Original Article
Copyright ©2012 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Jul 21, 2012; 18(27): 3527-3536
Published online Jul 21, 2012. doi: 10.3748/wjg.v18.i27.3527
Polo-like kinase 1, a new therapeutic target in hepatocellular carcinoma
Wei Chuen Mok, Shanthi Wasser, Theresa Tan, Seng Gee Lim
Wei Chuen Mok, Shanthi Wasser, Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore 138673, Singapore
Theresa Tan, Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore
Seng Gee Lim, Department of Gastroenterology and Hepatology, Yong Loo Lin School of Medicine, National University Health System, Singapore 117597, Singapore
Seng Gee Lim, Department of Medicine, National University of Singapore, Singapore 119074, Singapore
Author contributions: Mok WC performed the research and drafted the manuscript; Wasser S and Tan T contributed equally by troubleshooting and analyzing the work, editing the manuscript; Lim SG conceptualized the research, edited the manuscript and gave final approval for the manuscript.
Supported by The National University of Singapore Grants, No. R-172-000-001-731 and No. R-172-000-024-731
Correspondence to: Seng Gee Lim, Director of Hepatology, Department of Gastroenterology and Hepatology, Yong Loo Lin School of Medicine, National University of Singapore, 1E Kent Ridge Road, Singapore 119074, Singapore. mdclimsg@nus.edu.sg
Telephone: +65-67-724369 Fax: +65-67-724361
Received: October 28, 2010
Revised: March 30, 2012
Accepted: May 12, 2012
Published online: July 21, 2012
Abstract

AIM: To investigate the role of polo-like kinase 1 (PLK1) as a therapeutic target for hepatocellular carcinoma (HCC).

METHODS: PLK1 gene expression was evaluated in HCC tissue and HCC cell lines. Gene knockdown with short-interfering RNA (siRNA) was used to study PLK1 gene and protein expression using real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, and cell proliferation using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium (MTS) and bromodeoxyuridine (BrdU) assays. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and caspase-inhibition assay. Huh-7 cells were transplanted into nude mice and co-cultured with PLK1 siRNA or control siRNA, and tumor progression was compared with controls.

RESULTS: RT-PCR showed that PLK1 was overexpressed 12-fold in tumor samples compared with controls, and also was overexpressed in Huh-7 cells. siRNA against PLK1 showed a reduction in PLK1 gene and protein expression of up to 96% in Huh-7 cells, and a reduction in cell proliferation by 68% and 92% in MTS and BrdU cell proliferation assays, respectively. There was a 3-fold increase in apoptosis events, and TUNEL staining and caspase-3 assays suggested that this was caspase-independent. The pan-caspase inhibitor Z-VAD-FMK was unable to rescue the apoptotic cells. Immnofluorescence co-localized endonuclease-G to fragmented chromosomes, implicating it in apoptosis. Huh-7 cells transplanted subcutaneously into nude mice showed tumor regression in siPLK1-treated mice, but not in controls.

CONCLUSION: Knockdown of PLK1 overexpression in HCC was shown to be a potential therapeutic target, leading to apoptosis through the endonuclease-G pathway.

Keywords: RNA; Polo-like kinase 1; Apoptosis; Endonuclease G; Forkhead box transcription factors; Nude mice