-
Proceedings of the National Academy of... Jan 1996Purified NADPH:cytochrome c (P-450) reductase (FpT; NADPH-ferrihemoprotein oxidoreductase, EC 1.6.2.4) can reductively activate mitomycin antibiotics through a...
Purified NADPH:cytochrome c (P-450) reductase (FpT; NADPH-ferrihemoprotein oxidoreductase, EC 1.6.2.4) can reductively activate mitomycin antibiotics through a one-electron reduction to species that alkylate DNA. To assess the involvement of FpT in the intracellular activation of the mitomycins, transfectants overexpressing a human FpT cDNA were established from a Chinese hamster ovary cell line deficient in dihydrofolate reductase (CHO-K1/dhfr-). The parental cell line was equisensitive to the cytotoxic action of mitomycin C under oxygenated and hypoxic conditions. In contrast, porfiromycin was considerably less cytotoxic to wild-type parental cells than was mitomycin C in air and markedly more cytotoxic under hypoxia. Two FpT-transfected clones were selected that expressed 19- and 27-fold more FpT activity than the parental line. Levels of other oxidoreductases implicated in the activation of the mitomycins were unchanged. Significant increases in sensitivity to mitomycin C and porfiromycin in the two FpT-transfected clones were seen under both oxygenated and hypoxic conditions, with the increases in toxicity being greater under hypoxia than in air. These findings demonstrate that FpT can bioreductively activate the mitomycins in living cells and implicate FpT in the differential aerobic/hypoxic toxicity of the mitomycins.
Topics: Aerobiosis; Animals; Biotransformation; CHO Cells; Cricetinae; Glutathione Transferase; Humans; Hypoxia; Mitomycin; NADPH-Ferrihemoprotein Reductase; Oxidation-Reduction; Porfiromycin; Recombinant Proteins
PubMed: 8552660
DOI: 10.1073/pnas.93.1.456 -
Biochemical Pharmacology Jun 1996DT-Diaphorase catalyzes a two-electron reduction of mitomycin C (MC) and porfiromycin (POR) to reactive species. Many cell lines that overexpress DT-diaphorase and are...
DT-Diaphorase catalyzes a two-electron reduction of mitomycin C (MC) and porfiromycin (POR) to reactive species. Many cell lines that overexpress DT-diaphorase and are sensitive to the mitomycins are protected from the aerobic cytotoxicity of these drugs by the DT-diaphorase inhibitor dicumarol. The cytoprotective properties of this relatively non-specific inhibitor, however, vanish under hypoxic conditions. To ascertain the role of DT-diaphorase in mitomycin bioactivation and cytotoxicity in living cells, a rat liver DT-diaphorase cDNA was transfected into Chinese hamster ovary cells. MC was equitoxic to the parental cells under oxygenated and hypoxic conditions. In contrast, POR was less toxic than MC to these cells under aerobic conditions, but significantly more toxic than MC under hypoxia. Two DT-diaphorase-transfected clones displayed increases in DT-diaphorase activity of 126- and 133-fold over parental cells. The activities of other oxidoreductases implicated in mitomycin bioreduction were unchanged. MC was more toxic to both DT-diaphorase-transfected lines than to parental cells; the toxicity of MC to the transfected lines was similar in air and hypoxia. POR was also more toxic to the DT-diaphorase-elevated clones than to parental cells under oxygenated conditions. Under hypoxia, however, the toxicity of POR to the transfected clones was unchanged from that of parental cells. The findings implicate DT-diaphorase in mitomycin bioactivation in living cells, but suggest that this enzyme does not contribute to the differential toxicity of MC or POR in air and hypoxia.
Topics: Animals; CHO Cells; Cell Survival; Cricetinae; Dihydrolipoamide Dehydrogenase; Dose-Response Relationship, Drug; Enzyme Activation; Female; Hypoxia; Mitomycin; Ovary
PubMed: 8687482
DOI: 10.1016/0006-2952(96)00143-8 -
Radiotherapy and Oncology : Journal of... Apr 1996N-7[2-(4-nitrophenyldithio)-ethyl] mitomycin C, (BMS-181174; previously designated as BMY25067) is a mitomycin C analog now in initial clinical trials. The experiments...
N-7[2-(4-nitrophenyldithio)-ethyl] mitomycin C, (BMS-181174; previously designated as BMY25067) is a mitomycin C analog now in initial clinical trials. The experiments described in this report were performed to assess whether BMS-181174, like mitomycin C and porfiromycin, was selectively toxic to the hypoxic cells in solid tumors and might therefore prove valuable in combination with radiotherapy. In contrast to mitomycin C and porfiromycin, BMS-181174 was more toxic to aerobic EMT6 cells in vitro than to cells made acutely hypoxic. In vitro, BMS-181174 and radiation produced cytotoxicity compatible with either additive or slightly supra-additive cytotoxicity. In vivo, BMS-181174 was effective in killing cells in solid EMT6 tumors. The effects of regimens combining BMS-181174 and radiation in vivo were complex. Combinations of low doses of BMS-181174 plus a large dose of radiation were very effective in killing cells in solid tumors. However, the survival curve plateaued at high doses of BMS-181174, providing evidence for a subpopulation of tumor cells which were resistant to both BMS-181174 and radiation; this was hypothesized to be a hypoxic cell population.
Topics: Aerobiosis; Animals; Antibiotics, Antineoplastic; Antineoplastic Agents, Alkylating; Cell Hypoxia; Cell Survival; Chemotherapy, Adjuvant; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Drug Resistance, Neoplasm; Female; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Mitomycin; Mitomycins; Neoplasm Transplantation; Porfiromycin; Radiation-Sensitizing Agents; Radiotherapy Dosage; Skin Neoplasms; Specific Pathogen-Free Organisms; Tumor Cells, Cultured
PubMed: 8735495
DOI: 10.1016/0167-8140(95)01692-9 -
Proceedings of the National Academy of... Aug 1999The mitomycin C-resistance gene, mcrA, of Streptomyces lavendulae produces MCRA, a protein that protects this microorganism from its own antibiotic, the antitumor drug...
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.
Topics: Aerobiosis; Animals; Bacterial Proteins; Biotransformation; CHO Cells; Cell Hypoxia; Cell Survival; Cloning, Molecular; Cricetinae; Drug Resistance; Mitomycin; Oxidoreductases; Porfiromycin; Prodrugs; Recombinant Proteins; Streptomyces; Transfection
PubMed: 10468636
DOI: 10.1073/pnas.96.18.10489 -
The Journal of Antibiotics Mar 1986When the normal fermentation medium for the production of mitomycin C with Streptomyces caespitosus is supplemented with a number of primary amines, two new types of...
When the normal fermentation medium for the production of mitomycin C with Streptomyces caespitosus is supplemented with a number of primary amines, two new types of mitomycin analogs described as Type I and Type II are produced. Type I analogs are related to mitomycin C with the amine substitution at position C7 on the mitosane ring. Type II analogs also contain the same substitutions at C7 but the conformation of the mitosane ring is related to mitomycin B having an OH at positions C9a and a methyl substituted aziridine. The products obtained from the supplementation of the medium with methylamine, ethylamine, propylamine, propargylamine and 2-methylallylamine were isolated and characterized. In all cases the Type I analogs are more active in a prophage induction test and against L1210 lymphatic leukemia in mice. A number of other amines have been tested and shown to yield new products that have not yet been isolated. No secondary amines are incorporated.
Topics: Animals; Chemical Phenomena; Chemistry; Injections, Intraperitoneal; Leukemia L1210; Mice; Mice, Inbred DBA; Mitomycins; Porfiromycin; Stereoisomerism
PubMed: 3700245
DOI: 10.7164/antibiotics.39.437 -
Chemical & Pharmaceutical Bulletin Mar 2007The solvolysis rates for the substituted C(7)-cyclohexylamino- or C(8)-cyclohexyliminomitomycins 8-19 were determined in buffered methanolic solutions (0.06 M...
The solvolysis rates for the substituted C(7)-cyclohexylamino- or C(8)-cyclohexyliminomitomycins 8-19 were determined in buffered methanolic solutions (0.06 M bis-Tris.HCl, pH: 5.5) at 25 degrees C and then compared with mitomycin C (1) and porfiromycin. Kinetic studies showed that C(8)-cyclohexyliminomitomycins 8-13 underwent solvolysis 150-230 times faster than mitomycin C (1) to give C(1)-methoxymitosene products. The solvolysis rates were slightly faster than that reported for 6. The C(7)-(2'-hydroxy)cyclohexylaminomitomycins 16-19 exhibited comparable solvolysis rates with 1 and porfiromycin.
Topics: Hydrolysis; Mitomycins; Molecular Structure
PubMed: 17329899
DOI: 10.1248/cpb.55.482 -
British Journal of Cancer Mar 1989Two non-transformed human skin fibroblast strains, GM38 and 3437T, were found to be more sensitive to the bioreductive alkylating agents mitomycin C (MMC) and...
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.
Topics: Biotransformation; Cell Survival; Cells, Cultured; Dicumarol; Drug Resistance; Fibroblasts; Humans; Mitomycin; Mitomycins; Oxygen; Porfiromycin; Skin
PubMed: 2467684
DOI: 10.1038/bjc.1989.67 -
Biochemical Pharmacology Jan 1986
Topics: Anaerobiosis; Animals; Biotransformation; Cytochromes; Dicumarol; Drug Synergism; Female; Mammary Neoplasms, Experimental; Mitomycin; Mitomycins; NAD(P)H Dehydrogenase (Quinone); Neoplasms, Experimental; Porfiromycin; Quinone Reductases; Rats; Sarcoma, Experimental
PubMed: 2416320
DOI: 10.1016/0006-2952(86)90559-9 -
The Journal of Biological Chemistry Apr 1998NADH:cytochrome b5 reductase activates the mitomycins to alkylating intermediates in vitro. To investigate the intracellular role of this enzyme in mitomycin...
NADH:cytochrome b5 reductase activates the mitomycins to alkylating intermediates in vitro. To investigate the intracellular role of this enzyme in mitomycin bioactivation, Chinese hamster ovary cell transfectants overexpressing rat NADH:cytochrome b5 reductase were generated. An NADH:cytochrome b5 reductase-transfected clone expressed 9-fold more enzyme than did parental cells; the levels of other mitomycin-activating oxidoreductases were unchanged. Although this enzyme activates the mitomycins in vitro, its overexpression in living cells caused decreases in sensitivity to mitomycin C in air and decreases in sensitivity to porfiromycin under both air and hypoxia. Mitomycin C cytotoxicity under hypoxia was similar to parental cells. Because NADH:cytochrome b5 reductase resides predominantly in the mitochondria of these cells, this enzyme may sequester these drugs in this compartment, thereby decreasing nuclear DNA alkylations and reducing cytotoxicity. A cytosolic form of NADH:cytochrome b5 reductase was generated. Transfectants expressing the cytosolic enzyme were restored to parental line sensitivity to both mitomycin C and porfiromycin in air with marked increases in drug sensitivity under hypoxia. The results implicate NADH:cytochrome b5 reductase in the differential bioactivation of the mitomycins and indicate that the subcellular site of drug activation can have complex effects on drug cytotoxicity.
Topics: Animals; CHO Cells; Cell Nucleus; Cell Survival; Cricetinae; Cytochrome Reductases; Cytochrome-B(5) Reductase; Cytoplasm; Kinetics; Microsomes; Mitochondria; Mitomycin; Oxidation-Reduction; Rats; Recombinant Proteins; Succinate Dehydrogenase; Transfection
PubMed: 9535868
DOI: 10.1074/jbc.273.15.8875 -
Clinical Chemistry Apr 1997The aim of this study was to set up a method for quantification of plasma mitomycin C (MMC) concentrations during intravesical chemotherapy delivered in the presence of...
The aim of this study was to set up a method for quantification of plasma mitomycin C (MMC) concentrations during intravesical chemotherapy delivered in the presence of local bladder hyperthermia (HT). In comparison with existing methods, this assay, characterized by relative simplicity and efficiency, resulted in the facilitation of performance with nondedicated instrumentation or nonspecialized staff. Purification from plasma matrix was carried out by solid-phase extraction under vaccuum. The purified drug was then collected directly into the vials of the HPLC autosampler. Chromatographic analysis was performed on a reversed-phase C18 column with water:acetonitrile (85:15 by vol) as the mobile phase and the UV detector set at 365 nm. The use of porfiromycin as internal standard provided a method with good within-day precision (CV 6.0% at 5 micrograms/L, n = 6), linearity (0.5-50 micrograms/L), and specificity. The lower limit of detection (< or = 0.5 microgram/L) proved to be suitable for plasma pharmacokinetics monitoring in two tested patients treated with MMC + HT for superficial bladder cancer.
Topics: Antibiotics, Antineoplastic; Carcinoma, Transitional Cell; Chromatography, High Pressure Liquid; Humans; Hyperthermia, Induced; Kinetics; Mitomycin; Quality Control; Urinary Bladder Neoplasms
PubMed: 9105262
DOI: No ID Found