Preferential Activation of Mitomycin C to Cytotoxic Metabolites by Hypoxie Tumor Cells1

[CANCER RESEARCH 40. 2356-2360. July 1980] 0008-5472/80/0040-OOOOS02.00 Preferential Activation of Mitomycin C to Cytotoxic Metabolites by Hypoxie Tumor Cells1

Katherine A. Kennedy,2 Sara Rockwell, and Alan C. Salterelli

Departments of Pharmacology Â¡K.A. K.. A. C. S.Â¡and Therapeutic Radiology Â¡S.R.J and the Developmental Therapeutics Program, Comprehensive Cancer Center. Yale University School of Medicine, New Haven, Connecticut 06310

ABSTRACT rangement of the reduced molecule leads to the production of a highly reactive quinone methide intermediate (see Chart 1) Mitomycin C, a bioreductive alkylating agent with clinical which can alkylate DMA, RNA, protein, and other cellular utility against several human tumors, was found to be selec molecules. Schwartz (19) has demonstrated that the liver has tively toxic at a relatively low concentration (1.5 fiM) to EMT6 an enzymatic system capable of metabolizing mitomycin C tumor cells made chronically hypoxic by preincubation in 95% under anaerobic conditions. The enzyme system which carries N?-5% CO2 for 4 hr prior to drug exposure. This selective out the reduction is located in both the microsomal and nuclear cytotoxicity correlated well with the preferential activation and fractions (8). However, to exhibit selective cytotoxicity towards metabolism of mitomycin C by sonicated cell preparations. The hypoxic tumor cells, bioreductive alkylating agents such as bioactivation of mitomycin C to an alkylating agent by EMT6 mitomycin C must be activated by cancer cells in situ. and Sarcoma 180 cell sonicates required hypoxic conditions The data presented in this report demonstrate that mitomycin and a reduced nicotinamide adenine dinucleotide phosphate- C can be activated to an alkylating species under hypoxic generating system. Furthermore, the formation of reactive drug conditions in vitro by cells from 2 experimental mouse tumor metabolites and the disappearance of mitomycin C from the lines, Sarcoma 180 and EMT6. In addition, the cytotoxic effects reaction mixture were inhibited by carbon monoxide. The pres of mitomycin C were investigated using cells maintained under ence of potassium cyanide in the incubation mixture did not different states of oxygÃ©nation. At low drug concentrations, affect either the rate of overall metabolism or the rate of mitomycin C was found to be selectively toxic to hypoxic tumor formation of reactive metabolites. A high rate of disappearance cells in vitro. of mitomycin C from the medium of intact cultures of EMT6 cells was found only in those cultures which were made chron MATERIALS AND METHODS ically hypoxic. These data suggest that bioreductive alkylating agents like mitomycin C have the potential to attack selectively Drugs. Mitomycin was the gift of Dr. Maxwell Gordon of the the chemotherapeutically resistant hypoxic cell component of Bristol-Myers Co. (Syracuse, N. Y.). NADP+, glucose 6-phos- solid tumors. Thus, agents capable of bioreductive alkylation phate, and glucose-6-phosphate dehydrogenase were ob should be useful adjuncts to existing therapeutic regimens tained from Sigma Chemical Co. (St. Louis, Mo.). All other which are effective against well-oxygenated cells. reagents were obtained from standard chemical sources unless otherwise specified. INTRODUCTION Tumor Cells. Experiments using EMT6 mouse mammary tumor cells were performed in vitro. The techniques used for The bioreductive alkylating agents are a class of drugs which propagating the cells and measuring cell survival by colony require reductive biotransformation in order to exhibit alkylating formation have been described previously (16, 17). To obtain activity. Lin et al. (10, 11) postulated that hypoxic tumor cells sufficient quantities of cells for in vitro metabolism studies, the exist in environments which are conducive to the reductive EMT6 cells also were grown as spheroids. Approximately 3 x processes necessary to activate bioreductive alkylating agents. 106 cells were added to Optilux plastic Petri dishes containing As a result, hypoxic tumor cells would be expected to activate 20 ml of fresh Waymouth's medium (Grand Island Biological preferentially and therefore be selectively susceptible to drugs Co., Grand Island, N. Y.) supplemented with fetal bovine serum of this class. Because hypoxic tumor cells are relatively resis and antibiotics as specified previously (16). The medium was tant to radiotherapy and chemotherapy (9), agents directed changed twice a week for 3 weeks; the spheroids were then specifically against the hypoxic cell component of solid tumors harvested and washed with 0.9% NaCI solution before the cells would be extremely useful adjuncts to current cancer therapy. were prepared for use in metabolism studies. One clinically used agent which can function as a bioreduc Sarcoma 180 cells were collected as described previously tive alkylating agent is mitomycin C (Chart 1). It has been (1) from ascites fluid in the peritoneal cavities of female CD-1 shown previously that mitomycin C is activated to an alkylating mice (20 to 30 g) 7 days after implantation of 1 x 106 cells. species by reduction of the quinone moiety and subsequent The cells were washed 3 times with 0.9% NaCI solution to loss of the methoxy group (6, 7, 14, 20). Spontaneous rear- remove RBC and were then prepared for use in metabolism studies. ' This research was supported in part by USPHS Grants CA-02817 and CA- Metabolism Studies. EMT6 or Sarcoma 180 cells were 06519 from the National Cancer Institute and a grant from the Bristol-Myers suspended in 1 volume of distilled water and incubated for 10 Company. min on ice to facilitate disruption of the cells. After addition of ' Recipient of postdoctoral fellowship CA-06177 from the National Cancer Institute. To whom requests for reprints should be addressed. 1.8% NaCI solution to achieve isotonicity, the cell suspensions Received September 26. 1979; accepted April 3. 1980. were sonicated 3 times for 10 sec each using a Branson

2356 CANCER RESEARCH VOL. 40

zyDpyridine (Aldrich Chemical Co., Milwaukee, Wis.) were

enzyme added to the samples prior to incubation. After incubation, reduction each reaction was terminated by adding 2 ml of acetone and 1 ml of 1 M NaOH, and the sample was extracted immediately with 4 ml of ethyl acetate. The organic and aqueous phases were separated by centrifugation for 2 min at 1000 x g, and the absorbance of the organic layer at 540 nm was determined. The incubations and subsequent extractions for estimating the alkylating metabolites were carried out under subdued light. Metabolism studies were performed using exponentially growing monolayers of EMT6 cells, obtained by planting 5 x 105 cells in 25-sq cm Corning tissue culture flasks containing 5 ml of supplemented Waymouth's medium and incubating the flasks for 3 days in a humidified atmosphere of 95% air-5% CO?. The medium was changed just before the experiment. Mitomycin C was added to cells maintained under different states of oxygÃ©nationto produce a concentration of 0. 03 HIM, and the cells were incubated with the drug for 1 hr. At the end of the incubation period, the medium was removed and the protein was precipitated by the addition of 1 ml of saturated Ba(OH)? and 1 ml of 15% ZnSCv Mitomycin C concentrations in the supernatant were determined as described below. Cells Chart 1. Scheme for activation of mitomycin C to an alkylating species. were harvested by trypsinization and counted. The total number Reprinted from Ref. 9 with permission. of cells per flask was calculated, and the rates of metabolism were expressed as nmol of mitomycin C consumed per hr per sonicator at a setting of 25. The sonicated cell preparations 106 cells. were diluted with 0.1 M Tris-HCI buffer (pH 7.4) and used in Cytotoxicity Studies. Cytotoxicity studies were performed metabolism and alkylation assays. using exponentially growing monolayer cultures of EMT6 cells, Incubation mixtures for metabolism experiments with soni handled as described above. The medium was changed just cated cell preparations contained 0.72 /Â¿molNADP*, 5 /imol before the experiment. Cultures were incubated under different MgCI?, 5 /irnol glucose 6-phosphate, 1.25 units glucose-6- states of oxygÃ©nation and mitomycin C was added to the phosphate dehydrogenase in 0.1 M Tris-HCI buffer (pH 7.4), culture medium to produce a concentration of 1.5 /IM. After a and 2.0 mg cell sonicate protein. The total volume of the 1-hr incubation, the cells were harvested by trypsinization, and reaction mixture was 1.0 ml. Hypoxie conditions were achieved cell survival was assayed by colony formation in vitro as de by stoppering flasks and pregassing reaction mixtures for 10 scribed previously (16, 17). min with prepurified nitrogen which contained less than 10 ppm Hypoxic Conditions. Cells were made hypoxic by sealing oxygen (Matheson Gas, Rutherford, N. J.). Except as noted, 25-sq cm cell culture flasks with thick rubber sleeves. The all reactions were performed under a continuous flow of nitro flasks were then fitted with 20-gauge needles for inflow and gen. The incubation flasks were then warmed for 2 min to outflow of gases. The flasks were gassed continuously with a generate NADPH, and the reaction was initiated by the addition humidified mixture of 95% N?-5% CO? (Matheson Gas, Ruth of mitomycin C (0.30 rnw final concentration) dissolved in erford, N. J.). With this technique, drug could be added directly acetone. The NADPH-regenerating system continuously pro to cultures without breaking the hypoxia, by injecting a small vides sufficient quantities of NADPH for the reaction investi volume of drug through the sleeve. Normally aerated cultures gated in these experiments. Because the reaction catalyzed by were incubated in a humidified atmosphere of 95% air-5% glucose-6-phosphate dehydrogenase does not require O?, this CO?. Acutely hypoxic conditions were defined as those in method of producing NADPH is insensitive to the presence or which cultures were gassed with 95% N?-5% CO? for 1 hr, absence of O?. Furthermore, the absorbances of samples beginning immediately after the addition of drug to normally containing only the NADPH-regenerating system were similar aerated cultures. Chronically hypoxic cells were preincubated under aerobic and hypoxic (N?) atmospheres, suggesting that for 4 hr with a humidified atmosphere of 95% N?-5% CO? prior similar quantities of the cofactor were produced under these to the addition of drug. conditions. After 20 min, the incubations were stopped by Analyses. Protein was determined by the method of Lowry adding 1 ml of saturated Ba(OH)2 solution and 1 ml of 15% ef al. (12). Mitomycin C concentrations were estimated by ZnSO4 to precipitate protein. Mitomycin C concentrations in determining the absorbance of the quinone form of the drug at the supernatant were then determined as described below. 363 nm, and the measured absorbance was then related to Reactive metabolites of mitomycin C were estimated by a that obtained using known amounts of the drug. Any contribu modification of the method described by Wheeler et al. (23). tion from NADPH to the absorbance of mitomycin C at 363 nm Incubation mixtures used to estimate the relative amounts of was eliminated by determining drug-related absorbances biotransformation products with alkylating capabilities were the against appropriate samples containing only the NADPH-re same as those described for the metabolism experiments, generating system. The limit of sensitivity for the method was except that 10 /il of a 10% acetone solution of 4-(p-nitroben- 0.1 /ig/ml.

JULY 1980 2357

RESULTS

Mitomycin C was activated by sonicated cell preparations obtained from either Sarcoma 180 or EMT6 tumor cells as shown in Charts 2 and 3, respectively. The results are ex pressed as a percentage of the nitrogen control incubations. The rates of disappearance of mitomycin C from these control incubations were 1.00 Â±0.08 (S.E.) and 1.40 Â±0.22 nmol/ min/mg protein for the sonicated cell preparations obtained from Sarcoma 180 and EMT6 cells, respectively. Furthermore, the rates of formation of alkylating species were similar for Sarcoma 180 cell preparations (0.92 Â±0.10 AA64o/min/mg) Normal Acute Chronic Aeration Hypoxia Hypojuo and for EMT6 cell sonicates (1.13 Â±0.07 AA54o/min/mg). The disappearance of mitomycin C and the formation of alkylating Chart 4. Metabolism of mitomycin C by intact EMT6 cells in culture Cells metabolites, as measured with the trapping reagent 4-(p-nitro- were incubated with drug at a concentration of 0.03 mM under different states of oxygÃ©nation as described in the text. Results are expressed as the mean Â±S.E. benzyl)pyridine, were dependent upon both time and protein for 3 experiments. concentration being linear for 30 min under the conditions

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Untreated Normal Acute Chronic Control Aeration Hypoxia Hypoxio Atmosphere + + â€¢*â€¢ Mitomycin Mitomycin Mitomycin Conditions C C C Chart 5. Survival of EMT6 cells exposed to mitomycin C in vitro at a concen tration of 1.5 JIM under different states of oxygÃ©nation as described in the text. Chart 2 Metabolism and formation of alkylating metabolites of mitomycin C Results are expressed as the mean Â±S.E. tor 6 determinations. The cloning by sonicated preparations of Sarcoma 180 cells. Results are expressed as the efficiency of untreated cells exposed to air-5% CO2 was 61% mean Â±S.E. of at least 5 experiments for the formation of alkylating metabolites (hatched bars) and for the disappearance of mitomycin C from the reaction mixture (solid bars). Drug was present at a final concentration of 0.3 mM. Control used at protein concentrations up to 5 mg/ml (data not shown). values were 1.00 Â±0.08 nmol mitomycin C consumed per min per mg protein As shown in Charts 2 and 3, the metabolism of mitomycin C, for metabolism and 0.92 Â±0.10 AA54o per min per mg for the formation of alkylating metabolites. as determined by its disappearance from the incubation mix ture, required an NADPH-generating system, was inhibited

120 strongly by oxygen, was not affected by 0.5 mM potassium cyanide (Chart 3), and was inhibited by carbon monoxide lOO (Chart 3). The effect of carbon monoxide on mitomycin C metabolism could be reversed partially with high concentra 80 tions (1 mM) of drug (data not shown). Those manipulations which inhibited the rate of disappearance of mitomycin C also 5 60 depressed the rate of formation of metabolites capable of f alkylation. These results suggest that the generation of reactive 40 metabolites by sonicated tumor cell preparations was related

20 to, or was the direct consequence of, the metabolism of the antibiotic. 0 To examine the relationship between the utilization of mito Atmosphere "2 mycin C and the cytotoxic activity of the drug, the disappear Conditions +NAOPH + NADPH â€¢fNAOPH +NADPH +NADPH + ceil + cell + boned cell * cell + cell ance of mitomycin C from the medium and the cytotoxicity of tomcatÂ« somcott the antibiotic were determined using EMT6 cell cultures incu Chart 3. Metabolism and formation of alkylating metabolites of mitomycin C bated under different states of oxygÃ©nation. The spectropho- by sonicated preparations of EMT6 cells. Results are expressed as the mean Â± tometric assay for mitomycin C was relatively* insensitive; this S.E. of at least 3 experiments for the formation of alkylating metabolites (hatched bars) and for the disappearance of mitomycin C from the reaction mixture (solid necessitated studying the metabolism of mitomycin C at con bars) Drug was present at a final concentration of 0.3 mM. Control values were centrations of 0.03 mM in the medium. Only under conditions 1.40 Â±0.22 nmol mitomycin C consumed per min per mg protein for metabolism of chronic hypoxia was there a high rate of drug disappearance and 1.13 Â± 0.07 AA , per min per mg protein for formation of alkylating metabolites. from the culture medium (Chart 4). In contrast, under conditions

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1980 American Association for Cancer Research. Mitomycin Activation by Hypoxie Tumor Cells in which significant oxygen concentrations existed during at monoxide, however, supports the concept that a cytochrome least part of the incubation period of the cells with mitomycin P-450 enzyme system may be involved. Further work with C (i.e., normal aeration and acute hypoxia), relatively little drug other inhibitors will be required to elucidate conclusively the disappeared from the medium. enzymatic machinery responsible for the biotransformation of Chart 5 shows the fraction of EMT6 tumor cells that survived mitomycin C. exposure for 1 hr to 1.5 fiM mitomycin C under various states It had been hypothesized by Lin ef al. (10, 11) that, if of oxygÃ©nation. Results are expressed as a percentage of mitomycin C is activated preferentially under hypoxic condi untreated control cultures, which had a cloning efficiency of tions, the drug should be selectively toxic to hypoxic tumor 61%. Untreated hypoxic cell cultures (both acutely and chron cells. The data presented in this report show that cells made ically hypoxic cultures) were included in each experiment. In hypoxic in culture are much more sensitive to the cytotoxic the absence of mitomycin C, the cloning efficiencies of the actions of mitomycin C than are cells maintained under normal hypoxic cells were equivalent to those of the untreated normally aeration. Unpublished work in our laboratory has demonstrated aerated cells. As can be seen in Chart 5, the surviving fractions that the selective toxicity of mitomycin C towards hypoxic tumor of cells exposed to mitomycin C under normal aeration or acute cells is exhibited over a concentration range of 10~9 to 10~5 hypoxia were similar. However, cells exposed to mitomycin C M. Previous experiments have shown that EMT6 cell cultures under conditions of chronic hypoxia showed a significantly made hypoxic by the technique used in these experiments lower surviving fraction that did normally aerated or acutely have radiation dose-response curves similar to those of EMT6 hypoxic cells. cells rendered severely hypoxic in vitro by more rigorous techniques (18). Cultures gassed for 1 hr (acutely hypoxic DISCUSSION cells) become radiobiologically hypoxic only during the end of their incubation period. Neither the gassing procedure per se The hypoxic cells of solid tumors are known to limit the nor the moderate hypoxia that developed during the early part curability of experimental animal tumors by large doses of of these incubations altered the metabolism and cytotoxicity of radiation (9). Hypoxic cells may also be relatively resistant to mitomycin C from that seen in the aerobic cultures. Severe conventional chemotherapy for several reasons. Because hy hypoxia throughout the 1-hr incubation with the drug, however, poxic tumor cells exist at relatively great distances from blood resulted in increased metabolism and cytotoxicity. The similar vessels, drugs with physicochemical properties not conducive ity of the radiation dose-response curves for hypoxic EMT6 to diffusion through tissue may not reach the hypoxic regions cells in culture and in solid tumors (18) suggests that the of the tumor and, therefore, may be ineffective in attacking chronically hypoxic cells employed in the system used in these hypoxic cells. In addition, agents which are rapidly metabolized studies represent an acceptable model for the radiation-resist and cleared from the body may not reach hypoxic cells in ant, hypoxic cells of solid tumors. sufficient concentrations to produce biological effects. Fur The data obtained by examining the disappearance of the thermore, many hypoxic tumor cells are probably quiescent or drug from the culture medium of intact cells is consistent with are cycling with prolonged, abnormal cell cycle times. As a a markedly greater rate of metabolism of mitomycin C by cells result, these cells may not be susceptible to those anticancer maintained under conditions of chronic hypoxia. For method drugs which are active only against proliferating cells. Chemical ological reasons, these data were obtained at a much higher agents directed specifically against hypoxic tumor cells would drug level than that used in the cell survival studies. Although therefore be valuable adjuncts to both existing chemotherapy the generation of metabolites with alkylating potential could not and radiotherapy. be measured directly in the experiments with intact cells, the Bioreductive alkylating agents are envisioned to be a series results are consistent with the findings from the sonicated cell of drugs directed against hypoxic tumor cells. These agents preparations and indicate that an increase in the rate of drug were hypothesized (10, 11) to be activated selectively by disappearance from the culture medium occurred only under hypoxic tumor cells because these cells exist in environments conditions of chronic hypoxia and that oxygen strongly in which are conducive to reductive processes. The results pre hibited the reaction. Because drug uptake into cells maintained sented in this paper using the prototype bioreductive alkylating under normal aeration or hypoxia could not be determined, it agent, mitomycin C, strongly support this conceptual basis for is possible that differences in Â¡ntracellular steady-state levels a selective chemotherapeutic attack on hypoxic cells. Soni of drug under these 2 conditions may contribute to the overall cated preparations from EMT6 and Sarcoma 180 cells were rate of drug disappearance from the medium. Integration of all able to metabolize mitomycin C, and the biotransformation of of these results suggests that the enhanced cytotoxic activity the drug by the cell preparations appeared to result in the of mitomycin C under hypoxic conditions is the consequence production of a metabolite(s) with alkylating properties, as is of increased metabolic activation of this drug to an alkylating evidenced by the formation of an alkylated derivative of 4-(p- species. nitrobenzyl)pyridine. The enzyme system present in these ma Although mitomycin C is selectively toxic to hypoxic cells at lignant cells has properties similar to that responsible for mi low concentrations, this agent is capable of killing well-oxygen tomycin C metabolism and activation in the liver (8, 9, 19). The ated cells at higher levels of drug. Bachur ef al. (2, 3) have tumor cell enzyme(s) which activates mitomycin C requires reported that mitomycin C can undergo 1-electron reduction pyridine nucleotide reducing equivalents and is sensitive to and subsequent reoxidation by molecular oxygen to generate oxygen. The lack of effect by potassium cyanide on the metab Superoxide radicals. Superoxide anions, in turn, could lead to olism of the drug suggests that mitochondrial processes were other oxygen-containing species, such as hydrogen peroxide not involved in the activation of mitomycin C by neoplastic and the hydroxyl radical (4), and could result in significant cells. The inhibition of mitomycin C metabolism by carbon cytotoxicity if the cells could not effectively detoxify these

JULY 1980 2359

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1980 American Association for Cancer Research. K. A. Kennedy et al. reactive forms (15). This cyclic reduction and oxidation of mammalian cells in vitro. Cancer Res., 30. 644-649, 1978. mitomycin C by well-oxygenated cells may be responsible for 6. Iyer. V. N.. and Szybalski, W. A molecular mechanism of mitomycin action: linking of complimentary DMA strands. Proc. Nati. Acad. Sei. U. S A.. 50 the toxicity of the drug to aerated cells in tumors and normal 355-362. 1963. tissues. Because normal tissues are well oxygenated and un 7. Iyer, V. N.. and Szybalski, W. Mitomycin and porfiromycin: chemical mech anism of activation and cross-linking of DNA. Science (Wash. D. C.), 145: likely to activate the drug by bioreductive mechanisms, it is 55-58. 1964. possible that the clinical use of mitomycin C could be improved 8. Kennedy, K. A., and Sartorelli, A. C. Metabolic activation of mitomycin C by by the use of more appropriate treatment regimens which isolated liver nuclei and microsomes. Fed. Proc.. 30. 443. 1979. 9. Kennedy, K. A., Teicher, B. A., Rockwell, S., and Sartorelli. A. C. The would take advantage of the selectivity of this agent at low hypoxic tumor cell: a target for selective cancer chemotherapy. Biochem. concentrations for hypoxic cells. Pharmacol.. 29. 1-8, 1980. 10. Lin, A. J., Cosby, L. A., and Sartorelli. A. C. Potential bioreductive alkylating Agents other than mitomycin C are known to be activated to agents. In: A. C. Sartorelli (ed.). Cancer Chemotherapy, pp 71-86. Wash cytotoxic metabolites under hypoxic conditions. Studies by ington. D. C.: American Chemical Society. 1976. several workers have shown that CHO and Ehrlich ascites cells 11. Lin. A. J., Pardini, R S., Cosby. L. A.. Lillis. B. J.. Shansky. C. W.. and Sartorelli. A. C. Potential bioreductive alkylating agents. 2. Antitumor effect made hypoxic in vitro can reduce misonidazole to the corre and biochemical studies of naphthoquinone derivatives. J. Med. Chem.. 16: sponding amine and generate hydroxylamine and/or a nitro- 1268-1271. 1973. radical aniÃ³n as cytotoxic species (5, 13, 22). Although origi 12. Lowry, O. H., Rosebrough, N. J.. Farr, A. L., and Randall, R. J. Protein measurements with the Folin phenol reagent J. Biol. Chem.. 793 265-275. nally envisioned solely as agents which would sensitize hypoxic 1951. cells to radiation, recent experimental results have suggested 13. Mohindra, J. K., and Rauth. A. M. Increased cell killing by metronidazole and nitrofurazone of hypoxic compared to aerobic mammalian cells. Cancer a possible role for the nitroimidazoles as chemotherapeutic Res., 36. 930-936. 1976. agents directed against hypoxic tumor cells to be used in 14 Moore, H. W. 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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1980 American Association for Cancer Research. Preferential Activation of Mitomycin C to Cytotoxic Metabolites by Hypoxic Tumor Cells

Katherine A. Kennedy, Sara Rockwell and Alan C. Sartorelli

Cancer Res 1980;40:2356-2360.

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