Abstract
Purpose
Several clinical trials in human neoplasms have demonstrated the effectiveness of combination therapy with 5-fluorouracil (FUra), cisplatin (CDDP), and leucovorin (LV). Thymidylate synthase (TS), the target enzyme of FUra, and dihydropyrimidine dehydrogenase (DPD), the rate-limiting catabolic enzyme of pyrimidines, have both been reported to be predictors of the response to FUra-based chemotherapies. Therefore, we aimed to clarify the effects of a combination of the three drugs against hepatoma cells and to determine the role of these two enzymes using in vitro models.
Methods
Five human hepatoma cell lines (Hep3B, HepG2, HuH7, PLC/PRF/5 and Chang) were used. Cytotoxicity was determined after exposure to various concentrations and combinations of antitumor agents. The combination effects of FUra and CDDP in terms of synergy, additivity or antagonism were evaluated by median effect analysis. The mRNA levels of TS and DPD were measured by quantitative real-time PCR. Expression of TS and DPD proteins was also investigated.
Results
LV alone did not show any cytotoxicity, although it enhanced the cytotoxicity of FUra, but not that of CDDP. Synergistic enhancement was observed with the combination of FUra and CDDP against all cells. The median combination index at fraction 0.5 was 0.554 (range 0.273–0.616). All cells expressed TS and DPD with median relative quantities of mRNA normalized to that of HuH7 cells of 1.04 (range 1.00–1.32) and 1.18 (range 0.88–1.55), respectively. A strong correlation was found between the IC50 of FUra and the mRNA level of DPD (r=0.912, P=0.0295).
Conclusions
LV and CDDP enhanced the cytotoxicity of FUra, which provided a rationale for the regimen combining the three drugs for the treatment of hepatocellular carcinoma. DPD plays an important role in the sensitivity to FUra, and the DPD mRNA expression level may be used to predict the response to FUra-based chemotherapy for HCC.
Similar content being viewed by others
References
Ando E, Yamashita F, Tanaka M, Tanikawa K (1997) A novel chemotherapy for advanced hepatocellular carcinoma with tumor thrombosis of the main trunk of the portal vein. Cancer 79:1890
Aschele C, Debernardis D, Casazza S, Antonelli G, Tunesi G, Baldo C, Lionetto R, Maley F, Sobrero A (1999) Immunohistochemical quantitation of thymidylate synthase expression in colorectal cancer metastases predicts for clinical outcome to fluorouracil-based chemotherapy. J Clin Oncol 17:1760
Cascinu S, Aschele C, Barni S, Debernardis D, Baldo C, Tunesi G, Catalano V, Staccioli MP, Brenna A, Muretto P, Catalano G (1999) Thymidylate synthase protein expression in advanced colon cancer: correlation with the site of metastasis and the clinical response to leucovorin-modulated bolus 5-fluorouracil. Clin Cancer Res 5:1996
Chou TC (1974) Relationships between inhibition constants and fractional inhibition in enzyme-catalyzed reactions with different numbers of reactants, different reaction mechanisms, and different types and mechanisms of inhibition. Mol Pharmacol 10:235
Chou TC, Talalay P (1977) A simple generalized equation for the analysis of multiple inhibitions of Michaelis-Menten kinetic systems. J Biol Chem 252:6438
Chou TC, Talalay P (1984) Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 22:27
Chou TC, Motzer RJ, Tong Y, Bosl GJ (1994) Computerized quantitation of synergism and antagonism of taxol, topotecan, and cisplatin against human teratocarcinoma cell growth: a rational approach to clinical protocol design. J Natl Cancer Inst 86:1517
Dhir V, Swaroop VS, Mohandas KM, Dinshaw KA, Desai DC, Nagral A, Sharma V, Jagannath P, Desouza LJ (1992) Combination chemotherapy and radiation for palliation of hepatocellular carcinoma. Am J Clin Oncol 15:304
Ebara M, Ohto M, Sugiura N, Kita K, Yoshikawa M, Okuda K, Kondo F, Kondo Y (1990) Percutaneous ethanol injection for the treatment of small hepatocellular carcinoma. Study of 95 patients. J Gastroenterol Hepatol 5:616
Evans RM, Laskin JD, Hakala MT (1981) Effect of excess folates and deoxyinosine on the activity and site of action of 5-fluorouracil. Cancer Res 41:3288
Falkson G, MacIntyre JM, Moertel CG, Johnson LA, Scherman RC (1984) Primary liver cancer. An Eastern Cooperative Oncology Group trial. Cancer 54:970
Farmer DG, Rosove MH, Shaked A, Busuttil RW (1994) Current treatment modalities for hepatocellular carcinoma. Ann Surg 219:236
Fraile RJ, Baker LH, Buroker TR, Horwitz J, Vaitkevicius VK (1980) Pharmacokinetics of 5-fluorouracil administered orally, by rapid intravenous and by slow infusion. Cancer Res 40:2223
Gebbia V, Maiello E, Serravezza G, Giotta F, Testa A, Borsellino N, Pezzella G, Colucci G (1999) 5-Fluorouracil plus high dose levofolinic acid and oral hydroxyurea for the treatment of primary hepatocellular carcinomas: results of a phase II multicenter study of the Southern Italy Oncology Group (G.O.I.M.). Anticancer Res 19:1407
Grem JL, Danenberg KD, Behan K, Parr A, Young L, Danenberg PV, Nguyen D, Drake J, Monks A, Allegra CJ (2001) Thymidine kinase, thymidylate synthase, and dihydropyrimidine dehydrogenase profiles of cell lines of the National Cancer Institute’s Anticancer Drug Screen. Clin Cancer Res 7:999
Ikeda K, Saitoh S, Koida I, Arase Y, Tsubota A, Chayama K, Kumada H, Kawanishi M (1993) A multivariate analysis of risk factors for hepatocellular carcinogenesis: a prospective observation of 795 patients with viral and alcoholic cirrhosis. Hepatology 18:47
Ishikawa Y, Kubota T, Otani Y, Watanabe M, Teramoto T, Kumai K, Kitajima M, Takechi T, Okabe H, Fukushima M (1999) Dihydropyrimidine dehydrogenase activity and messenger RNA level may be related to the antitumor effect of 5-fluorouracil on human tumor xenografts in nude mice. Clin Cancer Res 5:883
Ishikawa Y, Kubota T, Otani Y, Watanabe M, Teramoto T, Kumai K, Takechi T, Okabe H, Fukushima M, Kitajima M (2000) Dihydropyrimidine dehydrogenase and messenger RNA levels in gastric cancer: possible predictor for sensitivity to 5-fluorouracil. Jpn J Cancer Res 91:105
Kato Y, Nakata K, Omagari K, Furukawa R, Kusumoto Y, Mori I, Tajima H, Tanioka H, Yano M, Nagataki S (1994) Risk of hepatocellular carcinoma in patients with cirrhosis in Japan. Analysis of infectious hepatitis viruses. Cancer 74:2234
Kim R, Tanabe K, Inoue H, Toge T (2002) Mechanism(s) of antitumor action in protracted infusion of low dose 5-fluorouracil and cisplatin in gastric carcinoma. Int J Oncol 20:549
Kornmann M, Link KH, Lenz HJ, Pillasch J, Metzger R, Butzer U, Leder GH, Weindel M, Safi F, Danenberg KD, Beger HG, Danenberg PV (1997) Thymidylate synthase is a predictor for response and resistance in hepatic artery infusion chemotherapy. Cancer Lett 118:29
Lai EC, Choi TK, Cheng CH, Mok FP, Fan ST, Tan ES, Wong J (1990) Doxorubicin for unresectable hepatocellular carcinoma. A prospective study on the addition of verapamil. Cancer 66:1685
Livraghi T, Goldberg SN, Lazzaroni S, Meloni F, Solbiati L, Gazelle GS (1999) Small hepatocellular carcinoma: treatment with radio-frequency ablation versus ethanol injection. Radiology 210:655
Matsui O, Kadoya M, Yoshikawa J, Gabata T, Arai K, Demachi H, Miyayama S, Takashima T, Unoura M, Kogayashi K (1993) Small hepatocellular carcinoma: treatment with subsegmental transcatheter arterial embolization. Radiology 188:79
Melia WM, Johnson PJ, Williams R (1987) Controlled clinical trial of doxorubicin and tamoxifen versus doxorubicin alone in hepatocellular carcinoma. Cancer Treat Rep 71:1213
Nishiyama M, Yamamoto W, Park JS, Okamoto R, Hanaoka H, Takano H, Saito N, Matsukawa M, Shirasaka T, Kurihara M (1999) Low-dose cisplatin and 5-fluorouracil in combination can repress increased gene expression of cellular resistance determinants to themselves. Clin Cancer Res 5:2620
O’Connell MJ, Hahn RG, Rubin J, Moertel CG (1988) Chemotherapy of malignant hepatomas with sequential intraarterial doxorubicin and systemic 5-fluorouracil and semustine. Cancer 62:1041
Okuda K, Ohtsuki T, Obata H, Tomimatsu M, Okazaki N, Hasegawa H, Nakajima Y, Ohnishi K (1985) Natural history of hepatocellular carcinoma and prognosis in relation to treatment. Study of 850 patients. Cancer 56:918
Okuda K, Tanaka M, Shibata J, Ando E, Ogata T, Kinoshita H, Eriguchi N, Aoyagi S, Tanikawa K (1999) Hepatic arterial infusion chemotherapy with continuous low dose administration of cisplatin and 5-fluorouracil for multiple recurrence of hepatocellular carcinoma after surgical treatment. Oncol Rep 6:587
Petrelli N, Herrera L, Rustum Y, Burke P, Creaven P, Stulc J, Emrich LJ, Mittelman A (1987) A prospective randomized trial of 5-fluorouracil versus 5-fluorouracil and high-dose leucovorin versus 5-fluorouracil and methotrexate in previously untreated patients with advanced colorectal carcinoma. J Clin Oncol 5:1559
Porta C, Moroni M, Nastasi G, Arcangeli G (1995) 5-Fluorouracil and d,l-leucovorin calcium are active to treat unresectable hepatocellular carcinoma patients: preliminary results of a phase II study. Oncology 52:487
Priest DG, Ledford BE, Doig MT (1980) Increased thymidylate synthetase in 5-fluorodeoxyuridine resistant cultured hepatoma cells. Biochem Pharmacol 29:1549
Rossana C, Gollakota Rao L, Johnson LF (1982) Thymidylate synthetase overproduction in 5-fluorodeoxyuridine-resistant mouse fibroblasts. Mol Cell Biol 2:1118
Rustum YM, Trave F, Zakrzewski SF, Petrelli N, Herrera L, Mittelman A, Arbuck SG, Creaven PJ (1987) Biochemical and pharmacologic basis for potentiation of 5-fluorouracil action by leucovorin. NCI Monogr (5):165
Sakon M, Nagano H, Dono K, Nakamori S, Umeshita K, Yamada A, Kawata S, Imai Y, Iijima S, Monden M (2002) Combined intraarterial 5-fluorouracil and subcutaneous interferon-alpha therapy for advanced hepatocellular carcinoma with tumor thrombi in the major portal branches. Cancer 94:435
Salonga D, Danenberg KD, Johnson M, Metzger R, Groshen S, Tsao-Wei DD, Lenz HJ, Leichman CG, Leichman L, Diasio RB, Danenberg PV (2000) Colorectal tumors responding to 5-fluorouracil have low gene expression levels of dihydropyrimidine dehydrogenase, thymidylate synthase, and thymidine phosphorylase. Clin Cancer Res 6:1322
Scanlon KJ, Newman EM, Lu Y, Priest DG (1986) Biochemical basis for cisplatin and 5-fluorouracil synergism in human ovarian carcinoma cells. Proc Natl Acad Sci U S A 83:8923
Seki T, Wakabayashi M, Nakagawa T, Itho T, Shiro T, Kunieda K, Sato M, Uchiyama S, Inoue K (1994) Ultrasonically guided percutaneous microwave coagulation therapy for small hepatocellular carcinoma. Cancer 74:817
Seno H, Ito K, Kojima K, Nakajima N, Chiba T (1999) Efficacy of an implanted drug delivery system for advanced hepatocellular carcinoma using 5-fluorouracil, epirubicin and mitomycin C. J Gastroenterol Hepatol 14:811
Stuart KE, Anand AJ, Jenkins RL (1996) Hepatocellular carcinoma in the United States. Prognostic features, treatment outcome, and survival. Cancer 77:2217
Takizawa K, Kamijo R, Ito D, Hatori M, Sumitani K, Nagumo M (1999) Synergistic induction of ICAM-1 expression by cisplatin and 5-fluorouracil in a cancer cell line via a NF-kappaB independent pathway. Br J Cancer 80:954
Tanaka T, Masuda H, Naito M, Tamai H (2001) Pretreatment with 5-fluorouracil enhances cytotoxicity and retention of DNA-bound platinum in a cisplatin resistant human ovarian cancer cell line. Anticancer Res 21:2463
Tetef M, Doroshow J, Akman S, Coluzzi P, Leong L, Margolin K, Morgan RJ Jr, Raschko J, Shibata S, Somlo G, et al (1995) 5-Fluorouracil and high-dose calcium leucovorin for hepatocellular carcinoma: a phase II trial. Cancer Invest 13:460
Toyoda H, Nakano S, Kumada T, Takeda I, Sugiyama K, Osada T, Kiriyama S, Suga T, Takahashi M (1995) The efficacy of continuous local arterial infusion of 5-fluorouracil and cisplatin through an implanted reservoir for severe advanced hepatocellular carcinoma. Oncology 52:295
Ueda M, Kitaura K, Kusada O, Mochizuki Y, Yamada N, Terai Y, Kumagai K, Ueki K, Ueki M (2000) Regulation of dihydropyrimidine dehydrogenase and pyrimidine nucleoside phosphorylase activities by growth factors and subsequent effects on 5-fluorouracil sensitivity in tumor cells. Jpn J Cancer Res 91:1185
Urabe T, Kaneko S, Matsushita E, Unoura M, Kobayashi K (1998) Clinical pilot study of intrahepatic arterial chemotherapy with methotrexate, 5-fluorouracil, cisplatin and subcutaneous interferon-alpha-2b for patients with locally advanced hepatocellular carcinoma. Oncology 55:39
van Eeden H, Falkson G, Burger W, Ansell SM (1992) 5-Fluorouracil and leucovorin in hepatocellular carcinoma. Ann Oncol 3:404
Washtien WL (1982) Thymidylate synthetase levels as a factor in 5-fluorodeoxyuridine and methotrexate cytotoxicity in gastrointestinal tumor cells. Mol Pharmacol 21:723
Yamasaki T, Kurokawa F, Shirahashi H, Kusano N, Hironaka K, Masuhara M, Okita K (2002) Novel arterial infusion chemotherapy using cisplatin, 5-fluorouracil, and leucovorin for patients with advanced hepatocellular carcinoma. Hepatol Res 23:7
Yin MB, Zakrzewski SF, Hakala MT (1983) Relationship of cellular folate cofactor pools to the activity of 5-fluorouracil. Mol Pharmacol 23:190
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Kogure, T., Ueno, Y., Iwasaki, T. et al. The efficacy of the combination therapy of 5-fluorouracil, cisplatin and leucovorin for hepatocellular carcinoma and its predictable factors. Cancer Chemother Pharmacol 53, 296–304 (2004). https://doi.org/10.1007/s00280-003-0725-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00280-003-0725-6