Radiol Oncol 1993 ; 27: 275-9.
Interactions of interferon and vinblastine on experimental tumor model melanoma B-16 in vivo
Barbara Jezersek,* Srdjan Novakovic,1 Gregor Sersa,1 Maja Cemazar,1 Marija Auersperg,1 W. Robert Fleischmann Jr.2
'Institute of Oncology, Ljubljana, Slovenia, 2University of Texas Medical Branch, Department of Microbiology, Galveston, Texas, USA
In the study, we investigated the in vivo interaction of two antitumor agents, that have different sites and different mechanisms of action. Vinblastine (VLB) in combination with human recombinant interferon a AID (rHuIFN-a AID) and in combination with human leukocyte interferon a (HuLIFN-a) was tested on intraperitoneal (i.p.) melanoma B-16 tumor model. The effect of the combination was determined with follow-up of animals' survival and the interaction defined by means of Spector's formula. Only subadditive enhancement of interferon's (IFN's) antitumor activity was observed when rHuIFN-a AID was combined with VLB and supraadditive, but not synergistic, interaction when HuLIFN-a was combined with VLB. Synergism between VLB and rHuIFN-a AID on B-16 melanoma in vitro, that had been observed in our previous study, did not come true in vivo.
Key words: melanoma, experimental-drug therapy; vinblastine; interferon alpha, recombinant;
Introduction
Chemotherapy and biotherapy are the two systemic modalities available for cancer treatment. However, because it is apparent that neither one nor the other are perfect treatments for cancer, the combination of cytotoxic drugs and cytokines offers a new approach to increase the therapeutic index in the treatment of neo-plastic diseases.1 2
Interferons (IFNs) are a complex group of cytokines with antiviral, antibacterial, antitu-
Correspondence to: Jezeršek Barbara MD, Institute of Oncology, Zaloška 2, 61105 Ljubljana, Slovenia, Tel. + 386 61 323 063 ext. 29 33, Fax + 386 61 131 41 80
UDC: 616-006.81-085
mor and immunomodulatory activities.3 '4 They exert antiproliferative effect on tumor cells, while IFNs p and y also have a direct cytotoxic activity.5 Antitumor activity of VLB is a consequence of its binding to microtubular proteins of the mitotic spindle, which causes metaphase arrest of cells in mitosis.6'7 VLB is, in higher concentrations, also directly cytotoxic for interphase cells.8
While in vitro studies have demonstrated both direct cytotoxic and cytokinetic effects of IFNs, a more interesting role derives from their ability to sinergistically potentiate the wide variety of cytotoxic agents against multiple human and rodent tumors, both in vitro and in animal models.9 The broad spectrum of cytotoxic drugs whose activity can be enhanced by cytokines argues for multiple levels of drug interaction in
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vitro: alteration of cellular drug uptake, modulation of drug target enzymes, and changes in metabolism or disposition of a drug. In vivo interaction between cytokines and cytotoxic drugs involves an additional layer of complexity because of the effects of cytokines on the host
immune system and on drug-metabolising enzy-
2
mes.
The ability of IFNs to directly modulate the biochemical effects of cytotoxic agents independent of immunomediated or host-protective effects has been evaluated in a variety of in vitro systems.9 Since synergistic cytotoxicity has been observed in vitro for IFN-a combination with VLB on BG-1 human ovarian carcinoma line,10 on RPMI 8226 human myeloma line, on MCF-7 human breast carcinoma line, on WiDr human colon carcinoma line11 and on murine B-16 melanoma line, 12 we wanted to define the interaction of VLB with rHuIFN-a AJD or Hu-LIFN-a in vivo on i.p. B-16 melanoma tumor model.
Materials and methods
Reagents
Recombinant HulFN-a A/D was provided by Hoffmann-LaRoche (Nutley, New Jersey) and HuLIFN-a by Immunological Institute (Zagreb, Croatia). Both were diluted with phosphate buffered saline (PBS).
Vinblastine sulfate (Lymphomed, Deerfield, Illinois) was used in combination with rHuIFN-a A/D and Velbe (Lilly, Firenze, Italy) with HuLIFN-a. Both were diluted with PBS.
Animals
Six to eight weeks old pathogen-free female C57BM6 mice were purchased from Jackson Laboratories, Bar Harbor, USA. Animals were maintained in a pathogen-free state in animal rooms with alternating cycles of 12 h light and 12 h darkness. Each experimental group consisted of 10 to 11 mice. These animals were used for experiments with rHuIFN-a A/D and Vin-blastine sulfate.
Female C57Bl/6 were purchased from Rudjer Boskovic Institute, Zagreb, Croatia. Animals were maintained at a natural day/night cycle in a standard animal colony. Eight to ten weeks old mice in good condition without any signs of fungal or other infections were used in the experiments. Each experimental group consisted of ten mice. These animals were used for experiments with HuLIFN-a and Velbe.
Tumor cells
Murine B-16 melanoma cells (done Fl, American Type Culture Collection, Rockville, Maryland) were grown in Eagle's Minimal Essential Medium supplemented with 10% fetal calf serum, penicillin (100 units/ml), streptomycin (100 ng/ml) and gentamycin (11 ng/ml). These cells were used for experiments with rHuIFN-a A/D and Vinblastine sulfate.
Murine B-16 melanoma cells (cone B6, Ru-djer Boskovic Institute, Zagreb, Croatia) were grown in RPMI 1640 medium supplemented with 10% fetal calf serum, gentamycin (500 Hg/ml) and 7,5% sodium bicarbonate (27 ml/l). These cells were used for experiments with HuLIFN-a and Velbe.
Experimental procedure
Mice were i.p. injected with 106 B-16 melanoma (done Fl or B6) cells on day 0 and randomly divided into four groups. All treatment was intraperitoneal and was administered as follows:
control group - PBS for five consecutive days, starting day 1
-	vinblastine group - Vinblastine sulfate or Velbe (30 ¡xg per animal) on day 4 only
-	interferon group - rHuIFN-a A/D (1 x 105 I.U. per animal) or HuLIFN-a (5 x 105 I.U. per animal) for five consecutive days, starting day 1
combination group - Vinblastine sulfate or Velbe (30 ¡xg per animal) on day 4 only + rHuIFN-a A/D (1 x 105 I.U. per animal) or HuLIFN-a (5 x 105 I.U. per animal) for five consecutive days, starting day l.
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The mice were monitored for the day of death and the average day of death was determined.
Statistical analysis
The Mantel - Cox test (BMDP Statistical Software, Los Angeles, California) was employed for comparison of the animals' survival and Spector's formula 13 to define the interaction of rHuIFN-a A/D or HuLIFN-a with VLB.
Results
Vinblastine sulfate and rHuIFN-a A/D as single agents or in combination were tested for their effect on survival of animals with i.p. B-16 melanoma (Fl). Intraperitoneal application of 30 |ig of Vinblastine sulfate on day 4 had a moderate (p =0,054) antitumor effect, while treatment with 1 x 105 I.U. of rHuIFN-a A/D for 5 consecutive days showed a more pronounced statistically significant (p<0,001) effect on
30	40
DAYS
Figure l. Survival of mice with i.p. B-16 melanoma (clone Fl) treated with Vinblastine sulfate (®), rHuIFN-a A/D (□) or combination of both agents (111); control (O). The antitumor effect of the combination was merely subadditive in comparison to the one expected on the basis of separate activities of VLB or IFN.
90 80
o::
O >
>
ir j
10
o
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DAYS
50
Figure 2. Survival of mice with i.p. B-16 melanoma (clone B6) treated with Velbe (®), HuLIFN-a (□) or combination of both agents (1111); control (O). The effect of the combination on survival of the animals was supraadclitive in comparison to the one expected on the basis of separate activities of VLB or IFN.
survival of mice with i.p. B-16 melanoma. The combination of both agents had a statistically significant (p<0,001) antitumor effect, but there was no significant difference (p = 0,497) in survival between the "interferon group" and the "combination group" of animals (Figure 1). According to Spector's formula the interaction of rHuIFN-a A/D with VLB was merely subadditive and the antitumor effect of the combination was 90% of the one expected on the basis of their separate activities.
Survival of animals with i.p. B-16 melanoma (B6) treated with HuLIFN-a or Velbe alone or in combination is presented in Figure 2. Treatment with 5 x 105 I.U. of HuLIFN-a for five consecutive days had a moderate (p = 0,059) antitumor effect, while i.p. application of 30 ¡ig of Velbe significantly (p<0,001) prolonged survival of the animals. The combination of both agents had a statistically significant (p<0,001) antitumor effect, but there was no significant difference (p = 0,058) in survival between the "vinblastine group" and the "combination group" of animals. The interaction of HuLIFN-a with VLB was supraadclitive ancl the antitu-
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mor effect of the combination was 115% of the one expected on the basis of their separate activities.
Discussion
The results of in vivo studies testing the combination of IFNs with VLB are controversial. Sidkey et al. report that murine IFN a/p increased survival in mice with P388 leukemia cells after treatment with VLB. 14 Harrison et al. on the other hand found no positive interaction when murine IFN a/p was combined with VLB on s.c. Meth A sarcoma tumor model and when recombinant murine IFN-y was combined with VLB on s.c. Meth A sarcoma and s.c. B-16 melanoma.15 Also Mitchell has pointed out that although type I IFN has been found to potentiate chemotherapy in cultured cells, "there is very little substantiation in vivo". 16
Our results clearly demonstrate that in vitro synergism between rHulFN-a A/D and VLB observed on Fl done of B-16 melanoma cells12 did not come true in vivo. However, there is an interesting difference between the antitumor activity of combination of rHulFN-a A/D with VLB and combination of HuLIFN-a with VLB. Even though HuLIFN-a alone has only moderate antitumor activity, the interaction with VLB was supraadditive, in comparison with rHuIFN-a A/D that has significant antitumor activity, but demonstrated only subadditive interaction with VLB. In part this difference could be explained with the fact that different clones of B-16 melanoma were used in the experiments, but Sklarin et al. report that in most of the cases where potentiation was observed, human IFN-a alone had only weak antitumor activity; however, IFN-a seemed to be most effective in combination with drugs that alone possessed substantial activity against the specific tumor. 17
The question of the mechanism of interaction between IFNs and VLB still cannot be resolved and it seems likely that multiple factors may be contributing to success or failure in these prec-linical models. The interactions observed are
not solely the consequence of the combined effect of two cytoreductive agents, since the enhanced activity of the drug-interferon combination was observed even in instances where IFNs alone lacked activity, and IFNs also failed to potentiate the activity of other efficacious drugs.9 There is also a complex relationship between the timing of interferon (IFN) with a cytotoxic agent, the doses used, and the efficacy of the regimen. In combination with cytotoxic drug, sequence and duration of exposure to IFN may play as significant a role as dose and dose intensity, and the maximum tolerated dose of IFN may not be the most biologically effective dose. Up till now, the lack of understanding of the biochemical interaction of these agents has prohibited a rational approach to design of schedule and sequence that allow translation of the positive in vitro data into effective preclini-cal treatment regimens.
Acknowledgement
This work was supported by U.S. Public Health Service Grant (National Cancer Institute) and by the Ministry of Science and Technology of Slovenia.
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