Virulizin, a novel immunotherapy agent, stimulates TNFalpha 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 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.