Antitumor Efficacy Of Oncolytic Vaccinia Virus That Harboring IL-24 Gene For Hepatocellular Carcinoma Cells Is Enhanced By Combination Therapy With Luteolin

Hepatocellular carcinoma(HCC)is a complex disease that characterized by both genetic and epigenetic alterations,and involves cross-link among several cell signaling,which makes HCC become a leading cause of cancer-related death in many regions of the world with high mortality.Therefore,it is of great social significance to explore effective methods for the treatment of liver cancer.Oncolytic vaccinia virus(VV)is a promising gene therapy vector which has a range of beneficial characteristics for anti-tumor efficacy.IL-24 gene can effectively inhibit the proliferation and metastasis of tumor cells by inducing apoptosis of tumor cells through multiple mechanisms.The experimental results showed that the oncolytic vaccinia virus carrying the IL-24 gene(VV-IL-24)could effectively inhibited the growth of liver cancer cells.In order to enhanced the inhibiting efficacy of VV-IL-24 in HCC cells,luteolin,an anti-tumor drug with synergistic effect with VV-IL-24,was selected for the study that combined with VV-IL-24 in inhibiting liver cancer cell growth.VV-IL-24 was constructed in our laboratory by homologous recombination method,in which IL-24 gene was inserted into the TK region of oncolytic vaccinia virus.Then,VVIL-24 was amplified and purified in large quantities after detection of having no wild-type vaccinia contamination and identification of the exogenous IL-24 gene expression in the recombinant virus by PCR;the effect of VV-IL-24 on the growth of liver cancer cells was detected by MTT assay,and VV-EGFP as the control virus,the results had proved that VVIL-24 more efficiently inhibited the viability of HCC cells than that of VV-EGFP;and the MTT assay was also used to detected the killing effect of VV-IL-24 combined with luteolin and each single treatment group on HCC cells,and the results had proved that the combination group of VV-IL-24 and luteolin have a better efficacy of killing HCC cells than VV-IL-24 or luteolin alone;Hoechst33342 staining and flow cytometry(FCM)were used to detected the effects of VV-IL-24 combined with luteolin and each single treatment group on the apoptosis of HCC cells and the results had proved that the combination group of VV-IL-24 and luteolin have a significantly higher percent of apoptosis than VV-IL-24 alone or luteolin alone;Western blot analysis was performed to detected the expression changes of related apoptotic proteins and anti-apoptotic proteins in HCC cells induced by VV-IL-24 combined with luteolin and each single treatment group,and the results had proved that the combination group of VV-IL-24 and luteolin increased the expression of related apoptotic proteins and decreased the expression of anti-apoptotic proteins;animal models further demonstrated that the combination of VV-IL-24 and luteolin inhibited the growth of HCC cells more effectively in vivo than each single treatment group.In conclusion,the combination of VV-IL-24 with luteolin inhibited the growth of HCC cells in vivo and in vitro more effectively than VV-IL-24 or luteolin alone.In addition,luteolin promoted the expression of the oncolytic vaccinia virus carrying IL-24 gene in HCC cells,and caused the up-regulation of apoptotic proteins and the down-regulation of anti-apoptotic proteins.Thus,the results could provide new ideas and directions for the clinical research on the therapy of liver cancer.