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. 2005 Jun 21;11(23):3498-503.
doi: 10.3748/wjg.v11.i23.3498.

Overexpression of Bax induces apoptosis and enhances drug sensitivity of hepatocellular cancer-9204 cells

Jian-Yong Zheng  1 , Guang-Shun Yang, Wei-Zhong Wang, Jiang Li, Kai-Zong Li, Wen-Xian Guan, Wen-Liang Wang
Affiliations

Overexpression of Bax induces apoptosis and enhances drug sensitivity of hepatocellular cancer-9204 cells

Jian-Yong Zheng et al. World J Gastroenterol. .

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 micromol/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.

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Figures

Figure 1
Figure 1
Inducible overexpression of Bax by ZnSO4 in HCC-9204/Bax cells SABC ×400.
Figure 2
Figure 2
Western blotting analysis of Bax expression in HCC-9204 cells.
Figure 3
Figure 3
Obvious morphological changes of HCC-9204/Bax cells TUNEL ×100.
Figure 4
Figure 4
HCC-9204/Bax cells apoptosis with sub- G peak by flow cytometer. A: vehicle-transfected HCC-9204/pMD cells; B: Bax-transfected HCC-9204/Bax cells.
Figure 5
Figure 5
HCC-9204 cells apoptosis after ADR with sub- G peak by flow cytometer.
Figure 6
Figure 6
MTT assay showed the viability of HCC-9204 cells.
See this image and copyright information in PMC

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