Combination Therapy of Chloroquine and C₂-Ceramide Enhances Cytotoxicity in Lung Cancer H460 and H1299 Cells.

Non-small cell lung cancer (NSCLC) is a type of malignant cancer, and 85% of metastatic NSCLC patients have a poor prognosis. C₂-ceramide induces G2/M phase arrest and cytotoxicity in NSCLC cells. In this study, the autophagy-inducing effect of C₂-ceramide was demonstrated, and cotreatment with the autophagy inhibitor chloroquine (CQ) was investigated in NSCLC H460 and H1299 cells. The results suggested that C₂-ceramide exhibited dose-dependent anticancer effects in H460 and H1299 cells and autophagy induction. Zebrafish-based acridine orange staining confirmed the combined effects in vivo. Importantly, the combination of a sublethal dose of C₂-ceramide and CQ resulted in additive cytotoxicity and autophagy in both cell lines. Alterations of related signaling factors, including Src and SIRT1 inhibition and activation of the autophagic regulators LAMP2 and LC3-I/II, contributed to the autophagy-dependent apoptosis. We found that C₂-ceramide continuously initiated autophagy; however, CQ inhibited autophagosome maturation and degradation during autophagy progression. Accumulated and non-degraded autophagosomes increased NSCLC cell stress, eventually leading to cell death. This study sheds light on improvements to NSCLC chemotherapy to reduce the chemotherapy dose and NSCLC patient burden.