Salinomycin inhibits proliferation and induces apoptosis of human hepatocellular carcinoma cells in vitro and in vivo

• Published in: PloS one Vol. 7; no. 12; p. e50638
• Main Authors: Wang, Fan, He, Lei, Dai, Wei-Qi, Xu, Ya-Ping, Wu, Dong, Lin, Chun-Lei, Wu, Shu-Mei, Cheng, Ping, Zhang, Yan, Shen, Miao, Wang, Chen-Feng, Lu, Jie, Zhou, Ying-Qun, Xu, Xuan-Fu, Xu, Ling, Guo, Chuan-Yong
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 20.12.2012; Public Library of Science (PLoS)
• Subjects: AC133 Antigen; Animals; Antibiotics; Anticancer properties; Antigens, CD - metabolism; Antineoplastic Agents - pharmacology; Apoptosis; Apoptosis - drug effects; Bax protein; Bcl-2 protein; beta Catenin - metabolism; Biology; Biotechnology; Breast cancer; Calcium (intracellular); Calcium - metabolism; Carcinoma, Hepatocellular - pathology; Cell cycle; Cell Cycle Checkpoints - drug effects; Cell Line, Tumor; Cell proliferation; Cell Proliferation - drug effects; Cytometry; Flow; Flow cytometry; Glycoproteins - metabolism; Hepatocellular carcinoma; Hepatoma; Humans; Immunohistochemistry; Intracellular signalling; Intracellular Space - drug effects; Intracellular Space - metabolism; Kinases; Liver; Liver cancer; Liver Neoplasms - pathology; Male; Medicine; Mice; Mutation; Peptides - metabolism; Proliferating cell nuclear antigen; Pyrans - pharmacology; Salinomycin; Signal Transduction - drug effects; Staining; Stem cells; Subpopulations; Tempering; Tumors; Wang, Lei; Wnt protein; Wnt Proteins - metabolism; Xenograft Model Antitumor Assays; β-Catenin; China
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0050638

The anti-tumor antibiotic salinomycin (Sal) was recently identified as a selective inhibitor of breast cancer stem cells; however, the effect of Sal on hepatocellular carcinoma (HCC) is not clear. This study aimed to determine the anti-tumor efficacy and mechanism of Sal on HCC. HCC cell lines (HepG2, SMMC-7721, and BEL-7402) were treated with Sal. Cell doubling time was determinated by drawing growth curve, cell viability was evaluated using the Cell Counting Kit 8. The fraction of CD133(+) cell subpopulations was assessed by flow cytometry. We found that Sal inhibits proliferation and decreases PCNA levels as well as the proportion of HCC CD133(+)cell subpopulations in HCC cells. Cell cycle was analyzed using flow cytometry and showed that Sal caused cell cycle arrest of the various HCC cell lines in different phases. Cell apoptosis was evaluated using flow cytometry and Hoechst 33342 staining. Sal induced apoptosis as characterized by an increase in the Bax/Bcl-2 ratio. Several signaling pathways were selected for further mechanistic analyses using real time-PCR and Western blot assays. Compared to control, β-catenin expression is significantly down-regulated upon Sal addition. The Ca(2+) concentration in HCC cells was examined by flow cytometry and higher Ca(2+) concentrations were observed in Sal treatment groups. The anti-tumor effect of Sal was further verified in vivo using the hepatoma orthotopic tumor model and the data obtained showed that the size of liver tumors in Sal-treated groups decreased compared to controls. Immunohistochemistry and TUNEL staining also demonstrated that Sal inhibits proliferation and induces apoptosis in vivo. Finally, the role of Sal on in vivo Wnt/β-catenin signaling was evaluated by Western blot and immunohistochemistry. This study demonstrates Sal inhibits proliferation and induces apoptosis of HCC cells in vitro and in vivo and one potential mechanism is inhibition of Wnt/β-catenin signaling via increased intracellular Ca(2+) levels.