Inhibition of EZH2 and EGFR produces a synergistic effect on cell apoptosis by increasing autophagy in gastric cancer cells

Abstract

Background: Numerous reports have shown that a combination of two or more drugs leads to better cancer treatment. Inhibitors of zeste homology 2 and epidermal growth factor receptor have been widely used in cancer treatments. However, the mechanisms of the combined use of these two drugs remain elusive. Methods: Sulforhodamine B assays and Alexa Fluor®-488 Annexin V/Dead Cell Apoptosis Kit were used to detect the cell proliferation and cell apoptosis in vitro, respectively. Western blotting analysis was used to detect the relative protein expression, and xenografted tumor was generated in nude mice to evaluate the effect in vivo. Results: Treatment with either Gefitinib ranging from 0 to 12.5 µM or GSK126 ranging from 0 to 8.3 µM caused a dose-dependent decrease in the cell survival fraction, and the combination of Gefitinib at 12.5 µM and GSK126 at 8.3 µM caused further significant decrease. The combination indexes were 0.061, 0.591, 0.713, and 0.371 for MGC803, A549, PC-3, and MDB-MA-231, respectively. In MGC803 cells, the combination of GSK126 and Gefitinib synergistically induced cell apoptosis (56.2%), which was markedly higher as compared to either drug alone (7.6% and 10.6%, P,0.05). Treatment with either Gefitinib or GSK126 alone induced a significant increase in cell apoptosis in LC3-II and p-ULK, whereas the combination of the two induced a further increase. Pretreatment with an autophagy inhibitor, 3-methyladenine, prevented the apoptosis induced by the combined use of Gefitinib and GSK126. In addition, the combined use of Gefitinib and GSK126 also inhibited the activation of mammalian target of rapamycin signaling pathway. Furthermore, the combined use of GSK126 and Gefitinib synergistically inhibited xenografted tumor proliferation. Conclusion: The combined use of GSK126 and Gefitinib exerts a synergic effect on tumor growth inhibition both in vitro and in vivo through inducing autophagy and promoting apoptosis. Therefore, GSK126 and Gefitinib in combination may be considered as a potential strategy in treating solid tumor clinically.