Zhiyong Shen1,2, Dong Xue1, Kun Wang1, Facai Zhang3,4, Jiaqi Shi4, Benzhong Jia2, Dan Yang5, Qianjin Zhang1,6, Shuai Zhang7, Hongyu Jiang7, Daiqin Luo4,7, Xueying Li7, Quliang Zhong4, Junhao Zhang4, Zheng Peng2, Yu Han2, Chongyang Sima2, Xiaozhou He8, Lin Hao9. 1. Department of Urology, The Third Affiliated Hospital of Soochow University, No.185, Juqian Street, Tianning District, Changzhou, 213000, Jiangsu Province, China. 2. Department of Urology, The Affiliated Cancer Hospital of Guizhou Medical University, Guiyang, Guizhou Province, China. 3. Department of Urology/Institute of Urology, West China Hospital, Sichuan University, Chengdu, Sichuan Province, China. 4. Department of Urology, The Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou Province, China. 5. Department of Clinic Research Center, The Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou Province, China. 6. Department of Urology, The Affiliated Suqian First People's Hospital of Nanjing Medical University, Nanjing, China. 7. Laboratory of the Affiliated Cancer Hospital of Guizhou Medical University, Guiyang, Guizhou Province, China. 8. Department of Urology, The Third Affiliated Hospital of Soochow University, No.185, Juqian Street, Tianning District, Changzhou, 213000, Jiangsu Province, China. czyyhxz2017@163.com. 9. Department of Urology, Xuzhou Central Hospital, No. 199 Jiefang Street, Quanshan District, Xuzhou, 221009, Jiangsu, China. haolinxuzhou@163.com.
Abstract
BACKGROUND: To observe and explore the effect of metformin on the migration and proliferation of bladder cancer T24 and 5637 cells in vitro. METHODS: Bladder cancer T24 and 5637 cell lines were cultured in vitro, and were divided into group A (blank control group) and group B (metformin group: 5, 10, 15, and 20 mmol/L); both groups were plated on 6-well plates at the same time. Culture in 24-well plates was used for wound healing assays and in 96-well plates for Transwell migration and invasion, and Cell Counting Kit-8 proliferation experiments. We observed and detected the cell migration and proliferation ability of each group at 48 h, and calculated the cell migration area and survival rate. Flow cytometry was used to detect cell apoptosis in the groups. The apoptosis-related proteins, cleaved-caspase 3, cleaved-PARP, and the PI3K/AKT/mTOR signaling pathway member proteins PI3K, phosphorylated (p)-PI3K, AKT, p-AKT, mTOR, and p-mTOR were detected using western blotting. RESULTS: After 48 h of treatment with different concentrations of metformin, the cell migration and proliferation capabilities were significantly lower than those in the blank control group. The proliferation and migration abilities of T24 and 5637 cells decreased in a metformin concentration-dependent manner (P < 0.05). The apoptosis rate under different concentrations of metformin, as detected by flow cytometry, showed a significantly higher rate in the metformin group than in the control group (P < 0.05). Compared with that in the control group, the level of cleaved-caspase 3 and cleaved-PARP protein in the metformin group was increased in each treatment group, and the levels of p-mTOR, p-AKT, and p-PI3K decreased significantly compared with those in the control group (P < 0.05). CONCLUSION: Metformin inhibited bladder cancer T24 and 5637 cell migration and proliferation, and induced their apoptosis. The mechanism might involve inhibition of the activation of the PI3K/AKT/mTOR signaling pathway.
BACKGROUND: To observe and explore the effect of metformin on the migration and proliferation of bladder cancer T24 and 5637 cells in vitro. METHODS: Bladder cancer T24 and 5637 cell lines were cultured in vitro, and were divided into group A (blank control group) and group B (metformin group: 5, 10, 15, and 20 mmol/L); both groups were plated on 6-well plates at the same time. Culture in 24-well plates was used for wound healing assays and in 96-well plates for Transwell migration and invasion, and Cell Counting Kit-8 proliferation experiments. We observed and detected the cell migration and proliferation ability of each group at 48 h, and calculated the cell migration area and survival rate. Flow cytometry was used to detect cell apoptosis in the groups. The apoptosis-related proteins, cleaved-caspase 3, cleaved-PARP, and the PI3K/AKT/mTOR signaling pathway member proteins PI3K, phosphorylated (p)-PI3K, AKT, p-AKT, mTOR, and p-mTOR were detected using western blotting. RESULTS: After 48 h of treatment with different concentrations of metformin, the cell migration and proliferation capabilities were significantly lower than those in the blank control group. The proliferation and migration abilities of T24 and 5637 cells decreased in a metformin concentration-dependent manner (P < 0.05). The apoptosis rate under different concentrations of metformin, as detected by flow cytometry, showed a significantly higher rate in the metformin group than in the control group (P < 0.05). Compared with that in the control group, the level of cleaved-caspase 3 and cleaved-PARP protein in the metformin group was increased in each treatment group, and the levels of p-mTOR, p-AKT, and p-PI3K decreased significantly compared with those in the control group (P < 0.05). CONCLUSION: Metformin inhibited bladder cancer T24 and 5637 cell migration and proliferation, and induced their apoptosis. The mechanism might involve inhibition of the activation of the PI3K/AKT/mTOR signaling pathway.
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