Metformin Sensitizes Non-small Cell Lung Cancer Cells To Epigallocatechin-3-gallate(EGCG) And Ionizing Radiation Treatment Via Regulating Nrf2

Part I Metformin Sensitizes Non-small Cell Lung Cancer Cells to EGCG Treatment via Regulating Nrf2Objectives: To study the mechanism of metformin sensitizing the non-small cell lung cancer cells to EGCG treatment.Methods:(1) Investigate the HO-1 expression in four NCSLC cell lines, namely A549, H1299, H1975 and H460, and perform MTT assay to examine the cell proliferation change after EGCG treatment.(2) cell apoptosis was detected by flow cytometry and PARP1 expression.(3) ROS level was detected using flow cytometry and flow cytometry after DCFH-DA staining.(4) Nrf2 and HO-1 protein expression and Nrf2 traslocation was detected by Western Blot and immunofluorescene.(5) HO-1 m RNA expression in 25 lung cancer tissue and adjacent normal tissue was detected by q RT-PCR.(6) The difference of HO-1 expression in lung cancer and normal lung tissue was analyzed and sensitivity to EGCG was investigated by MTT assay.(7) A549 xenograft model is established by subcutaneous injection into the right flank of nude mice. Metformin is dissolved in drinking water at the concentration of 200μg/m L. EGCG is delivered by intraperitoneal injection. Tumor volume is measured by vernier caliper. The protein level of Nrf2 is examined by immunohistochemistry.Results: The protein level of HO-1 was different in four cell lines of NSCLC. A549 expresses highest level of HO-1 while is most resistant to EGCG. Metformin can reduce the protein level of HO-1 in a dose depentent manner in A549. Metformin, at the concentration of 0.4mmol/L, inhibits the expression of HO-1, but has no effect on cell proliferation. Metformin can promote apoptosis and ROS induced by EGCG. Moreover, metformin can abrogate the nuclear translocation of Nrf2 by EGCG and thus inhibit Nrf2 signaling pathway and HO-1. Metformin can enhance the proliferation inhibition capacity of EGCG in vivo.Conclusions: This study demonstrated that metformin can inhibit the expression of HO-1 protein in A549. Metformin can inhibit cell proliferation of A549 by induction of apoptosis and ROS production. Metformin abrogates Nrf2 translocation and diminishes HO-1 expression. Moreover, the expression of HO-1 is different between lung cancer and normal lung tissue, which provide a new strategy to selectively kill lung cancer cells. Metformin can enhance the proliferation inhibition capacity of EGCG in vivo, inhibiting Nrf2 increasement induced by EGCG.PART III Metformin Sensitizes Non-small Cell Lung Cancer Cells to ionizing radiation via Regulating Nrf2Objectives: To study the mechanism of metformin sensitizing the non-small cell lung cancer cells to ionizing radiationMethods:(1) Cell apoptosis and cell cycle were detected by flow cytometry and Western Blot.(2) Cell proliferation was detected by cloning assay(3) Nrf2 and HO-1 protein expression and Nrf2 traslocation was detected by Western Blot and immunofluorescene.Results: metformin increase the proliferation inhibition and apoptosis of A549 caused by IR. Metformin deceases the number of cells in S phase, while IR induses G2/M cell cycle arrest. However, metformin abrogates the G2/M arrest by IR. Moreover, metformin can decrease the nuclear translocation of Nrf2 caused by IR.Conclusions: This study demonstrated that metformin can increase the inhibition of cell proliferation by ionizing radiation. Metformin also can reduce G2/M cell number induced by IR. Metformin abrogates Nrf2 translocation and diminishes HO-1 expression after IR.