The growth inhibition of hepatocellular and cholangiocellular carcinoma cells by gemcitabine and the roles of extracellular signal-regulated and checkpoint kinases

Okano Okano

doi:10.3892/or_00000084

Abstract

We examined the effects of gemcitabine, a pyrimidine analogue, on hepatocellular carcinoma (HCC) and cholangiocellular carcinoma (CCC) cells. After HCC cells (HepG2, Hep3B, HLF and PLC/PRF/5) and CCC cells (HuCCT-1) were treated with gemcitabine, cellular growth, cell cycle, nuclear morphology and activity of signaling molecules were evaluated by WST-8 assays, flow cytometry analysis, Hoechst 33258 staining and Western blotting, respectively. We found that gemcitabine significantly inhibited the growth of HCC and CCC cells in a dose- and time-dependent manner. Gemcitabine induced cell cycle arrest at the G1 phase, however, the sub-G1 fraction was not observed and nuclear morphology did not indicate the induction of apoptosis. Gemcitabine induced differential activation of checkpoint kinases, Chk2 and Chk1, in HCC and CCC cells, respectively and gemcitabine activated extracellular signal-regulated kinase (ERK)1/2 in both cell types. After the cells were pretreated with a MEK inhibitor U0126, activations of these checkpoint kinases were abrogated and the cell death was enhanced. These results demonstrate that gemcitabine inhibited the growth of HCC and CCC cells by cell cycle arrest without apoptosis and that the ERK/Chk1/2 signaling pathway was in part responsible for the resistance to gemcitabine. Our findings shed light on treating patients with HCC and CCC by gemcitabine, especially when combined with a MEK inhibitor and Chk1/2 inhibitors.

Highlights

Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world and the third-leading cause of death from cancer [1]
Increasing doses of gemcitabine inhibited the growth of both HCC and cholangiocellular carcinoma (CCC) cells in a dose- and timedependent manner (Fig. 1A-E). These results indicate that clinically relevant doses of gemcitabine inhibit the growth of primary liver cancer cells including HCC and CCC cells
Flow cytometry analysis revealed that cell cycle was arrested at the G0/G1 phase after Hep3B and HuCCT-1 cells were treated with gemcitabine and the subG1 fraction, a hallmark of apoptosis, was not induced after the gemcitabine treatment (Fig. 2A)

Summary

Introduction

Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world and the third-leading cause of death from cancer [1]. Gemcitabine (2',2'-difluorocytidine monohydrochloride), a pyrimidine analogue, has been clinically utilized for the treatment of patients with pancreatic cancer [4] and nonsmall cell lung cancer (NSCLC) [5]. In addition to these types of cancers, there is accumulating evidence that gemcitabine was effective in patients with advanced HCC [6,7,8,9] and intrahepatic CCC [10,11], when used as a single agent and combined with other chemotherapeutic drugs such as platinum.

Methods

Results

Conclusion