Ouabain induces apoptotic cell death in human prostate DU 145 cancer cells through DNA damage and TRAIL pathways

• Published in: Environmental toxicology 2019-12, Vol.34 (12), p.1329-1339
• Main Authors: Chang, Yi‐Ming, Shih, Yung‐Luen, Chen, Chao‐Ping, Liu, Ko‐Lin, Lee, Mei‐Hui, Lee, Ming‐Zhe, Hou, Hsin‐Tu, Huang, Hsieh‐Chou, Lu, Hsu‐Feng, Peng, Shu‐Fen, Chen, Kuo‐Wei, Yeh, Ming‐Yang, Chung, Jing‐Gung
• Format: Article
• Language: English
• Subjects: Apoptosis; Apoptosis - drug effects; Apoptosis-inducing factor; BAX protein; bcl-2-Associated X Protein - metabolism; Calcium (mitochondrial); Calcium ions; Cancer; Cardiotonic Agents - pharmacology; Caspase; Caspase 3 - metabolism; Cell cycle; Cell death; Cell Line, Tumor; Cell viability; Cells; CHOP protein; Cytochrome; Cytochrome c; Cytochromes; Cytotoxicity; Damage; Deoxyribonucleic acid; DNA; DNA damage; DNA Damage - drug effects; FADD protein; FasL protein; Flow cytometry; G1 phase; G1 Phase Cell Cycle Checkpoints - drug effects; Gene expression; human prostate cancer DU 145 cells; Humans; Initiation factor eIF-2; Male; Membrane potential; Membrane Potential, Mitochondrial - drug effects; Microscopy; Mitochondria; Morphology; Na+/K+-exchanging ATPase; Ouabain; Ouabain - pharmacology; Phase contrast; Prostate cancer; Prostatic Neoplasms - metabolism; Prostatic Neoplasms - pathology; Proteins; Proto-Oncogene Proteins c-bcl-2 - metabolism; Reactive Oxygen Species - metabolism; Steroids; TNF-Related Apoptosis-Inducing Ligand - metabolism; TRAIL pathway; TRAIL protein; Western blotting
• ISSN: 1520-4081; 1522-7278
• DOI: 10.1002/tox.22834

Ouabain, a cardiotonic steroid and specific Na+/K+‐ATPase inhibitor, has a potential to induce cancer cell apoptosis but the mechanisms of apoptosis induced by ouabain are not fully understand. The aim of this study was to investigate the cytotoxic effects of ouabain on human prostate cancer DU 145 cells in vitro. Cell morphological changes were examined by phase contrast microscopy. Cell viability, cell cycle distribution, cell apoptosis, DNA damage, the production of ROS and Ca2+, and mitochondrial membrane potential (ΔΨm) were measured by flow cytometry assay. Results indicated that ouabain induced cell morphological changes, decreased total cell viability, induced G0/G1 phase arrest, DNA damage, and cell apoptosis, increased ROS and Ca2+ production, but decreased the levels of ΔΨm in DU 145 cells. Ouabain also increased the activities of caspase‐3, ‐8, and ‐9. Western blotting was used for measuring the alterations of apoptosis‐associated protein expressions in DU 145 cells and results indicated that ouabain increased the expression of DNA damage associated proteins (pATMSer1981, p‐H2A.XSer139, and p‐p53Ser15) and ER‐stress‐associated proteins (Grp78, ATF6β, p‐PERKThr981, PERK, eIF2A, GADD153, CaMKIIβ, and caspase‐4) in time‐dependently. Furthermore, ouabain increased apoptosis‐associated proteins (DR4, DR5, Fas, Fas Ligand, and FADD), TRAIL pathway, which related to extrinsic pathway, promoted the pro‐apoptotic protein Bax, increased apoptotic‐associated proteins, such as cytochrome c, AIF, Endo G, caspase‐3, ‐8, and ‐9, but reduced anti‐apoptotic protein Bcl‐2 and Bcl‐x in DU 145 cells. In conclusion, we may suggest that ouabain decreased cell viability and induced apoptotic cell death may via caspase‐dependent and mitochondria‐dependent pathways in human prostate cancer DU 145 cells.