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 (CSF 2) expressing
granulocyte macrophage colony-stimulating factor (
GM-CSF/CSF 2) 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 CSF 2 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 1×10 6 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 (1×10 8 PFU), AdC6-htertE1A-ΔE3 (1×10 8PFU), and AdC6-htertE1A-ΔE3 (CSF 2) (1×10 8 PFU), respectively. When the
tumor size of PBS group reached 2 500 mm 3, 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 (CSF 2) and AdC6-ΔE1-ΔE3 were successfully constructed. Western blot confirmed that AdC6-htertE1A-ΔE3 (CSF 2) could infect different
tumor cells and stably express CSF 2, the exogenous gene. CCK8 results showed that AdC6-htertE1A-ΔE3 and AdC6-htertE1A-ΔE3 (CSF 2) 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 (CSF 2) 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 (CSF 2) 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: