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

Abstract

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 Ca 2+ 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. Wohn-Jenn Leu and Ching-Ting Wang equally contributed to this study. 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. K E Y W O R D S ascleposide, endocytosis, Na+/K+-ATPase α1-subunit, p38 MAPK, tubulin acetylation

Cardiac Glycosides as Immune System Modulators

“…Among such potential repurposed drug candidates belong, for example, cardiac glycosides (CGs), currently used in clinical practice for the treatment of heart diseases, such as congestive heart failure or cardiac arrhythmias. Furthermore, CGs also exert very promising anticancer activity both in vitro and in vivo [ 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 ], which is based mainly on the induction of complex signaling cascades in Na + /K + -ATPase (NKA) signalosome and induction of immune response. This fact could be utilized not only for cancer treatment but also in the treatment of a plethora of autoimmune and inflammatory diseases.…”

Study on the Drug Targets and Molecular Mechanisms of Rhizoma Curcumae in the Treatment of Nasopharyngeal Carcinoma Based on Network Pharmacology

“…e KEGG analyses showed that most of the enriched biological information was associated with the thyroid cancer pathway, small-cell lung cancer pathway, prostate cancer pathway, bladder cancer pathway, and other cancer pathways. According to an analysis of the literature, we found that multiple target proteins contained in these pathways can activate the MAPK signalling pathway [38][39][40][41]. erefore, we speculated that Rhizoma Curcumae may regulate the proliferation of nasopharyngeal carcinoma cells by regulating the MAPK signalling pathway.…”

Anti-tumor pharmacology of natural products targeting mitosis

“…Ascleposide, a natural cardiac steroid lactone isolated from Saussurea lappa , has been found to inhibit DU145 prostate cancer cell growth 149 . Ascleposide downregulates the expression of cyclin D1, cyclin A, and CDK4 in a dose-dependent manner, and also downregulaets c-Myc (an early identified oncogenes) in human prostate cancer cells 150 . C-myc indirectly regulates the cell cycle through the dissociation of the CDK repressor protein p21 from the CDK4/6-cyclin D complex 151 .…”

“…C-myc indirectly regulates the cell cycle through the dissociation of the CDK repressor protein p21 from the CDK4/6-cyclin D complex 151 . Therefore, ascleposide exerts indirect and direct effects on cell cycle regulation, and decreases the G1 distribution of cells while increasing the proportion of cells in G2/M and sub-G1 phase 150 . The p21 and p27 Cip/Kip proteins are crucial CDK-interacting protein/kinase inhibitory proteins in mitotis 152 .…”