ICANCER RESEARCH 34, 3258—3261,December 1974] Potentiation of Bleomycin by an Antifungal Polyene, Pentamycin, in Transformed Animal Cells'

Tadashi Nakashima, Michihiko Kuwano,2 Katsuko Matsui, Sohtaro Komiyama, Ikuichiro Hiroto, and Hideya Endo @ Cancer Research Institute fT. N., M. K., K. M., H. E.J and Depa@@. ‘tof Otorhinolaryngology IS. K., L H.J, Faculty ofMedicine, Kyushu University, Fukuoka 812, Japan

SUMMARY dine@4C (50 @.tCi/230 .tg; New England Nuclear, Boston, Mass.), and thymidine-3H (20 Ci/mmole; New England Bleomycin, a potent antitumor agent against human Nuclear) were used as isotopes. 5-Fluorouracil (Kyowa Hakko squamous cell carcinoma, was examined in vitro to determine Ind. Co., Tokyo, Japan), fusidic acid (Sankyo md. Co., Tokyo, whether its action is increased under synergistic conditions Japan), and a-amanitin (Lot A 32778; Boehringen/Mannheim, with a polyene antibiotic, pentamycin. The inhibition of DNA, Mannheim, Germany) were used for the study of synergism. RNA, and protein synthesis by bleomycin was greatly Bleomycin A2 was a gift from H. Umezawa. Amphotenicin B enhanced by combination with pentamycin in tissue culture. was purchased from Grand Island Biological Co., Grand Island, However, in comparison with bleomycin or a-amanitin, there N. Y., and pentamycin was a gift from Nikken Chemical Co., was less enhancement of action of both 5-fluorouracil and Tokyo, Japan. The antifungal activity of pentamycin was fusidic acid by pentamycin. stable over weeks in various solvents (20); in this case, pentamycin was dissolved in dimethyl sulfoxide. INTRODUCTION Cells and the Study of Synergi@n. Transformed rat fibroblastic cells, RFL-T, were used throughout this expeni The synergistic use of various chemical agents with polyene ment; characteristics of this cell line have been described antibiotics (5, 7), which alter membrane permeability, previously (10). Some 1 to 2 X i05 cells/ml of RFL-T cells provides a valuable approach to cancer chemotherapy. cultured in 3 ml Eagle's essential medium in Petni dishes were Amphotericin B, a heptane polyene, was shown to increase exposed to leucine-3H (1 @zCi/ml),unidine-'4C (0.05 j.tCi/ml), greatly the action of fusidic acid, 5-fluorouracil, chromomycin or thymidine-3H (1 j.zCi/ml) for 18 hr under the various A3 , or a-amanitin when each agent alone had little or no effect conditions described in the text, and 10% tnichloroacetic acid in tissue culture (9, 11—13). Independently, rifampicin, insoluble fraction was counted on glass filter paper as rifampicin analogs, and 1,3-bis(2-chloroethyl)-1-nitrosourea described earlier (9). each showed strengthened action by amphotericin B in leukemic and L-cells (4, 14—16). RESULTS In vitro synergism showed that the action of bleomycin, a potent anticancer agent (6, 21), alone or in combination with Enhancement of Action of Bleomycin by Pentamycin in amphotericin B, was inert against our transformed fibroblastic Vitro. Pentamycin alone inhibited DNA or RNA synthesisof cells, but bleomycin could be enhanced when combined with a RFL-T cells only slightly (less than 20% of control) within the high dose of polymyxin B, which is not a polyene compound concentration of 2 zg/ml, but almost complete blockage was (12). The loss of bleomycin action in the absence of observed at 5 pg/mI (Chart 2). We attempted to determine polymyxin B might be due to its inability to penetrate the whether this pentane polyene could enhance the action of membrane. Therefore, the experiment was designed to bleomycin in tissue culture using transformed fibroblastic determine whether other polyene antibiotics could enhance its RFL-T cells. Since bleomycin was known to inhibit DNA action in vitro, and in the present paper we report that synthesis in HeLa cells ( 19), the effect under synergistic pentamycin, an antifungal macrolide antibiotic (20), can conditions was assayed by measuring cellular DNA and RNA increase the action of bleomycin possibly through increasing formation (Chart 1). The dose-response curve of bleomycin its permeability. shows that the inhibition of both DNA and RNA formation by bleomycin was enhanced greatly only when it was combined MATERIALS AND METHODS with pentamycin (0.5 and 1.5 jig/mI). The extent of inhibition of DNA synthesis increased with increasing doses of pentamy Isotopes and Chemical Compounds. Leucine-3H (32 Ci! cin, as clearly seen in Chart lA (see, for example, the activity mmole; Daiichi Pure Chemical Co., Tokyo, Japan), un at bleomycin (50 or 75 j.tg/ml) with/without polyene). Then, in presence of bleomycin (50 ig/ml), the dose response of 1 Supported by a Grant-in-Aid for scientific research from the Ministry of Education, Japan. pentamycin to RNA synthesis was examined (Chart 2). The 2 To whom requests for reprints should be sent. inhibition of RNA synthesis by the antitumor agent could ReceivedApril16,1974;acceptedJuly8,1974. apparently be magnified when the concentration of pentamy

3258 CANCER RESEARCH VOL. 34

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Pentwnycin Potentiation of Bleomycin

cin was increased, although a high dose of polyene (5 jig/ml) the cells were detached, and fibroblastic cells transformed into alone was enough to inhibit RNA synthesis completely. These round ones only when both agents were present together. data strongly suggest that the pentane polyene enhances the Little change in cell morphology was observed when each action of bleomycin possibly as a function of the increase of agent was present alone (Fig. 1). intracellular concentration in transformed cells in vitro. Potentiation of Various Chemical Agents by Pentamycm. In Morphological examination of the synergistic combination

of bleomycin (50 @ig/ml)and pentamycin (1 .5 jig/ml) was I.—o------o carried out by microscope. As shown in Fig. 1, almost all of I 00 —S-. @O\

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0 4 50 ..-C 50 \ 41 z DI 18 C \\:@@@I 4, @1 @._•)¼@p_@\\@N \.0I @ 50 100 50 i@a 50 00 50 I 2 BI.omycin—A2 (pglml) Chart 1. Dose response of bleomycin in absence or presence of Pentamycin5 (pglml) pentamycin on DNA (A) and RNA (B) synthesis. RFL-T cells were Chart 2. Dose response of pentamycin in absence or presence of exposed to various concentrations of bleomycin A2 alone (o) or in bleomycin on RNA symthesis. RFL-T cells were exposed to various combination with pentemycin [0.5 @g/ml(@)or 1.5 j@g/ml(.)] for 18 doses of pentamycin in absence (o) or presence of bleomycin A3 (50 @ hr, and thymidine-3 H (A) or in4 C (B) incorporation into tg/ml) (.) with uridine-' C (0.5 @iCi/ml)for 18 hr. Normalized activity acid-insoluble fraction was measured as described in “Materialsand of RNA synthesis is shown when 100% correiponds to 1200 cpm in Methods.―The normalized activity is presented when 100% corre absence of drug and 1000 cpm in presence of bleomycin alone, sponds to cpm in absence of bleomycin A2. respectively, when background (50 cpm) is subtracted.

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Fig. 1. RFL-T cells (I X 10@cells/ml) in Petri dish treated for 18 hr under synergistic conditions. B, with bleomycin (50 @ig/ml)alone;C, with pentamycin (1.5 @g/ml)alone;D, with bleomycin (50 @sg/ml)andpentamycin (1.5 @g/ml)together. A, untreated control cells.

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Table 1 Effect of various chemical agents in combination with pentamycin upon protein and RNA synthesis

of(@tg/ml)Label0 at pentamycin concentration

Agents usedcpm j@g/mlNone3H @zg/ml0.5 pg/mI1.0

(loo)a (86) (70) (52)Fusidicacid(200)3H ‘4C4727 1154 (100)4092 768 (66)3399 606 (100) (89) (64) (63)Bleomycin ‘4C3464 909 (100)3112 661 (70)2275 577 (100)3H (100) (63) (34) (19)a-Amanitin ‘4C2993 448 (100)1915 170 (37)1041 86 (2)3H (100) (57) 1 (29) (11)5-Fluorouracil ‘4C48301123 (100)2771 405 (36)141 125 (5)H (100) (85) (60) ‘4C4937 444 (100)4205 449 (105)3000362 (85)

@ a Values indicate incorporated cpm of leucine-3 H and uridine-' C into acid-insoluble @ fraction; percentage in parentheses shows normalized activity of protein (3H) and RNA (‘C) synthesis when 100% corresponds to the activity in absence of pentamycin.

our system using RFL-T cells, previous study showed that the sensitivity to amphotericin B among established cell lines in action of fusidic acid, 5-fluorouracil, and a-amanitin, but not vitro suggested a positive correlation between levels of bleomycin, was increased markedly in each case by ampho density-dependent inhibition of cell growth and the sensitivity tenicin B (9, 11—13). Therefore, the membrane-activating (1). In addition, amphotericin B increased the therapeutic action of pentamycin was analyzed to determine whether it action of 1,3-bis(2-chloroethyl)-l-nitrosourea against a trans. enhanced these various chemical agents. As shown in Table 1, plantable AKR leukemia (17). These data could give us a clue pentamycin, unlike amphotenicin B, increased only slightly the to finding a specific control of tumor chemotherapy through action of fusidic acid or S-fluorouracil, although the action of modifying the permeation of antitumor agents. Synergistic a-amanitin and of bleomycin was enhanced by pentamycin. usage of antitumor agents with less toxic membrane-active Treatment with pentamycin did not alter the specificity of agents such as vitamin A was partly successful in vivo as well as 2nd agents because a-amanitin preferentially inhibited RNA in vitro (8, 16). In particular, therapeutic effects of synthesis without markedly affecting protein synthesis, and 1,3-bis(2-chloroethyl)-l-nitrosourea and cyclophosphamide bleomycin also rather specifically inhibited DNA or RNA were enhanced by vitamin A in mice bearing leukemia (3). synthesis (Table 1; Chart 1). The dosage of 5-fluorouracil or During our application over 1.5 years of the synergism of fusidic acid used here might have been too low to show 5-fluorouracil and vitamin A in combination with 60Co (8) to increasing effect by pentamycin, but amphotenicin B was clinical chemotherapy of laryngeal carcinoma, we have previously shown to increase the penetration when combined obtained satisfactory results thus far (manuscript in prepara with the same dose ofthese agents(12, 13). tion).

DISCUSSION ACKNOWLEDGMENTS

Bleomycin showed a much higher sensitivity to HeLa cells We thank Dr. Keitaro Kato and Dr. Yukio Ikehara for fruitful than to fibroblastic RFL-T cells (12), which might be discussion through this experiment. attributed in part to the differences in the abilities of the two cell lines to permeate the agent. When the membrane barrier to REFERENCES bleomycin could be overcome by polymyxin B (12) or pentamycin, fibroblastic cells were sensitive against the 1. Amati, P., and Lago, C. Sensitivity to Amphotericin B of in vitro anticancer agent. The mechanism involved here might be Established Cell Lines. Nature, 247: 466—469, 1974. similar to that in an Escherichia coli mutant sensitive to 2. Bittman, R., and Fishkoff, S. A. Fluorescence Studies of the bleomycin, which exhibited greater permeability to the agent Binding of the Polyene Antibiotics Filipin Ill, Amphotericin B, (22). Since amphotenicin B increased the action of 5-fluoro Nystatin, and Lagosin to CholesteroL Proc. Natl. Acad. Sci. U. S., uracil or fusidic acid more effectively than did pentamycin (9, 69: 3795—3799,1972. 3. Cohen, M. H., and Ca.rbone,P. P. Enhancement of the Antitumor 12, 13), these 2 polyenes seemed to control the permeability Effects of 1,3-Bis(2-chloroethyl)-1-Nitrosourea and Cyclophos differently. The differential abilities of polyene antibiotics to phamide by Vitamin A. J. Natl. Cancer Inst., 48: 921 —926,1972. enhance membrane permeability could be closely correlated 4. Hackett, A. J., Sylvester, S. S., Joss, Jr., and Calvin, M. Synergistic with the affinity of polyenes to sterols (2, 5, 7). Effect of Rifamycin Derivatives and Amphotericin B on Viral On the other hand, filipin III and lucensomycin, polyene Transformation of a Murine Cell Line. Proc. Natl. Acad. Sci. U. S., antibiotics, are shown to have rather preferential affinity for 69: 3653—3654,1972. the membrane of malignant cells ( 18). The differential 5. Hamilton-Miller, J. M. T. Chemistry and Biology of the Polyene

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Tadashi Nakashima, Michihiko Kuwano, Katsuko Matsui, et al.

Cancer Res 1974;34:3258-3261.

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