Autophagy contributes to the chemoresistance of non-small cell lung cancer

Abstract

Lung cancer is the leading cause of cancer-related death in the world. Chemotherapy plays a major role in treating patients with non-small cell lung cancer (NSCLC). However, even the best regimens available today are only capable of providing overall response rate of 30–50%, with the mechanisms involved in chemotherapy resistance remaining unknown. Although recent studies have suggested that autophagy, a catabolic digestion process of cellular organelles, plays a role in chemoresistance in hypoxic conditions, the underlying mechanisms remain elusive. In the present study, the role of hypoxia-induced autophagy in acquiring chemoresistance was evaluated in NSCLC. To confirm the existence of hypoxia in human NSCLC specimens, we evaluated the expression of hypoxia induced factor-1α (HIF-1α) in both solid tumor and normal lung tissues of patients who underwent therapeutic pulmonary resection for NSCLC. In vitro human lung cancer cells (A549) were grown in standard culture media. Cells were exposed to 1% oxygen for 2, 4, 6 and 8 hours and control cells were cultured in normoxia. We analyzed expressions of HIF-1α and autophagy related proteins by Western blot analysis. We also analyzed autophagosomes by transmission electron microscopy (TEM) and GFP-LC3 puncta staining. To confirm chemoresistance, cells were exposed to cisplatin at different concentrations for 8 hours under normoxia or hypoxia, and the percentage of viable cells was determined by MTT colorimetric assay. Finally, we compared the expression of autophagy- related proteins between the tumor tissues after neoadjuvant treatment and the tumor tissues with treatment-naïve, both of which underwent pulmonary resection.HIF-1α proteins were predominantly expressed in tumor tissue compared with normal tissue, as shown by immunohistochemical stainings and Western blot. In vitro hypoxia caused a time-dependent increase in the conversion of LC3B-I to LC3B-II as well as HIF-1α expression, and a decrease in p62/sequestosome1(SQSTM1) expression in A549 cells as determined by Western blots. Hypoxia also induced GFP-LC3 puncta staining and a marked accumulation of autophagosomes as determined by TEM. Compared with normoxia, cisplatin-induced cell death under hypoxia was significantly decreased. Finally, the tumor tissues after neoadjuvant treatment showed increased LC3B-I to LC3B-II conversion and decreased levels of p62/SQSTM1 compared with the treatment-naïve tumor tissue. The present study suggests that autophagy may play an important role in acquiring chemoresistance in NSCLC induced by hypoxia in solid tumors.