Virulizin®, a novel immunotherapy agent, stimulates TNFα expression in monocytes/macrophages in vitro and in vivo

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 TNFα 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 TNFα mRNA increased in peritoneal macrophages isolated from Virulizin®-treated mice as compared to the control group. An increase in TNFα protein expression was also observed, as assessed by flow cytometric analysis. Increased levels of TNFα mRNA were seen in human tumor xenografts following treatment of tumor-bearing mice with Virulizin®. In the presence of LPS, Virulizin® also stimulated TNFα 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 TNFα. Virulizin® also induced the phosphorylation of IκB, suggesting that induction of TNFα expression by Virulizin® is mediated by activation of NFκB. The results indicate that Virulizin®-induced TNFα expression contributes to modulation of immune responses and antitumor activities.