Radiol Oncol 1993; 27: 293-7. Anti-tumor effect of interferon alpha in combination with cisplatin - animal experiments Borut Stabuc Department of Medical Oncology, Institute of Oncology, Ljubljana, Slovenia Anti-tumor effect of human interferon-a and cisplatin was studied on B-16 melanoma bearing C57BI/6 syngeneic mice. When the tumors reached I mm in diameter, applications of cisplatin were started at a dosage of 0.001 mg/g of animal's body weight, or human interferon-a was given at a dosage of 5 X 104 IU. A control group of mice received normal saline solution. The treatment was applied every next day, altogether 12 times. Tumor volumes were measured every next day, and their mean values calculated; all 12 measurements confirmed that the mice treated with human interferon-a and cisplatin had smaller mean value than the control group, or the groups receiving either interferon or cisplatin alone, respectively (p < 0.05). The mean tumor volumes of cisplatin treated mice were lower than those of the controls or interferon-treated animals (p < 0.05). The obtained results indicate that human interferon-a enhances the antitumor effect of cisplatin. Key words: melanoma, experimental drug therapy; interferon-alpha; cisplatin Introduction Melanoma represents less than 5 % of all malignant diseases, though its incidence in the last few decades has been rapidly increasing. '' 2 3 Chemotherapy in melanoma has not been particularly effective. Although several drugs have a low order of antitumor activity, combination chemotherapy has not produced better results that those observed with the use of single agents such as dacarbazine, nitrosoureas, vinca-alkaloids, and cisplatin (CDDP). Moreover, none of these drugs as well as their Correspondence to: Borut Štabuc MD, PhD, Dcpart-ment of Medical Oncology, Institute of Oncology, Zaloška 2, 6110,5 Ljubljana, Slovenia. UDC: 616-006.81-085 combinations have been proved to definitely increase the survival of patients with metastatic melanoma.4' 5 A number of pre-clinical and clinical trials studying the effects of interferon alpha were consistent in confirming its antitumor activity against melanoma. 6 7 Even if the mechanism of antitumor effects of interferon are still insufficiently understood, it has become clear that in the majority of the experimental systems investigated, interferons act in a very different way from chemotherapy. Thus, the idea of using interferons in combination with other agents has interested investigators for a long time.8 Different experimental data have shown that combined treatment can improve the response rate and prolong the duration of response due 294 Stabuc B. to different actions of drugs on the tumor, interactions between the drugs and, perhaps, influences on the host immune system.9' 10 Therefore, the aim of this study was to investigate the possibility of enhancing the cisplatin antitumor effect by the application of human interferon alpha in suboptimal doses. Materials and methods Experimental animals The animals, 8-10 week old C57B1/6 mice were obtained from Rudjer Boskovic Institute, Zagreb. Mice used in the experiment were of the same sex and age. Animal colonies were maintained in accordance with the recommendations issued by the National Cancer Institute in Be-thesda, USA. B-16 melanoma was used as an experimental tumor model. Tumors were implanted to the animals by subcutaneous injection of 5 x 105 viable tumor cells given dorsola-terally. Tumor cell suspension was prepared by mechanical decomposition of viable tumor tissue. Treatment Mice were divided into four experimental groups as follows: 1) control group, 2) group treated with CDDP, and 3) group receiving human interferon-a (IFN-a) and 4) group receiving combined CDDP and IFN-a treatment. Each group consisted of 7 animals. Intraperito-neal applications of the cytotoxic agent and/or IFN-a and normal saline solution were started when the tumors reached 1 mm in diameter, or a volume of 0.5mm3. The injections of active substances were administered every next day, and the experiment was completed on the 25th day from the beginning of application. The solution of CDDP (Bristol-Myers Co.) and normal saline was injected at a dose of 0.001 mg/g b.w. or 0.01 ml of the solution per gram of the animal's body weight. IFN-a (human interferon alpha from the Institute of Immunology, Zagreb, Croatia)' dissolved in normal saline was injected at a dose of 5 x 104 IU which equalled to 0.25 ml of the solution per application. In one group CDDP injections were followed after one hour by IFN-a application; drug dosage was the same as in groups receiving either of the agents alone. Animals in the control group had the same quantity of normal saline solution injected intraperitoneally every next day. Tumor measurement and statistical analysis Tumor growth was followed up daily by the evaluation of tumor diameter and thickness. Tumor volume was calculated using the following formula: 0.523 X a X b X c, where a, b, c were tumor diameters. Mean volumes, as well as standard deviation and standard error of the mean values were calculated from the results of measurements performed on the same day. The data were statistically analysed by means of CIA softwa- Results Mean values of tumor volumes (MTV) expressed in mm3, measured every next day during the treatment with normal saline solution, IFN-a, CDDP, or combination of both are presented in Table l. In all measurements MTVs of CDDP-treated animals were found to be lower that those of the control group (p<0.05), and the values obtained after the 5th measurement were also statistically significantly lower that those of the IFN-a treated animals. In all 12 measurements MTV of the animals receiving combined CDDP and IFN-a treatment were statistically significantly lower than the relevant values obtained in all other experimental groups (p < 0.05). Figure 1 shows MTV values and 95 % confidence interval resulting from all 12 measurements performed in all experimental groups. In the groups receiving combined CDDP and IFN-a treatment or CDDP alone 2 animals died immediately after drug application. The Anti-tumor effect of IFN-a and cisplatin 295 Table l. Bl6 melanoma volume in mm3 in all 4 groups of C57Bl/6 mice. Controls IFN-a CDDP IFN-a and CDDP Measurement MTV(m3) MTV(mm3) MTV(mm3) MTV(mm3) 95-CI 95-CI 95-CI 95-CI 1 0.5 0.5 0.5 0.5 2 3.9 3.1 2.1 1.3 2.7-4.l 2.7-3.5 1.8-2.4 0.9-1.8 3 13 10.4 9.9 6.2 11-15 9.6-11.2 9.2-10.7 5-7.4 4 57.7 46.9 49.1 35.2 55-61 44-50 44.8-53.3 29-42 5 131 122 119 59.3 127-135 116-128 113-125 48-71 6 181 179 145 91.4 173-189 170-188 137-153 79-104 7 324 326 240 219 316-332 314-338 231-249 205-233 8 444 438 369 299 433-455 428-448 349-389 281-317 9 538 524 417 371 525-551 510-538 295-439 349-393 10 622 613 498 443 608-636 600-626 473-523 421-465 11 696 689 602 523 683-709 676-702 573-631 496-550 12 789 798 711 611 775-803 784-812 672-750 576-646 Days after treatment initiation Figure 1. The effect of IFN-a and CDDP on the growth of B-16 melanoma. Mice were treated every next day after the tumors reached 1mm in diameter. IFN-a (5 x 104 JU) and CDDP (0.00lmg/g) were injected intraperitoneally. Each experimental group consistcd of seven mice. The vertical bars represent 95% CI. animals in CDDP-treated group had for 1 g lower body weight on average, whereas the body weight of animals in other three groups did not differ from that of the control animals. Discussion According to the obtained results, IFN-a alone does not exert any direct statistically significant effect on the tumor. The antiproliferative effect of IFN-a depends on the dose applied, as well as on the mode of application, tumor size and the type of metastases. An indirect, immunomodulatory effect of interferon can be achieved at doses lower than those required for a direct antitumoral effect. In experimental animals human IFN-a does not influence the immune cells such as T-lymphocy-tes and NK cells. 14 Balkwill has reported on the interactions between human IFN-a and chemotherapeutic agents in human tumours grown in mice. The efficacy of sub-optimal doses of cyclophospha-mide and doxorubicin was greatly increased by interferon in a human breast cancer xenograft growing in nude mice. Even low doses of interferon, which alone had no effect on tumour growth, were able to potentiate the activity of anticancer drugs. 15 Numerous preclinical and clinical trials have shown synergistic or additive effects between 296 Stabuc B. interferon and at least 20 different cytotoxic agents including doxorubicim, vinca alkaloids, 5-fluorouracil and CDDP.9 16 The most striking synergy was demonstrated when low doses of IFN were used and, and it was associated predominantly with lymphoma cell lines.17 However, not all studies have established positive interaction between IFNs and chemotherapy. Antagonistic effects between some cytotoxic drugs and IFN have also been reported. 18 Little is known about the mode of IFN interaction with CDDP and other cytotoxic drugs. IFNs could potentially alter drug metabolism or act independently of the other agent. The cytochrom P-450 system was inhibited, thus significantly influencing the cell level of gluta-thione transferase. The decreased levels of cell glutathione result in increased cytotoxic effect of CDDP. IFN slows down the cell cycle by inhibiting the production of nucleic acids in its postmitotic 01 phase. IFN also slows down catabolism and elimination of some cytotoxic agents such as cyclophosphamide and doxorubi-cin, and influences cell membrane fluidity, i.e. the transport system for cytostatics.9 19 Combined IFN and chemotherapy has resulted in" clinical benefit in many patients with solid tumours.20 In general, however, significantly improved response rates were not observed. In many regimens, IFN are combined with cytotoxic drugs with different rationale: biochemical modulation, immunopotentiation, immunostimulation or host protection. Each approach is valid; however, the complexity of potential interactions requires close considera-tions.9' 22 Our study showed that IFN, even at a dose insufficient to influence the growth of B-16 melanoma when given as monotherapy, statistically significantly increased the antitumor effect of CDDP (95 % CI). Additional prospective clinical trials are of paramount importance for further explanation of the interaction mechanisms involved. References 1. Balch CM, Soong S-j, Shaw HM. A comparison of worldwide melanoma data. In: Balch CM, Milton GW eds. Cutaneous melanoma. Clinical management and treatment results worldwide. Philadelphia: Lippincott, 1985: 507-18. 2. English DR, Heenan PJ, Holman CDJ et al. Melanoma in Western Australia 1975-76 to 198081: Trends in demographic and pathological characteristics. Int J Cancer 1986; 37 : 209-15. 3. Silverberg E, Lubera J. Cancer statistics, 1988. Ca 1988; 38: 5-22. 4. Balch CM, Houghton A, Peters L. Cutaneous melanoma. In: DeVita VT Jr, Hellman S, Rosenberg SA eds. Cancer principles & practice of oncology. Vol 2. 3rd ed. Philadelphia: Lippincott 1989: 1499-542. 5. Glover DJ. New approaches to the chemotherapy of melanoma. Oncology 1991; 5: 101-2. 6. McLeod GR, Thomson DB, Hersey P. Clinical evaluation of interferons in malignant melanoma. J Invest Dermatol 1990; 96: 185S-7S. 7. Kirkwood JM. Studies of interferons in the therapy of melanoma. Semin Oncol 1991; 18: 83S-90S. 8. Welander CE, Muss HB, Morgan TM, Trotta PP, Spiegel RJ. Synergy in vitro and in clinical trials. In: Kisner DL, Smyth JF eds. Interferon alpha-2: pre-clinical and clinical evaluation. Boston: Marti-nus Nijhoff Pubi., 1985: 29-39. 9. Wadler S, Schwartz EL. Principles in the biomodulation of cytotoxic drugs by interferons. Semin Oncol 1992; 19: 45S-8S. 10. Richards JM, Mehta N, Ramming Km Skosey P. Sequential chemoimunotherapy in the treatment of metastatic melanoma. J Clin Oncol 1992; 10: 1338-43. 11. Gardner MJ, Altman DG eds. Statistics with confidence: confidence intervals and statistical guidelines. London: British Medical Journal 1989. 12. Jones A, Selby P. Biological therapies. Radiother Oncol 1991; 20: 211-23. 13. Creagan ET, Schaid DJ, Ahmann DL, Frytak S. Disseminated malignant melanoma and recombinant interferon: analysis of seven consecutive phase II investigations. J Invest Dermatol 1990; 95: S188-S92. 14. Balkwill FR, Moodie EM, Freedman V, Fantes KH. Human inter feron inhibits the growth of established human breast tumours in the nude mouse. Int J Cancer 1982; 30: 231-5. 15. Balkwill FR, Moodie EM. Positive interactions between human interferon and cyclophosphamide or adriamycin on a human model system. Cancer Res 1984; 44: 904-8. Anti-tumor effect of IFN-a. and cisplatin 297 16. Hoff von DD. In vitro data supporting interferon plus cytotoxic agent combinations. Semin Oncol 199l; 18: S58-S6 l. 17. Smyth JF, Balkwill FR, Fergusson RJ. Interferons combined with other anticancer agents - studies in experimental systems. In: Smyth JF ed. Interferons in oncology. Berlin: Springer-Verlag, 1987: 39-42. 18. Welander CE, Morgan TM, Homesley HD, Trotta PP, Spiegel RJ: Combined recombinant human interferon alpha 2 and cytotoxic agents studied in a clonogenic assay. Int J Cancer 1985; 35: 721-9. 19. Bonnem EM. Alpha interferon: the potential drug of adjuvant therapy: past achievements and future challenges. Eur J Cancer 1991; 27: S2-S6. 20. Hersey P, McLeod GR, Thomson DB. Treatment of advanced malignant melanoma with recombinant interferon alfa-2a in combination with DTIC: long term follow-up of two phase II studies. Br J Haemal 1991; 79: 60-6. 21. Grohn P, Kumpulainen E, Nuortio L et al. A phase II study of melanoma treated with a combination of interfcron-alfa 2b, dacarbazine ancl nimustine. Eur J Cancer 1992; 28: 441-3. 22. Gilewski TA, Golomb HM. Combination biothe-rapy studies: future goals ancl challenges. 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