Ascleposide, a natural cardenolide, induces anticancer signaling in human castration‐resistant prostatic cancer through Na+/K+‐ATPase internalization and tubulin acetylation

• Published in: The Prostate Vol. 80; no. 4; pp. 305 - 318
• Main Authors: Leu, Wohn‐Jenn, Wang, Ching‐Ting, Hsu, Jui‐Ling, Chen, Ih‐Sheng, Chang, Hsun‐Shuo, Guh, Jih‐Hwa
• Format: Journal Article
• Language: English
• Published: United States Wiley Subscription Services, Inc 01-03-2020
• Subjects: Acetylation; Antitumor activity; Apoptosis; Arrhythmia; ascleposide; BAK protein; Calcium (intracellular); Cancer; Cardiac glycosides; Caspase; Castration; Cell cycle; Cell growth; Cell proliferation; Congestive heart failure; Cyclin-dependent kinase inhibitor p27; Cyclins; Endocytosis; Flow cytometry; G1 phase; Glycosides; Immunofluorescence; Internalization; Intracellular; MAP kinase; Membrane potential; Mitochondria; Na+/K+‐ATPase α1‐subunit; p38 MAPK; Prostate cancer; Protein expression; Proteins; Reactive oxygen species; tubulin acetylation; ascleposide; endocytosis; tubulin acetylation; Na+/K+-ATPase α1-subunit; p38 MAPK
• ISSN: 0270-4137; 1097-0045
• DOI: 10.1002/pros.23944

Background
Cardiac glycosides, which inhibit Na+/K+‐ATPase, display inotropic effects for the treatment of congestive heart failure and cardiac arrhythmia. Recent studies have suggested signaling downstream of Na+/K+‐ATPase action in the regulation of cell proliferation and apoptosis and have revealed the anticancer activity of cardiac glycosides. The study aims to characterize the anticancer potential of ascleposide, a natural cardenolide, and to uncover its primary target and underlying mechanism against human castration‐resistant prostate cancer (CRPC).
Methods
Cell proliferation was examined in CRPC PC‐3 and DU‐145 cells using sulforhodamine B assay, carboxyfluorescein succinimidyl ester staining assay and clonogenic examination. Flow cytometric analysis was used to detect the distribution of cell cycle phase, mitochondrial membrane potential, intracellular Na+ and Ca2+ levels, and reactive oxygen species production. Protein expression was examined using Western blot analysis. Endocytosis of Na+/K+‐ATPase was determined using confocal immunofluorescence microscopic examination.
Results
Ascleposide induced an increase of intracellular Na+ and a potent antiproliferative effect. It also induced a decrease of G1 phase distribution while an increase in both G2/M and apoptotic sub‐G1 phases, and downregulated several cell cycle regulator proteins, including cyclins, Cdk, p21, and p27 Cip/Kip proteins, Rb and c‐Myc. Ascleposide decreased the expression of antiapoptotic Bcl‐2 members (eg, Bcl‐2 and Mcl‐1) but upregulated proapoptotic member (eg, Bak), leading to a significant loss of mitochondrial membrane potential and activation of both caspase‐9 and caspase‐3. Ascleposide also dramatically induced tubulin acetylation, leading to inhibition of the catalytic activity of Na+/K+‐ATPase. Notably, extracellular high K+ (16 mM) significantly blunted ascleposide‐mediated effects. Furthermore, ascleposide induced a p38 MAPK‐dependent endocytosis of Na+/K+‐ATPase and downregulated the protein expression of Na+/K+‐ATPase α1 subunit.
Conclusion
Ascleposide displays antiproliferative and apoptotic activities dependent on the inhibition of Na+/K+‐ATPase pumping activity through p38 MAPK‐mediated endocytosis of Na+/K+‐ATPase and downregulation of α1 subunit, which in turn cause tubulin acetylation and cell cycle arrest. Cell apoptosis is ultimately triggered by the activation of caspase cascade attributed to mitochondrial damage through the downregulation of Bcl‐2 and Mcl‐1 protein expressions while upregulation of Bak protein levels. The data also suggest the potential of ascleposide in anti‐CRPC development.