Original Articles
Copyright ©2009 The WJG Press and Baishideng. All rights reserved.
World J Gastroenterol. Mar 28, 2009; 15(12): 1443-1451
Published online Mar 28, 2009. doi: 10.3748/wjg.15.1443
Down-regulation of extracellular signal-regulated kinase 1/2 activity in P-glycoprotein-mediated multidrug resistant hepatocellular carcinoma cells
Feng Yan, Xiao-Min Wang, Chao Pan, Quan-Ming Ma
Feng Yan, Department of Hepatobiliary Surgery, Postdoctoral Station, Zhongshan Hospital, Xiamen University, Xiamen 361004, Fujian Province, China
Xiao-Min Wang, Department of Hepatobiliary Surgery, Digestive Diseases Institute, Xiamen University, Xiamen 361004, Fujian Province, China
Chao Pan, Department of Pathology, Zhongshan Hospital, Xiamen University, Xiamen 361004, Fujian Province, China
Quan-Ming Ma, Department of Hepatobiliary Surgery, Zhongshan Hospital, Xiamen University, Xiamen 361004, Fujian Province, China
Author contributions: Yan F, Wang XM and Pan C designed the research; Yan F and Ma QM performed the research; Yan F and Ma QM analyzed the data; Yan F, Wang XM and Pan C wrote the paper.
Correspondence to: Xiao-Min Wang, MD, PhD, Professor of Medicine, Chief, Department of Hepatobiliary Surgery, Digestive Diseases Institute, Xiamen University, Xiamen 361004, Fujian Province, China. wxm@xmzsh.com
Telephone: +86-592-2292203
Fax: +86-592-2212328
Received: January 17, 2009
Revised: February 17, 2009
Accepted: February 24, 2009
Published online: March 28, 2009
Abstract

AIM: To study the expression and phosphorylation of extracellular signal-regulated kinase (ERK) 1 and ERK2 in multidrug resistant (MDR) hepatocellular carcinoma (HCC) cells.

METHODS: MDR HCC cell lines, HepG2/adriamycin (ADM) and SMMC7721/ADM, were developed by exposing parental cells to stepwise increasing concentrations of ADM. MTT assay was used to determine drug sensitivity. Flow cytometry was employed to analyze cell cycle distribution and measure cell P-glycoprotein (P-gp) and multidrug resistant protein 1 (MRP1) expression levels. ERK1 and ERK2 mRNA expression levels were measured by quantitative real-time PCR (QRT-PCR). Expression and phosphorylation of ERK1 and ERK2 were analyzed by Western blot.

RESULTS: MTT assay showed that HepG2/ADM and SMMC7721/ADM were resistant not only to ADM, but also to multiple anticancer drugs. The P-gp expression was over 10-fold higher in HepG2/ADM cells than in HepG2 cells (8.92% ± 0.22% vs 0.88% ± 0.05%, P < 0.001) and over 4-fold higher in SMMC7721/ADM cells than in SMMC7721 cells (7.37% ± 0.26% vs 1.74% ± 0.25%, P < 0.001). However, the MRP1 expression was not significantly higher in HepG2/ADM and SMMC7721/ADM cells than in parental cells. In addition, the percentage of MDR HepG2/ADM and SMMC7721/ADM cells was significantly decreased in the G0/G1 phase and increased in the the S phase or G2/M phase. QRT-PCR analysis demonstrated that the ERK1 and ERK2 mRNA expression increased apparently in HepG2/ADM cells and decreased significantly in SMMC7721/ADM cells. Compared with the expression of parental cells, ERK1 and ERK2 protein expressions were markedly decreased in SMMC7721/ADM cells. However, ERK2 protein expression was markedly increased while ERK1 protein expression had no significant change in HepG2/ADM cells. Phosphorylation of ERK1 and ERK2 was markedly decreased in both HepG2/ADM and SMMC7721/ADM MDR cells.

CONCLUSION: ERK1 and ERK2 activities are down-regulated in P-gp-mediated MDR HCC cells. ERK1 or ERK2 might be a potential drug target for circumventing MDR HCC cells.

Keywords: Multidrug resistance, Extracellular signal-regulated MAP kinases, Hepatocellular carcinoma, P-glycoprotein, Multidrug resistance-associated protein