Oncolytic abilities of vaccinia virus (VACV) served as a basis for

Oncolytic abilities of vaccinia virus (VACV) served as a basis for the development of varied recombinants for treating cancer; nevertheless “organic” oncolytic properties from the disease are not analyzed in detail. from the blood. The L-IVP strain caused decrease of sizes in both tumors however in different ways. Direct cell destruction by replicating virus plays a main role in regression of A431 carcinoma xenografts while in Ehrlich carcinoma which poorly supported VACV replication the virus induced decrease of mitoses by pushing tumor cells into S-phase of cell cycle. Our study showed that genetically unmodified VACV possesses at least two mechanisms of antitumor effect: direct destruction of tumor cells and suppression of mitoses in tumor cells. mice after intratumoral injection of both viruses [20]. The L-IVP strain clearly demonstrated oncolytic effects via direct destruction of tumor cells (signs of inflammatory reactions and leukocyte accumulation in tumor tissue and viral destruction of blood vessels were not observed). The question arose: what are other mechanisms may contribute to the antitumor effects of the VACV? In this study we examined antitumor effect of the L-IVP strain using murine Ehrlich carcinoma in C57Bl mice and compared that with oncolytic effect of this Cabozantinib virus in human A431 carcinoma xenografts in mice. In contrast with human cells murine cells are not naturally susceptible to VACV so it was interesting to compare viral antitumor effects in these two models. Our study showed that the L-IVP strain of VACV possesses antitumor activity towards murine tumor which is mainly related with mitotic arrest in murine tumor cells. 2 Materials and Methods 2.1 Virus and Cells The L-IVP strain of VACV was obtained from the State Collection of Viral and Rickettsial Disease Agents of the State Research Center of Virology and Biotechnology “Vector” (SRC VB “Vector” Koltsovo Russia). The strain was cloned and has been passed 6 times in CV-1 cells and purified by centrifugation in sucrose density gradient (25%-45%). The viral preparation was sonicated and titrated using the plaque formation Cabozantinib assay in CV-1 cell monolayers. Virus titers were expressed as plaque forming units (PFU) per mL. The viral stock represented 109 PFU/mL in sterile saline and aliquots were stored at ?80 °C. Human cancer cell lines (A549 A431 C33A U87MG RD DU145 MCF7 Mel8 SW480 HeLa) of different origin were grown in DMEM (Invitrogen Waltham MA USA) supplemented with 10% fetal calf serum (FCS HyClone Logan UT USA). Diploid human embryonic LECH-240 cells were grown Cabozantinib in F-12 medium (Invitrogen) supplemented with 10% fetal calf serum (FCS HyClone). MCF10A cells were grown in a specialized culture medium for mammary epithelial cells MEGM Bullet Kit (Lonza Allendale NJ USA). 2.2 Cytotoxic Activity of VACV Strain L-IVP toward Human Tumor Cell Lines Cytotoxic activity of VACV strain L-IVP toward human tumor cell lines was evaluated by XTT microassay (using 2 3 Sigma-Aldrich St. Louis MO USA) in 96-well plates (Greiner Pleidelsheim Germany) [8]. This method employs the fact that mitochondrial dehydrogenases can convert soluble XTT into formazan which crystallizes within the cell. Formazan can be solubilized by phenazine methosulfate (PMS) treatment and the optical density of the solution determined by spectrophotometry accurately reflects the changes Mouse monoclonal to CD16.COC16 reacts with human CD16, a 50-65 kDa Fcg receptor IIIa (FcgRIII), expressed on NK cells, monocytes/macrophages and granulocytes. It is a human NK cell associated antigen. CD16 is a low affinity receptor for IgG which functions in phagocytosis and ADCC, as well as in signal transduction and NK cell activation. The CD16 blocks the binding of soluble immune complexes to granulocytes. of formazan quantities in viable cells. The specific rate of cell death in infected cultures was assessed in relation to uninfected control cells (100% viabilityπ). Cytolytic activity was evaluated as the 50% cytotoxic dose (CD50) that is the virus concentration causing death of 50% of cells. To determine CD50 cells growing in a 50% monolayer were infected with sequential tenfold dilutions of viral suspension in 100 μL of 199 medium supplemented with 2% FCS (0.001 to 10 PFU/cell). Following 72 h incubation at 37 °C; in an atmosphere of 5% CO2 and 85% humidity 50 μL of XTT/PMS mixture were added to each well (the mixture Cabozantinib was prepared with 20 μL of 1 1.25 mM PMS (Fluka St. Louis MO USA) per 1 mL of 1 1 mg/mL XTT working solution). Plates were incubated for additional 4 h and optical densities (and multiplicity of infection data obtained from five replicates were used to calculate the mean and SD values for each point corresponding to different virus concentrations. This curve was used to determine CD50 as the virus concentration at which the mice (Nursery for Laboratory Animals Institute of Cabozantinib Bioorganic Chemistry Moscow Russia) were used for the xenograft model of human A431 carcinoma. Tumors were.