Cardenolide glycosides sensitize gefitinib-induced apoptosis in non-small cell lung cancer: inhibition of Na+/K+-ATPase serving as a switch-on mechanism

Abstract

The treatment of non-small cell lung cancer (NSCLC) is known as a significant level of unmet medical need in spite of the progress in targeted therapy and personalized therapy. Overexpression of the Na⁺/K⁺-ATPase contributes to NSCLC progression, suggesting its potentiality in antineoplastic approaches. Epi-reevesioside F, purified from Reevesia formosana, showed potent anti-NSCLC activity through inhibiting the Na⁺/K⁺-ATPase, leading to internalization of α1- and α3-subunits in Na⁺/K⁺-ATPase and suppression of Akt-independent mTOR-p70S6K-4EBP1 axis. Epi-reevesioside F caused a synergistic amplification of apoptosis induced by gefitinib but not cisplatin, docetaxel, etoposide, paclitaxel, or vinorelbine in both NCI-H460 and A549 cells. The synergism was validated by enhanced activation of the caspase cascade. Bax cleavage, tBid formation, and downregulation of Bcl-xL and Bcl-2 contributed to the synergistic apoptosis induced by the combination treatment of epi-reevesioside F and gefitinib. The increase of membrane DR4 and DR5 levels, intracellular Ca²⁺ concentrations, and active m-calpain expression were responsible for the caspase-8 activation and Bax cleavage. The increased α-tubulin acetylation and activation of MAPK (i.e., p38 MAPK, Erk, and JNK) depending on cell types contributed to the synergistic mechanism under combination treatment. These signaling pathways that converged on profound c-Myc downregulation led to synergistic apoptosis in NSCLC. In conclusion, the data suggest that epi-reevesioside F inhibits the Na⁺/K⁺-ATPase and displays potent anti-NSCLC activity. Epi-reevesioside F sensitizes gefitinib-induced apoptosis through multiple pathways that converge on c-Myc downregulation. The data support the inhibition of Na⁺/K⁺-ATPase as a switch-on mechanism to sensitize gefitinib-induced anti-NSCLC activity.

Keywords: Bax cleavage; Gefitinib; Na+/K+-ATPase; Non-small cell lung cancer; Tubulin acetylation.