The in vivo antitumor effect of the apoptin-producing recombinant vaccinia virus strain is associated with blockage of mitotic division of cancer cells

Abstract

Vaccinia virus (VACV) possesses a natural oncolytic activity, for the enhancement of which genes encoding various effector molecules, for example, those inducing apoptotic death of tumor cells, are introduced into the virus genome. One such transgene is the gene encoding apoptin protein. The aim of the current work was to study antitumor activity of the apoptin-producing recombinant VACV strain VVdGF-ApoS24/2 in a syngenic murine tumor model. An Ehrlich carcinoma was implanted subcutaneously or intraperitoneally into C57B1 line mice. After tumor development, mice were intratumorally injected with 107 PFU/mouse of VVdGF-ApoS24/2 virus in a single dose, while control mice received 0.9% NaCl solution. Animals were sacrificed at different times after VACV injection. Tumor samples and ascetic fluid cells were fixed in 4% paraformaldehyde and further studied by light microscopy, immunohistochemistry, and electron microscopy. Virus titers in tumor cells were determined by the PFU method. VVdGF-ApoS24/2 VACV strain caused a significant reduction in the volumes of both solid and ascites Ehrlich carcinomas compared to the tumors in the control group of mice, although the virus reproduction rate in the tumor cells was rather low. The antitumor effect of VVdGF-ApoS24/2 could neither be attributed to virus-induced destruction, necrosis, or apoptosis of tumor cells nor to accumulation of the immune-effector cells in the tumors. A decrease in the number of tumor cells undergoing mitosis was observed when examining carcinoma sections, while counting Ki-67 and PCNA positive cells and analysis of their numbers showed that VVdGF-ApoS24/2 strain arrests cell cycle in the S-phase, thereby blocking tumor-cell division and slowing down tumor growth. The obtained results indicate that the insertion of the gene encoding apoptin into the genome of the original L-IVP VACV strain improved the ability of the virus to arrest the cell cycle in Ehrlich carcinoma cells.