| Background Pheochromocytoma(PHEO),originated from neuroectoderm,is a kind of endocrine tumors.Due to overproduction of a large number of catecholamines,it can cause blood pressure,headache and palpitation and serious injuries in heart,brain and kidney.For its treatment,resection remains the major method of radical adrenal pheochromocytoma.About 90% of patients can get 5 years of disease-free survival.However,due to current lack of effective identification factors,the prognosis of patients with metastatic adrenal pheochromocytoma remains very poor with 5-year survival rate less than <35%.Therefore,treatment of adrenal pheochromocytoma is still a major clinical challenge.In recent years,epidemiologic studies have shown that metformin could exert an inhibitory effect on the risk of tumorigenesis and tumor-related mortality in diabetic patients,which is independent of its hypoglycemic effect.Therefore,the anti-tumor effect of metformin attracts more and more attention.Besides,experimental studies found that metformin treatment could inhibit proliferation and growth of many types of human cancers,including breast cancer,prostate cancer,pancreatic cancer,lung cancer,liver cancer.However,whether metformin inhibits the development and progression of pheochromocytoma remains unknown.In the present study,we aimed to examine whether metformin exerts an antitumor effect in PC12 cells,a rat pheochromocytoma cell line,and to analyze the underlying mechanisms.Objective 1.We investigated the effect of metformin on the growth of pheochromocytoma in cells cultured in vitro;2.We investigated the effect of metformin on the growth of pheochromocytoma in vivo 3.We explored the molecular mechanisms of metformin on pheochromocytoma.Methods 1.Rat pheochromocytoma cells(PC12 cells)were cultured and treated with metformin or vehicle control.CCK-8 assays were used to detect the effect of different concentrations of metformin on the viability and growth curve of PC12 cells.The effect of different concentrations of metformin on the proliferation of PC12 cells was detected by EDU staining.Annexin V-FITC / PI staining was performed under fluorescence microscope and flow cytometry to detect the effect of different concentrations of metformin on the apoptosis of PC12 cells.The effects of different concentrations of metformin on PC12 cell cycle were detected by PI staining.2.For in vivo animal experiment,the adrenal pheochromocytoma cell PC12 was constructed by subcutaneously implanted tumor model of nude mice to detect the effect of metformin on the growth of adrenal pheochromocytoma xenografts.3.To investigate the molecular mechanisms of metformin,genes expression and the signaling pathways involved were analyzed in PC12 cells by quantitative real-time PCR and western blots,respectively.Results 1.In vitro studies found that metformin treatment can inhibit the viability and proliferation of adrenal pheochromocytoma cells.CCK-8 assays showed that metformin can inhibit the viability of PC12 cells in a dose-and time-dependent manner.Consistently,the Ed U flow cytometry analysis showed that the cell proliferation potential was suppressed by metformin treatment.2.In vitro studies found that metformin can promote the apoptosis of adrenal pheochromocytoma cells.For apoptosis analysis,flow cytometry was performed after staining with Annexin V/PI,showing that the metformin treatment induced a higher apoptosis rate.3.Flow cytometry analysis also revealed a cell-cycle arrest at the G0/G1 phase of the PC12 cells treated with metformin.4.Metformin can inhibit the growth of adrenal pheochromocytoma transplanted tumor in the nude mice.PC12 cells were inoculated subcutaneously in nude mice to form tumor blocks.After 14 days,the growth rate,weight and volume of subcutaneous tumor in the nude mice was suppressed by metformin treatment.5.Ccna2,Ccnb2,and PCNA were significantly downregulated by metformin treatment,whereas other cell-cycle regulators,including Cdkn1 b,Cdkn1c,Ccnd1,and Ccnd3,remained unaffected.Therefore,the expression levels of certain cell-cycle regulators may be modulated by metformin treatment of PC12 cells.6.The Western blot analyses in our study revealed that AMPK phosphorylation was enhanced in PC12 cells treated with metformin.Moreover,phosphorylated ACC,a downstream target of AMPK,was also induced by metformin.On the other hand,phosphorylated m TOR and ERK1/2 levels were reduced,suggesting that these downstream signaling pathways were inhibited in the PC12 cells upon metformin treatment.Conclusion In this study,we for the first time,report the roles of metformin in the regulation of adrenal pheochromocytoma growth.Firstly,in vitro cellular experiments found that metformin can inhibit proliferation and promote apoptosis of PC12 cells.Secondly,in vivo animal experiments found that metformin can inhibit the growth of adrenal pheochromocytoma in the nude mice.Thirdly,at the molecular level,we showed that metformin treatment downregulated the expression of Ccna2 and Ccnb2 in PC12 cells to inhibit cell-cycle progression.Besides,the AMPK signaling pathway was activated,whereas the m TOR and ERK1/2 signaling pathways were inhibited by metformin.Taken together,our results suggest a previously unknown role of metformin in pheochromocytoma development,which might provide a novel option for future cancer therapy. |
