Anti-Tumor Effect of a Novel Oncolytic Virus Based on Chimpanzee Adenovirus Type 6

WANG Qi, WANG Yi-han, LI Ying, XING Man, ZHOU Dong-ming

Abstract

To construct, with chimpanzee adenovirus serotype 6 (AdC6) as the vector, a novel oncolytic adenovirus, enabling it to selectively replicate intratumorally, to test its tumor suppressive effect in vitro and in vivo, and to study its oncolytic mechanism.  Methods  Based on the AdC6 vector, the human telomerase reverse transcriptase (hTERT) promoter was used to drive the expression of E1A, the adenovirus replication-related gene, and the recombinant oncolytic virus AdC6-htertΔE1A-ΔE3 was thus obtained. The oncolytic virus AdC6-htertE1A-ΔE3 (CSF2) expressing granulocyte macrophage colony-stimulating factor (GM-CSF/CSF2) and replication-deficient adenovirus AdC6-ΔE1-ΔE3 were constructed by homologous recombination, respectively. The recombinant adenovirus was packaged in HEK293 cells, purified and then identified with restriction enzyme digestion. Different types of tumor cells, including RD, SW-620, HeLa, Huh7, RM-1 and MC-38 were infected with the three adenoviruses. Twenty-four hours after infection, Western blot was used to determine the expression of CSF2 24 hours after infection. CCK8 assay was used to determine the survival rate of tumor cells 72 hours after infection. HeLa cells were infected with the three adenoviruses, and the expression levels of apoptosis signaling pathway proteins were examined with Western blot at 12 h, 24 h, and 48 h. C57BL/6 mice were subcutaneously injected with cell suspension containing 1×106 MC38 murine colon cancer cells and RM-1 murine prostate cancer cells to construct two tumor-bearing mice models. The tumor-bearing mice were divided into 4 groups, receiving intratumoral injection of 50 μL of PBS, AdC6-ΔE1-ΔE3 (1×108 PFU), AdC6-htertE1A-ΔE3 (1×108 PFU), and AdC6-htertE1A-ΔE3 (CSF2) (1×108 PFU), respectively. When the tumor size of PBS group reached 2 500 mm3, all the mice were sacrificed and the tumor tissue was collected for TUNEL staining. Then, apoptosis-positive cells were observed and counted under a microscope.  Results  Restriction digestion revealed that the oncolytic viruses AdC6-htertE1A-ΔE3, AdC6-htertE1A-ΔE3 (CSF2) and AdC6-ΔE1-ΔE3 were successfully constructed. Western blot confirmed that AdC6-htertE1A-ΔE3 (CSF2) could infect different tumor cells and stably express CSF2, the exogenous gene. CCK8 results showed that AdC6-htertE1A-ΔE3 and AdC6-htertE1A-ΔE3 (CSF2) had obvious killing effects on RD, SW-620, HeLa, Huh7, RM-1and MC-38. Compared with the replication-deficient adenovirus AdC6-ΔE1-ΔE3, AdC6-htertE1A-ΔE3 and AdC6-htertE1A-ΔE3 (CSF2) at a multiplicity of infection of 100 MOI had extremely obvious killing effects on tumor cells (P<0.05). Western blot showed that AdC6-htertE1A-ΔE3 and AdC6-htertE1A-ΔE3 (CSF2) induced tumor cell apoptosis by activating the P53-dependent pathway. Injection of oncolytic virus in tumor-bearing mouse models of prostate cancer and colorectal cancer could significantly inhibit the tumor growth and even clear the tumor.  Conclusion  Oncolytic virus based on AdC6 could eliminate tumor in vivo and in vitro through mechanisms that induced apoptosis, showing great potential for the treatment of tumors.

 

Keywords: Chimpanzee adenovirus type 6, Oncolytic virus, Human telomerase reverse transcriptase promoter

 

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