Adenovirus vector expressing mda-7 selectively kills hepatocellular carcinoma cell line Hep3B.
BACKGROUND
Melanoma differentiation associated gene-7 (mda-7) is a novel tumor suppressor gene, which has suppressor activity in a broad spectrum of human cancer cells both in vitro and in vivo through activation of various intracellular signaling pathways. In this study, we investigated the potential effect of mda-7 on human hepatocellular carcinoma (HCC) in vitro.


METHODS
Cells from the human HCC cell line Hep3B and the human liver cell line L-02 were assigned to three groups. One was cultured in Dulbecco's modified Eagle's medium without serum (control). The others were transfected with adenovirus expressing the mda-7 gene (Ad.mda-7) or adenovirus vector serving as negative control (Ad.vec). The expression of MDA-7 and Bcl-2 proteins in Hep3B and L-02 cells was confirmed by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay. The methyl thiazolyl tetrazolium colorimetric assay and flow cytometry were used to assess tumor cell proliferation and the cell cycle. Hoechst and Annexin-V/propidium iodide staining were used to study mda-7 gene expression in Hep3B and L-02 cells. The expression of MDA-7, Bcl-2 and Bax proteins were detected by Western blotting.


RESULTS
The mda-7 gene was expressed in Hep3B and L-02 cells. The protein concentrations of MDA-7 in supernatants were 790 and 810 pg/ml, respectively. mda-7 induced Hep3B growth suppression and apoptosis, compared with Ad.mda-7 and control (P<0.01). In addition, cell block in G2/M was identified by exposure of HCC cells to secreted MDA-7 protein, but this was not found in L-02. The gene expression of Bcl-2 was markedly decreased in Hep3B but not in L-02.


CONCLUSIONS
mda-7 selectively induces growth inhibition and apoptosis in the HCC cell line Hep3B but not in the normal liver cell line L-02 via downregulating the anti-apoptosis protein Bcl-2. It could be an ideal gene for gene therapy in HCC.
Xue
Xin-Bo
XB
Department of Biliary and Pancreatic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China. xiangfanck@163.com.
Chen
Kun
K
Wang
Cong-Jun
CJ
Zheng
Jian-Wei
JW
Yu
Yuan
Y
Peng
Zhi-Hai
ZH
Wu
Zai-De
ZD
D000256:Adenoviridae / Q000235:genetics*; D017209:Apoptosis / Q000235:genetics; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D007378:Interleukins / Q000235:genetics / Q000378:metabolism*; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors; D014161:Transduction, Genetic; D051028:bcl-2-Associated X Protein / Q000378:metabolism; D000099180:Interleukin-24
D007378:Interleukins; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D051028:bcl-2-Associated X Protein; D000099180:Interleukin-24; C494826:BAX protein, human