Study Of Oncolytic Adenovirus Expressing IL-24 Gene In Inhibition Of Metastasis Of Hepatocellular Carcinomaition

Hepatocellular carcinoma (HCC) is the sixth prevalent malignant tumor, which is ranked the third of tumor-related death in the world and the second in China. The recurrence and metastasis lead to the poor prognosis. Surgery remains the best option of treatment for HCC although the rate of recurrence and metastasis is 61.5% after radical resection and 43.5% even for small HCC. It's very important to find some effective therapies. Oncolytic adenoviruses are thought as a promising novel therapeutic option for cancer gene therapy. However, the effect and safety are far from satisfactory at present and the selectivity and antitumor effect need to be improved. IL-24 is a new anti-cancer gene which can suppress many kinds of malignant tumors, but its effect in lung metastasis of HCC has not been investigated.Part one:Construct the new recombinant adenovirus with human alpha-fetoprotein (AFP) promoter expressing IL-24 gene The recombinant oncolytic adenoviruses Ad. HS4.AFP. E1A/IL-24 and Ad.HS4. AFP. E1A/GFP were constructed by using modified human alpha-fetoprotein (AFP) promoter to drive adenovirus E1A gene and IL-24 gene. Segment HS-4-AFP-E1A-PolyA from vector HS4-AFP-E1A-PolyA-T was connected to recombinant shuttle plasmid pShuttle-Basic-IL-24, then transferred into adenovirus skeletal plasmid pAdxsi to construct adenovirus plasmid pAd-HS4-AFP. E1A-IL-24, and pAd-HS4-AFP. E1A-IL-24-7 was transferred into HEK293 cells to produce the recombinant adenovirus Ad. HS4. AFP. E1A/IL-24. The recombinant adenoviruses were analyzed to confirm to be correct. CCK-8 kit assay was to determine killing specificity in HCC cell lines SMMC-7721,Hep 3B,MHCC97-H and the human liver cell line L02. The expression of IL-24 and adenovirus E1A proteins in MHCC97-H cells was confirmed by the reverse transcriptase-polymerase chain reaction and western blotting. The results showed that the recombinant adenoviruses were constructed correctly, and the IL-24 gene was expressed in MHCC97-H cells. Ad. HS4. AFP. E1A/IL-24 induced SMMC-7721, Hep 3B and MHCC97-H cells growth suppression with no reaction on L02, which suppressed MHCC97-H much more than the other HCC cell lines (P<0.01).Part two:Oncolytic adenovirus vector expressing IL-24 gene suppresses hepatocellular carcinoma in vitroTo investigate the selective oncolytic role and antitumor action of a novel recombinant adenovirus Ad. HS4. AFP. E1A/IL-24 on hepatocellular carcinoma. Cell counting and Annexin V/propidium iodide assay were used to study cell proliferation and apoptosis of the recombinant adenovirus expressing IL-24 (Ad. HS4. AFP. E1A/IL-24) on MHCC97-H cells. The antitumor effects of the recombinant adenovirus were evaluated in tumor growth, apoptosis, cell adhesion, migration, and cell motion. mRNA of E-cadherin and Matrix metalloproteinase 2 (MMP-2) was examined by the reverse transcriptase-polymerase chain reaction and expression of MMP-2 protein was detected by zymography while that of E-cadherin by Western blotting. Selective growth suppression of Ad. HS4. AFP. E1A/IL-24 was observed in over expression AFP cell line MHCC97-H, a highly metastatic potential HCC cell line but not in hepatocyte cell line LO2. Ad.HS4.AFP. E1A/IL-24 could induce apoptosis in MHCC97-H cells as well (P<0.05). Besides, the recombinant adenovirus significantly inhibited MHCC97-H metastatic potential such as cell adhesion, migration and invasion as well (P<0.05). The gene expression of E-cadherin was markedly increased, but expression of MMP-2 was inhibited in MHCC97-H cells infected by Ad. HS4. AFP. E1A/IL-24Part three:Oncolytic adenovirus vector expressing IL-24 gene suppress hepatocellular carcinoma invasion and metastasis in vivoTo evaluate the effect of Ad. HS4. AFP. E1A/IL-24 on lung metastasis of HCC and study the underlying mechanisms.48 nude mice were randomized distributed to 6 groups,2 for each different administration. Adenoviruses injection through intraperitoneum(i.p.) started at the forth day after tumor implantation. Ad. HS4.AFP. E1A/IL-24 and Ad. HS4. AFP. E1A/GFP were administrated 108 PFU each time, with 0.2ml normal saline injected as control, once every three days for totally 6 times. After six weeks,24 mice of different experimental groups were sacrificed to observe the growth and metastasis of MHCC97-H. E-cadherin, MMP-2 and VEGF protein expression were assessed by immunohistochemistry. Pulmonary metastases were detected by H. E. stains. Another 3 groups of mice were maintained to evaluate life span. The results showed that there was no difference of the tumor weight and volume and the survival of nude mice with MHCC97-H among 3groups, but the incidence of lung metastases in nude mice treated by Ad. HS4. AFP. E1A/IL-24 were significantly inhibited compared to the other groups. Immunohistochemistry stain showed E-cadherin expression were enhanced significantly though VEGF and MMP-2 decreased markedly. IL-24 gene may inhibit lung metastases through down regulation on VEGF and MMP-2 genes and up regulation on E-cadherin gene.Conclusions1. The selective oncolytic adenovirus Ad. HS4. AFP. E1A/IL-24, can specifically express therapeutic gene IL-24 in AFP-expressing HCC cell lines to improve its HCC killing specificity and efficiency.2. IL-24 selectively induces growth inhibition and apoptosis in the MHCC97-H cell line but not in the normal liver cell line L02, and it also inhibits invasive potential of MHCC97-H cells via downregulating the VEGF and MMP-2 gene expression but upregulating E-cadherin gene expression.3. Ad. HS4. AFP. E1A/IL-24, with highly selective and markedly antitumor effect, may serve as a novel therapeutic strategy for HCC.