Liver Cancer
Copyright ©2005 Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jun 21, 2005; 11(23): 3498-3503
Published online Jun 21, 2005. doi: 10.3748/wjg.v11.i23.3498
Overexpression of Bax induces apoptosis and enhances drug sensitivity of hepatocellular cancer-9204 cells
Jian-Yong Zheng, Guang-Shun Yang, Wei-Zhong Wang, Jiang Li, Kai-Zong Li, Wen-Xian Guan, Wen-Liang Wang
Jian-Yong Zheng, Guang-Shun Yang, Department of Laparoscope, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China
Wei-Zhong Wang, Wen-Xian Guan, Department of Gastrointestinal Surgery, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province, China
Jiang Li, Wen-Liang Wang, Department of Pathology, Fourth Military Medical University, Xi’an 710032, Shaanxi Province, China
Kai-Zong Li, Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi’an 710032, Shaanxi Province, China
Author contributions: All authors contributed equally to the work.
Correspondence to: Jian-Yong Zheng, Department of Laparoscope, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China
Telephone: +86-21-65564166 Fax: +86-21-65564166
Received: November 16, 2004
Revised: November 18, 2004
Accepted: November 24, 2004
Published online: June 21, 2005
Abstract

AIM: To investigate the role of overexpression of Bax in apoptotic pathways and the response of human hepatocellular cancer (HCC)-9204 cells to cell death induced by adriamycin.

METHODS: The whole length of Bax cDNA was transfected into human HCC-9204 cells by the method of lipofectamine transfection. An inducible MT-II regulatory system was constructed, which allowed controlled expression of protein upon addition of ZnSO4 (100 μmol/L) as an external inducer. Stable transfecting inducible expression vector containing Bax gene was performed. Expression of Bax in protein was analyzed by immunohistochemistry and Western blotting. TUNEL and flow cytometry were used to assess the effect of Bax on apoptosis. Colony assay and tetrazolium blue (MTT) assay were used to evaluate the difference in drug sensitivity of HCC-9204 cells after Bax-transfection.

RESULTS: Immunohistochemistry and Western blotting demonstrated that the expression of Bax protein markedly increased in Bax-transfected cells 4 h after the addition of ZnSO4. Bax positive signal was frequently found on the cytoplasm and perinuclear region of HCC-9404 cells, and there was ectopic expression in cells with marked condensation of chromatin and cytoplasm (apoptotic cells). Apoptotic index significantly increased in Bax-transfected HCC-9204/Bax cells (3.6 vs 27.2, 4.2 vs 32.3, P<0.05). Flow cytometry analysis showed a significant sub-G1 peak and apoptosis in 15.4% HCC-9204/Bax cells 24 h after treatment. Furthermore, colony survival rate decreased from 66% (HCC-9204/pMD) to 45% (HCC-9204/Bax) 2 d after ADR withdrawal. MTT assay result showed that the effects of Bax on cell viability following ADR exposure were significant as compared to the vehicle-transfected HCC-9204/pMD cells (21% vs 44%, P<0.01).

CONCLUSION: Overexpression of Bax not only induces apoptosis, but also sensitizes HCC-9204 cells to cell death induced by adriamycin.

Keywords: Bax; Apoptosis; Hepatocellular carcinoma