ACTIVATION OF HUMAN KUPFFER CELLS BY THYMOSTIMULIN (TP-1) TO PRODUCE CYTOTOXICITY AGAINST HUMAN HEPATOCELLULAR CANCER

Background: In a small pilot study, thymostimulin (TP-1) produced tumo r regression in almost 50% of patients with hepatocellular cancer (HCC ) who were treated with TP-1 alone. However, the mechanism of the TP-1 -mediated antitumor effect against HCC is unknown. Methods: Human hepa tocytes and Kupffer cells were isolated from liver biopsy specimens by collagenase infusion and counterflow elutriation. Hepatocytes and Kup ffer cells were incubated in vitro with clinically relevant doses of T P-1. Cell-free supernatants were collected at various time points afte r incubation. Hepatocyte and Kupffer cell supernatant levels for a pan el of growth factors and monokines were determined by enzyme-linked im munosorbent assay. The cytotoxic activity of TP-1 alone and of TP-1-st imulated hepatocyte and Kupffer cell supernatants against Hep G2 and H ep 3B human HCC cells in vitro was measured by MTT assay. Results: Dos es of TP-1 up to 100 mu g/ml produced no cytotoxicity against Hep G2 o r Hep 3B cells. Furthermore, supernatants from TF-1-treated hepatocyte s produced no cytotoxicity against Hep G2 or Hep 3B cells, and TP-1 di d not stimulate the release of transforming growth factor (TGF)-alpha, TGF-beta, or hepatocyte growth factor. TP-1-treated Kupffer cell supe rnatants produced significant cytotoxicity against Hep G2 cells but pr oduced no cytotoxicity against Hep 3B cells. Kupffer cells stimulated by TP-1 released significant amounts of tumor necrosis factor-alpha (T NF-alpha), interleukin (IL)-1 alpha, and IL-6 compared with control Ku pffer cells (p < 0.01). The activity of TP-1-treated Kupffer cell supe rnatants against Hep G2 cells was blocked by anti-TNF-alpha antibodies , whereas neither anti-IL-1 alpha nor anti-IL-6 antibodies blocked cyt otoxicity. Conclusions: These results demonstrate that TP-1 cytotoxici ty against human HCC cells is not mediated directly or through hepatoc ytes, but occurs through activation of Kupffer cells and release of TN F-alpha. Understanding the mechanism of TP-1 cytotoxicity against huma n HCC has been used to plan a phase I trial of TP-1 combined with regi onal infusion of doxorubicin to treat unresectable HCC.