Abstract 3795: Cardiac glycoside digitoxin and its monosaccharide analogue MonoD inhibit NFκB to induce apoptotic cell death in ER+ MCF7 and triple-negative MDAMB-468 breast cancer cells

Abstract

Abstract Digitoxin, a cardiac glycoside well appreciated in the treatment of congestive heart failure, has been poorly understood and appreciated as an anti-cancer therapeutic due to its narrow therapeutic potential. Using a patented palladium-catalyzed de novo synthesis method, we have developed a library of digitoxin analogs, which alleviate the toxic side effects of digitoxin while maintaining its chemical activity. We have evaluated MonoD, a digitoxin analog with a single sugar moiety, in a variety of NCI-60 panel of cell lines, with a significant tumoristatic effect observed in estrogen receptor positive and triple negative breast cancer cells. Therefore, in order to determine the efficacy of digitoxin and MonoD, delineate its mode of action and identify molecular targets we chose to test this novel compound in ER+ MCF7 and TN MDAMB-468 breast cancer cells. Digitoxin and MonoD showed a dose dependent decrease in cell viability in MCF7 and MDAMB-468 cell lines. Further characterization of cell death using the Hoechst assay showed that MonoD induced significantly higher apoptosis at the same dose as digitoxin. Both drugs induce oxidative stress in the form of superoxide as assayed using Dihydroethidium (DHE). In vitro scratch migration assay showed a five-fold decrease in rate of cell migration in the presence of test drugs. MCF7 and MDAMB-468 cells were analyzed for cell cycle progression in the presence of digitoxin and MonoD. We observed that the drugs induced cell cycle arrest in G0/G1 phase at the expense of S and G2/M phases. Increased expression of p27Kip1 in the presence of drugs provided further validation, as increased p27 expression is known to block cell cycle progression through G1-S transition. In addition to cell cycle arrest, apoptosis is another widely accepted mechanism by which anti-cancer drugs inhibit tumor formation. We observed that these drugs induced apoptosis as indicated by cleaved PARP and cleaved caspase-9, in addition to down-regulation of pro-survival factor Bcl-2. NFκB has been reported to be constitutively active in many types of cancer by controlling the expression of several genes involved in cellular proliferation and apoptosis. We observed that NFκB expression was down-regulated in both cell lines with digitoxin and MonoD treatment. IKKβ, a critical kinase involved in regulating NFκB pathway showed significant down-regulation, thereby suggesting that these drugs exert their anti-cancer therapeutic potential through the inhibition of NFκB pathway. While additional work is required to establish their mode of action, we have sufficient evidence to hypothesize that down-regulation of NFκB is critical to the mode of action of these drugs. Further, it is evident from densitometric analyses of western blots, MTT and Hoechst assays that MonoD shows higher efficacy as compared to digitoxin in inducing cancer cell death. Citation Format: Yogesh Kulkarni, Vivek Kaushik, Raj Venkatadri, Clayton Wright, Juan Sebastian Yakisich, George O'Doherty, Neelam Azad, Anand Iyer. Cardiac glycoside digitoxin and its monosaccharide analogue MonoD inhibit NFκB to induce apoptotic cell death in ER+ MCF7 and triple-negative MDAMB-468 breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3795. doi:10.1158/1538-7445.AM2015-3795