Study On Autophagy And Related Signaling Pathways Induced By Digitalis Drugs

Digitalis drugs, as a specifiac ligand of Na+/K+-ATPases, have been in clinical use for many years to treat heart failure. In recent years, they are reported to show obvious anti-cancer activity. Our previous work found that autophagic cell death was induced by digitalis drugs, represented by digoxin and ouabain, in human non-small cell lung cancer (NSCLC) cells. However, whether autophagy could be also induced in other cancer cells and how related molecular signaling pathways actually involved, remain to be clarified. In this study, the role of AMPK, Src and ERK1/2in signaling pathways as well as autophagy induced by digitalis was examined in A549and MCF-7cells.Cell viability and colony formation ability were assessed by MTT and clone formation assay, and ouabain caused significant proliferation inhibition in cancer cells at IC50level. Morever, autophagy was markly induced by ouabain in A549and MCF-7cells, but not MRC5cells, as evidenced by positive acridine orange staining of intracellular acidic vesicles, time-dependent increase in FL3/FL1ratio, p62decrease and conversion of LC3-I to autophagosome-associated LC3-II in the presence of lysosomal acidification blockers, as well as the formation of LC3punctae detected by immunofluorescence analysis.Simultaneously, a dose-and time-dependent increase of intracellular reactive oxygen species (ROS) levels was found in ouabain-treated cells. Interestingly, pretreatment with Src inhibitor PP2could significantly attenuate the generation of ROS induced by the drugs, and this confirms a positive regulation of Src during ROS generation. The positive role of ROS during autophagy induction is further supported by the fact that N-Acetyl-L-cysteine (NAC), a well-known ROS scavenger, was found to reduce LC3-II conversion upon co-treatment with ouabain, just like PP2did.Importantly, activation of AMPK, Src, ERK1/2, PERK and e1F2a as well as up-regulation of AMPK activity in vitro and intracellular ATP depletion were found after exposure to oubain. Co-treatment with PP2or siRNA-Src could block drug-induced ERK1/2phosphorylation, together with autophagic phenotypes in the cells, and ultimately alleviate the drugs’ cytotoxicity. More importantly and interestingly, the interaction between AMPK and Src was examined by co-immunoprecipitation, and AMPK may function as an upstream regulator for Src activation.In summary, this work provides solid evidences showing that digitalis drugs have selective killing activity in cancer cells. ATP-mediated AMPK phosphorylation and Src-mediacted activation of ERK1/2signaling pathway, as well as ER stress signaling and ROS generation, are both found to be involved in the autophagy induced by drugs at IC50level. Morever, AMPK functions as an upstream regulator of Src activation during autophagy induction.