Abstract 1331: MonoD, a novel analogue of digitoxin, induces superoxide mediated autophagic cell death in H460 lung cancer cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA While the efficacy of cardiac glycosides such as digitoxin in the treatment of congestive heart failure is well documented, its potential use as anti-cancer therapeutics is not well understood due to its narrow therapeutic index. We have synthesized a derivative of digitoxin, MonoD, which demonstrates cytotoxic effects on the NCI-60 panel of cell lines. In this study, we have characterized the efficacy of MonoD, delineated its mode of action and identified molecular targets that mediate action of this novel compound in H460 lung cancer cells. Staining with Cyto-ID®, a dye that selectively labels LC3-II specific autophagic vacuoles showed increased accumulation of the dye with MonoD treatment. Further evaluation of autophagic markers using immunoblotting revealed increased expression of both Beclin1 and LC3-II. In addition, MonoD caused oxidative stress and induced higher expression levels of NF-κB and p38 MAPK, proteins that are involved in positive regulation of autophagy mediated by Beclin1. Cells pretreated with MnTBAP, a superoxide dismutase mimic and •O2- scavenger not only inhibited •O2- production, but showed inhibition of both LC3-II and Beclin1, which was confirmed using immunofluorescence. Fluorometric caspase assays showed that MonoD exerted its pro-apoptotic effect mainly through the intrinsic caspase-9 apoptotic pathway. In response to prolonged 8h treatment with MonoD, global protein analysis of apoptotic proteins demonstrated a decrease in the levels of several pro-survival proteins, including Bcl-2, c-IAP, NF-κB and Survivin, and an increase in levels of pro-apoptotic proteins Bax, SMAC and p53. MonoD also inhibited Akt and HIF-1α, and had a strong anti-angiogenic effect as assessed by in vitro tube formation assays. These data suggest a multipotent effect for MonoD wherein autophagic mechanisms are triggered early, transitioning into a pronounced pro-apoptotic effect at later time point. To elucidate the potential crosstalk between autophagy and apoptosis, we investigated MonoD induced regulation of Akt and Bcl2. Inhibitors of Akt and Bcl2, when combined with MonoD showed higher expression of autophagy marker LC3-II, Beclin, NF-κB and p38 MAPK as compared to MonoD alone. This effect was validated using Cyto-ID®, which indicates translocation of LC3 from cytosol (LC3-I) to autophagosome membrane (LC3-II). In conclusion, we find that MonoD exerts its tumoristatic effects in H460 cancer cells by initiating autophagic cell death triggered by the production of superoxide, eventually leading to activation of several pro-apoptotic mediators. Most current therapies target individual rather than multiple cell death pathways thereby increasing the probability of cancer recurrence. Therefore, therapeutic entities such as MonoD that target multiple pathways such as autophagy and apoptosis, leading to sustained tumor regression are extremely desirable, and will be further investigated in vivo. Citation Format: Yogesh Kulkarni, Vivek Kaushik, Clayton Wright, George O'Doherty, Neelam Azad, Anand Iyer. MonoD, a novel analogue of digitoxin, induces superoxide mediated autophagic cell death in H460 lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1331. doi:10.1158/1538-7445.AM2014-1331