Effect of Novobiocin on the Antitumor Activity and Tumor Cell and Bone Marrow Survivals of Three Alkylating Agents1

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(CANCER RESEARCH 49, 595-598, February 1, 1989] Effect of Novobiocin on the Antitumor Activity and Tumor Cell and Bone Marrow Survivals of Three Alkylating Agents1

J. Paul Eder,2 Beverly A. Teicher, Sylvia A. Holden, Kathleen N. S. Cathcart, Lowell E. Schnipper, and Emil Frei III

Department of Medicine, Beth Israel Hospital [J. P. E., L. E. S.], and Dana-Farber Cancer Institute [B. A. T., S. A. H., K. N. S. C, E. F.j, Boston, Massachusetts 02115

ABSTRACT Novobiocin has myriad effects which occur at pharmacolog ically relevant concentrations and likely involve multiple mech Our previous in vitro studies demonstrated marked synergy with anisms. Novobiocin inhibits the bacterial topoisomerase II by alkylating agents when novobiocin was present during and after alkylating agent exposure. To determine whether this effect is observed in vivo, interfering with its ATPase and energy transduction processes novobiocin was administered daily for 3 days prior to alkylating agent (5). Novobiocin also inhibits replicative and repair DNA syn treatment, during alkylating agent treatment, and for 2 days after com thesis, but perhaps by different mechanisms (8, 9). The DNA pletion of alkylating agent treatment. When combined with cu-diam- repair target of novobiocin is not the a-polymerase and may be minedichloroplatinum(II), l,3-bis(2-chloroethyl)-l-nitrosourea, or cyclo- topoisomerase (9). Novobiocin also affects mitochondria! struc phosphamide, there was significant enhancement of the growth delay of ture and ATP metabolism (10), directly precipitates chromo the FSalIC fibrosarcoma implanted s.c. in C3H mice when compared somes, and impairs RNA polymerase (11, 12). Thus, an un with alkylating agents alone. In a second assay using ex vivo studies of ambiguous explanation of novobiocin activity is difficult. tumor cells exposed in vivo, single doses of 100 mg/kg of novobiocin In this study, we have attempted to extend these promising followed by cÂ»-diamminedichloroplatinum(II) resulted in a 3- to 4-fold increase in tumor cell killing by m-diamminedichloroplatinum(II). At a in vitro observations to an in vivo setting. The effect of novo dose of 100 mg/kg of l,3-bis(2-chloroethyl)-l-nitrosourea there was about biocin in combination with any of three bifunctional alkylating a 7-fold increase in tumor cell kill upon addition of novobiocin. Cyclo- agents, BCNU, CDDP, and cyclophosphamide, on the TGD phosphamide showed a dose response effect with novobiocin, reaching and ex vivo tumor cell survival of the FSall fibrosarcoma and 13-fold at a dose of 300 mg/kg of cyclophosphamide. In all cases bone on the toxicity of the drugs to the bone marrow has been marrow elements were affected less than were neoplastic cells, suggesting investigated. The TGD assay was performed at optimal sched that the combination of novobiocin and alkylating agents may be a ule with novobiocin administered before, during, and for several clinically useful strategy. days after chemotherapy administration. The doses of novobio cin were tolerable to the animals. The tumor excision assay was INTRODUCTION performed after only a single dose of novobiocin and chemo- The emergence of drug-resistant tumor clones in clinical therapeutic drug, but allowed chemotherapy dose escalations to 3- to 5-fold and concomitant measurement of vital organ func cancer therapy is a major cause of treatment failure. Strategies tion-committed bone marrow progenitors. to overcome this problem include the use of drug combinations, escalation to maximally tolerated drug doses, and short treat ment intervals (1). Despite these strategies, most tumors ulti MATERIALS AND METHODS mately exhibit clinical drug resistance which results in progres Drugs. Novobiocin was purchased from Sigma Chemical Co. (St. sive disease and death from cancer. Louis, MO). BCNU (Carmustine) and cyclophosphamide (Cytoxan) The alkylating agents are among the most useful clinical were obtained from the Dana-Farber Cancer Institute pharmacy. CDDP antineoplastic agents, with cytotoxic activity in many human was obtained as a gift from Dr. Donald Picker and Dr. Michael Abrams, tumors. These compounds differ in their mechanisms of uptake, Johnson Matthey, Inc. (West Chester, PA). intracellular drug binding and metabolism, and site and timing Tumor. The FSall fibrosarcoma (13) adapted for growth in culture of DNA adduci formation (2). Furthermore, the alkylating (FSalIC) (14) was carried in male C3H/FeJ mice (Jackson Laboratory, agents demonstrate a lack of cross-resistance (3,4). Novobiocin, Bar Harbor, ME). For the experiments, 2 x IO6 tumor cells prepared a coumeromycin antibiotic which inhibits the DNA gyrase from a brei of several stock tumors were implanted i.m. into the legs of (topoisomerase II) of eubacteria (5), potentiated the cytotoxicity male C3H/HeJ mice 8 to 10 wk of age. Tumor Growth Delay Experiments. Treatment was initiated when the of a number of alkylating agents in vitro (6, 7) and was syner- tumors were approximately 50 mm-' in volume. Novobiocin (50 mg/kg gistically cytotoxic in combination with CDDP3 and BCNU in or 100 mg/kg) was administered i.p. once daily on Days 6 through 15 Chinese hamster ovary cells. This increased cytotoxicity ac post tumor implantation. CDDP (4.5 mg/kg) was administered i.p. crued in association with increased number of DNA interstrand once daily on Days 9 through 13. BCNU (8 mg/kg) was also adminis cross-links being formed from monoadducts. Continuous no tered i.p. once daily on Days 9 through 13. Cyclophosphamide (100 vobiocin exposure was necessary to maximize the cross-link mg/kg) was administered i.p. once daily on Days 9, 11, and 13. The formation. Novobiocin has also been shown to increase the progress of each tumor was measured with calipers 3 times weekly until number of DNA interstrand cross-links formed in a nitrogen it reached a volume of 500 mm3. Tumor growth delay was calculated mustard-resistant human Raji lymphoblastoid cell line which as the days taken by each individual tumor to reach 500 mm' compared has elevated topoisomerase II activity (7). to the untreated controls. Each treatment group had 7 animals, and the experiment was repeated 3 times. Days of tumor growth delay are the Received 6/21/88; revised 10/6/88; accepted 10/25/88. mean Â±SE for the treatment group compared to the control. The costs of publication of this article were defrayed in part by the payment Tumor Excision Assay. When the tumors were approximately 50 of page charges. This article must therefore be hereby marked advertisement in mm3 in volume (about 1 wk after tumor cell implantation), single doses accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by National Cancer Institute Grant 5PO1-CA38493 of novobiocin (100, 200, or 300 mg/kg), CDDP (15, 20, or 25 mg/kg), (B. A. T.). BCNU (50, 100, or 200 mg/kg), or cyclophosphamide (100, 200, or 2To whom requests for reprints should be addressed, at Department of 300 mg/kg) were administered i.p., or novobiocin (100 mg/kg) was Medicine, Beth Israel Hospital, 330 Brookline Avenue, Boston, MA 02115. 3The abbreviations used are: CDDP, as-diamminedichloroplatinum(II); given l h prior to each of the alkylating agents to ensure the presence BCNU, 1,3-bis(2-chloroethyl)-l -nitrosourea; TGD, tumor growth delay; a-MEM, of novobiocin in the circulation at the time of alkylating agent treat a-minimal essential medium; FBS, fetal bovine serum. ment. Mice were sacrificed and soaked briefly in 95% ethanol 24 h 595

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1989 American Association for Cancer Research. NOVOBIOCIN AND ALKYLATING AGENTS after treatment to allow for full expression of drug cytotoxicity and Table 1 Tumorgrowth delay of the fSal 1Cfibrosarcoma treated with novobiocin repair of potentially lethal damage. The tumors were excised under alone or in combination with CDDP, BCNU, or cyclophosphamide sterile conditions in a laminar flow hood and minced to a fine brei with Tumor growth delay 2 scalpels. Four tumors were pooled to make each treatment group. Treatment group (days) Approximately l g of tumor brei was used to make each single-cell Novobiocin (50 mg/kg)" 1.0 Â±0.5* suspension. All reagents were sterilized with 0.22-ftm Millipore mem Novobiocin (100 mg/kg) 1.0 Â±0.5 branes and were added aseptically to the tumor cells. Each sample was CDDI* 10.6 Â±0.7 washed in 20 ml of a-MEM (Grand Island Biological Co., Grand Novobiocin (50 mg/kg) + CDDP 14.0 Â±1.2 Island, NY) in a SO-mlcentrifuge tube, after which the liquid was gently Novobiocin (100 mg/kg) + CDDP 29.6 Â±2.5 decanted and discarded. The samples were resuspended in 450 units/ BCNU* 2.9 Â±0.5 ml of collagenase (Sigma) and 0.1 mg/ml of DNase (Sigma) and Novobiocin (50 mg/kg) + BCNU 4.1 Â±0.6 incubated for 10 min at 37Â°Cin a shaking water bath. The samples Novobiocin (100 mg/kg) + BCNU 26.2 Â±2.0 were centrifugea at 200 x g, and the supernatant was discarded. The Cyclophosphamide' 6.8 Â±1.0 samples were resuspended as above and incubated for another 15 min at 37Â°C.One ml of 1 mg/ml of DNase was added, and incubation was Novobiocin (50 mg/kg) + cyclophosphamide 9.0 Â±1.5 continued for 5 min at 37Â°C.The samples were then filtered through 2 Novobiocin (100 mg/kg) + cyclophosphamide 14.4 Â±1.7 "Novobiocin was given once daily on Days 6 to 15 post tumor cell implanta layers of sterile gauze. The samples were washed twice and then tion. resuspended in a-MEM supplemented with 10% FBS (Sterile Systems, * Mean Â±SEof the days required for treated tumor to reach 500 mm3compared Logan, UT). These single-cell suspensions were counted and plated at to untreated controls. Controls reached 500 mm3 in 15.0 Â±0.2 days. CCDDP (4.5 mg/kg) was given once daily on Days 9 to 13 post tumor cell three different cell concentrations in duplicate for the colony-forming implantation. assay. One wk later the plates were stained with crystal violet, and d BCNU (8 mg/kg) was given once daily on Days 9 to 13 post tumor cell colonies of more than 50 cells were counted. The untreated tumor cell implantation. ' Cyclophosphamide (100 mg/kg) was given once daily on Days 9, 11, and 13 suspensions had a plating efficiency of 10 to 16%. The results are expressed as the surviving fraction Â±SE of cells from treated groups post tumor cell implantation. compared to untreated controls. Bone Marrow Toxicity. Bone marrow was taken from the same 1.000 r animals used for the tumor excision assay. A pool of marrow from the femurs of 2 animals was obtained by gently flushing the marrow through a 23-gauge needle using ice-cold McCoy's Medium 5A (Grand Island Biological Co.), supplemented with 2% FBS and nonessential amino 0.100 acids (lOOx; 2 ml/liter) (Grand Island Biological Co.), sodium pyruvate U (50 Mg/ml), L-glutamine (146 Mg/ml), L-asparagine (8 Mg/ml), L-serine < K (4.2 Mg/ml), vitamins (lOOx; 2 ml/liter), penicillin (50 units/ml), and (9 streptomycin (50 Mg/ml) (Grand Island Biological Co.) (15). The cells Z were washed and resuspended in supplemented medium. Granulocyte- macrophage colony-forming units were measured as follows. Bone > 0.0 l O marrow cells were suspended in supplemented McCoy's Medium 5A containing 15% FBS, 0.3% agar (Difco, Detroit, MI), and 10% L-cell- conditioned medium as a source of colony-stimulating activity. The colony-stimulating activity supplement was prepared by incubating L 929 mouse fibroblasts (2500 cells/ml; Microbiological Associates, Be- 0.001 thesda, MD) with 30% FBS in McCoy's Medium 5A for 7 days in a 100 200 300 humidified 5% CO2 atmosphere at 37Â°C.The colony-stimulating activ NOVOBIOCIN, mg/kg ity-containing supernatant was obtained by centrifugation of the me dium at 10,000 x # for 10 min at 4'C and then nitration under sterile Fig. 1. Survival of FSalIC cells (â€¢)from FSallC tumors and bone marrow (O) from animals treated with various doses of novobiocin. Points, mean of three conditions (16). The bone marrow cell cultures were incubated for 7 independent determinations; bars, SE. days in a humidified 5% CO2 atmosphere at 37Â°Cand then fixed with 10% glutaraldehyde. Colonies of at least 50 cells were scored on an Acculile Colony counter (Fisher, Springneid, NJ). The results from 3 in the FSalIC fibrosarcoma, there was significant enhancement experiments, in which each group was measured in triplicate, were of the tumor growth delay produced by the alkylating agent. averaged. The results are expressed as the surviving fraction of treated With each of the alkylating drugs, it appeared that the higher groups compared to untreated controls. dose of novobiocin was much more effective in increasing the growth delay of the tumor. There was about a 3-fold increase in the tumor growth delay produced by CDDP, about a 9-fold RESULTS increase in tumor growth delay produced by BCNU, and about Our previous studies with Chinese hamster ovary cells in a 2-fold increase in the tumor growth delay produced by cyclo tissue culture indicated that the presence of novobiocin was phosphamide with the addition of novobiocin (100 mg/kg) to required during and after alkylating agent exposure to obtain the drug treatment. enhancement in the cell killing by the alkylating agent (6). To When single doses of novobiocin were administered to tumor- mimic this effect in vivo, novobiocin was administered daily for bearing animals and tumor cell survival was measured in vitro, 3 days prior to alkylating agent treatment, during alkylating some toxicity of novobiocin toward tumor cells was seen (Fig. agent treatment, and for 2 days after completion of alkylating 1). There was only a slight level of toxicity of this drug seen agent treatment. In humans, steady-state levels are achieved in toward bone marrow at the highest dose examined. The dose 48 h with twice daily p.o. preparations (17). Novobiocin at of 100 mg/kg of novobiocin was chosen to use in combination doses of 50 mg/kg or 100 mg/kg had very little effect on tumor with the alkylating agents to be comparable to the tumor growth growth (Table 1). The dose of 100 mg/kg of novobiocin daily delay studies. There was a 3- to 4-fold increase in tumor cell for Days 6 to 15 was the maximum tolerated dosage of this killing by CDDP when administration of CDDP was preceded drug. However, when combined with CDDP, BCNU, or cyclo- by a dose of novobiocin (100 mg/kg) (Fig. 2). In the bone phosphamide at optimal dosage and schedules for these drugs marrow, even at the highest dose of CDDP there was less than 596

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> 0.010 > 0.00100 > K D (n 0.00010 0.001 15 20 25 15 20 25 CDDP, mg/kg 0.0000 l 100 200 300 100 200 300 Fig. 2. Survival of FSallC cells from FSallC tumors (left) and bone marrow (right) from animals treated with various doses of CDDP (â€¢)or with various CYCLOPHOSPHAMIDE, mg/kg doses of CDDP preceded l h earlier by a single dose of novobiocin (100 mg/kg) (O). Points, mean of three independent determinations; bars, SE. Fig. 4. Survival of FSallC cells from FSallC tumors (left) and bone marrow (right) from animals treated with various doses of Cyclophosphamide (â€¢)orwith various doses of Cyclophosphamide preceded l h earlier by a single dose of TUMOR BONE MARROW novobiocin (100 mg/kg) (O). Points, mean of three independent determinations; oars, SE. 1.000 icity of Cyclophosphamide to the bone marrow by 4- to 5-fold over the dosage range of Cyclophosphamide examined. z O 0.100 DISCUSSION Â£e L. Chemotherapeutic drug resistance had emerged as a major unresolved problem in cancer chemotherapy. The determina tion of specific mechanisms of resistance in a specific tumor > 0.010 cell type or the ability to determine the presence of a specific mechanism of resistance for an individual tumor would allow for specific countermeasures to be taken to increase the thera peutic effectiveness of available cancer Chemotherapeutic agents. Known mechanisms of resistance include elevated levels 0.001 50 100 200 50 100 200 of pleiotropic resistance due to enhanced expression of the mdr gene, increased glutathione levels, amplification of target genes BCNU, mg/kg like dihydrofolate reducÃase,and enhanced DNA repair (18). Fig. 3. Survival of FSallC cells from FSallC tumors (left) and bone marrow (right) from animals treated with various doses of BCNU (â€¢)or with various Recent work indicates that elevated activity or altered enzyme doses of BCNU preceded l h earlier by a single dose of novobiocin (100 mg/kg) properties of topoisomerase II may mediate some forms of drug (O). Points, mean of three independent determinations; bars, SE. resistance (7, 19). While experimental protocols may produce a substantial a 2-fold increase in cell killing with the addition of novobiocin amount of resistance by virtue of a single mechanism, other to the drug treatment. protocols produce multiple mechanisms of resistance. Where a BCNU gave a more complex pattern (Fig. 3). At the lowest high degree of resistance is due to intrinsic expression of a gene dose of BCNU, there was no significant effect of novobiocin. constitutively present in a cell type (such as mdr in the liver, At a dose of 100 mg/kg of BCNU there was about a 7-fold kidney, or intestinal cells), a specific pharmacological modula increase in tumor cell kill upon addition of novobiocin to the tion may have therapeutic benefit (20). In human cells made resistant to alkylating agents by gradual, incremental exposure, drug treatment; however, at the highest dose of BCNU (200 mg/kg), this effect fell to about a 2-fold increase in tumor cell however, multiple mechanisms of resistance are observed and may limit the ability of any single intervention to overcome it kill with the combination treatment. As with CDDP, there was (3, 21). Thus, multiple pharmacological interventions may be overall less than a 2-fold increase in bone marrow killing with required. the combination of BCNU and novobiocin compared to BCNU These recent discoveries regarding the nature and mecha alone. nisms of drug resistance offer new treatment strategies directed Cyclophosphamide showed the increasing effect of novobio at specific mechanisms. The use of calcium channel blockers to cin with the increasing dose of alkylating agent (Fig. 4). The reverse the rapid efflux associated with the multiple drug re presence of novobiocin increased the tumor cell kill of 100 mg/ sistance phenotype is already in clinical trials (22). Lipid-soluble kg of Cyclophosphamide by approximately 3-fold. However, at analogues can overcome resistance due to decreased drug trans a dose of 300 mg/kg of Cyclophosphamide there was a 13-fold port (23). Buthionine sulfoxime, which inhibits glutathione increase in tumor cell kill when the alkylating agent was pre synthesis, can restore drug sensitivity in cell lines with enhanced ceded by a dose of novobiocin. Novobiocin increased the tox- glutathione levels as a mechanism of resistance (24). 597

H. Patterns of resistance and therapeutic synergism among alkylating agents. Novobiocin inhibits eukaryotic topoisomerase II and specif Antibiot. Chemother., 23: 200-215, 1978. ically overcomes resistance in an alkylating agent-resistant hu 5. Geliert, M., O'Dea, M. H., Itoh, T., and Tomizawa, J. Novobiocin and man tumor cell line (7). We were able to demonstrate therapeu coumeromycin inhibit DNA supercoiling catalyzed by DNA gyrase. Proc. Nati. Acad. Sci. USA, 73:4474-4478, 1976. tic synergism between novobiocin and the alkylating agents 6. Eder, J. P., Teicher, B. A., Holden, S. A., Cathcart, K. N. S., and Schnipper, CDDP and BCNU (6). We then chose to see if any therapeutic L. E. Novobiocin enhances alkylating agent cytotoxicity and DNA interStrand benefit could be found in vivo in experimental tumors. The cross-links in a murine model. J. Clin. Invest., 79: 1524-1528, 1987. 7. Tan, K. B., Muttern, M. R., Boyce, R. A., Hertzberg, R. P., and Schein, P. TGD and tumor excision assays were used so that studies could S. Elevated topoisomerase II activity and altered chromatin in nitrogen be performed in solid tumors. The TGD assay was done at mustard-resistant human cells. NCI Monogr., 4:95-98, 1987. 8. Edenberg, H. J. Novobiocin inhibition of simian virus 40 DNA replication. therapeutically active doses which allow normal animal survival Nature (Lond.), 286: 529-531, 1980. and allowed an optimal schedule of novobiocin. The doses of 9. Mattern, M. R., Paone, R. F., and Day, R. S., III. Eukaryotic DNA repair is novobiocin used (50 to 100 mg/kg) can be safely given to blocked at different steps by inhibitors of DNA topoisomerases and of DNA polymerases a and ÃŸ.Biochim.Biophys. Acta, 697: 6-13, 1982. humans. The therapeutic effect seen was significant and sug 10. Downes, C. S., Ord, M. J., Mulligan, A. M., Collins, A. R. S., and Johnson, gested a dose response effect for the alkylating agent. Moreover, R. T. Novobiocin inhibition of DNA excision repair may occur through since topoisomerase II is a proliferation-dependent enzyme effects on mitochondria! structure and ATP metabolism, not on repair topoisomerases. Carcinogenesis (Lond.), 6: 1343-1352, 1985. which could be affected by an antiproliferative but not cytotoxic 11. Catten, M., Bresnahan, D., Thompson, S., and Chalkly, R. Novobiocin effect of the drug combination, the tumor excision assay directly precipitates histones at concentrations normally used to inhibit eukaryotic measures cell kill. type II topoisomerase. Nucleic Acids Res., 14: 3671-3686, 1986. 12. Gottesfeld, J. Novobiocin inhibits RNA polymerase III transcription in vitro By treating animals with novobiocin prior to, during, and by a mechanism distinct from DNA topoisomerase II. Nucleic Acids Res., after treatment with each of three alkylating agents, we observed 14: 2075-2088, 1986. significant increases in tumor growth delay. In a single dose 13. Rice, L., Urano, M., and Suit, H. D. The radiosensitivity of a murine fibrosarcoma as measured by three cell survival assays. Br. J. Cancer, 41: protocol measuring tumor cell survival, again a significant effect 240-245, 1980. of the addition of novobiocin was seen. Since there was, in 14. Teicher, B. A., and Rose, C. M. Perfluorochemical emulsion can increase tumor radiosensitivity. Science (Wash. DC), 223:934-936, 1984. general, less increase in the toxicity to the bone marrow than 15. Pike, B. L., and Robinson, W. A. Human bone marrow colony growth in to the tumor, the addition of novobiocin to treatment with these agar-gel. J. Cell Physiol., 76: 77-84, 1970. alkylating drugs represents an increase in the therapeutic effect 16. Bradley, T. R., and Summer, M. A. Stimulation of mouse bone marrow colony growth in vitro by conditioned medium. Aust. J. Exp. Biol. Med. Sci., of the drugs if bone marrow is the dose-limiting tissue. 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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1989 American Association for Cancer Research. Effect of Novobiocin on the Antitumor Activity and Tumor Cell and Bone Marrow Survivals of Three Alkylating Agents

J. Paul Eder, Beverly A. Teicher, Sylvia A. Holden, et al.

Cancer Res 1989;49:595-598.

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