Melanoma differentiation-associated gene-7, MDA-7/IL-24, selectively induces growth suppression, apoptosis in human hepatocellular carcinoma cell line HepG2 by replication-incompetent adenovirus vector.
OBJECTIVE
To investigate the effect of replication-incompetent adenovirus vector expressing MDA-7/IL-24 on tumor growth and apoptosis in human hepatocellular carcinoma (HCC) cell line HepG2 and normal liver cell line L02.


METHODS
We constructed the recombinant replication-incompetent Ad.mda-7 virus vector and infected it into the human HCC cell line HepG2 and normal liver cell line L02. RT-PCR was performed to detect the mRNA expressing in cells. by ELISA was used to detect MDA-7/IL-24 protein expression in the culture supernatant. The effect of apoptosis induced by Ad.mda-7 was confirmed by Hoechst staining and flow cytometry assay with Annexin-V and PI staining. MTT assay was used to determine growth inhibition of HepG2 cells, and cell-cycle and hypodiploidy analyses were performed by flow cytometry.


RESULTS
Recombinant replication-defective virus expressing MDA-7/IL-24 was constructed successfully. RT-PCR showed that the Ad.mda-7 could mediate the expression of the exogenous gene MDA-7/IL-24 into HepG2 and L02. The concentration of MDA-7/IL-24 protein in supernatant was 130 pg/mL and 110 pg/mL in Ad.mda-7-infected L02 and HepG2 cells, respectively. Ad.mda-7 infection obviously induced apoptosis (from 2.60%+/-0.72% to 33.6%+/-13.2%, P=0.00012) and growth suppression in HepG2 (inhibition ratio IR=68%) and an increase in the percentage of specific cancer cell types at the G2/M phase of the cell cycle (from 6.44% to 32.29%, P<0.01), but not in L02 cells.


CONCLUSIONS
These results confirm selectively induction of apoptosis and growth suppression by the mda-7/IL-24 gene with replication-incompetent adenovirus vector in human hepatocellular carcinoma cell line HepG2.
Wang
Cong-Jun
CJ
Department of Biliary and Pancreatic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China.
Xue
Xin-Bo
XB
Yi
Ji-Lin
JL
Chen
Kun
K
Zheng
Jian-Wei
JW
Wang
Jian
J
Zeng
Jian-Ping
JP
Xu
Rong-Hua
RH
D000256:Adenoviridae / Q000235:genetics; D017209:Apoptosis / Q000235:genetics*; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000473:pathology* / Q000628:therapy; D002453:Cell Cycle / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D015967:Gene Expression Regulation, Viral; D015316:Genetic Therapy; D005822:Genetic Vectors; D022781:Hepatocytes; D006801:Humans; D007378:Interleukins / Q000235:genetics*; D008113:Liver Neoplasms / Q000235:genetics / Q000473:pathology* / Q000628:therapy; D008856:Microscopy, Fluorescence; D012333:RNA, Messenger; D012334:RNA, Neoplasm / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014779:Virus Replication; D000099180:Interleukin-24
D007378:Interleukins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D012367:RNA, Viral; D000099180:Interleukin-24