Mechanism of chloroquine modulated autophagy in EGFR-TKI resistant lung cancer cell lines

Objective To investigate the mechanism of chloroquine intervene the epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) resistant lung cancer cell lines. Methods EGFR-TKI sensitive lung cancer cell line (PC9) and resistant cell lines (A549, H1960, H1975) were used in the study.Cell Counting Kit-8 (CCK8) assay was applied to measure the proliferation and viability of four cell lines.Western blot was used to detect the expression of autophagy protein and activity of mammalian traget of rapamycin (mTOR) signaling pathway.Further, all four cell lines were treated by erlotinib, an EGFR-TKI, combined with chloroquine, an autophagy inhibitor, in order to observe cell viability and autophagy protein expression. Results Western blot analysis showed that phosphorylation of S6 in four cell lines was increased and P62 protein was decreased in EGFR-TKI resistant cell lines (A549, H1650, H1975). Whereas, the expression of P62 in EGFR-TKI sensitive cell lines (PC9) was increased.Erlotinib markedly inhibited the PC9 cells growth, contrast to other cell lines by CCK8 assay.Furthermore, four cell lines were treated with Torin2, an inhibitor of mTOR pathway.It was found that cell growth of PC9 is sensitive to Torin2, but not in other cell lines.Then, Western blot analysis showed that chloroquine didn′t change the phosphorylation of S6, but strongly inhibited the cell viability in all cell lines, especially in PC9 and A549.At last, the combination of chloroquine with erlotinib significantly inhibit the PC9, A549, H1975 cell proliferation, while it was little effective to H1650 by CCK8 assay. Conclusions mTOR is activated and autophagy is increased in EGFR-TKI resistant cell lines.Moreover, Torin2 has no effect on autophagy expression and lung cancer cells growth.Chloroquine inhibits EGFR-TKI resistant lung cancer cell lines viability.Also, the combination of chloroquine and EGFR-TKI increases efficacy of inhibition in all four cell lines.It is demonstrated that combining autophagy inhibitor and EGFR-TKI represents a novel strategy to overcome resistance to EGFR-TKI. Key words: Epidermal growth factor receptor-tyrosine kinase inhibitor; Autophagy; Non-small cell lung cancer; Resistance