The antidiabetic drug metformin inhibits the proliferation of bladder cancer cells in vitro and in vivo

Abstract

Recent studies suggest that metformin, a widely used antidiabetic agent, may reduce cancer risk and improve prognosis of certain malignancies. However, the mechanisms for the anti-cancer effects of metformin remain uncertain. In this study, we investigated the effects of metformin on human bladder cancer cells and the underlying mechanisms. Metformin significantly inhibited the proliferation and colony formation of 5637 and T24 cells in vitro; specifically, metformin induced an apparent cell cycle arrest in G0/G1 phases, accompanied by a strong decrease of cyclin D1, cyclin-dependent kinase 4 (CDK4), E2F1 and an increase of p21waf-1. Further experiments revealed that metformin activated AMP-activated protein kinase (AMPK) and suppressed mammalian target of rapamycin (mTOR), the central regulator of protein synthesis and cell growth. Moreover, daily treatment of metformin led to a substantial inhibition of tumor growth in a xenograft model with concomitant decrease in the expression of proliferating cell nuclear antigen (PCNA), cyclin D1 and p-mTOR. The in vitro and in vivo results demonstrate that metformin efficiently suppresses the proliferation of bladder cancer cells and suggest that metformin may be a potential therapeutic agent for the treatment of bladder cancer.

Figure 1.

Metformin inhibits the proliferation of bladder cancer cells. (A) 5637 (a) and T24 (b) cells (5 × 10³ cells/well) were seeded in 96-well culture plates. After 24 h, cells were treated with metformin (0, 2, 5, 10, 20 mM) for another 48 h. Cell viability was measured by MTT assay. The results were expressed as percent of cell viability compared with control (0 mM). Columns, means of three independent experiments; bars, SEs; (B) 5637 (a) and T24 (b) cells (5 × 10⁴ cells/well) were seeded in 12-well culture plates. After treatment as in panel A, cell numbers were determined using a hemocytometer. The results were expressed as percent of viable cells compared with control. Columns, means of three independent experiments; bars, SEs; (C,D) 5637 (a) and T24 (b) cells were treated with metformin (Met) at different concentrations for 24, 48 and 72 h. Cell proliferation was measured by MTT (C) or cell count assay (D). Data, means of three independent experiments; bars, SEs. *p < 0.05 versus control; **p < 0.01 versus control.

Figure 2.

Metformin inhibits colony formation of bladder cancer cells. (A) 5637 and T24 cells grown in 6-well culture plates were treated with the indicated concentrations of metformin, every third days for two weeks. The pictures of 6-well culture plates with colonies were taken by a digital camera on day 14; and (B) The bar graph was obtained by calculating the percentages of colony numbers relative to controls, defined as 100%, measured by 1-D gel quantity software Quantity One. Columns, means of three independent experiments; bars, SEs. *p < 0.05 versus control; **p < 0.01 versus control.

Figure 3.

Metformin blocks the cell cycle in G0/G1 phases and affects the expression level of the cell cycle proteins. (A,B) Proliferating 5637 (A) and T24 cells (B) were treated with 5 mM metformin (Met) for the indicated time and cell cycle distributions were analyzed by flow cytometry; and (C,D) Western blot analysis of related cell cycle proteins in 5637 (C) and T24 cells (D) treated or not with 5 mM metformin (Met) for the indicated time. Data shown are representative of three independent experiments with similar results.

Figure 4.

Metformin activates AMP-activated protein kinase (AMPK) and inhibits mammalian target of rapamycin (mTOR) signaling in bladder cancer cells. 5637 and T24 cells were treated with 5 mM metformin for the indicated time and the lysates were immunoblotted with the indicated antibodies. (A,C) Effects of metformin on the phosphorylation of AMPK in 5637 (A) and T24 cells (C); and (B,D) Effects of metformin on mTOR signaling in 5637 (B) and T24 cells (D). Data shown are representative of three independent experiments.

Figure 5.

In vivo antitumor effects of metformin on bladder cancer xenograft model. Xenografts were generated by implantation of 2 × 10⁶ cells of 5637 cells subcutaneously into the right flanks of nude mice. When the tumors reached a mean diameter of 6 mm, the animals were randomized into control and treated groups (five mice per group). Metformin (Met, 100 mg/kg) was injected once daily intraperitoneally for three weeks, control mice received purified water only. (A) Graphs represent the average tumor volumes of 5637 xenografts in mice from the control and metformin-treated groups; (B) Weight of the excised tumors from the two groups; (C) Representative microphotograph of PCNA, cyclin D1 and p-mTOR staining on tumor sections from the control and metformin-treated groups. The scale bars equal 50 μm; and (D) Quantitative data for percentage of PCNA, cyclin D1 and p-mTOR positive cells in tumors from the two groups. Data, means of five mice in each group; bars, SEs. *p < 0.05 versus control; **p < 0.01 versus control.

Figure 5.

In vivo antitumor effects of metformin on bladder cancer xenograft model. Xenografts were generated by implantation of 2 × 10⁶ cells of 5637 cells subcutaneously into the right flanks of nude mice. When the tumors reached a mean diameter of 6 mm, the animals were randomized into control and treated groups (five mice per group). Metformin (Met, 100 mg/kg) was injected once daily intraperitoneally for three weeks, control mice received purified water only. (A) Graphs represent the average tumor volumes of 5637 xenografts in mice from the control and metformin-treated groups; (B) Weight of the excised tumors from the two groups; (C) Representative microphotograph of PCNA, cyclin D1 and p-mTOR staining on tumor sections from the control and metformin-treated groups. The scale bars equal 50 μm; and (D) Quantitative data for percentage of PCNA, cyclin D1 and p-mTOR positive cells in tumors from the two groups. Data, means of five mice in each group; bars, SEs. *p < 0.05 versus control; **p < 0.01 versus control.