Abstract 2265: Antitumorigenic effects of a novel digitoxin analogue.
Authors: Azad Neelam , Rojanasakul Yon , O'Doherty George , Iyer Anand 
Published: August 2014

Cardiac glycosides (cardenolides) such as digitoxin have been used to regulate heart function for centuries; however, recent studies have reported their potential use as anti-cancer agents. Although detailed investigation of their anti-cancer properties have been tempered by concerns relating to a narrow therapeutic index, their versatility in inhibiting multiple pathways critical to tumorigenesis has sustained interest in evaluating their potential to treat cancer. An approach to ameliorate potential side effects associated with cardenolides would be to synthesize chemical analogs with much higher anti-tumor efficacy, while still maintaining the pathway-specificities of their parent compounds. To this end, we have developed a library of digitoxin analogues using a patented palladium-catalyzed de novo synthesis methodology. One novel non-natural monosaccharide derivative (α-L-digitoxin or MonoD) in particular demonstrates strong cytotoxic effects on a wide variety of tumors. Using several ex vivo and in vitro techniques, we have further characterized this compound to determine its efficacy and mode of action in lung cancer cells. We first established using SAR studies that MonoD exhibited much stronger tumor-cytostatic effect than digitoxin across the NCI panel of sixty cell lines, with most potent activity against non small-cell cancer NCI-H460 cells. Further characterization of cell death using the Hoechst assay showed that MonoD induced apoptosis at doses almost ten-times lower than digitoxin (DTX), which could be overcome using the pan-caspase inhibitor ZVAD; non-tumorigenic lung epithelial Beas-2B cells were not significantly affected at such doses. Since MonoD is a cardenolide derivative, we assessed its safety profile using the Langendorff assay with intact rat hearts. Robust cardiac activity was observed even at very high MonoD levels, validating its safety. The mechanistic details of MonoD action were characterized using various functional assays. Fluorometric caspase assays showed that MonoD exerted its pro-apoptotic effect mainly through the intrinsic caspase-9 apoptotic pathway. In response to MonoD, global protein analysis of apoptotic proteins using immunoassays demonstrated a decrease in the levels of pro-survival proteins such as 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. Luciferase reporter assays demonstrated cross-talk between Bcl-2 and HIF-1α, suggesting that anti-angiogenic effects of MonoD may be mediated at least in part by the apoptosis pathway, and further studies are underway. Our long-term goal is to generate, using a rational drug-design approach, a class of more potent MonoD derivatives that act as tumor-vascularization disruptors, leading to regression in lung carcinogenesis via apoptotic cell death.

Citation Format: Neelam Azad, Yon Rojanasakul, George O'Doherty, Anand Iyer. Antitumorigenic effects of a novel digitoxin analogue. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2265. doi:10.1158/1538-7445.AM2013-2265