Annals of Surgical Oncology

, Volume 4, Issue 2, pp 149–155 | Cite as

Activation of human Kupffer cells by thymostimulin (TP-1) to produce cytotoxicity against human hepatocellular cancer

  • Glen Balch
  • Francesco Izzo
  • Paul Chiao
  • Jim Klostergaard
  • Steven A. Curley
Original Articles


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.

Key Words

Thymostimulin Kupffer cells Hepatocellular cancer 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Curley SA, Levin B, Rich TA. Liver and bile ducts. In: Abeloff MD, Armitage JO, Lichter AS, Niederhuber JE, eds.Clinical oncology. New York: Churchill Livingstone, 1995:1305–72.Google Scholar
  2. 2.
    Rustgi V. Epidemiology of hepatocellular cancer.Ann Intern Med 1989;108:390–7.Google Scholar
  3. 3.
    Shoham J, Theodor E, Brenner HJ, Goldman B, Lusky A, Chaitchick S. Enhancement of the immune system of chemotherapy-treated cancer patients by simultaneous treatment with thymic extract, TP-1.Cancer Immunol Immunother 1980;9:173–80.CrossRefGoogle Scholar
  4. 4.
    Klein AS, Fixler R, Shoham J. Antiviral activity of a thymic factor in experimental viral infections.J Immunol 1984;132:3159–63.PubMedGoogle Scholar
  5. 5.
    Palmieri G, Gravina A, Frasci G, Montesarchio V, Leo L, Bianco AR. Thymostimulin treatment of hepatitis and cirrhosis.Proceedings of the First International Conference on Hepatobiliary Tumors. Rome: Monduzzi Editore, 1992;1:51.Google Scholar
  6. 6.
    Mustacchi G, Pevise L, Milani S, et al. High-dose folinic acid (FA) and fluorouracil (FU) plus or minus thymostimulin (TS) for treatment of metastatic colorectal cancer: results of a randomized multicenter clinical trial.Anticancer Res 1994;14:617–20.PubMedGoogle Scholar
  7. 7.
    Tanaka H, Hiyama T, Tsukuma H, et al. Cumulative risk of hepatocellular carcinoma in hepatitis C virus carriers: statistical estimations from cross-section data.Jpn J Cancer Res 1994;85:485–90.PubMedGoogle Scholar
  8. 8.
    Ikeda K, Saitoh S, Koida I, et al. A multivariate analysis of risk factors for hepatocellular carcinogenesis: a prospective observation of 795 patients with viral and alcoholic cirrhosis.Hepatology 1993;18:47–53.CrossRefPubMedGoogle Scholar
  9. 9.
    Curley SA, Izzo F, Gallipoli A, De Bellis M, Cremona F, Parisi V. Identification and screening of 416 patients with chronic hepatitis at high risk to develop hepatocellular cancer.Ann Surg 1995;222:375–83.PubMedGoogle Scholar
  10. 10.
    Palmieri G, Cimmino L, Gravina A, Topa M, Budillon G, Bianco AR. Thymostimulin treatment of hepatocellular carcinoma: preliminary data.Proceedings of the First International Conference on Hepatobiliary Tumors. Rome: Monduzzi Editore, 1992;1:52.Google Scholar
  11. 11.
    Lanford RE, Carey KD, Estlack LE, Smith GC, Hay RV. Analysis of plasma protein and lipoprotein synthesis in long-term primary cultures of baboon hepatocytes maintained in serum-free medium.In Vitro Cell Dev Biol 1989;25:174–82.PubMedGoogle Scholar
  12. 12.
    White AL, Rainwater DL, Lanford RE. Intracellular maturation of apolipoprotein[a] and assembly of lipoprotein[a] in primary baboon hepatocytes.J Lipid Res 1993;34:509–17.PubMedGoogle Scholar
  13. 13.
    Strom SG, Jirtle RL, Jons RS, et al. Isolation, culture, and transplantation of human hepatocytes.J Natl Cancer Inst 1982;68:771–8.PubMedGoogle Scholar
  14. 14.
    Heuff G, Steenbergen JJE, Van de Loosdrecht AA, et al. Isolation of cytotoxic Kupffer cells by a modified enzymatic assay: a methodological study.J Immunol Methods 1993;159:115–23.CrossRefPubMedGoogle Scholar
  15. 15.
    Shoham J, Cohen M. Thymic hormonal activity on human peripheral blood lymphocytes, in vitro. V. Effect on induction of lymphocytotoxicity.Int J Immunopharmacol 1983;6:523–32.Google Scholar
  16. 16.
    Carmichael J, DeGraff WG, Gazdar AF, Minna JD, Mitchell JB. Evaluation of a tertrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing.Cancer Res 1987;47:943–6.PubMedGoogle Scholar
  17. 17.
    Falchetti R, Bergesi G, Eshkol A, Cafiero C, Adorini L, Caprino L. Pharmacological and biological properties of a calf thymus extract (TP-1).Drugs Exp Clin Res 1977;3:39–47.Google Scholar
  18. 18.
    Shoham J, Eshel I, Aboud M, Salzberg S. Thymic hormonal activity on human peripheral blood lymphocytes in vitro.J Immunol 1980;125:54–8.PubMedGoogle Scholar
  19. 19.
    Aiuti F, Papetti C. Pharmacodynamics of thymic hormones. In: Byrom NA, Hobbs JR, eds.Thymic factor therapy. New York: Academic, 1990:33–43.Google Scholar
  20. 20.
    Palmisano L, Biglino A, Panichi G, et al. Combined chemoimmunotherapy (AZT-thymostimulin) in HIV positive patients: a multicenter, randomized, controlled study. Presented at IXth International Conference on AIDS, Berlin, June 7–11, 1993.Google Scholar
  21. 21.
    Pavesi L, Italina Cooperative Trials Group. Fluorouracil (F), with and without high dose folinic acid (HDFA) plus epirubicin (E) and cyclophosphamide (C): FEC versus HDGAFEC plus or minus thymostimulin (TS) in metastatic breast cancer: results of a multicentre study [Abstract].Eur J Cancer 1993;29(suppl):77.Google Scholar
  22. 22.
    Macchiarini P, Danesi R, Del Tacca M, Angeletti CA. Effects of thymostimulin on chemotherapy-induced toxicity and long-term survival in small cell lung cancer patients.Anticancer Res 1989;9:193–6.PubMedGoogle Scholar
  23. 23.
    Federico M, Gobbi P, Moretti G, et al. Effects of thymostimulin with combination chemotherapy in patients with aggressive non-Hodgkin's lymphoma.Am J Clin Oncol 1995;18:8–14.PubMedGoogle Scholar
  24. 24.
    Molto LM, Carballido JA, Manzano L, Olivier C, Lapuerta M, Alvarez-Mon M. Thymostimulin enhances the natural cytotoxic activity of patients with transitional cell carcinoma of the bladder.Int J Immunopharmacol 1993;15:335–41.PubMedGoogle Scholar
  25. 25.
    Elias L, Sandoval JM. Interferon effects upon fluorouracil metabolism by HL-60 cells.Biochem Biophys Res Commun 1989;163:867–74.CrossRefPubMedGoogle Scholar
  26. 26.
    Walder S, Wiernick PH. Clinical update on the role of fluorouracil and recombinant interferon alfa-2α in the treatment of colorectal carcinoma.Semin Oncol 1990;17:16–21.Google Scholar
  27. 27.
    Curley SA, Roh MS, Feig B, Oyedeji C, Kleinerman ES, Klostergaard J. Mechanisms of Kupffer cell cytotoxicity in vitro against the syngeneic murine colon adenocarcinoma line MCA26.J Leukoc Biol 1993;53:714–21.Google Scholar
  28. 28.
    Curley SA, Roh MS, Kleinerman ES, Klostergaard J. Human recombinant macrophage colony stimulating factor activates murine Kupffer cells to a cytotoxic state.Lymphokine Res 1990;9:355–63.PubMedGoogle Scholar
  29. 29.
    Roh MS, Wang L, Oyededi C, LeRoux ME, Curley SA, Pollock RE, Klostergaard J. Human Kupffer cells are cytotoxic against human colon adenocarcinoma.Surgery 1990;108:400–5.PubMedGoogle Scholar
  30. 30.
    Ismail T, Howl J, Wheatley M, McMaster P, Neuberger JM, Strain AJ. Growth of normal human hepatocytes in primary culture: effect of hormones and growth factors on DNA synthesis.Hepatology 1991;14:1076–82.CrossRefPubMedGoogle Scholar
  31. 31.
    McMahon JB, Richards WL, del Camp AA, Song MH, Thorgeirsson SS. Differential effects of transforming growth factor-α on the proliferation of normal and malignant rat liver epithelial cells in culture.Cancer Res 1986;46:4665–71.PubMedGoogle Scholar
  32. 32.
    Hill DB, Schmidt J, Shedlofsky SI, Cohen DA, McClain CJ. In vitro tumor necrosis factor cytotoxicity in Hep G2 liver cells.Hepatology 1995;21:1114–9.CrossRefPubMedGoogle Scholar

Copyright information

© The Society of Surgical Oncology, Inc 1997

Authors and Affiliations

  • Glen Balch
    • 2
  • Francesco Izzo
    • 2
  • Paul Chiao
    • 2
  • Jim Klostergaard
    • 1
  • Steven A. Curley
    • 2
  1. 1.Department of Tumor BiologyThe University of Texas M. D. Anderson Cancer CenterHoustonUSA
  2. 2.Department of Surgical OncologyThe University of Texas M. D. Anderson Cancer CenterHoustonUSA

Personalised recommendations