly enhance power of cisplatin on tumor growth inhibition and angiogenesis.