Metformin Regulates The Proliferation Of Prostate Epithelial Cells Inhibition Of Insulin-like Growth Factor1Receptor

Objective:Benign prostatic hyperplasia (BPH) is the most common age-related proliferative abnormality of the prostate affecting elderly men throughout the world. Epidemiologic studies suggest that diabetes significantly increases the risk of developing BPH. However, it is unclear whether anti-diabetic medications may be protective against development of BPH. Here we demonstrate that Metformin, a first line medication for treatment of type2diabetes inhibits the proliferation of BPE cells and affects the effect of IGF-1on cell proliferation.Method:Benign prostate epithelial cell lines BPH-1and P69were used. Cellular proliferation was completed by MTS assay and Ki-67immunofluorescence staining. Cell migration was determined by wound assay; Cells in sub-GO phase was measured by flow cytometry.Results:Proliferation ratio changes of the cells were observed after being treated by0.5,1,5,10mM metformin for6h～48h. With the increase of concentration of metformin or the prolongation of the treating time, the proliferation ratio was significantly decreased. Meanwhile growth promoting effect of IGF-1(100ng/ml) was compeletly abrogated by Metformin (5mM). Cell migration was also markedly inhibited by increasing doses of metformin. In addition, the precentage of cells in sub-GO phase was not affected by metformin treatment.Conclusion:Metformin inhibits the proliferation and migration of prostate epithelial cells, and attenuates the effect of IGF-1on cell growth. Metformin may have a protective role in prostatic proliferation. Objective:Since our previous study showed that IGF-1signaling acts a critical role in the progress of BPH signaling pathway, we asked if metformin could regulate the activation of IGF-1R and downstream signaling transduction pathway.Method:Western blotting analyses detected the protein level of pho-IGF-1R and downstream regulators of cell proliferation in prostate epithelial cells. Immunocytochemistry was used to measure the changes of the activation of IGF-1R by metformin treatment in presence or absence of IGF-1(100ng/ml) in BPH-1and P69cells.Results:As shown by western blotting analyses, the protein level of pho-IGF-1R was significantly decreased after metformin treatment in a time-and dose-dependent manner in BPH-1cells. Metformin also inhibited the phosphorylation of IRS-1, which is one of the primary substrates of the IGF-1R. The ratio between pho-IGF-1R and IGF-1R and the ratio between pho-IRS-1and GAPDH were30-100folds decreased in metformin-treated BPH-1cells compare to control cells. Similar results were obtained in P69cells (data not shown). We further confirmed the decreasing of pho-IGF-1R by Immunocytochemistry. In addition, Metformin inhibited the activation of Erk, AKT and cell cycle proteins in a dose-dependence manner in BPH-1cells.Conclusion:inhibition of IGF-1R involves the effects of metformin on benign prostate epithelial cell growth. This study will help us have a better understanding of the effects of IGF-1axis in the development of BPH. Objective:To study the effect of metformin on cell cycle status and cyclin D expression in benign prostate epithelial cells, further to invest the melocular mechanism for treating metformin.Method:The changes of cell cycle in BPH-1and P69cells treated with metformin and IGF-1were analysised by flow cytometric. The protein levels of cyclin D in BPH-1cells after treatments of different doses(0.5,1,5,10mM) metformin were detected by Western-Blot.Results:IGF-1led to significant reduction of cells in G0/G1and an increase of the percentages of cells in the S and G2/M phase. However, cells failed to enter the S phase and were blocked in G0/G1in presence of metformin. The S and G2/M phase cell population was low ered from41.8%to26.96%in P69cells and from31.1%to24.7%in BPH-1cells. In consistence with the results of cell cycle, metformin prevented the increase of cyclin D protein levels, especially cyclin D1. Cyclin D1protein level is no longer detectable48h after adding5mM metformin with or without100ng/ml IGF-1.Conclusion:These results indicate that metformin prevents the mitotic effect of IGF-1and arrests the cell cycle of prostate epithelial cells in G0/G1phase. This effect may be explained by the down-regulation of cyclin D levels. Objective:To investigate the interactions of stromal cells and epithelial cells involved in the proliferation of human prostate epithelial cell lines, and the effects of metformin on this interaction.Method:Benign prostate epithelial cell lines BPH-1and P69and stromal cell line3T3were used in this study. The conditioned medium of3T3cell (3T3-CM) was obtained after24h culture of3T3cells;3T3-CM was detected by enzyme-linked immunosorbent assay (ELISA) for IGF-1. BPH-1and P69cells were incubated with3T3-CM in presence or absence of5mM metformin, cell proliferation ratios were analysised by MTS assay Results:3T3-CM markedly enhanced the proliferation of BPH-1and P69cells. The proliferative effect of CM was aborted by metformin, metformin decrease the percentage of viable cells to the same level as control group. Furthermore, in3T3-CM, the concentration of IGF-1was robustly inhibited by metformin from574.31pg/ml to197.61pg/ml.Conclusion:We therefore suppose metformin downregulates the proliferative effect of stromal cells on epithelial cells, at least partially, via inhibiting IGF axis. The effects of metformin on stromal cells are still unclear and need further study.