Antimicrobial metallopeptides with broad nuclease and ribonuclease activity.

Metallopeptides containing both the complex Cu(2+)-glycyl-glycyl-histidine (Cu-GGH) and the sequence WRWYCR were shown to possess antimicrobial activity against a variety of pathogenic bacteria, as well as bind to and cleave a variety of nucleic acids, suggesting potential mechanisms for antimicrobial activity that involve binding and/or irreversible cleavage of bacterial nucleic acids.

Reversal of mitomycin C resistance by overexpression of bioreductive enzymes in Chinese hamster ovary cells.

The clinical utility of antineoplastic agents is limited by the development of drug resistance by tumors. Mitomycin C (MC) is a bacterial product that must be enzymatically reduced to exert anticancer activity. We have demonstrated that expression of the bacterial MC resistance-associated (MCRA) protein in Chinese hamster ovary (CHO) cells confers profound resistance to this antibiotic under aerobic conditions, but not under hypoxia. MCRA produces resistance to MC by redox cycling of the activated hydroquinone intermediate back to the prodrug form. A CHO cell line developed by stepwise exposure to increasing concentrations of MC likewise expressed high level resistance to MC in air, but not under hypoxia. The overexpression of DT-diaphorase and NADPH:cytochrome c (P-450) reductase, two enzymes known to activate MC, restored sensitivity to MC in both MCRA-transfected and drug-selected cell lines. The level of sensitization was proportional to the quantity of enzyme activity expressed, supporting the concept that the levels of these two activating enzymes are important for sensitivity to MC. The findings of resistance to MC in air but not under hypoxic conditions and of restoration of sensitivity to MC by increasing levels of DT-diaphorase activity, properties not adequately explained by other resistance mechanisms (i.e., decreases in MC activation, repair of DNA lesions, and/or drug efflux), support the hypothesis that a functional mammalian homologue of MCRA may be involved in producing resistance to MC.

Inhibition of DNA cross-linking by mitomycin C by peroxidase-mediated oxidation of mitomycin C hydroquinone.

Mitomycin C requires reductive activation to cross-link DNA and express anticancer activity. Reduction of mitomycin C (40 microm) by sodium borohydride (200 microm) in 20 mm Tris-HCl, 1 mm EDTA at 37 degrees C, pH 7.4, gives a 50-60% yield of the reactive intermediate mitomycin C hydroquinone. The hydroquinone decays with first order kinetics or pseudo first order kinetics with a t(12) of approximately 15 s under these conditions. The cross-linking of T7 DNA in this system followed matching kinetics, with the conversion of mitomycin C hydroquinone to leuco-aziridinomitosene appearing to be the rate-determining step. Several peroxidases were found to oxidize mitomycin C hydroquinone to mitomycin C and to block DNA cross-linking to various degrees. Concentrations of the various peroxidases that largely blocked DNA cross-linking, regenerated 10-70% mitomycin C from the reduced material. Thus, significant quantities of products other than mitomycin C were produced by the peroxidase-mediated oxidation of mitomycin C hydroquinone or products derived therefrom. Variations in the sensitivity of cells to mitomycin C have been attributed to differing levels of activating enzymes, export pumps, and DNA repair. Mitomycin C hydroquinone-oxidizing enzymes give rise to a new mechanism by which oxic/hypoxic toxicity differentials and resistance can occur.

Pharmaceutical development and manufacturing of a parenteral formulation of a novel antitumor agent, VNP40101M.

The objective of this study was to develop and manufacture a stable parenteral formulation for Phase I clinical trials of VNP40101M (1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(2-methylamino)carbonyl] hydrazine), a novel antitumor agent. The solubility and stability of the drug was determined. Solubility studies suggested that VNP40101M exhibited poor aqueous solubility but showed appreciable solubility in nonaqueous solvents. The aqueous solubility of the drug could not be increased by adjusting the pH. At a pH above 7, base-catalyzed decomposition of VNP40101M occurred. The low octanol-water partition coefficient of 0.75 suggested poor solubility in lipophilic solvents. Based on these preformulation observations, a parenteral formulation containing 10 mg/mL of VNP40101M was prepared in a solvent system consisting of 30% ethyl alcohol and 70% polyethylene glycol-300 (PEG-300). To minimize base-catalyzed hydrolytic degradation, citric acid at 0.6% concentration was included to acidify the formulation. Rubber closures, filter membranes, and liquid transfer tubing were selected on the basis of compatibility studies and absence of loss of drug due to adsorption of these components. The formulation was subjected to accelerated stability studies and dilution studies with large volume parenteral (LVP) solutions, normal saline, and 5% dextrose injection (D5W). The results of the dilution study indicated that the formulation could be diluted in these solutions up to 2 mg/mL for 8 hours without drug precipitation and degradation. Accelerated stability studies suggested that the product should be kept at 2 degrees C to 8 degrees C for long-term storage. The developed formulation was successfully scaled up and manufactured for use in clinical trials.

Mitomycin resistance in mammalian cells expressing the bacterial mitomycin C resistance protein MCRA.

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.

Exploring the mechanistic aspects of mitomycin antibiotic bioactivation in Chinese hamster ovary cells overexpressing NADPH:cytochrome C (P-450) reductase and DT-diaphorase.

We have directly demonstrated the involvement of human NADPH: cytochrome c (P-450) reductase in the aerobic/hypoxic differential toxicity of mitomycin C and porfiromycin in living cells by varying only this enzyme in a transfected cell line. In the same manner, we have implicated rat DT-diaphorase in the aerobic and hypoxic activation of mitomycin C, but found only a minor role for this enzyme in the aerobic activation of porfiromycin. DT-Diaphorase does not cause the production of an aerobic/hypoxic differential toxicity by mitomycin C, but rather activates this agent through an oxygen insensitive pathway. The evidence suggests that DT-diaphorase activates mitomycin C more effectively than porfiromycin, with porfiromycin being preferentially activated through a one-electron reductive pathway. The therapeutic potential of mitomycin antibiotics in the treatment of cancer can be envisioned to be enhanced for those tumors containing elevated levels of the bioreductive enzymes. However, cytogenetic heterogeneity within the tumor cell population and the various environmental factors which impact on bioreductive enzyme function, including pH and oxygen tension, may subvert this approach. Moreover, if high tumor levels of a drug activating enzyme reflect high levels in the normal tissues of the patient, normal tissue damage may also be enhanced with possibly no improvement in the therapeutic ratio. Approaches utilizing gene therapy, whereby a specific bioreductive catalyst is introduced into the tumor cell population via a targeting vehicle to activate a particular prodrug, may be more effective in that not only will the prodrug of choice be specifically activated in the tumor, but the source of the catalyst, be it bacterial, rodent, or human, will not be important. In fact, in the case of DT-diaphorase and mitomycin C, the rat form of the enzyme could be advantageous because it is more effective in activating mitomycin C than is the human form of this enzyme. Assuming targeted gene delivery to malignant cells, a non-host enzyme which is more effective at activating mitomycin C than the analogous host enzyme might also result in less drug activation in normal tissue and, hence, less normal tissue toxicity.

The intracellular location of NADH:cytochrome b5 reductase modulates the cytotoxicity of the mitomycins to Chinese hamster ovary cells.

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.

Measurement of dicumarol-sensitive NADPH: (menadione-cytochrome c) oxidoreductase activity results in an artifactual assay of DT-diaphorase in cell sonicates.

Purified DT-diaphorase can be assayed as either dicumarol-inhibitable NAD(P)H:menadione oxidoreductase or dicumarol-inhibitable NAD(P)H:dichlorophenolindophenol reductase. Both of these methods have been utilized to assay DT-diaphorase activity in tissue and cell homogenates. When DT-diaphorase activity was measured as dicumarol-inhibitable NADPH:dichlorophenolindophenol reductase in sonicates of two cell lines previously shown to not have any measurable activity of this enzyme, no enzymatic activity was detected. However, when the water-soluble bisulfite addition product of menadione was used as the electron acceptor, an artifactual activity for DT-diaphorase was detected in these cell lines. When another cell line was assayed utilizing menadione bisulfite, an apparent activity of about three times that found with dichlorophenolindophenol was measured, and thus, may overestimate DT-diaphorase activity in cells having activity. When menadione was used in place of menadione bisulfite, an artifactual DT-diaphorase activity was also detected, but was about one-half that obtained with menadione bisulfite. Polarographic determinations of the midpoint potentials for menadione and menadione bisulfite indicated that the latter compound was easier to reduce and may account for the greater apparent DT-diaphorase activity measured with this compound.

Induction of the differentiation of HL-60 promyelocytic leukemia cells by vitamin E and other antioxidants in combination with low levels of vitamin D3: possible relationship to NF-kappaB.

Epidemiological studies have provided evidence that diets rich in antioxidant nutrients may reduce the risk of cancer. To evaluate the possibility that dietary phytochemicals with antioxidant potential would create an environment capable of affecting the differentiation of HL-60 leukemia cells, we measured the effects of vitamin E and other dietary antioxidants on the differentiation produced by low levels of vitamin D3 and analogs thereof. Vitamin E succinate and other antioxidant compounds (ie butylated hydroxyanisole, beta-carotene and lipoic acid) used alone had no significant effect on the differentiation of HL-60 cells; however, these agents markedly increased the differentiation produced by vitamin D3. Previous studies from this laboratory have shown that a sequence-specific antisense phosphorothioate oligonucleotide to the Rel A subunit of NF-kappaB enhanced the differentiation of HL-60 cells produced by several inducing agents. Consistent with these observations, vitamin E succinate caused a marked reduction in the nuclear content of NF-kappaB both in the presence and absence of vitamin D3. These findings suggest that NF-kappaB may be a factor in regulating the differentiation of myeloid leukemia cells. The results also indicate that combinations of vitamin D3 and analogs thereof with dietary antioxidants may be useful in overcoming the differentiation block present in acute promyelocytic leukemia cells.

Bioactivation of mitomycin antibiotics by aerobic and hypoxic Chinese hamster ovary cells overexpressing DT-diaphorase.

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.

Differential toxicity of mitomycin C and porfiromycin to aerobic and hypoxic Chinese hamster ovary cells overexpressing human NADPH:cytochrome c (P-450) reductase.

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.

Preferential kill of hypoxic EMT6 mammary tumor cells by the bioreductive alkylating agent porfiromycin.

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.

The pH-dependent reduction of Adriamycin catalysed by NADH:cytochrome b5 reductase.

Adriamycin is a redox active antineoplastic antibiotic that upon reduction can, in the presence of oxygen, redox cycle to form reactive oxygen species, while in anaerobiosis can generate a reactive quinone methide. NADH:cytochrome b5 reductase catalysed the reduction of adriamycin at pH 6.6 with an apparent Km of 1.8 microM; at pH 7.6, no measurable reduction of adriamycin occurred. Aerobically, in the presence of enzyme and NADH, adriamycin stimulated oxygen consumption and concomitant accumulation of hydrogen peroxide. At pH 7.6, no discernible oxygen consumption nor detectable hydrogen peroxide generation was observed. The findings demonstrate that NADH:cytochrome b5 reductase is capable of reducing adriamycin, in a pH-dependent manner, to species that can redox cycle in the presence of oxygen to form reactive oxygen molecules and thus may contribute to the generation of oxidative stress, a phenomenon suggested to be involved in both the toxicity and the antineoplastic activity of adriamycin.

Inhibition of mitochondrial respiration and cyanide-stimulated generation of reactive oxygen species by selected flavonoids.

A continuation of our structure-activity study on flavonoids possessing varied hydroxyl ring configurations was conducted. We tested six additional flavonoids for their ability to inhibit beef heart mitochondrial succinoxidase and NADH-oxidase activities. In every case, the IC50 observed for the NADH-oxidase enzyme system was lower than for succinoxidase activity, demonstrating a primary site of inhibition in the complex I (NADH-coenzyme Q reductase) portion of the respiratory chain. The order of potency for inhibition of NADH-oxidase activity was robinetin, rhamnetin, eupatorin, baicalein, 7,8-dihydroxyflavone, and norwogonin with IC50 values of 19, 42, 43, 77, 277 and 340 nmol/mg protein, respectively. Flavonoids with adjacent tri-hydroxyl or para-dihydroxyl groups exhibited a substantial rate of auto-oxidation which was accelerated by the addition of cyanide (CN-). Flavonoids possessing a catechol configuration exhibited a slow rate of auto-oxidation in buffer that was stimulated by the addition of CN-. The addition of superoxide dismutase (SOD) and catalase in the auto-oxidation experiments each decreased the rate of oxygen consumption, indicating that O2- and H2O2 are generated during auto-oxidation. In the CN(-)-stimulated oxidation experiments, the addition of SOD also slowed the rate of oxygen consumption. These findings demonstrate that the CN-/flavonoid interaction generated O2- non-enzymatically, which could have biological implications.

Mitomycin C: a prototype bioreductive agent.

Hypoxic cells of solid tumors represent a therapeutically resistant population that limits the curability of many solid tumors by x-irradiation and by most chemotherapeutic agents. The oxygen deficit, however, creates an environment conducive to reductive processes that results in a major exploitable difference between normal and neoplastic tissues. Mitomycin C (MC) can be reductively activated by a number of oxidoreductases, in a process required for the production of its therapeutic effects. This enzymatic reduction results in preferential activation of MC under hypoxia and, in most instances, the production of greater toxicity to oxygen-deficient cells than to their oxygenated counterparts. DNA appears to be the most important target of the reactive species generated from MC, with both mono- and bis-adducts of DNA being formed in drug-treated cells. The demonstration that MC, used to kill the hypoxic fraction, in combination with x-irradiation, to eradicate the oxygenated portion of the tumor, produced enhanced cytodestructive effects on solid tumors of animals has led to the clinical evaluation of the mitomycin antibiotics in combination with x-rays in patients with cancers of the head and neck. The findings from these clinical trials have demonstrated the utility of directing a concerted therapeutic attack on the hypoxic fraction of solid tumors as an approach toward enhancing the curability of localized neoplasms by x-irradiation.

Reductive activation of mitomycin C by NADH:cytochrome b5 reductase.

Mitomycin C requires bioreduction in order to exert its cytotoxic action. Activation of mitomycin C to an electrophile was equally supported by NADPH and NADH in EMT6 tumor cell sonicates under hypoxia. No alkylation was observed under aerobic conditions. Purified NADH:cytochrome b5 reductase catalyzed the reduction of mitomycin C with a Km of 23 microM at pH 6.6. At pH 7.6, the rate of enzymatic reduction of mitomycin C was 61% of that at pH 6.6. NADH:cytochrome b5 reductase catalyzed the activation of mitomycin C to alkylating metabolites under both hypoxic and aerobic conditions, with alkylation being 1.5 times greater in hypoxia. Dicumarol at 100 microM inhibited the NADH:cytochrome b5 reductase-catalyzed reduction of mitomycin C by 24% and by 57% at 300 microM. The degree of inhibition of the enzyme by dicumarol was the same at both pH 6.6 and 7.6. NADH:cytochrome b5 reductase exhibited a small but measurable NADH-oxidase activity, which was unaffected by 300 microM dicumarol. These findings demonstrate that (a) NADH:cytochrome b5 reductase can metabolically activate mitomycin C and (b) dicumarol is capable of inhibiting this enzymatic activity.

Electrochemistry of flavonoids. Relationships between redox potentials, inhibition of mitochondrial respiration, and production of oxygen radicals by flavonoids.

We have investigated the redox behavior of a series of structurally related flavonoids employing cyclic voltammetry under physiological conditions. The flavonoids that auto-oxidized and produced oxygen radicals had oxidation potentials (E 1/2) significantly lower [-30 to +60 mV vs (SCE)] than those that did not undergo auto-oxidation (+130 to +340 mV vs SCE). The range of E 1/2 values for the auto-oxidizable flavonoids was comparable to the E 1/2 range reported for the optimum quinone induced production of superoxide (O2 pi) in mitochondrial NADH-CoQ reductase (complex I). The most potent flavonoid inhibitors of mitochondrial succinate-CoQ reductase (complex II) possessed hydroxyl configurations capable of supporting redox reactions. For a series of 3,5,7-trihydroxyflavones that differed by b-ring hydroxylation it was found that decreasing E 1/2 of the flavonoids was associated with decreasing I50 values towards succinoxidase. These findings suggest that the electrochemical properties of the flavonoids may contribute to their biological activity.