Activity Of Mitomycin C Research Articles

Targeting of pegylated liposomal mitomycin-C prodrug to the folate receptor of cancer cells: Intracellular activation and enhanced cytotoxicity

Mitomycin C (MMC) is a powerful anti-bacterial, anti-fungal and anti-tumor antibiotic, often active against multidrug resistant cells. Despite a broad spectrum of antitumor activity, MMC clinical use is relatively limited due to its fast clearance and dose-limiting toxicity. To exploit the potential antitumor activity of MMC and reduce its toxicity we have previously developed a formulation of pegylated liposomes with a lipophilic prodrug of MMC (PL-MLP), activated by endogenous reducing agents which are abundant in the tumor cell environment in the form of different thiols. PL-MLP has minimal in vitro cytotoxicity unless reducing agents are added to the cell culture to activate the prodrug. In the present study, we hypothesized that targeting PL-MLP via folate receptors will facilitate intracellular activation of prodrug and enhance cytotoxic activity without added reducing agents. We grafted a lipophilic folate conjugate (folate-PEG(5000)-DSPE) to formulate folate targeted liposomes (FT-PL-MLP) and examined in vitro cell uptake and cytotoxic activity in cancer cell lines with high folate receptors (HiFR). 3H-cholesterol-hexadecyl ether (3H-Chol)-radiolabeled liposomes were prepared to study liposome-cell binding in parallel to cellular uptake of prodrug MLP. 3H-Chol and MLP cell uptake levels were 4-fold and 9-fold greater in KB HiFR cells when FT-PL-MLP is compared to non-targeted PL-MLP liposomes. The cytotoxic activity of FT-PL-MLP liposomes was significantly increased up to ~5-fold compared with PL-MLP liposomes in all tested HiFR expressing cell lines. The enhanced uptake and intracytoplasmic liposome delivery was confirmed by confocal fluorescence studies with Rhodamine-labeled liposomes. In vivo, no significant differences in pharmacokinetics and biodistribution were observed when PL-MLP was compared to FT-PL-MLP by the intravenous route. However, when liposomes were directly injected into the peritoneal cavity of mice with malignant ascites of J6456 HiFR lymphoma cells, the tumor cell levels of MLP were significantly greater with the folate-targeted liposomes. Thus, folate targeting enhances liposome uptake by tumor cells enabling intracellular activation of prodrug in the absence of exogenous reducing agents, and leading to increased cytotoxicity. These results may be particularly relevant to the application of folate-targeted PL-MLP in intracavitary or intravesical treatment of cancer.

DNA damage protective effect of honey-sweetened cashew apple nectar in Drosophila melanogaster.

Fruits and derivatives, such as juices, are complex mixtures of chemicals, some of which may have mutagenic and/or carcinogenic potential, while others may have antimutagenic and/or anticancer activities. The modulating effects of honey-sweetened cashew apple nectar (HSCAN), on somatic mutation and recombination induced by ethyl methanesulfonate (EMS) and mitomycin C (MMC) were evaluated with the wing spot test in Drosophila melanogaster using co- and post-treatment protocols. Additionally, the antimutagenic activity of two HSCAN components, cashew apple pulp and honey, in MMC-induced DNA damage was also investigated. HSCAN reduced the mutagenic activity of both EMS and MMC in the co-treatment protocol, but had a co-mutagenic effect when post-administered. Similar results were also observed with honey on MMC mutagenic activity. Cashew apple pulp was effective in exerting protective or enhancing effects on the MMC mutagenicity, depending on the administration protocol and concentration used. Overall, these results indicate that HSCAN, cashew apple and honey seem capable of modulating not only the events that precede the induced DNA damages, but also the Drosophila DNA repair processes involved in the correction of EMS and MMC-induced damages.

P-glycoprotein Blockers Augment the Effect of Mitomycin C on Human Tenon's Fibroblasts

Mitomycin C (MMC), which induces apoptosis in human Tenon's fibroblasts (HTF), is frequently used to retard wound healing after glaucoma surgery. The aim of this in vitro study was to examine whether adjunctive Verapamil and Cyclosporine could augment the cytotoxic effect of MMC on HTF. Fibroblast cell lines were established by explant culture from human tissue biopsy samples obtained during trabeculectomy procedures. Cells were exposed to MMC at varying concentrations (0.01-0.4 mg/ml) for 3 minutes, prior to washing in the presence or absence of the following drugs: Staurosporine (0.003mg/ml), Verapamil (2.5-0.25 mg/ml), or Cyclosporine (50-0.5 mg/ml). Following exposure, cells were cultured for 6 hours and surviving cells quantitated by haemocytometer counts. Both Verapamil and Staurosporine exhibited mild toxic effects on their own, but greatly enhanced the apoptotic effect of MMC. Staurosporine is too toxic to be considered clinically, so its augmentive effect on the activity of MMC was not studied further here. Doses as low as 0.25 mg/ml of Verapamil continued to show significant augmentation of the apoptotic effect of MMC Cyclosporine at a clinically used concentration (5 mg/ml) exhibited modest augmentation of the effect of MMC. Verapamil and Cyclosporine in clinically acceptable concentrations potentiate the effect of MMC and may obviate the need for high dose antimetabolites in trabeculectomy; however, further preclinical study is required. Adjunctive Verapamil or Cyclosporine may allow lower dose MMC to be used in glaucoma filtration surgery while maintaining the same antifibrotic effects.

A multicenter, multinational analysis of mitomycin C in refractory metastatic colorectal cancer

BackgroundA considerable number of metastatic colorectal cancer (mCRC) patients who progress on standard treatment with 5-fluorouracil (5FU), oxaliplatin, irinotecan and monoclonal antibodies, still have adequate performance status and desire further treatment. Mitomycin C (MMC) has been widely used in this context, and despite good tolerability, there are doubts regarding its true benefit. MethodsIn order to assess the activity of MMC in the refractory mCRC setting, we retrospectively evaluated 109 heavily pre-treated patients who received MMC as single agent or in combination for mCRC at three different institutions in two countries. ResultsMedian patient’s age was 54years old, 57% were male and 94% had performance status ECOG 0 or 1. MMC was used in second line in 11%, third line in 38% and fourth line or beyond in 51% of patients. 58% received MMC combinations, mainly with capecitabine. Grade 3 or 4 toxicity was observed in 5% of patients and 6% required dose reductions. Median time to treatment failure (TTF) was 1.7months with MMC and 3.6months on the regimen prior to MMC, with a ratio between these TTF below 1 in 82% of patients. Median survival was only 4.5months (95% confidence interval (CI) of 3.48–5.56). ConclusionsThis retrospective data represent the largest reported series of unselected refractory mCRC patients treated with MMC. The median survival of 4.5months is similar to the survival expected for best supportive care. This lack of activity strongly suggests that MMC should not be routinely used in refractory mCRC.

2A Peptide-based, Lentivirus-mediated Anti-death Receptor 5 Chimeric Antibody Expression Prevents Tumor Growth in Nude Mice

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, induces tumor cell death via death receptors on target cells, without adverse effects on most normal cells. Its receptors are therefore an attractive target for antibody-mediated tumor therapy. Here, we report the creation of a lentivirus vector constructed by linking the heavy chain and the light chain of the antibody with a 2A/furin self-processing peptide in a single open reading frame that expresses a novel chimeric antibody (named as zaptuximab) with tumoricidal activity, which is consisted of the variable region of a mouse anti-human DR5 monoclonal antibody, AD5-10, and the constant region of human immunoglobulin G1. Lentivirus-expressed zaptuximab bound specifically to its antigen, DR5, and exhibited significant apoptosis-inducing activity in various tumor cell lines. The packaged recombinant virus lenti-HF2AL showed strong apoptosis-inducing activity in vitro. Meanwhile, inoculated subcutaneous human colon HCT116 tumor formation in nude mice were inhibited significantly. Moreover, there was a synergistic effect of mitomycin C (MMC) on the observed tumoricidal efficacy, prolonging the life span of nude mice with orthotopic human lung tumor cancers. These data suggest that lentivirus-mediated, 2A peptide-based anti-DR5 chimeric antibody expression may have clinical utility as an anticancer treatment and may represent a rational adjuvant therapy in combination with chemotherapy.

Arsenite pretreatment enhances the cytotoxicity of mitomycin C in human cancer cell lines via increased NAD(P)H quinone oxidoreductase 1 expression

Arsenic is an effective therapeutic agent for the treatment of patients with refractory or relapsed acute promyelocytic leukemia. The use of arsenic for treating solid tumors, particularly in combination with other chemotherapeutic agents, has been extensively studied. Here, we report that arsenite-resistant human lung cancer CL3R15 cells constitutively overexpress NAD(P)H quinone oxidoreductase 1 (NQO1), an enzyme responsible for activation of mitomycin C (MMC), and are more susceptible to MMC cytotoxicity than parental CL3 cells. The effects of arsenite pretreatment on NQO1 induction were examined in CL3, H1299, H460, and MC-T2 cells. Arsenite pretreatment significantly enhanced the expression of NQO1 and susceptibility to MMC in CL3, H1299, and MC-T2 cells, but not in H460 cells that express high endogenous levels of NQO1. Alternatively, arsenic pretreatment reduced adriamycin sensitivity of CL3 cells. Arsenite-mediated MMC susceptibility was abrogated by dicumarol (DIC), an NQO1 inhibitor, indicating that NQO1 is one of the key regulators of arsenite-mediated MMC susceptibility. Various cancer cell lines showed different basal levels of NQO1 activity and a different capacity for NQO1 induction in response to arsenite treatment. However, overall, there was a positive correlation between induced NQO1 activity and MMC susceptibility in cells pretreated with various doses of arsenite. These results suggest that arsenite may increase NQO1 activity and thus enhance the antineoplastic activity of MMC. In addition, our results also showed that inhibition of NQO1 activity by DIC reversed the arsenite resistance of CL3R15 cells.

LOW DOSE SURAMIN AS A CHEMOSENSITIZER OF BLADDER CANCER TO MITOMYCIN C

We have reported that acidic and basic fibroblast growth factors expressed in solid tumors induce broad-spectrum chemoresistance and their nonspecific inhibitor suramin (Sigma Chemical Co., St. Louis, Missouri) at nontoxic concentrations enhances the activity of chemotherapeutic agents. In the current study we evaluated whether low dose suramin enhances tumor sensitivity to mitomycin C (MMC) (Bristol-Myer Squibb Co., Wallingford, Connecticut). Three-dimensional histocultures of RT4 (American Type Culture Collection, Rockville, Maryland) xenograft tumors and tumors from patients with bladder cancer were treated with MMC with or without 20 microM suramin. For in vivo studies mice bearing RT4 tumors received physiological saline, MMC (3 mg/kg), suramin (10 mg/kg) or MMC/suramin combination every 3 or 4 days. Two MMC schedules were studied, namely subtherapeutic (3 treatments) and therapeutic (6 treatments) schedules. Suramin enhanced MMC activity in xenograft and patient tumor histocultures by greater than 2-fold. The chemosensitization effect was observed in all patient tumors (2 superficial and 9 muscle invading lesions). In animals tumors in the control and suramin groups increased to approximately 3 to 5 times the initial size. The subtherapeutic MMC schedule retarded tumor growth but did not produce tumor shrinkage, whereas the therapeutic MMC schedule reduced tumor size by 27%. The addition of suramin enhanced MMC activity. Respective tumor sizes were 40% and 63% of the sizes after subtherapeutic and therapeutic MMC alone (p <0.01). The combination groups showed body weight losses similar to those of MMC alone. Low dose suramin enhanced the in vitro and in vivo activity of subtherapeutic and therapeutic MMC without enhancing host toxicity, thus, providing proof of concept for evaluating low dose suramin as a chemosensitizer with MMC in bladder cancer.

Anti-clastogenic activity of two structurally related pterocarpans purified from Bituminaria bituminosa in cultured human lymphocytes

Plant-derived isoflavones are currently receiving much attention because of their phyto-estrogenic, antioxidant, anti-mutagenic, and anti-tumor activities. In this study we have evaluated the clastogenic and anti-clastogenic activities in human lymphocytes of two structurally related pterocarpans, iso-flavonoid derivatives, termed erybraedin C and bitucarpin A, recently purified from Bituminaria bituminosa and chemically characterized. Mitomycin C (MMC) and the radio-mimetic bleomycin (BL) were used as reference clastogens. The end point studied was micronucleus formation. The results obtained in this study indicate that erybraedin C and bitucarpin A, when assayed alone, do not affect either the mitotic index or the cell-proliferation index of human lymphocytes. Interestingly, both compounds appear to be non-clastogenic in the range of concentrations used. In contrast, both substances seem to affect significantly the clastogenic effects induced by BL and MMC. A 1-h pre-exposition of the cell culture to erybraedin C was necessary to display its anti-clastogenic potential against BL, whereas bitucarpin A was inactive in this respect, with a structure-activity relationship. In contrast, the clastogenic activity of MMC was significantly reduced by both erybraedin C and bitucarpin A, using either a pre-incubation schedule or simultaneous treatment. These results suggest that the protective effects displayed by the two anti-clastogenic compounds against MMC could be due to the induction or inhibition of cellular reductive metabolic enzymes.

Possible modulating actions of plant extracts on the chromosome breaking activity of MMC and Ara-C in human lymphocytes in vitro

Plants popularly used as medicine have been seen as promising natural agents by the pharmaceutical industry. In the present study the action of Psidium guajava L. ( Pg) and Achillea millefolium L. ( Am) infusions on chromosomal aberration formation in human lymphocyte system in vitro was assessed, associating them with the alkylating agent mitomycin C (MMC) and the DNA repair inhibitor cytosine-β-arabin-furanoside (Ara-C). The cells were cultivated for 72 h and treated continuously with Pg and the Am infusions at dosages of 2.62 × 10 −4 g and 3.5 × 10 −4 g/ml culture medium, respectively. Treatments with MMC (0.30 μg/ml) or Ara-C (5 × 10 −7 μg/ml) were administered after 48 h of cell culture. Each samples (five individual) were exposed to nine treatments (control with PBS; Pg; Am; MMC; MMC + Pg; MMC + Am; Ara-C; Ara-C + Pg; and Ara-C + Am) and 100 cells were analyzed per cell culture. The used doses of each infusion did not cause clastogenic effects significantly different to the negative control (control=1%; Pg=2.2%; Am=1.8%). Nevertheless, the aberrant cell frequency after MMC treatment was significantly increased by the Am infusion (MMC=32.4%; MMC + Pg=36.2%; MMC + Am=44%), especially when the chromatid break types number was scored (MMC=151; MMC + Pg=173; MMC + Am=249). Regarding DNA repair inhibition by Ara-C, the Pg infusion caused a significant reduction in aberrant cell frequency (Ara-C=15.8%; Ara-C + Pg=11%; Ara-C + Am=14.4%), only when the chromatid break types number was scored (Ara-C=63; Ara-C + Pg=40; Ara-C + Am=58). These results indicate that the plant infusions per se do not have clastogenic activity, but can influence the clastogenic action of MMC and Ara-C on DNA break induction, in vitro.

Mitomycin antitumor compounds. 2. Interaction of transition metal ions with mitomycin C. Solution structure and biological activity of a Pd(2+)-MMC complex.

Interactions of Cu(2+), Zn(2+), and Pd(2+) ions with the antitumor compound mitomycin C (MMC) have been investigated by UV-vis, circular dichroism, and (13)C NMR spectroscopy. While Zn(2+) and Cu(2+) neither interacted with MMC nor catalyzed the formation of mitosenes, Pd(2+) induced strong MMC spectral modifications, suggesting the formation of a major complex, in which MMC acted as a bidentate ligand through N(1) and N(4) atoms. The coordination mode in this complex was solvent dependent: in MeOH, the NH(2) of the carbamate function was also involved as a third coordination site whereas, in H(2)O, Pd(2+) hydrolysis was more effective, leading to the replacement of the carbamoyl NH(2) function with either H(2)O or OH(-) ligands. Although coordination of the indoline nitrogen prevented methanol elimination and consequent aziridino ring opening, Pd(2+) complexation maintained MMC biological activity against cancer cells, as shown by IC(50) values. This suggests that alternative mechanisms in the biological activity of MMC should be explored.

Activity of dissolved mitomycin C after different methods of long-term storage.

The cytostatic substance mitomycin C (MMC) is used in trabeculectomy to enhance the success rate in problematic cases and is usually dissolved immediately before application, but only approximately 1% of the substance is needed for treatment. The authors evaluated different methods of long-time storage of MMC for a period of 6 months. MMC in concentrations of 0.02, 0.05, 0.1, 0.2, and 0.4 mg/mL was prepared at the local pharmaceutical department and stored at +22 degrees C (room temperature), +4 degrees C (refrigerator), -20 degrees C (freezer compartment), and -196 degrees C (liquid N2). The activity of MMC was evaluated with a microagar diffusion method (bioassay) after 30 minutes, and 1, 3, 7, 14, 30, 90, and 180 days for the different concentrations and storage methods. There was no difference in the long-term stability of the investigated MMC concentrations. Ninety percent of the initial activity was preserved after storage at +22 degrees C for 1 week, -20 degrees C for 1 month, or +4 degrees C for 3 months. At 6 months the activities were 16%, 48%, and 78% of the initial values, respectively. The activity of MMC stored at -196 degrees C is not reduced after storage for 6 months. Dissolved MMC can be stored in the refrigerator for up to 3 months without significant loss of activity. Storage at room temperature is not recommended. Costs of trabeculectomy can be reduced by storage of reconstituted solutions rather than dissolving MMC before each glaucoma surgery.

Enhanced cytotoxicity of mitomycin C in human tumour cells with inducers of DT-diaphorase.

DT-diaphorase is a two-electron reducing enzyme that activates the bioreductive anti-tumour agent, mitomycin C (MMC). Cell lines having elevated levels of DT-diaphorase are generally more sensitive to MMC. We have shown that DT-diaphorase can be induced in human tumour cells by a number of compounds, including 1,2-dithiole-3-thione. In this study, we investigated whether induction of DT-diaphorase could enhance the cytotoxic activity of MMC in six human tumour cell lines representing four tumour types. DT-diaphorase was induced by many dietary inducers, including propyl gallate, dimethyl maleate, dimethyl fumarate and sulforaphane. The cytotoxicity of MMC was significantly increased in four tumour lines with the increase ranging from 1.4- to threefold. In contrast, MMC activity was not increased in SK-MEL-28 human melanoma cells and AGS human gastric cancer cells, cell lines that have high base levels of DT-diaphorase activity. Toxicity to normal human marrow cells was increased by 50% when MMC was combined with 1,2-dithiole-3-thione, but this increase was small in comparison with the threefold increase in cytotoxicity to tumour cells. This study demonstrates that induction of DT-diaphorase can increase the cytotoxic activity of MMC in human tumour cell lines, and suggests that it may be possible to use non-toxic inducers of DT-diaphorase to enhance the efficacy of bioreductive anti-tumour agents. © 1999 Cancer Research Campaign

Interferon gamma increases the antitumor activity of mitomycin C against human colon cancer cells in vitro and in vivo.

A combined antitumor activity of mitomycin C (MMC) and interferon gamma-1a (IFN-gamma) was evaluated to be synergistic by the 3-(4, 5-dimethylthiazol-2yl)-2,5-diphenyl-2H tetrazolium bromide assay and human tumor xenografts/severe combined immunodeficient (SCID) mouse system using colon cancer cell lines. The experimental metastasis of WiDr cells in SCID mouse was inhibited by MMC and IFN-gamma with their synergism. Intracellular uptake of MMC in WiDr cells in vitro was significantly increased by IFN-gamma, suggesting the mode of synergism of these agents. This model may also partly explain the antitumor activity of combined MMC, 5-fluorouracil and interleukin-2 treatment on hepatic metastasis of colon cancer.

Induction of DT-diaphorase by 1,2-dithiole-3-thiones in human tumour and normal cells and effect on anti-tumour activity of bioreductive agents.

DT-diaphorase is a two-electron-reducing enzyme that is an important activator of bioreductive anti-tumour agents, such as mitomycin C (MMC) and EO9, and is inducible by many compounds, including 1,2-dithiole-3-thiones (D3Ts). We showed previously that D3T selectively increased DT-diaphorase activity in mouse lymphoma cells compared with normal mouse marrow cells, and also increased MMC or EO9 cytotoxic activity in the lymphoma cells with only minor effects in the marrow cells. In this study, we found that D3T significantly increased DT-diaphorase activity in 28 of 38 human tumour cell lines representing ten tissue types with no obvious relationships between the tumour type, or the base level of DT-diaphorase activity, and the ability of D3T to increase the enzyme activity. Induction of DT-diaphorase activity in human tumour cell lines by 12 D3T analogues varied markedly with the D3T structure. D3T also increased DT-diaphorase activity in normal human bone marrow and kidney cells but the increases were small in these cells. In addition, D3T increased the level of enzyme activity in normal human lung cells. Pretreatment of human tumour cells with D3T analogues significantly increased the cytotoxic activity of MMC or EO9 in these cells, and the level of enhancement of anti-tumour activity paralleled the level of DT-diaphorase induction. In contrast, D3T did not effect the toxicity of EO9 in normal kidney cells. These results demonstrate that D3T analogues can increase DT-diaphorase activity in a wide variety of human tumour cells and that this effect can enhance the anti-tumour activity of the bioreductive agents MMC and EO9.

Potentiation of antitumor activity of mitomycin C by estradiol: Studies of human breast carcinoma xenografts serially transplanted into nude mice

The effect of experimental cancer chemotherapy with mitomycin C (MMC) was studied using three estrogen-receptor (ER)-positive (MCF-7, R-27, and Br-10) and one ER-negative (MX-1) human breast carcinoma xenograft serially transplanted into nude mice, and the effect of estradiol (E2) priming on the antitumor activity of MMC was investigated. Intramuscular injection of E2 at 1 mg/kg changed the ER state and increased the growth fraction detected by flow cytometry, although the growth rate of ER-positive tumors was not effective by E2 priming. MMC suppressed the growth of the four xenografts in a dose-dependent manner. When 1 mg/kg E2 was administered 1 h before MMC treatment, which was given intraperitoneally at a dose of 3 mg/kg, the antitumor activity of MMC was increased in comparison with MMC alone in ER-positive strains, although the effect of MMC on MX-1 was not changed by E2-priming. Priming with E2 at this dose increases the growth fractions of ER-positive breast carcinoma cells, which are sensitive to MMC, resulting in increased antitumor activity of MMC. This E2-primed MMC chemotherapy may be of value in the treatment of ER-positive human breast cancer.

Characteristics of the antitumor activity of M-16 and M-18, major metabolites of a new mitomycin C derivative KW-2149, in mice.

The cell growth inhibitory activity, antitumor activity and toxicity of M-16 and M-18, the major metabolites of a new mitomycin C (MMC) derivative KW-2149, in mouse and human were compared with those of KW-2149 or MMC in vitro and in vivo. The growth inhibitory activity of M-18, a symmetrical disulfide dimer, against human uterine cervix carcinoma HeLa S3 cells was almost equivalent to that of KW-2149 and their IC50 values were about 10-fold smaller than that of MMC. The activity of M-16, a methyl sulfide form, was almost equivalent to that of MMC. The cell-killing activity of MMC and M-16 was augmented in the hypoxic condition, whereas that of KW-2149 and M-18 was reduced. M-16 also exhibited almost equivalent activities to MMC in vivo in terms of many biological profiles, i.e., antitumor activity against murine P388 leukemia, ascitic or solid B16 melanoma or human lung carcinoma xenograft L-27, and bone marrow toxicity in mice. These in vitro and in vivo results indicate that the antitumor activity and toxicity of KW-2149 might not be mediated by M-16 in mice. On the other hand, M-18 exhibited almost equivalent activities to KW-2149 in these regards, suggesting the involvement of M-18 in the biological activities of KW-2149. However, the small values of the area under the curve of M-18 in mice make this unlikely. Thus the biological activities of KW-2149 in mice are not explained by the M-16 or M-18 concentration in plasma and are postulated to be manifested by KW-2149 itself.

Synergistic antitumor activity of combination chemotherapy with mitomycin C and cisplatin against human gastric cancer xenografts in nude mice

A new combined cancer chemotherapy regimen of mitomycin C (MMC) and cisplatin (DDP) showed synergistic antitumor activity against human gastric cancer xenografts St-40 and SC-1-NU in BALB/c nu/nu mice. The drugs were administered intraperitoneally at doses of 2 or 4 mg/kg for MMC and 3 or 6 mg/kg for DDP, respectively. To clarify the schedule-dependent antitumor activity of MMC and DDP against St-40 and SC-1-NU, different sequential therapies were conducted. Simultaneous administration of these agents showed the highest antitumor activity against SC1-NU among the three regimens used, whereas the sequence of MMC followed by DDP showed higher antitumor activity than the reverse sequence against St-40. The intratumoral concentration of platinum was significantly increased in St-40 treated with the sequence MMC to DDP, in comparison with the sequence DDP to MMC. The maximum tolerated dose (MTD) of this combination was 4 mg MMC plus 6 mg DDP per kg in all the combinations, and these MTDs were 2/3 of the corresponding values for their single use. Since this combination increased the antitumor activity of each single agent without any increase in their toxicity, it would appear to be useful clinically.

Suppressive effects of urine on the SOS responses induced by UV and chemical mutagens

We studied the antimutagenic effects of urine on the SOS-inducing activity of mutagenic substances by using a novel test system (umu-test) for detecting DNA damaging agents, which uses a new tester strain ( Salmonella typhimurium TA1535 pSK1002 ). The SOS-inducing activity of chemicals was detected in terms of the level of umu operon expression by measuring β-galactosidase activity. We first examined the effects of various amounts of urine on SOS responses caused by mitomycin C (MMC). As a result, the urine suppressed β-galactosidase activity of MMC dose-dependently. A urine concentration of 50 μl/ml in the medium also suppressed 89.9% of SOS response induced by 0.1 μg/ml of PEP, 75.6% of that induced by 0.02 μg/ml of AF2 and 60.9% of that induced by 0.1 μg/ml of AFB 1. In addition, a urine concentration of 50 μl/ml in the medium also suppressed 85.6% of SOS response by 5 J/m 2 of UV irradiation. The observed suppression seemed to be directly related to the SOS responses at a cellular level, rather than to interaction between urine and mutagens, because the urine suppressed SOS responses induced not only by various mutagens but also by UV irradiation. These results suggest that urine works as a strong antimutagen against UV and chemical substances.

Phase II trial of mitomycin C (MMC) in advanced gallbladder and biliary tree carcinoma. An EORTC Gastrointestinal Tract Cancer Cooperative Group Study

Based on a recent pharmacokinetic study suggesting that high biliary levels of mitomycin C (MMC) may be achieved as a result of an entero-hepatic recycling mechanism, we conducted a Phase II EORTC trial which involved MMC administration to patients with non-resectable biliary tract carcinoma. Of the 34 patients entered in the study, 30 were eligible (11m + 19f, median age 58 yrs). I.V. bolus injections of 15 mg/m2 MMC were administered at six-week intervals. The tumors were confined to the liver in 17 patients and 13 had extra-hepatic localizations. All 30 eligible patients were evaluable for toxicity and response. Mild thrombocytopenia was the main toxic side effect. Severe, WHO grade III/IV thrombocytopenia was limited to 4 patients. The haemolytic uraemic syndrome was not observed and there were no toxic deaths. Of 30 patients, 3 had partial remissions (overall response 10%, 95% confidence interval 2%-27%). This study, the largest such ongoing phase II trial, shows no significant activity of single-agent MMC in patients with advanced biliary tract carcinoma.

The concomitant detection of gene mutation and micronucleus induction by mitomycin C in vivo using lacZ transgenic mice

A new assay system that can simultaneously provide gene mutagenicity and clastogenicity data in vivo is described. Transgenic mice (Muta TM Mouse) harboring the lacZ gene as a target for mutation analysis were injected intraperitoneally with mitomycin C (MMC), either once or on 5 successive days. Micronucleuc assays were performed with small amounts of peripheral blood collected from a tail vessel. The spontaneous frequency of micronucleated reticulocytes was 0.42%. For the mutation analysis, DNA was extracted from bone marrow and liver cells at several harvest times. The lacZ gene was rescued by lambda packaging and infection of E. coli C ( lac −), followed by plating on agarose plates containing X-gal. The spontaneous lacZ mutant frequencies were 37 and 29×10 −6 in bone marrow and liver, respectively. In the micronucleus assay, single treatments with 1.0 and 2.0 mg/kg of MMC induced micronuclei in 3.6 and 5.8% of reticulocytes, respectively, peaking 48 h after treatment. Muta TM Mouse sensivitiy to micronucleus induction was similar to nontransgenic strains used routinely for the micronucleus test. On the other hand, single treatments with MMC at 1.0 and 2.0 mg/kg did not induce any significant increases in the frequency of lacZ − mutants in bone marrow or liver. N-Ethyl- N-nitrosourea, used at 100 mg/kg as a positive control, yielded a 5-fold increase in mutant frequency above untreated animals in bone marrow only. After 5-day treatments, MMC induced approximately a 2-fold increase in mutant frequency in bone marrow only for the sublethal dose of 2 mg/kg. Therefore, this study indicated that the strong clastogenic activity of MMC in bone marrow was not accompanied by significant gene mutagenic activity.