Antitumor Effects Of Metformin In Bladder Cancer And The Mechanisms

BACKGROUNDBladder cancer is the most common malignancy of the urinary tract and one of the major fatal cancers in adult men. Over70%of the patients are nonmuscle invasive bladder cancer (NMIBC) at initial diagnosis, which would be managed with transurethral resection (TUR). However, approximately60%-70%of these cases will develop recurrent tumors, with25%showing progression to a higher stage or grade. Intravesical chemotherapy or immunotherapy has been widely used to prevent the recurrence and progression of superficial disease after TUR. Although certain intravesical agents have shown some evidence of activity, unfortunately, strong adverse reactions and limited response rate remain major clinical problems. For advanced bladder cancer, cisplatin-based combination chemotherapy is the current standard therapeutic regimen. Although bladder cancer is relatively chemotherapy sensitive, unfortunately, the responses are not typically durable and the majority of these patients experience subsequent disease progression, with a5-year survival rate of approximately20%-40%. Since the existing treatment options are hardly satisfactory, new effective therapeutic agents with fewer side effects and sensitizers for intravesical therapy are urgently required for the treatment of bladder cancer.Metformin (1,1-dimethylbiguanide hydrochloride), an oral biguanide agent, has been widely used for the treatment of type2diabetes. Its antihyperglycemic effect mainly relies on the ability to activate the intracellular enzyme AMP-activated protein kinase (AMPK), which leads to reduction of hepatic gluconeogenesis and increase of glucose uptake in peripheral tissues. In addition to the anti-hyperglycemic effects, metformin could inhibit the proliferation of various cancer cells and potentiate the chemotherapeutics-induced cytotoxicity in vitro and in vivo. Epidemiological studies also revealed that metformin indeed decreased the incidence of cancer and cancer-related mortality in diabetic patients compared with those treated with other antidiabetic agents. Moreover, previous studies demonstrated that metformin use was associated with a better survival in diabetic patients with breast cancer or lung cancer, indicating that metformin may improve the efficiency of chemotherapy. Interestingly, recent studies revealed that metformin could exert a protective effect on disease recurrence in patients with bladder cancer, providing a strong rationale to evaluate metformin as an anti-cancer drug or as an adjuvant in combination therapy for bladder cancer. However, the anti-cancer effects and the potential chemotherapy-sensitizing effects of metformin on bladder cancer are not entirely elucidated, which need to be investigated in bladder cancer cell lines and animal models at preclinical level. On the other hand, recent studies have indicated that Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, is the effector molecule in intravesical Bacillus Calmette-Guerin (BCG) immunotherapy, which significantly reduces the risk of recurrence and progression in high-risk NMIBC patients. Given that metformin use was associated with a decreased disease recurrence in diabetic patients with bladder cancer, we speculate that metformin may be an optimal adjuvant of TRAIL-based therapy for bladder cancer. However, this should also be verified at preclinical level.In the present study, we investigated the effects of metformin on the proliferation of human bladder cancer cells in vitro and in vivo, and further determined whether the underlying mechanism was involved in AMPK activation and mammalian target of rapamycin (mTOR) inhibition. We also investigated the effects of metformin on TRAIL-induced apoptosis and explored the molecular mechanisms by which metformin-sensitized TRAIL-sensitive bladder cancer cells to TRAIL-induced apoptosis.OBJECTIVES1. To investigate the effects of metformin on the proliferation of bladder cancer cells and the underlying mechanisms.2. To investigate the effects of metformin on the growth of human bladder tumor xenografts in nude mice and the underlying mechanisms..3. To investigate the effects of metformin on TRAIL-induced apoptosis in human bladder cancer cells and the underlying mechanisms.MATERIALS AND METHODS1. The human bladder cancer cell lines,5637, T24, RT4and253J were used as the object to investigate the anti-cancer effects of metformin in vitro. Four-week-old male athymic BALB/c nu/nu mice were used to investigate the anti-cancer effects of metformin in vivo.2. Cell proliferation assay:cell viability was assessed using a tetrazolium-based assay. Cells were seeded in96-well plates and treated the next day with the indicated agents. MTT assay was used to determine the cell viability of each group. The percentages of surviving cells from each group relative to controls were calculated.3. Colony formation assay:cells were seeded in6-well culture plates at a density of1000cells/well and incubated with metformin at the indicated concentrations. Fourteen days later, the colonies were stained and counted.4. Assay for apoptosis:cells were treated with the indicated agents for24hours and the apoptosis were analyzed by flow cytometry after Annexin Ⅴ-FITC/PI staining.5. Assay for cell cycle:cells were treated with5mM metformin for the indicated time. Cell cycle distribution was then analyzed by flow cytometry.6. Western blotting analysis:cell lysates were subjected to SDS-PAGE on8or12%Tris-glycine gels and transferred onto nitrocellulose membranes. Blots were probed with specific primary antibodies and protein signal was then detected with the ECL chemiluminescent detection system.7. Tumor xenograft model:5637cells (2×106) were implanted subcutaneously into the left flank regions of each mouse to initiate tumor growth. The metformin-treated group was injected intraperitoneally at100mg/kg body weight per day for3weeks and the control group received vehicle only. Body weight, diet consumption and tumor volume were recorded and analyzed for each group.8. Immunohistochemistry staining:IHC was conducted using a Dako Autostainer Plus system. Sections were deparaffinized, rehydrated, subjected to heat-induced antigen retrieval and endogenous enzyme block, and further incubated with primary antibody at4℃overnight. Signals were detected the next day using diaminobenzidine as a chromogen.9. RNA isolation and quantitative real-time PCR:total RNA was extracted and reverse transcribed into cDNA, which was then subjected to a25μL real-time PCR carried out in a CFX96TM Real-Time PCR Detection System.10. siRNA and transfection:cells were transfected with the indicated siRNAs (human S6K1siRNA or a negative control siRNA) using Lipofectamine2000. Cellular lysates were prepared48hours post-transfection for further experiments.RESULTS1. Metformin inhibits the proliferation and the colony formation of bladder cancer cells in vitro.Metformin significantly inhibited the proliferation of bladder cancer cells5637and T24in a dose-and time-dependent manner, and reduced the colony formation of these two cells in a dose-dependent manner, even at concentrations as low as2mM.2. Metformin does not induce apoptosis but induces cell cycle arrest in G0/G1phases. Metformin treatment did not affect the percentage of Annexin Ⅴ-positive cells and the cleavage of PARP, but induced an apparent cell cycle arrest in G0/G1phases, accompanied by a strong decrease of cyclin D1, CDK4, E2F1and an increase of p21.3. Metformin activates AMPK/mTOR signaling in bladder cancer cells.Metformin activated the phosphorylation of AMPKa (Thr172) and suppressed the phosphorylation of mTOR (Ser2448), S6K1(Thr389),4E-BP1(Thr37/49) in a time-dependent manner in bladder cancer cells.4. Metformin inhibits the growth of human bladder tumor xenografts in nude mice.Daily treatment of metformin led to a substantial inhibition of tumor growth in a xenograft model with concomitant decrease in the expression of PCNA, cyclin D1and p-mTOR (Ser2448).5. Metformin potentiates the TRAIL-induced apoptosis in TRAIL-sensitive bladder cancer cells, but not TRAIL-resistant cells.Metformin markedly potentiated the TRAIL-induced apoptosis in TRAIL-sensitive bladder cancer cells (5637,253J and RT4), evidenced by the increased Annexin V-positive cells and the activation of caspases. But these effects were not observed in the TRAIL-resistant bladder cancer cells T24.6. Metformin sensitizes bladder cancer cells to TRAIL-induced apoptosis through downregulation of c-FLIPL.Metformin did not alter the expression levels of DR4and DR5, but significantly reduced the levels of c-FLIPL, contributing toward the sensitization to TRAIL.7. Metformin downregulates c-FLIPL expression through the mTOR/S6K1pathway. Metformin treatment did not affect the mRNA level, proteasomal degradation and protein stability of c-FLIPL, but inhibited the mTOR/S6K1pathway in bladder cancer cells. Knockdown of S6K1effectively reduced the levels of c-FLIPL, indicating metformin downregulated c-FLIP through inhibition of mTOR/S6K1pathway.CONCLUSIONS1. Metformin effectively inhibits bladder cancer cell proliferation and tumor growth in vitro and in vivo. 2. Metformin exerts the inhibitory effects possibly by inducing G0/G1cell cycle arrest and activating AMPK/mTOR pathway.3. Metformin could effectively downregulate c-FLIP1expression in bladder cancer cells through inhibition of mTOR/S6K1pathway, which renders these cancer cells more sensitive to TRAIL-induced apoptosis.4. Metformin could be a potential anti-cancer drug or as an adjuvant of TRAIL-based therapeutic therapy for bladder cancer...