IBT/031 Synergestic Effect of HN Gene of NDV and TNF-alpha in Oncolysis of Experimentally Induced Tumor in Rat

HN gene of NDV is known to possess the inherent ability to induce apoptosis in transformed cells. It is reported that HN is a potent inducer of interferons and capable of upregulating the TNFα related apoptosis inducing ligand (TRAIL). Tumor necrosis factor-alpha has been shown to exhibit direct anti tumor activity, killing some tumor cells and reducing the rate of proliferation of others while sparing normal cells. TNF-α is an important regulator of immune response that influences cell differentiation, survival and apoptosis. Apoptotic response of TNF-α is mediated by the activation of caspase-8. In the present study, the HN gene of Newcastle disease virus and TNF-α of rat were cloned with Kozak sequence for better expression in eukarayotic expression vector pcDNA 3.1(+). For the assessment of oncolytic effect of HN and TNF-α, skin tumor were induced (papilloma and squamous cell carcinoma) in 30 male wistar rats (age 6–7 weeks) by applying 1% DMBA (7, 12dimethyl benz (a) anthracene, M.W.-256.3, Sigma USA) topically on shaved skin of hind back region of rats. In 3 to 5 months, all rats developed benign tumor (papilloma like growth) and squamous cell carcinoma. The rats were grouped in 5 groups viz I, II, III, IV and V (having 6 rats in each group). The recombinant pcDNA-hn-Kozak was treated intratumorally @100 μg/rat in group I. and group II rats were treated with pcDNA.TNF-α Kozak. The group III rats were treated with both the recombinant clones. The group IV rats were taken as vector control (pcDNA 3.1(+) @100 μg/rat) while group V was taken as mock control (treated with PBS alone). Two rats from each group were sacrificed humanely at day 3, 7 and 14 post treatment. Spleen, peripheral blood and tumor tissues were collected from these rats for the assessment of immune response and oncolytic effect by assessing proliferation of CD4⁺, CD8⁺ and NK cells (Flow cytometry, immunohistochemistry and histopathology). The flow cytometry analysis showed a significant increase in CD4⁺ CD8⁺ and NK⁻ cell population in group III followed by group I and II while there was no significant increase in group IV and V. Immunohistochemistry and histopathological analysis of tumor tissues also revealed the synergestic effect of HN and TNF-alpha in oncolysis. Further studies are being designed to assess the expression pattern of different cytokines in in vivo tumor model after treatment with recombinant pcDNA nd.hn-Kozak and pcDNA rat INFα -Kozak in combination.