| BackgroundRenal cell carcinoma (RCC) is the third most prevalent urologic malignancy, and the sixth leading cause of cancer deaths in the United States. RCC cells are highly proliferative and metastatic, and accounts for 3.8% of all new cancers in adults. Surgical intervention is the primary treatment for RCC, which includes radical nephrectomy and nephron sparing surgery (NSS). However, these treatment options are not suitable for all patients since there are subsets of patients that develop metastases or recurrent disease. Furthermore, RCC is resistant to conventional treatment (chemotherapy, hormonal therapy and radiotherapy). Thus, there is a tremendous need for the development of new treatment strategies. Currently, targeted therapies, which interfere with specific signal transduction pathways of tumor formation and progression, is an area of great clinical and research interest. For example, sorafenib and sunitinib, which target the vascular endothelial growth factor (VEGF) pathways, and temsirolimus, a mammalian target of rapamycin (mTOR)-inhibitor, have been approved for treatment of metastatic RCC (mRCC). Treatment using these targeted agents has showed impressive improvements in time to progression and survival. Unfortunately, these drugs are not effective for all mRCC patients. They are expensive, and only a small population of patients will respond to such treatment. It is necessary to develop new targeted agents to manage RCC.Metformin (1,1-dimethylbiguanide hydrochloride) is used worldwide to treat type-2 diabetes and pre-diabetic syndromes by modulating glucose and fatty acid metabolism. In particularly, metformin can improve glucose utilization, reduce hepatic glucose production and free fatty acid utilization, increase insulin sensitivity, and modify serum lipid profile. Metformin is generally considered safe with a large therapeutic window, and rare cases of hypoglycemia and lactic acidosis have been reported. Importantly, recent evidence indicates that metformin may reduce the risk of cancer and inhibited growth of several cancers, including prostate, breast, liver, and colon. However, the effects of metformin on RCC have not been reported.Objective1. To investigate the potential effects of metformin on the proliferation, migration, and apoptosis of RCC cell lines.2. To investigate the potential effects of metformin on the growth of RCC tumor xenograft.3. To explore the possible signaling mechanisms involved by which metformin exerts its effects.Materials and Methods1. The human RCC cell lines, 786-O and OS-RC-2 were used as the object to investigate the therapeutic effects of metformin on RCC in vitro. Four-week-old female athymic nude mice were used as the object to investigate the therapeutic effects of metformin on RCC in vivo, which were housed in a specific pathogen–free facility.2. Cell proliferation assayCells were seeded in 96-well plates and were treated with metformin (0, 0.1, 0.2, 0.5, 1, 5, 10, 20, 50 mM). At the indicated intervals, MTT assay was used to determine the proliferation level for each group. The percentages of surviving cells from each group relative to controls were calculated. And the cell growth curve were drawn.3. Colony formation assayCells were seeded in 6-well plates in triplicates at a density of 200 cells/well and different concentrations of metformin were added to each well. 14d later, the formed colonies were stained and the number of them were counted.4. Assay for RCC cell migrationThe scratch wound assay was used to determine RCC cell migratory activity. Cells seeded in 6-well cell culture plates were carefully scratched with a fine pipette tip to create a gap. The gap width were computed and were compared to determine the possible effect of metformin on the migratory ability of RCC cells.5. Assay for RCC cell death Cells were were incubated in the presence or absence of 10 mM metformin with or without 10% FBS for 24 h . By using Annexis/PI staining the cells were analyzed by flow cytometric analysis and fluorescent microscopy.6. Assay for cell cycleCells were synchronized at the G1/S boundary after starvation with basal medium for 24 h, followed by incubation in the presence or absence of 5 mM metformin with 10% FBS for 48 h . Cell cycle distribution was analyzed by flow cytometry.7. Western blotting analysisCell lysates were resolved by SDS/PAGE and transferred electrophoretically to Nitro cellulose membrane. Blots were probed with specific antibodies and immunoreactive proteins were revealed by the enhanced chemiluminescence (ECL) kit.8. Tumor xenograft modelEarly passage 786-O cells were harvested and 3×106 cells were implanted subcutaneously into the left flanks of each mouse to form xenograft model. The treatment group received metformin daily i.p. at 250 mg/kg (50?l per mouse). Tumor volume (V) and the cyclin D1 level in tumors were analyzed for each group.Results1. Metformin inhibits RCC cell proliferationMetformin significantly inhibited the proliferation of both RCC cell lines in a dose- and time-dependent manner (Fig. 1A, B). At 0.2 mM and 0.5 mM, metformin inhibited the growth of 786-O and OS-RC-2 cells by 10% and 14%, respectively (Supplemental figure 1). This indicates that metformin is effective to inhibit RCC cell growth.2. Metformin inhibits RCC cell colony formationMetformin prevented the colony formation of RCC cells in a dose-dependent manner. When used at 1 mM, metformin inhibited cell colony formation of 786-O and OS-RC-2 by 37% and 25%, respectively. Additionally, 10 mM metformin decreased colony formation of 786-O and OS-RC-2 cells by 78% and 86%, respectively, compared to control.3. Metformin inhibits RCC cell migrationThe scratch wound assay indicated that metformin treatment led to a decrease in migration index of 786-O and OS-RC-2 cells.4. Metformin induces RCC cell death in low serum medium Metformin can not induce RCC cell death in medium containing 10% FBS. While in medium containing 0.5% FBS, metformin induces RCC cell death demonstrated by flow cytometric analysis and fluorescent microscopy.5.Metformin down-regulates cyclin D1 expression and induces a G0/G1 cell cycle arrest in RCC cellsMetformin-treated cells were arrested at G0/G1, whereas FBS-treated cells progressed to the S-phase These results indicate that metformin prevents the mitotic effects of FBS. Accordingly, cyclin D1, a key protein implicated in the transition of the G0/G1 phase, was significantly reduced in a time-dependent manner in metformin-treated cells.6. Metformin activates AMPK and inhibits mTOR signaling in RCC cellsMetformin activated AMPK time-dependently. Metformin decreased the phosphory- lation of S6K1 (Thr389) in a time-dependent manner, and consequently reduced the phosphorylation of the ribosomal S6 protein (Ser235/236). Metformin treatment also de-phosphorylated 4E-BP1 and reduced Akt phosphorylation. These results suggest that metformin activates AMPK and inhibits mTOR signaling in RCC cells.7 Metformin inhibits the growth of 786-O cell xenografts in nude miceAdministration of metformin strikingly decreased the growth of 786-O cells xenografts in nude mice. Consistent with the effects of metformin on cyclin D1 expression in RCC cells, cyclin D1 expression also significantly decreased in metformin-treated tumors compared to control-treated animals. These results suggest that metformin inhibits the growth of 786-O cell xenografts in nude mice.ConclusionsMetformin was able to inhibit RCC growth in vitro and in vivo. Metformin was also able to induce RCC cell death in low serum medium and inhibit RCC cell migration. Metformin exerts the effects by inducing G0/G1 cell cycle arrest, activating AMPK signaling and inhibiting mTORC1 and mTORC2 signaling. In a word, metformin has great potential to become a therapeutic agent for the treatment of RCC. |
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