The Proliferation-inhibition, Apoptosis-inducing,and Gefitinib Sensitizing Effect Of Metformin On Human Lung Adenocarcinoma Cell Lines PC9and PC9/GR

Background:Lung Cancer is the leading cause of death in China, whose incidence ranks No.1in male cancer patients and No.2in female cancer patients. Non-Small Cell Lung Cancer (NSCLC) is the major pathological subtype of lung cancer. Most of the patients are diagnosed at a comparatively late stage and lose the chance to take an operation. Thus, chemotherapy and radiotherapy is the major approaches for treatment. However, the efficacy of these approaches reaches a plateau; even though researchers from all over the world have given great effort to improve it. The5-year survival rate of NSCLC is less than15%. With the deepening understanding of NSCLC tumorgenesis, the importance of Epidermal Growth Factor Receptor (EGFR) signal pathway is unveiled. The target therapy based on EGFR signal pathway (eg. EGFR-TKI) is now widely used in the treatment of EGFR mutant positive NSCLC patients and achieves a great success. Nowadays, target therapy is a new and important treatment approach of NSCLC patients. However, nearly all the patients become resistant to the target therapy, which results in the progression of disease, with a median time-to-progression of about10months. Therefore, it is a challenging and important issue for both physician and researcher to find ways to delay or reverse the resistance.Metformin is a widely used insulin-sensitizer for the treatment of type II diabetes mellitus. However, recent studies showed that, besides its role in diabetes, metformin seems to play a protective role in the cardiovascular disease, poly-cystic ovary syndrome (PCOS) and cancer. The relation between metformin and cancer is emphasized. There have been pre-clinical evidences supporting that metformin can inhibit the proliferation of some kinds of cancer, including breast cancer, ovary cancer, and endometrial cancer, and increase their sensitivity to certain kinds of chemotherapy drugs. However, the exact mechanism of metformin’s effect is not fully understood yet. It is now widely accepted that metformin can influent the intracellular proliferative signal transduction and induce apoptosis via the activation of adenosine monophosphate activated protein kinase (AMPK). The influence of metformin on the sensitivity of lung adenocarcinoma to EGFR-TKI is not reported. In this study, we use human lung adenocarcinoma cell lines PC9and PC9/GR as a model to evaluate the anti-proliferation, apoptosis-inducing, and EGFR-TKI sensitivization effect of metfotmin. Our study may provide a basis for the further study of the exact mechanism of metformin’s effect and the possibility of using metformin as a EGFR-TKI sensitizer for NSCLC patient. Part I Anti-proliferation and apoptosis-inducing effect of metformin on PC9and PC9/GR cellsObjective:To evaluate the effect of proliferation and apoptosis of metformin on PC9and PC9/GR cellsMethods:Various concentrations of metformin were given to both PC9and PC9/GR cells. Tetrazolium blue (MTT) assay was used to evaluate proliferation inhibition rate. Flow cytometry was used to analyze the apoptosis rate.Results:1. MTT assay showed that metformin had proliferation inhibition effect on both PC9and PC9/GR manner in a dose and time-dependent manner, and the difference showed statistical significance. And the anti-proliferation effect of metformin showed no difference between PC9and PC9/GR.2. Flow cytometry assay showed that the apoptosis rate of both PC9and PC9/GR increased after48h exposure to metformin. And the apoptosis rate increased accompanied with the increase of metformin concentration.Conclusion:Metformin has anti-proliferation and apoptosis-inducing effect on both PC9and PC9/GR cells. Part ⅡEGFR-TKI sensitization effect of metformin on PC9/GR cell and its possible mechanismObjective:To study the EGFR-TKI sensitization effect of metformin on PC9/GR cell and its possible mechanismMethods:MTT assay was used to evaluate the proliferation inhibition rate of PC9/GR cell after treated with5μmol/L gefitinib combined with various concentration of metformin for48h. Flow cytometry was used to analyze the apoptosis rate of PC9/GR cell and western blot was used to evaluate the expression of AMPK、p-AMPK、mTOR and p-mTOR after treated with2mmol/L metformin,5μmol/L gefitinib and2mmol/L metformin+5μmol/L gefitinib for48h.Results:1. MTT assay showed that gefitinib combined with metformin, compared with gefitinib alone, exerted stronger proliferation inhibition effect on PC9/GR cell. And the difference is statistical significant.2. Flow cytometry assay showed that the apoptosis rate is higher after the treatment of2mmol/L metformin+5μmol/L gefitinib for48h, compared with5μmol/L gefitinib alone.3.Wstern Blot showed that metformin can increase the expression of AMPK、p-AMPK、mTOR, and down-regulate the expression of p-mTOR. While gefitinib could down-regulate the expression of AMPK、p-AMPK、 mTOR, and increase the expression of p-mTOR. The combination of metformin and gefitinib induced a even higher expression of AMPK、 P-AMPK、mTOR, and lower expression of p-mTOR compared with gefitinib alone.Conclusion:Metformin may sensitize PC9/GR cell to EGFR-TKI via affecting the AMPK/mTOR pathway and synergetic apoptosis-inducing effect.