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The clinical success of mitomycin C (1) and its associated toxicities and resistance have led to efforts to prepare semisynthetic analogues (i.e., KW-2149 (3), BMS-181174 (4)) that have improved pharmacological profiles. In this study, we report the preparation and evaluation of the novel 7-N-(1'-amino-4',5'-dithian-2'-yl)porfiromycin C(8) cyclized imine (6) and its reference compound, 7-N-(1'-aminocyclohex-2'-yl)porfiromycin C(8) cyclized imine (13). Porfiromycin 6 contains a disulfide unit that, upon cleavage, may provide thiol(s) that affect drug reactivity. We demonstrated that phosphines dramatically accelerated 6 activation and solvolysis in methanolic solutions ("pH 7.4") compared with 13. Porfiromycins 6 and 13 efficiently cross-linked EcoRI-linearized pBR322 DNA upon addition of Et3P. We found enhanced levels of interstrand cross-link (ISC) adducts for 6 and 13 compared with porfiromycin (7) and that 6 was more efficient than 13. The large Et3P-mediated rate enhancements for the solvolysis of 6 compared with 13 and a N(7)-substituted analogue of 1, and the increased levels of ISC adducts for 6 compared with 13 and 7 are attributed to a nucleophile-assisted disulfide cleavage process that permits porfiromycin activation and nucleophile (MeOH, DNA) adduction. The in vitro antiproliferative activities of 6 and 13 using the A549 tumor cell line (lung adenocarcinoma) were determined under aerobic and hypoxic conditions and then compared with 7. Both 6 and 13 were more cytotoxic than 7, with 13 being more potent than 6. The C(8) iminoporfiromycins 6 and 13 displayed anticancer profiles similar to 3.

The mitomycin C-resistance gene, mcrA, of Streptomyces lavendulae produces MCRA, a protein that protects this microorganism from its own antibiotic, the antitumor drug mitomycin C. Expression of the bacterial mcrA gene in mammalian Chinese hamster ovary cells causes profound resistance to mitomycin C and to its structurally related analog porfiromycin under aerobic conditions but produces little change in drug sensitivity under hypoxia. The mitomycins are prodrugs that are enzymatically reduced and activated intracellularly, producing cytotoxic semiquinone anion radical and hydroquinone reduction intermediates. In vitro, MCRA protects DNA from cross-linking by the hydroquinone reduction intermediate of these mitomycins by oxidizing the hydroquinone back to the parent molecule; thus, MCRA acts as a hydroquinone oxidase. These findings suggest potential therapeutic applications for MCRA in the treatment of cancer with the mitomycins and imply that intrinsic or selected mitomycin C resistance in mammalian cells may not be due solely to decreased bioactivation, as has been hypothesized previously, but instead could involve an MCRA-like mechanism.

Mitomycin C (1) is the prototypical bioreductive alkylating agent. Studies have shown that mitomycin C and its derivatives selectively alkylate guanine residues within di- and trinucleotide DNA sequences. This investigation sought to improve the selective DNA bonding properties of the mitomycins by coupling them with antisense oligodeoxynucleotides. Two procedures were developed that allowed the attachment of a phosphorothioate oligodeoxynucleotide containing a hexylamino spacer at the 5' terminus with a C(10)-activated mitomycin. In the first procedure, decarbamoylation of 1 (NaOCH3/ benzene) afforded 10-decarbamoylmitomycin C (10), which was treated with either dimethyl sulfate or methylthiochloroformate and base to yield 10-decarbamoylporfiromycin (11) and N(1a)-[(methylthio)-carbonyl]-10-decarbamoylmitomycin C (12), respectively. Activation of the C(10) site in 11 and 12 with 1,1'-carbonyldiimidazole or with 1,1'-thiocarbonyldiimidazole provided the N(1a)-substituted mitomycin 10-decarbamoyl-10-O-carbonylimidazoles (5, 7) and 10-decarbamoyl-10-O-thiocarbonylimidazoles (6, 8), respectively. Compounds 5-8 were reacted with glycine methyl ester hydrochloride (17) and base in both methylene chloride and aqueous buffered solutions to determine the ease and efficiency in which these C(10)-activated mitomycin derivatives coupled to amines. It was found that 5-8 all reacted with 17 in methylene chloride to give the coupled products 18-21 but that improved amine coupling yields in water were observed for the 10-decarbamoyl-10-O-thiocarbonylimidazoles 6 and 8 as compared with the 10-decarbamoyl-10-O-carbonylimidazoles 5 and 7. This finding led to the coupling of the phosphorothioate oligodeoxynucleotide, H2N(CH2)6-P(S)(OH)-GGCCCCGTG-GTGGCTCCAT (22) to 8. Compound 22 complemented a 19-base sequence in the translation initiation region of the human A-raf-1 gene. Use of excess 8 (28 equiv) with 22 gave only a 36% yield of the coupled product 23, which proved difficult to separate from 22. In the second procedure, phosphorothioate oligodexynucleotides that contained a hexylamino spacer at the 5'termini were coupled to 10-des(carbamoyloxy)-10-isothiocyanatoporfiromycin (9). Compound 9 was prepared in four steps from 11. Mesylation (methanesulfonyl chloride/pyridine) of 11 gave the C(10) mesylate 13, which was then treated with NaN3 (dimethylformamide, 90 degrees C) to give 10-des(carbamoyloxy)-10-azidoporfiromycin (14). Catalytic reduction (PtO2, H2) of 14 in pyridine afforded C(10) amine 15. Treatment of 15 with di-2-pyridyl thionocarbonate provided the desired 10-des(carbamoyloxy)-10-isothiocyanatoporfiromycin (9). Compound 9 readily coupled with 17 and base in both methylene chloride and aqueous buffered solutions to give 25. Use of the 5'hexylaminophosphorothioate oligodeoxynucleotides 32-35 in place of 17 gave the conjugated adducts 28-31, respectively, in a 12% to near-quantitative yield. The products were purified by semipreparative HPLC. Antisense agents 28-31 were designed to target a 30-base-long region from the coding region of the human FGFR1 gene. One adduct, 29, reduced the number of FGFR1 receptors in human aortic smooth cells for bFGF on the cell surface, which suggested down-regulation of FGFR1 gene expression. Further, 29 inhibited cultured human aortic smooth muscle cell proliferation and was less cytotoxic than porfiromycin (2). The biological assay data suggest that the phosphorothioate oligodexynucleotide porfiromycin conjugates may be more target selective and less toxic than either mitomycin or porfiromycin and thus be promising therapeutic agents.

Hypoxic cells in solid tumors represent a therapeutically resistant population that limits the curability of many solid tumors by irradiation and by most chemotherapeutic agents. The oxygen deficit, however, creates an environment conducive to reductive processes; this results in a major exploitable difference between normal and neoplastic tissues. The mitomycin antibiotics can be reductively activated by a number of oxidoreductases, in a process required for the production of their therapeutic effects. Preferential activation of these drugs under hypoxia and greater toxicity to oxygen-deficient cells than to their oxygenated counterparts are obtained in most instances. The demonstration that mitomycin C and porfiromycin, used to kill the hypoxic fraction, in combination with irradiation, to eradicate the oxygenated portion of the tumor, produced enhanced cytodestructive effects on solid tumors in animals has led to the clinical evaluation of the mitomycins in combination with radiation therapy in patients with head and neck cancer. The findings from these clinical trials have demonstrated the value of directing a concerted therapeutic attack on the hypoxic fraction of solid tumors as an approach toward enhancing the curability of localized neoplasms by irradiation.

Laboratory studies and clinical trials are exploring the use of hypoxia-directed cytotoxic agents as adjuncts to radiotherapy. Because hypoxia and the microenvironmental inadequacies associated with hypoxia in solid tumours inhibit cell proliferation, an essential requirement for the successful use of hypoxia-directed drugs in cancer therapy is that these drugs be toxic to quiescent tumour cells, as well as tumour cells progressing rapidly through the cell cycle. The experiments reported here compared the cytotoxicities of mitomycin C and porfiromycin to exponentially growing and plateau phase cultures of EMT6 mouse mammary tumour cells. The proliferative status of the cultures did not influence the cytotoxicity of mitomycin C under either aerobic or hypoxic conditions, or the cytotoxicity of porfiromycin in air. Exponentially growing cultures were slightly more sensitive than plateau phase cultures to porfiromycin in hypoxia, but the difference between the sensitivities of proliferating and quiescent cells was much smaller than the difference between aerobic and hypoxic cells. No evidence for repair of potentially lethal damage was found after treatment with porfiromycin in air or in hypoxia; this is in agreement with previous findings for mitomycin C. Mitomycin C and porfiromycin therefore exhibit the toxicity to quiescent cells needed for effective use as hypoxia-directed drugs for the treatment of solid tumours.

Mitomycin C and porfiromycin were found to inactivate rat hepatic DT-diaphorase. Inactivation was pH dependent; little inactivation was detected at pH 5.8, but inactivation increased as the pH was raised to 7.8. Inactivation was concentration and time dependent and displayed pseudo-first-order kinetics. Inactivation was NADH dependent, indicating that reductive metabolism was necessary for inhibition. [3H]Mitomycin C was covalently bound to DT-diaphorase during inhibition, and the stoichiometry for inactivation of DT-diaphorase by mitomycin C was approximately 0.8 nmol of mitomycin C bound/nmol of enzyme. A higher molecular mass product (60 kDa) was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis of DT-diaphorase preincubated with NADH and mitomycin C at pH 7.8, suggesting that mitomycin C is capable of cross-linking DT-diaphorase. The kinetics of inhibition, requirement for NADH for inhibition, covalent binding of [3H] mitomycin C to DT-diaphorase, and approximate 1:1 stoichiometry suggest that this inactivation process may be mechanism based. Inhibition of DT-diaphorase by mitomycin C and porfiromycin is not limited to a cell-free system and could also be observed in HT-29 cells in culture at pH 7.2. Bioactivation of mitomycin C or porfiromycin by DT-diaphorase is favored at lower pH, whereas at higher pH values enzyme alkylation and inactivation of DT-diaphorase occur. These data suggest that the success of attempts to exploit the elevated DT-diaphorase content of certain human tumors for improved chemotherapeutic response using mitomycin C or porfiromycin will depend on intracellular pH.

The mitomycin antibiotics, because of their preferential toxicities for hypoxic cells, have significant potential as adjuncts to ionizing radiation in the treatment of solid tumors. To gain information on the mechanism by which these agents exert their cytotoxicities to hypoxic and aerobic cells, the effects of MC, POR and several of their analogs were studied in EMT6 mammary carcinoma cells. The rate of uptake of POR by these cells was directly correlated with the cytotoxicity produced by this agent under both hypoxia and aeration. At equivalent concentrations, uptake of POR into hypoxic cells was more rapid than into aerobic cells. Hypoxic cells also accumulated the antibiotic in concentrations well in excess of that present in the extracellular medium, presumably as a result of reductive activation and covalent binding of POR to cellular structures. Such activation and binding occur to a much lesser degree in aerated cells, resulting in the rapid efflux of POR from these cells when the antibiotic is removed from the extracellular environment. To gain information on the reaction of POR with DNA, mono- and bis-adducts formed in EMT6 cells exposed to this agent were measured. Three major adducts were formed. Two were mono-adducts consisting of deoxyguanosine linked at its N2-position to the C-1 of POR and of 10-decarbamoyl POR. The third was a bis-adduct in which POR was cross-linked to two deoxyguanosines at their N2-positions. More adducts were formed in hypoxia than in air, and more bis-adducts were present in hypoxic cells. Simultaneous exposure of cells to both POR and DIC reduced the total adduct level and a new unknown adduct was formed, primarily under hypoxia. Several mitomycins were evaluated for their capacity to kill EMT6 cells and to produce DNA cross-links in both hypoxia and aeration. The number of cross-links required to produce a given amount of cell kill was similar, regardless of the mitomycin employed or the degree of oxygenation. The findings support the concept that DNA is a critical target in the action of the mitomycins and that cross-linking of the DNA creates an important lesion for cytodestruction.

15 human tumour cell lines (lung, breast and colon) have been evaluated for their sensitivity to the quinone based anti-cancer drugs Mitomycin C, Porfiromycin, and EO9 (3-hydroxymethyl-5-aziridinyl-1-methyl-2-(IH-indole-4,7-dione)prop-beta- en-alpha-ol). Sensitivity has been compared with the intra-cellular levels of DT-diaphorase, an enzyme thought to be important in the reductive activation of these quinones. No correlation exists between levels of DT-diaphorase and sensitivity to Mitomycin C or Porfiromycin. However, for EO9 those cell lines showing highest levels of DT-diaphorase activity tend to be the most sensitive.

A mitomycin C (MMC)- and porfiromycin (PFM)-resistant subline of the HCT 116 human colon-cancer cell line was isolated after repeated exposure of HCT 116 cells to increasing concentrations of MMC under aerobic conditions. The MMC-resistant subline (designated HCT 116-R30A) was 5 times more resistant than the parent cells to MMC and PFM under aerobic conditions. Both the MMC-resistant cells and the parent HCT 116 cells accumulated similar amounts of PFM by passive diffusion, but levels of macromolecule-bound PFM were about 50% lower in the resistant cell line, implying a decrease in PFM reductive activation in the resistant cells. The finding that microsomes from either sensitive or resistant cells showed an equal ability to reduce MMC and PFM indicated that the activity of NADPH cytochrome P-450 reductase (EC 1.6.2.4) was not changed in the resistant subline. Soluble extracts of HCT 116 cells reduced MMC and PFM more effectively at pH 6.1, and NADH and NADPH were utilized equally well as electron donors under both aerobic and anaerobic conditions. These data suggest that quinone reductase (EC 1.6.99.2; DT-diaphorase) in soluble extracts is responsible for the reduction of MMC. Quinone reductase activities in soluble extracts of HCT 116-R30A cells for the reduction of dichlorophenol indophenol (DCPIP) and menadione-cytochrome c at optimal pHs were decreased by 95% as compared with those obtained in parent cells. However, the MMC-reducing activity of HCT 116-R30A soluble extracts was only 50% lower than that of the parent cell extracts. The kinetic constants (Km, Vmax) found for quinone reductase in the two cell lines with respect to the substrates DCPIP and menadione differed. Two species of mRNA for quinone reductase (2.7 and 1.2 kb) were detected in both cell lines, and there was no detectable difference between parent and resistant cells in the steady-state level of either of these mRNA species. Furthermore, incubation with the quinone reductase inhibitor dicoumarol rendered HCT 116 cells more resistant to MMC. Alteration of the quinone reductase activity in HCT 116-R30A cells appears to be the mechanism responsible for their resistance to MMC and PFM.

Stable acquired resistance to etoposide (VP-16) or teniposide (VM-26) in HCT116 human colon carcinoma cells and A549 human lung adenocarcinoma cells, was previously obtained by weekly 1-h exposures to either drug (B. H. Long, Natl. Cancer Inst. Monogr., 4: 123-127, 1987). The purpose of this study was to identify possible mechanisms of resistance present in these cells by using human mdr1 and topoisomerase II DNA probes, antibodies to these gene products, and P4 phage unknotting assay for topoisomerase II activities. HCT116(VP)35 cells were 9-, 7-, and 6-fold resistant to VP-16, VM-26, and Adriamycin, respectively, and showed no cross-resistance to colchicine and actinomycin D. These cells had no differences in mdr1 gene, mdr1 mRNA, or P-glycoprotein levels but displayed decreased levels of topoisomerase II mRNA and enzyme activity without any alteration of drug sensitivity displayed by the enzyme. HCT116(VM)34 cells were 5-, 7-, and 21-fold resistant to VP-16, VM-26, and Adriamycin; were cross-resistant to colchicine (7-fold) and actinomycin D (18-fold); and possessed a 9-fold increase in mdr1 mRNA and increased P-glycoprotein without evidence of mdr1 gene amplification. No alterations in topoisomerase II gene or mRNA levels, enzyme activity, or drug sensitivity were observed. A549(VP)28 and A549(VM)28 cells were 8-fold resistant to VP-16 and VM-26 and 3-fold resistant to Adriamycin. Both lines were not cross-resistant to colchicine or actinomycin D but were hypersensitive to cis-platinum. No alterations in mdr1 gene, mdr1 mRNA, or P-glycoprotein levels, but lower topoisomerase II mRNA levels and decreased enzyme activities, were observed. Of the four acquired resistant cell lines, resistance is likely related to elevated mdr1 expression in one line and to decreased topoisomerase II expression in the other three lines.

The mechanism of aerobic resistance to the quinone-containing anti-tumour agents mitomycin C (MMC) and porfiromycin (PM) has been investigated using non-transformed human cells. One of the cell strains used (3437T) was derived from an afflicted member of a cancer-prone family. This cell strain had been shown previously to be six times more resistant to the cytotoxic effects of these agents under aerobic but not hypoxic conditions when compared to a cell strain derived from an unrelated, normal donor (GM38). Differences could not be detected in the ability of cell sonicates prepared from either cell strain to produce alkylating species under aerobic conditions using a 4-(p-nitrobenzyl)pyridine assay. However, using 3H-labelled PM to monitor rapid drug uptake and subsequent accumulation due to drug metabolism, results were obtained indicating that the resistant cell strain (3437T) was deficient in an enzymatic pathway capable of metabolizing these compounds under aerobic but not hypoxic conditions. Dicumarol, an inhibitor of the quinone reductase DT-diaphorase (EC 1.6.99.2), decreased aerobic drug accumulation and cytotoxicity in the control cell strain, but did not alter the lack of accumulation noted in the resistant cell strain. Under hypoxic conditions, dicumarol increased cytotoxicity and drug accumulation in both cell strains. The mechanism of this enhanced cytotoxicity remains unclear. These results suggested that the resistant cells were deficient in the enzyme DT-diaphorase, a potential activator of PM. Enzymatic assays confirmed this and revealed no alterations in cytochrome P450 reductase (EC 1.6.2.4) activity or glutathione content. No protein characteristic of DT-diaphorase was detected in the resistant cell strain using a polyclonal rabbit-anti-rat antibody raised against this enzyme. Southern blot analysis using a rat DT-diaphorase cDNA probe demonstrated differences between the normal and resistant cell strains in the restriction fragment patterns. The present results are consistent with the hypothesis that decreased DT-diaphorase levels are causally associated with PM and MMC resistance in these cells under aerobic exposure conditions.

In an effort to develop effective combination treatments for use with radiation against solid tumors, the cytotoxic effects of the addition of mitomycin C or porfiromycin on treatment with Fluosol-DA/carbogen (95% O2/5% CO2) breathing and radiation in the FSaIIC tumor system were studied. In vitro mitomycin C and porfiromycin were both preferentially cytotoxic toward hypoxic FSaIIC cells. After in vivo exposure, however, the cytotoxicity of mitomycin C toward single cell tumor suspensions obtained from whole tumors was exponential over the dose range studied, but for porfiromycin a plateau in cell killing was observed. With Fluosol-DA/carbogen breathing and single dose radiation, addition of either mitomycin C or porfiromycin increased the tumor cell kill achieved at 5 Gy by approximately 1.2 and 1.0 logs, respectively. Less effect was seen with addition of the drugs at the 10 and 15 Gy radiation doses. In tumor growth delay experiments, the addition of either mitomycin C or porfiromycin to Fluosol-DA/carbogen breathing and radiation resulted in primarily an additive increase in tumor growth delay. The survival of Hoechst 33342 dye-selected tumor cell subpopulations indicated that Fluosol-DA/carbogen breathing increased the cytotoxicity of radiation (10 Gy) more in the bright cell subpopulation (4-fold) than in the dim cell subpopulation (2-fold) resulting in an overall 4-fold sparing of the dim subpopulation. Mitomycin C and porfiromycin were both more toxic toward the dim cell subpopulations. Addition of mitomycin C or porfiromycin to Fluosol-DA/carbogen breathing and radiation (10 Gy) resulted in a primarily additive effect of the drugs and radiation killing in both tumor cell subpopulations. Thus, with mitomycin C/Fluosol-DA/carbogen and radiation there was a 2-fold sparing of dim cells and with porfiromycin in the combined treatment a 1.6-fold sparing of the dim cell population. Our results indicate that treatment strategies directed against both oxic and hypoxic tumor subpopulations can markedly increase the tumor cell kill achieved by radiation.

The cytotoxicity, metabolism, and DNA alkylation of porfiromycin (PFM) under aerobic and hypoxic conditions were evaluated in P388 murine leukemia cells. Clonogenic assays showed that the IC50 value for a 1-h exposure to PFM was 4 microM for aerobic cells and 0.5 microM for hypoxic cells. After a 1-h exposure to concentrations of 1, 5, and 10 microM [14C]-PFM, the accumulation of total radioactivity in hypoxic cells was 10 to 20 times that in aerobic cells. The disposition of radioactivity in cells that had been treated for 1 h with 5 microM PFM under aerobic or hypoxic conditions showed that (a) under either condition, internal free-PFM concentration equalled the external drug concentration; (b) DNA-, RNA-, and protein-bound radioactivity were at least 10 times greater in hypoxic cells than in aerobic cells; and (c) known metabolites and unidentified radioactive products were also generated in greater amounts in hypoxic cells than in aerobic cells. Thus, the increased amounts of radioactivity accumulated by hypoxic P388 cells after exposure to [14C]-PFM resulted from the accumulation of nonexchangeable protein and nucleic-acid adducts and metabolites rather than free PFM. Determinations of DNA adducts formed in P388 cells revealed five possible adducts: (1) N2-(2'-deoxyguanosyl)-7-methylaminomitosene, (2) a second monofunctional PFM-guanine adduct, (3) a PFM cross-linked dinucleotide, (4) possibly a nucleoprotein-related adduct, and (5) an unknown. We conclude that the enhancement of PFM-induced cytotoxicity by hypoxia appears to be primarily due to increased alkylation of macromolecules.

Mitomycin C and its methylated analogue porfiromycin (Por) have significant potential as adjuncts to regimens presently used for treating solid tumors because of their preferential toxicity to cells existing in an hypoxic environment. An understanding of the factors producing the differential activity of these drugs under aerobic and hypoxic conditions would facilitate the development of new agents of this class. Previous studies have focused on the enzymes that reductively activate the mitomycins and on the interaction of these drugs with DNA; none of these studies has fully explained the differences in cytotoxicity observed under hypoxic and aerobic conditions. The present investigation demonstrates that the rate of Por uptake is directly correlated with cytotoxicity under both aerobic and hypoxic conditions. Uptake of Por into hypoxic cells is more rapid than into aerobic cells at equal drug concentrations. Hypoxic cells also accumulate drug in concentrations well in excess of those in the extracellular medium; this is apparently a reflection of drug sequestration in these cells. This sequestration of Por, which affects the rate and extent of uptake in hypoxic cells, does not take place in aerobic cells. The failure of aerobic cells to sequester drug is evidenced by the very rapid efflux of Por from these cells upon removal of extracellular Por and by the fact that aerobic cells attain a state of equilibrium between the intracellular and extracellular drug concentrations. The findings demonstrate that differences in the uptake and retention of Por are associated with the preferential toxicity of Por to hypoxic cells.

We have demonstrated previously that dicoumarol (DIC) increased the generation of reactive metabolites from mitomycin C (MC) in EMT6 cells under hypoxic conditions in vitro. This increased reaction rate was associated with an increased toxicity of MC to hypoxic EMT6 cells. In contrast, aerobic cells treated with DIC in vitro were protected from MC toxicity. We now demonstrate that DIC sensitizes EMT6 cells to two MC analogues, porfiromycin (POR) and the 7-N-dimethylaminomethylene analogue of mitomycin C (BMY-25282), in hypoxia and protects cells from these agents in air, despite the fact that POR is preferentially toxic to hypoxic cells and BMY-25282 is preferentially toxic to aerobic cells. In contrast, DIC increases menadione cytotoxicity in both air and hypoxia and has no effect on the cytotoxicity of Adriamycin. We have also shown previously that the preferential toxicity of POR to hypoxic cells is associated with an increased rate of drug uptake. In the present study, DIC had no measurable effect on the uptake of [3H]POR but increased the extent of efflux of this agent. MC-induced DNA cross-links, which have been proposed as the lesions responsible for the lethality of MC, are decreased by DIC in air and increased by DIC in hypoxia, in concert with the observed modifications of MC cytotoxicity by DIC. However, in aerobic cells treated with DIC and MC, the decrease in DNA interstrand cross-links is not directly associated with a decrease in cytotoxicity. L1210 cells, which have no measurable quinone reductase activity, demonstrate increased toxicity when treated with DIC and MC in hypoxia, as observed with EMT6 cells. Unlike EMT6 cells, however, L1210 cells are not protected by DIC from MC toxicity in air. Taken together, these findings suggest that DIC is altering the intracellular metabolism of MC and that quinone reductase or another, unidentified, enzyme sensitive to DIC may be involved in activating MC to a toxic product in aerobic EMT6 cells.

Solid neoplasms may contain deficient or poorly functional vascular beds, a property that leads to the formation of hypoxic tumor cells, which form a therapeutically resistant cell population within the tumor that is difficult to eradicate by ionizing irradiation and most existing chemotherapeutic agents. As an approach to the therapeutic attack of hypoxic cells, we have measured the cytotoxicity and DNA lesions produced by the bioreductive alkylating agents mitomycin C and porfiromycin, two structurally similar antibiotics, in oxygen-deficient and aerobic cells. Mitomycin C and porfiromycin were preferentially cytotoxic to hypoxic EMT6 cells in culture, with porfiromycin producing a greater differential kill of hypoxic EMT6 cells relative to their oxygenated counterparts than did mitomycin C. Chinese hamster ovary cells were more resistant to these quinone antibiotics; although in this cell line, porfiromycin was significantly more cytotoxic to hypoxic cells than to aerobic cells, and the degree of oxygenation did not affect the toxicity of mitomycin C. Alkaline elution methodology was utilized to study the formation of DNA single-strand breaks and DNA interstrand cross-links produced by mitomycin C and porfiromycin in both EMT6 and Chinese hamster ovary cells. A negligible quantity of DNA single-strand breaks and DNA interstrand cross-links were produced in hypoxic and aerobic Chinese hamster ovary cells by exposure to mitomycin C or porfiromycin, a finding consistent with the considerably lower sensitivity of this cell line to these agents. In EMT6 tumor cells, no single-strand breaks appeared to be produced by these antitumor antibiotics under both hypoxic and aerobic conditions; however, a significant number of DNA interstrand cross-links were formed in this cell line following drug treatment, with substantially more DNA interstrand cross-linking being produced under hypoxic conditions. Mitomycin C and porfiromycin caused the same amount of cross-linking under conditions of oxygen deficiency; however, mitomycin C produced considerably more DNA cross-linking than did porfiromycin in oxygenated cells. DNA interstrand cross-links were observed in hypoxic EMT6 cells throughout a 24-h period following removal of mitomycin C and porfiromycin, with a decrease in DNA interstrand cross-links observed at 24 h. An increase in DNA interstrand cross-links occurred in aerobic EMT6 cells treated with mitomycin C and porfiromycin at 6 h after drug removal, with a decrease in these lesions being observed by 24 h, suggesting that the rate of formation of the cross-links may be slower and the removal of cross-links more rapid under aerobic conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Since the cure of solid tumors is limited by the presence of cells with low oxygen contents, we have approached the development of treatment regimens and of new drugs for these tumors by investigating agents which are preferentially bioactivated under hypoxia. Major emphasis has been directed at studying the mode of action of the mitomycin antibiotics, as bioreductive alkylating agents. Using primarily the EMT6 mouse mammary carcinoma as a solid tumor model, we have found that mitomycin C and porfiromycin are preferentially toxic to cells with low oxygen contents. The mitomycin analog BMY-25282 is more toxic to hypoxic cells than are mitomycin C and porfiromycin; however, unlike these antibiotics, BMY-25282 is preferentially toxic to well-oxygenated cells. With these three mitomycins, we have observed a correlation between cytotoxicity to hypoxic cells, the rate of generation of reactive products, and the redox potentials of the drugs. Investigations of the enzymes in EMT6 cells that could possibly activate mitomycin C have revealed that cytochrome P-450 and xanthine oxidase are not present in measurable quantities and therefore are not responsible for activation of mitomycin C. Activities representative of NADPH-cytochrome c reductase and DT-diaphorase are present in these neoplastic cells. Comparison of these enzymatic activities in EMT6, CHO, and V79 cells with the rate of generation of reactive products under hypoxia shows a direct correlation between these two parameters, but there is no quantitative correlation between these two parameters and the amount of cytotoxicity. Use of purified NADPH-cytochrome c reductase and inhibitors of this enzyme demonstrated that NADPH-cytochrome c reductase can activate mitomycin C, but that it is probably not the only enzyme participating in this bioactivation in EMT6 cells. The DT-diaphorase inhibitor dicoumarol was employed to show that this enzyme is not involved in the activation of mitomycin C to a cytotoxic agent. Instead, DT-diaphorase appears to metabolize mitomycin C to a nontoxic product. This property has been exploited to develop a new treatment regimen for solid tumors. Using X-rays to eliminate well oxygenated cells of a solid tumor implant of the EMT6 carcinoma, we have found that the combination of dicoumarol plus mitomycin C is more toxic to hypoxic tumor cells in vivo than mitomycin C alone. Furthermore, knowledge of the biochemical mechanism of mitomycin C activation permits a prediction of which tumors can best be treated with this combination of drugs by measuring enzymatic activities in biopsy specimens.

New mitomycin C and porfiromycin analogues were prepared by treating mitomycin A and N-methylmitomycin A with a variety of amines, including aziridines, allylamines, propargylamines, chloroalkylamines, hydroxyalkylamines, glycine derivatives, aralkylamines, and heterocyclic amines. All analogues were evaluated against P-388 murine leukemia and selected ones were examined for their leukopenic properties. Certain analogues were found to be superior to mitomycin C in potency, efficacy, and therapeutic ratio in the P-388 assay. The most active substituents at the mitosane 7 position included aziridine, 2-methylaziridine, propargylamine, furfurylamine, methyl glycinate, and 3-aminopyridine. Mitomycin A and the 7-aziridino, 7-(2-methylaziridino), and 3-aminopyridine analogues were less leukopenic than mitomycin C. Certain other analogues, including propargylamino and methyl glycinate, were highly leukopenic. The three compounds tested against B-16 melanoma in mice were significantly more effective than mitomycin C in this assay. Previously established structure--activity relationships were found inadequate to account for all of the new data.

Some factors affecting the cytotoxicity of porfiromycin (PM), an analogue of mitomycin C (MMC), were investigated in suspension cultures of wild-type (AA8-4) and repair-deficient (UV-20) Chinese hamster ovary cells. Oxygen was an important modulator of PM toxicity in AA8-4 cells. The aerobic toxicity was significantly less, and toxicity under extremely hypoxic conditions was significantly greater for PM than MMC. Porfiromycin cytotoxicity at intermediate O2 levels was similar to that observed previously for MMC. While the aerobic/hypoxic ratio was greater for PM than MMC, survival at intermediate oxygen concentrations could limit the therapeutic utility of these drugs as adjuncts to radiotherapy. Ascorbic acid was found to increase the aerobic, but not hypoxic, cytotoxicity of PM in AA8-4 cells, as was observed previously for MMC. Investigation of various exposure times and drug concentrations revealed that drug toxicity for both aerobic and hypoxic cells was dependent on the product of drug concentration and time, and that the aerobic/hypoxic differential observed in AA8-4 cells was constant over a broad range of exposure conditions. The sensitivity of UV-20 cells was also a linear function of concentration and time, but no aerobic/hypoxic differential was observed in these cells. It is suggested that the sensitivity of UV-20 to PM and MMC, and its lack of an hypoxic/aerobic differential could result from lethality being due to a different lesion than in wild-type cells.

A series of 7-(2-substituted-ethyl)amino analogues of mitomycin C and porfiromycin was prepared and screened in standard antitumor systems. Certain of these analogues showed better activity than mitomycin C against P-388 leukemia, L-1210 leukemia, and/or B-16 melanocarcinoma in mice. Compounds also tested for their leukopenic effects in mice, the limiting toxicity of mitomycin C. Some of them were less leukopenic and some were more leukopenic than this clinical agent. No statistically significant correlations could be made between physicochemical properties and antitumor activities of the analogues.

Mitomycin C (MC) and its structural analogs porfiromycin (PM), BMY-25282 and BL-6783 are toxic to EMT6 cells under aerobic and hypoxic conditions. The mitomycin antibiotics are hypothesized to exert cytotoxicity under hypoxic conditions by cross-linking DNA following reductive activation, while aerobic cytotoxicity may involve DNA cross-linking by these agents and/or damage due to the generation of oxygen radicals. Previous findings (Pritsos and Sartorelli, 1986) indicated that the rank order of cytotoxicity for a series of mitomycins was the same as the rank order for the rate of oxygen consumption induced by these agents. As an additional approach to explore the role of oxygen radicals in the aerobic cytotoxicity of the four agents studied, EMT6 cells were treated with the mitomycins in the presence of the superoxide dismutase inhibitor diethyldithiocarbamate (DETC). DETC, which decreased superoxide dismutase activity in EMT6 cells, increased the cytotoxicity of BMY-25282 and BL-6783 by half an order of magnitude, but did not affect the toxicity of PM or MC to these cells. DNA cross-links, a proposed cytotoxic lesion induced by BMY-25282, however, were not detectably increased in EMT6 cells exposed to this agent in the presence of DETC in spite of the large increase in cytotoxicity under these treatment conditions. No single strand breaks were detected in cells exposed to either BMY-25282 or BMY-25282 plus DETC. The findings support the concept that oxygen radicals may have a role in the aerobic cytotoxicity of some of the mitomycin antibiotics, and that the lesions responsible for cytotoxicity produced by oxygen radicals may not reside entirely at the level of DNA.

The effects of dicoumarol (DIC) on the cytotoxicity of porfiromycin (POR) were studied in vitro using EMT6 mammary tumor cells in monolayer cultures and in vivo using solid EMT6 tumors and bone marrow stem cells. In vitro, POR was more toxic to hypoxic EMT6 cells than to aerobic cells. Exposure of aerobic cultures to DIC protected against POR; in contrast, DIC sensitized hypoxic cells to POR. Treatment of mice with DIC produced a slight increase in the toxicity of POR to cells in solid tumors. The toxicity of POR to marrow stem cells (CFU-GM and CFU-MK) was not altered by DIC. Pretreatment of mice with DIC therefore produced a small improvement in the therapeutic ratio.

Two non-transformed human skin fibroblast strains, GM38 and 3437T, were found to be more sensitive to the bioreductive alkylating agents mitomycin C (MMC) and porfiromycin (PM) under hypoxic compared to aerobic conditions. One of these strains, 3437T, was 6-7 times more resistant to these agents under aerobic exposure conditions, but was identical in sensitivity to the normal strain, GM38, under hypoxic conditions. Aerobic 3437T cells demonstrated no increased resistance to cisplatin compared to the normal strain, arguing against enhanced ability to repair DNA interstrand cross-links as the underlying explanation for the mitomycin resistance. The aerobic resistance of 3437T was not altered by dicumarol, an inhibitor of the enzyme DT-diaphorase which is believed to be involved in aerobic activation of MMC and PM. Dicumarol did increase the resistance of GM38, but not to the same level of resistance demonstrated by 3437T. These results suggest that the aerobic MMC and PM resistance of 3437T may arise, in part, from a deficiency in DT-diaphorase activity. The identical sensitivities under hypoxic conditions indicate that drug activation pathways operative in the absence of oxygen are similar in both the normal and 3437T cells.

Hypoxic cells may limit the curability of solid tumors by conventional chemotherapeutic agents and radiotherapy. Agents which are preferentially toxic to cells with low oxygen contents could therefore be useful as adjuncts to the regimens now used to treat these cancers. To date, the best agent of this type that we have tested is porfiromycin. Porfiromycin is similar to mitomycin C in its toxicity to hypoxic EMT6 cells in vitro but has much less toxicity than mitomycin C to well-oxygenated EMT6 cells. EMT6 cell sonicates reduce mitomycin C and porfiromycin to reactive electrophiles at similar rates under hypoxic conditions, a finding that correlates with cytotoxicity, whereas the rate of production of reactive species from both drugs is very slow under aerobic conditions. We also show that porfiromycin is capable of killing hypoxic radiation-resistant cells in solid EMT6 tumors. Appropriate regimens combining porfiromycin (which preferentially kills hypoxic cells) and radiation (which preferentially kills aerated cells) may therefore be especially efficacious for the treatment of solid tumors.