| Objective: To explore the inhibitory effect and mechanism of metformin alone on bladder cancer cells and evaluate the impact of various administration modes for bladder cancer growth in mice. To investigate the combination efficacy of Gefitinib-the representative drug of EGFRTKI and metformin both in vitro and in vivo.Methods: MTT method and clonogenic assay were used to detect the impact of these drugs on cell proliferation and colony formation. Western blot was applied to detect the alterations of cell signaling pathway including AMPK and EGFR proteins. The orthotopic bladder cancer mouse model was applied for measuring the efficacy of anti-tumor activities of these drugs by comparing the difference between oral and intravesical administrations. The plasma lactate concentrations were measured by lactate detection kit. Approaching completion of experiments, immunohistochemistry of bladder tissues was applied for detecting the levels of H&E and Ki67 in each group. In the meantime, the antitumor efficacy of combination of metformin and gefitinib was evaluated using intravesical treatment in orthotopic model.Results: MTT assay showed that metformin inhibited the growth of various bladder cancer cells in dose dependent fashion. In clonogenic assay, it was found that 0.5m M metformin favored the colony formation and suppressive effect increased significantly when the concentration of metformin was 4m M. To mimic intravesical treatment, we designed an intermittent treatment and dramatic suppression on colony formation was observed, implying that intravesical treatment is applicable to cause signifiant cell killing effects. For the mechanisms of metformin, western blot results suggested metformin exerts antitumor effects via activating the AMPK cell signaling pathway and reducing the action of some downstream proteins. The inhibitory effect of combination of metformin with AMPK specific inhibitor Compound C was evaluated in various bladder cancer cell lines using MTT assay, it was found that the ability of metformin to inhibit bladder cancer cell proliferation was reduced after adding compound C, suggesting that inhibition of AMPK accelerates proliferation of cancer cells, consistent with western blot results. On the other hand, metformin decreased the phosphorylation of cell growth- and proliferation- associated AKT and ERK proteins. While combining metformin with AKT specific inhibitor or ERK specific inhibitor, MTT assay found that these two drugs synergistically inhibited bladder cancer cell proliferation, respectively and western blot analysis confirmed that combined drugs further decreased phosphorylation levels of AKT, ERK protein respectively. Intravesical administration route dramatically inhibited the bladder cancer growth without any sign of toxicity comparing with oral administration, using orthotopic model. Combination of metformin and gefitinib exhibited synergistic inhibition, showing by MTT and clonogenic assays in various bladder cancer cells. On the one hand, metformin enhanced the anti-tumor effect of gefitinib by activating AMPK cell signaling pathway. On the other hand, metformin and gefitinib exerted a synergistic effect against bladder cancer via jointly inhibiting AKT/ERK protein phosphorylation. In vivo study using orthotopic model for evaluating these drug activities exhibited the consistent result comparing with the in vitro results mentioned above.Conclusion:1 In vitro and in vivo studies show that metformin inhibits the growth of bladder cancer and the combination of metformin with gefitinib shows a synergistic effect.2 Metformin exhibits the inhibitory effect on bladder cancer through activating AMPK signaling pathway and reducing the phosphorylation of AKT, ERK. The combination of these two drugs displays the synergistic anti-cancer effect on bladder cancer cells depending on the joint decrease of phosphorylation of AKT, ERK. |
