Cardiac Glycoside Activities Link Na⁺/K⁺ ATPase Ion-Transport to Breast Cancer Cell Migration via Correlative SAR

Abstract

The cardiac glycosides ouabain and digitoxin, established Na+/K+ ATPase inhibitors, were found to inhibit MDA-MB-231 breast cancer cell migration through an unbiased chemical genetics screen for cell motility. The Na+/K+ ATPase acts both as an ion-transporter and as a receptor for cardiac glycosides. To delineate which function is related to breast cancer cell migration, structure–activity relationship (SAR) profiles of cardiac glycosides were established at the cellular (cell migration inhibition), molecular (Na+/K+ ATPase inhibition), and atomic (computational docking) levels. The SAR of cardiac glycosides and their analogs revealed a similar profile, a decrease in potency when the parent cardiac glycoside structure was modified, for each activity investigated. Since assays were done at the cellular, molecular, and atomic levels, correlation of SAR profiles across these multiple assays established links between cellular activity and specific protein–small molecule interactions. The observed antimigratory effects in breast cancer cells are directly related to the inhibition of Na+/K+ transport. Specifically, the orientation of cardiac glycosides at the putative cation permeation path formed by transmembrane helices αM1–M6 correlates with the Na+ pump activity and cell migration. Other Na+/K+ ATPase inhibitors that are structurally distinct from cardiac glycosides also exhibit antimigratory activity, corroborating the conclusion that the antiport function of Na+/K+ ATPase and not the receptor function is important for supporting the motility of MDA-MB-231 breast cancer cells. Correlative SAR can establish new relationships between specific biochemical functions and higher-level cellular processes, particularly for proteins with multiple functions and small molecules with unknown or various modes of action.

Therapeutic concentrations of digitoxin inhibit endothelial focal adhesion kinase and angiogenesis induced by different growth factors

“…Digitoxin and ouabain blocked HUVEC migration towards different growth factors: VEGF, bFGF, EGF, FBS plus ECGS. Inhibition of cell migration by several cardiac glycosides (ouabain, arenobufagin, bufalin, the cardenolide analogue UNBS1450) has been demonstrated in other cell types, in particular cancer cells, indicating that it might be a common biological effect of this class of drugs (Mijatovic et al , ; Li et al , ; Chueh et al , ; Magpusao et al , ). Interfering with cell migration in the tumour context affects several key processes for cancer development and progression such as angiogenesis, recruitment of inflammatory cells, tumour cells invasion and metastasis.…”

Effect of bafilomycin and NAADP on membrane‐associated ATPases and respiration of isolated mitochondria of the murine Nemeth‐Kellner lymphoma

“…Na + /K + ‐ATPase is highly expressed in cancer cells. The inhibition of Na + /K + ‐ATPase also exhibits antimigratory activity of breast cancer cells . The SERCA pump is identified as an endoplasmic reticulum (EPR) protein.…”

“…The inhibition of Na + /K + -ATPase also exhibits antimigratory activity of breast cancer cells. 15 The SERCA pump is identified as an endoplasmic reticulum (EPR) protein. Its normal function is required for all cells and represents a potential therapeutic target for cancer therapy too.…”

Nigericin decreases the viability of multidrug-resistant cancer cells and lung tumorspheres and potentiates the effects of cardiac glycosides

“…27 However, DIG is a well-characterized Na + /K + -ATPase inhibitor. 44 At relatively high concentrations (0.5-5 µM), DIG has the ability to inhibit the Na + /K + -ATPase pump, but at low concentrations (10-100 nM) may activate several transcriptional regulatory cascades via the Na + /K + -ATPase signalosome. 45 We recently found that DIG downregulates the expression of proteins involved in chemoresistance, survival, and stemness maintenance such as MDR1, ABCG2, Bcl-2, pAKT, and key proteins of the Wnt signaling pathway.…”