Aniline TFPA enhances camptothecin-induced anti-NSCLC by modulating oxidative stress and impairing autophagy.

Abstract

Background: Camptothecin (CPT) derivatives are widely used in cancer therapies, but their efficacy can be attenuated by resistance mechanisms such as autophagy. We recently showed that the aniline compound 4-[4-(4-aminophenoxy)-2,3,5,6-tetrafluorophenoxy] aniline (TFPA) can potently increase CPT cytotoxicity against non-small cell lung cancer (NSCLC) cells. The purpose of this study was to evaluate whether TFPA improves CPT-based chemotherapy by modulating autophagy and other cell death pathways in NSCLC models.

Methods: Two NSCLC cell lines, A549 and H1299, were tested. The synergism of CPT and TFPA was evaluated by trypan blue exclusion and colony formation assays. Annexin V staining was used for the detection of apoptosis, and autophagy was assessed by acridine orange staining and immunofluorescence. Flow cytometry-based dihydroethidium staining was used to assess oxidative stress. Changes in the expression of apoptosis-associated factors and autophagy-associated factors were determined by Western blot assays. The synergism of CPT and TFPA was validated using a zebrafish xenograft assay.

Results: The accumulation of markers for lysosomal expansion (LAMP2) and degradation (cathepsin D) and markers for autophagosome formation (LC3B-II) suggested that blockage of autolysosome formation might impair autophagy in CPT-treated NSCLC cells and subsequently lead to autophagic cell death. Cotreatment with TFPA and CPT induced cell death by increasing the production of reactive oxygen species, which contributed to autophagic impairment and eventually apoptotic cell death in NSCLC cells.

Conclusions: Our present work suggests that increased autophagic impairment induced by the combination of CPT and TFPA contributes to the apoptotic cell death of lung cancer cells.