Ouabain, a Cardiac Glycoside, Inhibits the Fanconi Anemia/BRCA Pathway Activated by DNA Interstrand Cross-Linking Agents

Abstract

Modulation of the DNA repair pathway is an emerging target for the development of anticancer drugs. DNA interstrand cross-links (ICLs), one of the most severe forms of DNA damage caused by anticancer drugs such as cisplatin and mitomycin C (MMC), activates the Fanconi anemia (FA)/BRCA DNA repair pathway. Inhibition of the FA/BRCA pathway can enhance the cytotoxic effects of ICL-inducing anticancer drugs and can reduce anticancer drug resistance. To find FA/BRCA pathway inhibitory small molecules, we established a cell-based high-content screening method for quantitating the activation of the FA/BRCA pathway by measuring FANCD2 foci on DNA lesions and then applied our method to chemical screening. Using commercial LOPAC1280 chemical library screening, ouabain was identified as a competent FA/BRCA pathway inhibitory compound. Ouabain, a member of the cardiac glycoside family, binds to and inhibits Na+/K+-ATPase and has been used to treat heart disease for many years. We observed that ouabain, as well as other cardiac glycoside family members―digitoxin and digoxin―down-regulated FANCD2 and FANCI mRNA levels, reduced monoubiquitination of FANCD2, inhibited FANCD2 foci formation on DNA lesions, and abrogated cell cycle arrest induced by MMC treatment. These inhibitory activities of ouabain required p38 MAPK and were independent of cellular Ca2+ ion increase or the drug uptake-inhibition effect of ouabain. Furthermore, we found that ouabain potentiated the cytotoxic effects of MMC in tumor cells. Taken together, we identified an additional effect of ouabain as a FA/BRCA pathway-inhibiting chemosensitization compound. The results of this study suggest that ouabain may serve as a chemosensitizer to ICL-inducing anticancer drugs.

FANCD2 re-expression is associated with glioma grade and chemical inhibition of the Fanconi Anaemia pathway sensitises gliomas to chemotherapeutic agents

“…To date, four separate FAPi have been described that are commercially available; curcumin, its monoketone analogues EF-24 & 4H-TTD, and the menadione analogue DDN [13-16]. However, in initial toxicity studies, we found that 4H-TTD was toxic to both U87 and U138 cells even at nM concentrations (data not shown), and was therefore excluded from our further analyses, as was the recently reported FAPi Ouabain [24]. …”

Ouabain and Digoxin Activate the Proteasome and the Degradation of the ERα in Cells Modeling Primary and Metastatic Breast Cancer

“…Accordingly, the fact that within 24 h, OU and Digo reduce the expression of the proteasome subunit genes, while within 8 h they induce the activation of the proteasome enzymatic activities, leads us to speculate that cells activate a rapid compensatory mechanism to buffer the CGs-dependent proteasome activation. Interestingly, the ability of OU to activate the proteasome can also explain some of the previously reported effects of this CG on specific components of the ubiquitination pathway [ 52 ].…”

Digoxin use and the risk for colorectal cancer

“…Cardiac glycosides also affect several pathways central for tumor formation. Digoxin was shown to: suppress growth of hypoxic NSCLC and breast cancer cells (2) (3) (4) and possibly decrease the risk for castrate resistant prostate cancer through inhibition of hypoxia inducible factor 1 (5, 6); reduce protein synthesis of the tumor suppressor gene P53 through the Src/MAPK pathway (7); inhibit the FA/BRCA pathway (8); induce autophagy through mTOR and ERK1/2 signaling pathways in NSCLC cells (9) (10); and induce immunogenic cell death in immune-competent but not immune-deficient mice through inhibition of the Na+/K+-ATPase (11). Furthermore, the alpha 1 isoform of the sodium potassium ATPase mediates cell migration and growth in addition to the pumping activities and is down regulated in colon, pancreas, kidney, bladder and prostate cancers (12, 13).…”