Reductive activation of mitomycins A and C by vitamin C.

The anticancer drug mitomycin C produces cytotoxic effects after being converted to a highly reactive bis-electrophile by a reductive activation, a reaction that a number of 1-electron or 2-electron oxidoreductase enzymes can perform in cells. Several reports in the literature indicate that ascorbic acid can modulate the cytotoxic effects of mitomycin C, either potentiating or inhibiting its effects. As ascorbic acid is a reducing agent that is known to be able to reduce quinones, it could be possible that the observed modulatory effects are a consequence of a direct redox reduction between mitomycin C and ascorbate. To determine if this is the case, the reaction between mitomycin C and ascorbate was studied using UV/Vis spectroscopy and LC/MS. We also studied the reaction of ascorbate with mitomycin A, a highly toxic member of the mitomycin family with a higher redox potential than mitomycin C. We found that ascorbate is capable to reduce mitomycin A efficiently, but it reduces mitomycin C rather inefficiently. The mechanisms of activation have been elucidated based on the kinetics of the reduction and on the analysis of the mitosene derivatives formed after the reaction. We found that the activation occurs by the interplay of three different mechanisms that contribute differently, depending on the pH of the reaction. As the reduction of mitomycin C by ascorbate is rather inefficiently at physiologically relevant pH values we conclude that the modulatory effect of ascorbate on the cytotoxicity of mitomycin C is not the result of a direct redox reaction and therefore this modulation must be the consequence of other biochemical mechanisms.

DNA adducts of decarbamoyl mitomycin C efficiently kill cells without wild-type p53 resulting from proteasome-mediated degradation of checkpoint protein 1.

The mitomycin derivative 10-decarbamoyl mitomycin C (DMC) more rapidly activates a p53-independent cell death pathway than mitomycin C (MC). We recently documented that an increased proportion of mitosene1-beta-adduct formation occurs in human cells treated with DMC in comparison to those treated with MC. Here, we compare the cellular and molecular response of human cancer cells treated with MC and DMC. We find the increase in mitosene 1-beta-adduct formation correlates with a condensed nuclear morphology and increased cytotoxicity in human cancer cells with or without p53. DMC caused more DNA damage than MC in the nuclear and mitochondrial genomes. Checkpoint 1 protein (Chk1) was depleted following DMC, and the depletion of Chk1 by DMC was achieved through the ubiquitin proteasome pathway since chemical inhibition of the proteasome protected against Chk1 depletion. Gene silencing of Chk1 by siRNA increased the cytotoxicity of MC. DMC treatment caused a decrease in the level of total ubiquitinated proteins without increasing proteasome activity, suggesting that DMC mediated DNA adducts facilitate signal transduction to a pathway targeting cellular proteins for proteolysis. Thus, the mitosene-1-beta stereoisomeric DNA adducts produced by the DMC signal for a p53-independent mode of cell death correlated with reduced nuclear size, persistent DNA damage, increased ubiquitin proteolysis and reduced Chk1 protein.

Relative toxicities of DNA cross-links and monoadducts: new insights from studies of decarbamoyl mitomycin C and mitomycin C.

Mitomycin C (MC), a cytotoxic anticancer drug and bifunctional DNA DNA alkylating agent, induces cross-linking of the complementary strands of DNA. The DNA interstrand cross-links (ICLs) are thought to be the critical cytotoxic lesions produced by MC. Decarbamoyl mitomycin C (DMC) has been regarded as a monofunctional mitomycin, incapable of causing ICLs. Paradoxically, DMC is slightly more toxic than MC to hypoxic EMT6 mouse mammary tumor cells as well as to CHO cells. To resolve this paradox, EMT6 cells were treated with MC or DMC under hypoxia at equimolar concentrations and the resulting DNA adducts were analyzed using HPLC and UV detection. MC treatment generated both intrastrand and interstrand cross-link adducts and four monoadducts, as shown previously. DMC generated two stereoisomeric monoadducts and two stereoisomeric ICL adducts, all of which were structurally characterized; one was identical with that formed with MC, the other was new and unique to DMC. Overall, adduct frequencies were strikingly higher (20-30-fold) with DMC than with MC. Although DMC monoadducts greatly exceeded DMC cross-link adducts ( approximately 10:1 ratio), the latter were equal or higher in number than the cross-link adducts from MC. DMC displayed a much higher monoadduct:cross-link ratio than MC. The similar cytotoxicities of the two drug show a correlation with their similar DNA cross-link adduct frequencies, but not with their total adduct or monoadduct frequencies. This provides specific experimental evidence that the ICLs rather than the monoadducts are critical factors in the cell death induced by MC. In vitro, overall alkylation of calf thymus DNA by DMC was much less efficient than by MC. Nevertheless, ICLs formed with DMC were clearly detectable. The chemical pathway of the cross-linking was shown to be analogous to that occurring with MC. These results also suggest that the differential sensitivity of Fanconi's Anemia cells to MC and DMC is related to factors other than a selective defect in cross-link repair.

Reversion mutation in dark variants of luminous bacteria and its application in gene toxicant monitoring.

The luminous intensity of dark variant (S1) separated from photobacterium phosphoreum (A2) was 1/10,000 less than that of wild-type. Ethidium bromide (EB) (0.6 mg/L), Mytomycin C (MC, 0.05 mg/L), 2-amino fluorene (2-AF, 1.0 mg/L) all could strongly induce reversion mutation for S1 within 24 h and increase reversion ratio significantly. The results of experiments indicated that these revertants had stable genetic characteristic and the mutation may take place at gene levels. The mutagenesis to S1 caused by EB, MC and 2-AF was detected and it may be used as a new rapid, simple and sensitive method for gene toxicant monitoring.

Reversion mutation in dark variants of luminous bacteria and its application in gene toxicant monitoring / 华中科技大学学报（医学）（英德文版）

The luminous intensity of dark variant (S1) separated from photobacterium phosphoreum (A2) was 1/10,000 less than that of wild-type. Ethidium bromide (EB) (0.6 mg/L), Mytomycin C (MC, 0.05 mg/L), 2-amino fluorene (2-AF, 1.0 mg/L) all could strongly induce reversion mutation for S1 within 24 h and increase reversion ratio significantly. The results of experiments indicated that these revertants had stable genetic characteristic and the mutation may take place at gene levels. The mutagenesis to S1 caused by EB, MC and 2-AF was detected and it may be used as a new rapid, simple and sensitive method for gene toxicant monitoring.

Inhibitory effects of subcutaneous dexamethasone treatment on rat pulmonary toxicity of KW-2149, a new mitomycin C analogue.

An experimental model for pulmonary toxicity of KW-2149, a new mitomycin C analogue, was established and the inhibitory effects of dexamethasone (DM) were investigated. KW-2149 was given to male rats 3 or 5 times at weekly intervals by intravenous injection of 3.28 or 8.2 mg/kg. As a suitable model for pulmonary toxicity, the dose of 3.28 mg/kg per week for 3 weeks was selected, this causing exudative pleural effusion in all animals but no deaths. For preventing this toxicity, DM was injected subcutaneously 3 times every week at 0.5, 1.0, 2.5 or 5.0 mg/kg. The 0.5 mg/kg dose was sufficient to completely prevent development of pleural effusions. Combined DM treatment may be an effective chemotherapy for KW-2149 induced pulmonary toxicity.

99mTc-MIBI scintigraphy as an indicator of the chemosensitivity of anthracyclines in patients with breast cancer.

BACKGROUND: Chemoresistance of tumor cells is involved with many factors, one of which is the P-glycoprotein function to pump anthracyclines out of cells. 99mTc-MIBI accumulates in several tumors, and some of these cells wash out 99mTc-MIBI through P-glycoprotein. MATERIALS AND METHODS: We investigated if the wash-out of 99mTc-MIBI from the tumor in fifteen female patients with breast cancer could be related with the chemosensitivity of anticancer agents; doxorubicin (DOX), epirubicin (FAM), pinorubicin (PINO), mitomycin (MMC), cisplatin (CDDP), and 5-fluorouracil (5-FU), in each tumor tissues. The wash-out of 99mTc-MIBI, defined as retention index, was quantified from an early and delayed 99mTc-MIBI imaging. The chemosensitivity of the anticancer agent, and inhibition ratio, was determined in vitro assay by using surgical specimens obtained from patients who underwent 99mTc-MIBI imaging. P-glycoprotein in the surgical specimen was studied by immunohistochemical staining on its paraffin section using a monoclonal antibody. RESULTS: Inhibition ratio of anthracycline agent, DOX, FAM or PINO, was well correlated with retention index of 99mTc-MIBI with coefficient of 0.75, 0.60, or 0.62, respectively, whereas a poor relationship was observed for MMC and CDDP. The retention indices of 99mTc-MIBI were remarkably small for patients in the P-glycoprotein positive group. CONCLUSION: 99mTc-MIBI retention index quantified from its early and delayed scintigraphy is a good indicator to predict the chemosensitivity of anthracyclines in untreated breast cancer.

In vitro toxicity studies with mitomycins and bleomycin on endothelial cells.

Pulmonary side effects are increasingly observed as dose-limiting toxicity (DLT) of cancer treatment. The available preclinical models have a limited predictive value for lung toxicity in humans. We have attempted to elucidate potential mechanisms involved in these reactions, by studying the effects on cells, possibly involved in these reactions after in vitro exposure to drugs with known lung toxic effects. We have investigated the effects of bleomycin (BLM), mitomycin C (MMC), KW-2149 and its two known metabolites, M16 and M18, on oxygen radical production by granulocytes, on cytokine production: interleukin (IL)-6, transforming growth factor (TGF)-beta, tumor necrosis factor (TNF)-alpha by a human macrophage cell line (THP-1), by human endothelial cells (HVEC and HMEC) and a human colorectal cancer cell line (DLD-1), and on the cytotoxicity on endothelial cells in both confluent and non-confluent culture. The generation of oxygen radicals by normal and pre-stimulated granulocytes was not increased after preincubation with any of the drugs, at the concentrations tested. None of the cytokines (IL-6, TNF-alpha or TGF-beta) was found significantly increased in culture medium after exposure to any of the mitomycins. This was in contrast with the effect of BLM incubation, causing a rise in TGF-beta concentration. Both types of endothelial cells showed a dose-dependent, exposure duration-dependent, proliferation inhibition for all agents tested. This inhibitory effect was clearly proliferation dependent as shown by the increased inhibition in semi-confluent as opposed to confluent endothelial cell cultures. Both mitomycins tested were more cytotoxic than BLM to both confluent and proliferating endothelial cells.

Suppression of P-glycoprotein expression and multidrug resistance by DNA cross-linking agents.

Overexpression of the trans-membrane drug efflux pump P-glycoprotein is one of the major mechanisms by which cancer cells develop multidrug resistance. We demonstrated previously that noncytotoxic doses of various genotoxic chemicals, particularly DNA cross-linking agents, preferentially altered expression of inducible genes. These effects occurred principally at the transcriptional level and were closely correlated temporally with DNA damage. Because the mdr1 gene coding for P-glycoprotein has been reported to be highly inducible, we were interested in the effects of genotoxic cancer chemotherapy agents on its expression. We report that the DNA cross-linking agent mitomycin C significantly suppressed mRNA and protein expression of P-glycoprotein and decreased the rate of drug efflux. Mitomycin C pretreatment also significantly increased the sensitivity of cancer cells to subsequent killing by the P-glycoprotein substrate doxorubicin, decreasing the ED50 by 5- to 10-fold. Suppression of P-glycoprotein expression was also observed with subtoxic doses of the DNA cross-linking agents cisplatin, BMS181174, and chromium(VI). These effects occurred in both human and rodent cell lines; in cell lines derived from colon, breast, leukemia, neuroblastoma, and hepatoma tumors; and under both monolayer and "spheroid" culture conditions. These results suggest the basis for novel clinical cancer chemotherapy regimens aimed at drug-resistant tumors, in which a sub-chemotherapeutic dose of a DNA cross-linking agent is used to modulate the multidrug resistance phenotype prior to treatment with a second cytotoxic agent.

Determination of DNA sequences required for regulated Mycobacterium tuberculosis RecA expression in response to DNA-damaging agents suggests that two modes of regulation exist.

The recA gene of Mycobacterium tuberculosis has previously been cloned and sequenced (E. O. Davis, S. G. Sedgwick, and M. J. Colston, J. Bacteriol. 173:5653-5662, 1991). In this study, the expression of this gene was shown to be inducible in response to various DNA-damaging agents by using a transcriptional fusion to the reporter gene encoding chloramphenicol acetyltransferase. A segment of DNA around 300 bp upstream of the coding region was shown to be required for expression. However, primer extension analysis indicated that the transcriptional start sites were 47 and 93 bp upstream of the translation initiation codon. Sequence motifs with homology to two families of Escherichia coli promoters but also with significant differences were located near these proposed transcription start sites. The differences from the E. coli consensus patterns would explain the previously described lack of expression of the M. tuberculosis recA gene from its own promoter in E. coli. In addition, the M. tuberculosis LexA protein was shown to bind specifically to a sequence, GAAC-N4-GTTC, overlapping one of these putative promoters and homologous to the Bacillus subtilis Cheo box involved in the regulation of SOS genes. The region of DNA 300 bp upstream of the recA gene was shown not to contain a promoter, suggesting that it functions as an upstream activator sequence.

Assessment of the ability of propoxur, methomyl, and aldicarb, three carbamate insecticides, to induce micronuclei in vitro in cultured Chinese hamster ovary cells and in vivo in BALB/c mice.

Three carbamate insecticides (propoxur, methomyl, and aldicarb) were evaluated for their ability to induce micronuclei (MN) in vitro using cultured Chinese hamster ovary (CHO) cells, and in vivo in mouse bone marrow erythrocytes. In vitro, all three insecticides induced a significant increase in micronucleated binucleate cells, which was generally both dose and sample time dependent. The in vivo studies involved treating male BALB/c mice by different routes, either once or on 3 consecutive days, followed by multiple or single sampling. Treatment by intraperitoneal injection or oral gavage induced a significant increase in micronucleated reticulocytes (MNRETs) in peripheral blood. For all three chemicals, the MN response depended on sample time and the number of treatments, while for aldicarb, the response depended also on the route of exposure. These positive results demonstrate that propoxur, methomyl, and aldicarb are capable of inducing structural and/or numerical chromosomal aberrations in mammalian cells either in vitro or in vivo. Furthermore, based on the results obtained, on optimal in vivo MN protocol for carbamate insecticides is a single treatment followed by blood sampling at 24 and 48 hr after treatment.

Chirality of a 1,10-bisacetoxymitosene compound. Impact on reductive activation, DNA interstrand cross-linking and antitumour activity.

The absolute configuration at the C-1 position of a 1,10-bisacetoxymitosene (WV15) appears to be important for enzymatic reduction, DNA interstrand cross-linking and in vitro antitumour activity of this compound. DNA cross-linking by the (-)-(S)-enantiomer of WV15 upon reduction with sodium dithionite (Na2S2O4) was more efficient than cross-linking by the (+)-(R)-enantiomer. Also, following enzymatic two-electron reduction by DT-diaphorase or one-electron reduction by xanthine oxidase, (-)-(S)-WV15 was more efficient in DNA cross-linking than (+)-(R)-WV15. However, the difference in cross-linking efficiency was less than upon chemical reduction, and in the case of enzymatic reduction that higher amount of DNA cross-links formed by (-)-(S)-WV15 can be explained by more efficient enzymatic activation of this enantiomer as compared to (+)-(R)-WV15. The enantiomeric preference upon chemical reduction can be explained by a second chemical reduction of DNA-bound WV15, which presumably does not occur upon enzymatic reduction. (-)-(S)-WV15 appeared to be more active than its (+)-(R) counterpart in A204 and L1210 tumour cell lines, with (+)-(R)/(-)-(S) toxicity ratios as high as 200 and 68, respectively. In Chinese hamster V79 cell lines, toxicity of the enantiomers was measured under oxic and hypoxic conditions. The oxic/hypoxic toxicity ratios of (+)-(R)-and (-)-(S)-WV15 in the Chinese hamster V79 cell line were 5.5 and 2.4, respectively. These different oxic/hypoxic toxicity ratios may indicate that different reducing enzymes are involved in the activation of the enantiomers. Generally, in biological systems, different activities of (+)-(R)- and (-)-(S)-WV15 appear not to be caused by different intrinsic cross-linking capacities of the enantiomers, but by more efficient enzymatic activation of (-)-(S)-WV15, as compared to (+)-(R)-WV15. The (-)-(S)-enantiomer of WV15 appears to be more active both in in vitro tumour models and in DNA cross-linking assays, and therefore the absolute configuration of mitosenes is indicated to be important for the antitumour activity of these compounds.

Lipopolysaccharide-induced DNA damage is greatly reduced in rats treated with the pineal hormone melatonin.

The ability of melatonin to influence lipopolysaccharide (LPS)-induced genotoxicity was tested using micronuclei as an index in both bone marrow and peripheral blood cells of rats. LPS was given as a single dose of 10 mg/kg. Melatonin (5 mg/kg) was injected prior to LPS administration and thereafter at 6 h intervals to the conclusion of the study (72 h). The number of micronucleated polychromatic erythrocytes increased significantly after LPS administration both in cells from peripheral blood and bone marrow. Melatonin administration to LPS-treated rats highly significantly reduced micronuclei formation in both peripheral blood and bone marrow cells beginning at 24 h after LPS administration and continuing to the end of the study. In blood the increase in micronuclei formation was time-dependent in LPS-treated rats with peak values being reached at 36-48 h. The ability of melatonin to reduce LPS-related genotoxicity is likely related to its antioxidant activity.

Potential antitumour mitosenes: relationship between in vitro DNA interstrand cross-link formation and DNA damage in Escherichia coli K-12 strains.

This investigation was aimed at determining the possible relationship between DNA interstrand cross-linking and the cytotoxic activity of potential antitumour mitosene compounds. Mitosenes, possessing two good leaving groups at C-1 and C-10, were found to be able to cross-link calf thymus DNA under hypoxic conditions following sodium dithionite (Na2S2O4) reduction at pH 7.0 and pH 5.5. DNA interstrand cross-linking was pH dependent for most of the mitosenes used, with a higher amount of cross-links formed at pH 5.5 compared to pH 7.0. Without reduction or under aerobic conditions no cross-link formation was detected. The importance of DNA damage for the toxic effect of these mitosenes was assayed by comparing the survival in a DNA repair deficient and a DNA repair proficient E. coli K-12 strain. A correlation between the number of cross-links formed in calf thymus DNA in vitro and the IC50 values in the DNA repair deficient E. coli strain was found. The effect of hypoxia on toxicity of mitosenes was studied in Chinese hamster V79 cells. In these cells, mitosenes appeared to be very active. Under severe hypoxic conditions toxicity of these mitosenes increased, most likely due to the increased lifetime of the activated mitosene species as compared to aerobic conditions. The results suggest that DNA cross-linking following reductive activation is important for the eventual activity of mitosenes in a bacterial system. Increased activity of mitosenes under hypoxic conditions in the V79 cells indicates that these mitosenes may be more active in hypoxic parts of tumours.

Structure-activity relationships for mitomycins. Application of the distance and charge analysis method.

Molecular orbital calculations based on coordinates from X-ray analysis have been performed for a set of 24 mitomycins, of which eight compounds have not been isolated so far. To prioritize the order of synthesis of these missing compounds, a new method named DISCA (distance and charge analysis) has been developed. DISCA screens correlations between spatial distribution of charge in molecules and their biological activity. The spatial distribution of charge is represented by several indexes in DISCA. LD50 and ED50 were used as measures of biological activity. DISCA has successfully extracted indexes which have significantly high correlation coefficients. The indexes with the highest correlation coefficient were common to both LD50 and ED50. By use of the correlation functions with high correlation coefficients DISCA has predicted that 9-epi-1a-N-demethylmitomycin D should have the best ED50 and a modest LD50 among the missing mitomycins.

Experience with mitomycin in the treatment of non-small cell lung cancer.

Mitomycin has proven to be among the most active drugs available for the single-agent treatment of non-small cell lung cancer (NSCLC). In combination with vinca alkaloids and cisplatin, mitomycin can produce response rates greater than or equal to 50% in properly selected patients. In our experience, such responses were achieved using moderate doses (7 or 8 mg/m2) of mitomycin, which also resulted in fewer hematologic and other toxicities. Delivery of MVP (mitomycin/vinca alkaloid/cisplatin) to 150 patients with stages III and IV NSCLC during the last decade showed maximal response was achieved after two or three cycles of therapy. A comparative analysis of results reported using MVP regimens suggests that high response rates are associated with greater dose-intensive use of cisplatin and lesser dose-intensive use of mitomycin. Although the role of MVP in the treatment of advanced NSCLC is unclear, use of mitomycin-containing regimens as part of a multidisciplinary approach to stage IIIA NSCLC has yielded high response rates and has successfully downstaged patients prior to surgery. Randomized clinical trials will be required to validate these findings, but the focus of future research should be on discovering new agents with greater activity and on developing new approaches wherein these agents can be delivered with maximum efficacy and minimum toxicity.

Mitomycin, ifosfamide, and cisplatin in non-small cell lung cancer.

Mitomycin, ifosfamide, and cisplatin have demonstrated the best single-agent activity thus far in patients with non-small cell lung cancer (NSCLC), the most common malignant disease in the western world. For this reason, we initiated a phase II study, giving these three agents in combination (designated MIC) to 74 patients with inoperable NSCLC. Sixty-six patients were evaluable for response, of whom 30 (45%) demonstrated a partial response and 7 (11%) a complete response. These results, along with those obtained in two other phase II trials of MIC in NSCLC, promoted us to begin a large-scale, multicenter, phase III study of MIC in patients with inoperable limited-stage NSCLC. In this ongoing study, patients have been randomized to receive treatment with MIC and radiotherapy or radiotherapy alone. We hope to resolve the issue of whether a survival advantage is conferred on NSCLC patients treated with radiotherapy in combination with this promising chemotherapeutic regimen.

Mitomycin in anal canal carcinoma.

One hundred and ten patients with primary epidermoid cancers of the anal canal were treated in a series of prospectively designed, nonrandomized protocols of split-course radiation therapy with concurrent administration of 5-fluorouracil (5-FU) with or without mitomycin. The addition of mitomycin was associated with improved primary tumor control rates (87 vs. 58% at 4 years, p = 0.005) and improved 4-year actuarial cause-specific survival (80 vs. 64%, p = 0.02). Hematologic toxicity was the most frequent acute side effect of mitomycin use. No long-term toxicity was attributed to mitomycin only. Mitomycin appeared to benefit patients principally through improved control of cancer in the irradiated volume; there was no evidence of reduced risk of extrapelvic metastases. Several investigators have reported high rates of control of epidermoid anal cancers with preservation of anorectal function following concurrent treatment with mitomycin, 5-FU, and radiation. Mitomycin's role in anal cancer is being evaluated in a randomized clinical trial by the Radiation Therapy Oncology Group. The mechanisms of any interactions between mitomycin and radiation or other cytotoxic drugs in clinical practice remain to be determined.

Spontaneous and induced levels of chromosomal aberration and sister-chromatid exchange in neurofibromatosis: no evidence of chromosomal hypersensitivity.

Chromosomal aberration (CA) and sister-chromatid exchange (SCE) frequencies have been assessed in 9 patients with von Recklinghausen's neurofibromatosis (NF1) and 8 apparently healthy controls. In separate experiments over a 5-year period, blood lymphocytes, skin fibroblast cell strains, and lymphoblastoid lines from both groups were treated with X-rays or mitomycin C (MMC) to determine whether the NF1 group was more sensitive to these agents than the control group. No difference between cells from NF1 patients and controls was observed with respect to spontaneous or X-ray-induced CA. Spontaneous or X-ray- and MMC-induced SCE frequencies were also similar in NF1 patients and controls.

Inhibition of the recBCD-dependent activation of Chi recombinational hot spots in SOS-induced cells of Escherichia coli.

Nucleotide sequences called Chi (5'-GCTGGTGG-3') enhance homologous recombination near their location by the RecBCD enzyme in Escherichia coli (Chi activation). A partial inhibition of Chi activation measured in lambda red gam mutant crosses was observed after treatment of wild-type cells with DNA-damaging agents including UV, mitomycin, and nalidixic acid. Inhibition of Chi activation was not accompanied by an overall decrease of recombination. A lexA3 mutation which blocks induction of the SOS system prevented the inhibition of Chi activation, indicating that an SOS function could be responsible for the inhibition. Overproduction of the RecD subunit of the RecBCD enzyme from a multicopy plasmid carrying the recD gene prevented the induced inhibition of Chi activation, whereas overproduction of RecB or RecC subunits did not. It is proposed that in SOS-induced cells the RecBCD enzyme is modified into a Chi-independent recombination enzyme, with the RecD subunit being the regulatory switch key.