| Construction and identification of Oncolytic Adenovirus SG600-IL24 Expressing Human MDA-7/IL-24Objective:We constructed oncolytic adenovirus SG600-IL24 vector which carrying human MDA-7/IL-24, and observed its expression in hepatocellular carcinoma cell lines and the normal liver cell lines, in order to investigate its theoretical value of gene therapy in hepatocellular carcinoma.Methods:Digesting pZD55-IL24 to get IL24, then connected to pClon9-INS-IL24, and digesting pClon9-INS-IL24 to get INS+IL24, loading into SG502-ACR2 vector, and then to get recombinant plasmid pSG600-IL24, Using pSG600-IL24 and adenovirus plasmid backbone ppE3 to polyfect mediated cotransfected into 293 cells, to obtain oncolytic adenovirus SG600-IL24 vector which carrying human MDA-7/IL-24 by intracellular homologous recombination. The vector SG600-EGFP was constructed using the same methods. The human hepatocullular carcinoma lines HepG2, Hep3B, SMMC7721 and normal liver cell line L02 were infected with oncolytic adenovirus SG600-IL24, the expression of mda-7/IL-24 gene was verified by RT-PCR.Results:HEK293 cells was appeared characteristic CPE after infection, HEK293 Cells become large and round, floating, were gathered like grapes. To take the virus DNA in Clone 1 and clone 2 each 2μl as a template, the specific fragments of PCR amplification is about 640bp, and its size is consistent with the positive control, while the negative control SG600-EGFP has no PCR amplification products. RT-PCR results suggest that the exogenous MDA-7/IL-24 gene was highly expressed in hepatocullular carcinoma lines HepG2, Hep3B, SMMC7721 and normal liver cell line L02 after infected with SG600-IL24.Conclusion:We constructed oncolytic adenovirus SG600-IL24 vector which carrying human MDA-7/IL-24 successfully. SG600-IL24 vector which carrying MDA-7/IL-24 gene was highly expressed in human hepatocullular carcinoma lines and normal liver cell line.Oncolytic adenovirus SG600-IL24 selectively kills hepatocellular carcinoma cell lines in vitroObjective:We investigate the selective antitumor activity of oncolytic adenovirus SG600-IL24 vector which carrying human MDA-7/IL-24 on human hepatocullular carcinoma lines HepG2, Hep3B, SMMC7721, MHCC97L, HCCLM3 and normal liver cell line L02 in vitro, to provide a theoretical basis for hepatocullular carcinoma gene therapy.Methods:The human hepatocullular carcinoma lines HepG2, Hep3B, SMMC7721, MHCC97L, HCCLM3 and normal liver cell line L02 were infected with oncolytic adenovirus SG600-IL24. MDA-7/IL-24 mRNA and protein expressions in infected cells were detected by reverse transcription-polymerase chain reaction (RT-PCR), enzymelinked immunosorbent assay (ELISA), and Western-blot respectively. MTT assay was used to investigate the proliferation of HCC cell lines and normal liver cell lines. Antitumor activity of SG600-IL24 on HCC cell lines was detected by Hoechst33258 staining. Annexin-V and PI staining was studied to indicate the apoptosis effect and the flow cytometry was used to assess the cell cycle.Results:RT-PCR and Western blotting showed that the exogenous MDA-7/IL-24 gene was highly expressed in hepatocullular carcinoma lines HepG2, Hep3B, SMMC7721, MHCC97L, HCCLM3 and normal liver cell line L02 after infected with SG600-IL24. The protein product was confirmed by assay the supernatant with ELISA. MTT and Hoechst33258 staining indicated that SG600-IL24 induced growth suppression, promoted apoptosis, and blocked cancer cell lines in the G2/M phase in hepatocellular carcinoma cell lines but not in the normal liver cell line.Conclusion:Oncolytic adenovirus SG600-IL24 vector which carrying human MDA-7/IL-24 can selectively kill different metastatic potential HCC cell lines and induce apoptosis, block cell proliferation but not normal liver cell line L02.The mechanism of Oncolytic adenovirus SG600-IL24 selectively kills hepatocullular carcinoma cell linesObjective:To explore the mechanism of oncolytic adenovirus SG600-IL24 selectively kills hepatocullular carcinoma cell lines HepG2 and HCCLM3 in vitro, and in order to provide a theoretical basis for hepatocullular carcinoma gene therapy.Methods:The human hepatocullular carcinoma cell lines HepG2, HCCLM3 and normal liver cell line L02 were intervened by oncolytic adenovirus SG600-IL24. RT-PCR and Western-blot were used to detected the protein expressions of total STAT3 and the its signaling pathway downstream signal molecule C-myc, bax, bcl-2, bcl-xl, CyclinD2, Survivin, XIAP, OPN, MMP-2, MMP-9 and VEGF in HepG2, HCCLM3 and normal liver cell line L02 after intervened by oncolytic adenovirus SG600-IL24, also examined the expression of p-STAT3 after infection with SG600-IL24.Results:It was confirmed by RT-PCR and western-blot method that the STAT3 and the its signaling pathway downstream signal molecule C-myc, Bcl-xl, bcl-2, CyclinD2, Survivin, MMP-2, MMP-9, XIAP, OPN, VEGF were downregulated while upregulation of Bax. The phosphorylation of STAT3 after infection was increased and then decreased, and reach to the peak in 2 hours.Conclusion:Oncolytic adenovirus SG600-IL24 may achieve the role of anti-hepatoma through inhibiting STAT3 and the its signaling pathway downstream signal molecule while not normal liver cell line, which make oncolytic adenovirus SG600-IL24 has significant selectively antitumor effects. p-STAT3.Treatment of hepatoma in nude mice combined oncolytic adenovirus SG600-IL24 with InterferonObjective:To study the effects of oncolytic adenovirus SG600-IL24 which carrying human MDA-7/IL-24 and Interferon on hepatoma xenografts in nude mice and explore a new way for hepatoma gene therapy combined with neoadjuvant therapy.Methods:First established subcutaneous hepatoma nude mice model, when the tumors had grown to 100-150 mm3(day 0), the animals were randomized into four groups(each group has eight nude mice) as follows:(1) Control group:Saline 0.3 ml, intraperitoneal injection to the right side,1/d,3 times. (2) Neoadjuvant therapy group (IFN group):IFN 1.5 x104 IU/g, intraperitoneal injection to the right side,1/d,10 times. (3) SG600-IL24 group:SG600-IL24 2 x 108 PFU, multi-point injection to the local tumor,5 times every other day. (4) SG600-IL24+IFN group:IFN 1.5 x104 IU/g, intraperitoneal injection to the right side,1/d, 10 times, and the next day SG600-IL242x108 PFU, multi-point injection to the local tumor, 5 times every other day. And the expression of mda-7/IL-24 gene and protein was detected in tumor tissue after intervented for 30d, the survival time and the tumor volume of the nude mice were observed in each group subsequently.Results:The exogenous MDA-7/IL-24 gene was highly expressed in nude mice after treatment with SG600-IL24 or SG600-IL24 plus IFN. The mice treated with SG600-IL24 plus Interferon had significantly longer survival time (3 mice>120) days and smaller tumor volume than in other groups(P<0.01).Conclusion:Oncolytic adenovirus SG600-IL24 has stronger synergistic antitumor effect on metastastic hepatoma nude mice model combined with Interferon, and pave a new way for HCC virus gene therapy. |
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