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 tumor 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 hepatocytes and Kupffer cells were isolated from liver biopsy specimens by collagenase infusion and counterflow elutriation. Hepatocytes and Kupffer cells were incubated in vitro with clinically relevant doses of TP-1. Cell-free supernatants were collected at various time points after incubation. Hepatocyte and Kupffer cell supernatant levels for a panel of growth factors and monokines were determined by enzyme-linked immunosorbent assay. The cytotoxic activity of TP-1 alone and of TP-1-stimulated hepatocyte and Kupffer cell supernatants against Hep G2 and Hep 3B human HCC cells in vitro was measured by MTT assay. Results: Doses of TP-1 up to 100 μg/ml produced no cytotoxicity against Hep G2 or Hep 3B cells. Furthermore, supernatants from TP-1-treated hepatocytes produced no cytotoxicity against Hep G2 or Hep 3B cells, and TP-1 did not stimulate the release of transforming growth factor (TGF)-α, TGF-β, or hepatocyte growth factor. TP-1-treated Kupffer cell supernatants produced significant cytotoxicity against Hep G2 cells but produced no cytotoxicity against Hep 3B cells. Kupffer cells stimulated by TP-1 released significant amounts of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1α, and IL-6 compared with control Kupffer cells (p < 0.01). The activity of TP-1-treated Kupffer cell supernatants against Hep G2 cells was blocked by anti-TNF-α antibodies, whereas neither anti-IL-1α nor anti-IL-6 antibodies blocked cytotoxicity. Conclusions: These results demonstrate that TP-1 cytotoxicity against human HCC cells is not mediated directly or through hepatocytes, but occurs through activation of Kupffer cells and release of TNF-α. Understanding the mechanism of TP-1 cytotoxicity against human HCC has been used to plan a phase I trial of TP-1 combined with regional infusion of doxorubicin to treat unresectable HCC.