Virulizin

Virulizin has demonstrated strong antitumor efficacy in a variety of human tumor xenograft models including melanoma, pancreatic cancer, breast cancer, ovarian cancer and prostate cancer. Our previous studies have demonstrated that macrophages, NK cells, and cytokines are important in the antitumor mechanism of Virulizin. Virulizin treatment of tumor bearing mice results in the expansion as well as increased activity of monocytes/macrophages and production of cytokines IL-12 and TNFalpha and activation of NK cells. In this study we show that the inflammatory cytokine IL-17E (IL-25) is induced by Virulizin treatment and is part of its antitumor mechanism. IL-17E is a proinflammatory cytokine, which induces a T(H)2 type immune response, associated with eosinophil expansion and infiltration into mucosal tissues. IL-17E was increased in sera of Virulizin-treated mice bearing human melanoma xenografts, compared to saline-treated controls, as shown by 2D gel electrophoresis and ELISA. Treatment of splenocytes in vitro with Virulizin resulted in increased IL-17E mRNA expression, which peaked between 24 and 32 h post-stimulation. Both in vitro and in vivo experiments demonstrated that B cells produced IL-17E in response to Virulizin treatment. Furthermore, Virulizin treatment in vivo resulted in increased blood eosinophilia and eosinophil infiltration into tumors. Finally, injection of recombinant IL-17E showed antitumor activity towards xenografted tumors, which correlated with increased eosinophilia in blood and tumors. Taken together, these results support another antitumor mechanism mediated by Virulizin, through induction of IL-17E by B cells, leading to recruitment of eosinophils into tumors, which may function in parallel with macrophages and NK cells in mediating tumor destruction.

Virulizin, a novel biological response modifier, has demonstrated broad antitumor efficacy in a variety of human tumor xenograft models including melanoma, pancreatic cancer, breast cancer, ovarian cancer and prostate cancer. Previous studies have demonstrated a significant role of macrophages and NK cells in the antitumor mechanism of Virulizin. Increased activity and expansion of macrophages and NK cells has been observed in mice treated with Virulizin. In the present study, the effects of Virulizin on TNFalpha expression were investigated in vitro and in vivo. CD-1 nude mice were treated with Virulizin daily for 5 days. Quantitative RT-PCR demonstrated that the level of TNFalpha mRNA increased in peritoneal macrophages isolated from Virulizin-treated mice as compared to the control group. An increase in TNFalpha protein expression was also observed, as assessed by flow cytometric analysis. Increased levels of TNFalpha mRNA were seen in human tumor xenografts following treatment of tumor-bearing mice with Virulizin. In the presence of LPS, Virulizin also stimulated TNFalpha protein secretion and mRNA expression in human monocytic U937 cells and mouse macrophage RAW264.7 cells in vitro in a time- and dose-dependent manner. U937 cells treated with Virulizin showed a significantly enhanced cytotoxicity that was eliminated upon neutralization of TNFalpha. Virulizin also induced the phosphorylation of IkappaB, suggesting that induction of TNFalpha expression by Virulizin is mediated by activation of NFkappaB. The results indicate that Virulizin-induced TNFalpha expression contributes to modulation of immune responses and antitumor activities.