Cytotoxicity of ketoconazole in malignant cell lines

Summary The cytotoxic effects of ketoconazole, an antifungal agent known to have some activity against human prostate cancer, adrenal cancer, and male metastatic breast cancer, were evaluated using colony-growth and clonogenic assays in eight malignant cell lines. The cytotoxicity of ketoconazole showed a dose-and time-dependent pattern, with the following concentrations, inhibiting 90% of the growing colonies (IC₉₀): MCF 7 (human breast cancer) 7.25 g/ml, T 47 D (human breast cancer) 9.0 g/ml, MiaPaCa (human pancreatic carcinoma) 10.0 g/ml, COLO 357 (human pancreatic carcinoma) 9.5 g/ml, HCT 8 (human colonic adenocarcinoma) 27.1 g/ml, DU 145 (human prostatic cancer) 40.0 g/ml, AR 42 J (rat pancreatic carcinoma) 9.0 g/ml, and L1210 (murine leukemia) 8.6 g/ml. Since a concentration of 10 g/ml can be achieved in humans, the use of ketoconazole in human malignancies might be worthy of clinical evaluation.This investigation was supported in part by a grant from the German Volkswagen Foundation, Hannover, Federal Republic of Germany and by a gift from Dr Virgil Gianelli, Stockton, California     

Adenosine and deoxyadenosine toxicity in colony assay systems for human T-lymphocytes,B-lymphocytes,and granulocytes

Summary Adenosine and deoxyadenosine toxicity was examined in colony assay systems for human T lymphocytes, B lymphocytes, and granulocytes. In the absence of deoxycoformycin, an adenosine deaminase inhibitor, no growth inhibition was observed in the three systems with concentrations of adenosine or deoxyadenosine of at least 200 M. Deoxycoformycin itself had no growth-inhibitory effect at concentrations of at least 10 g/ml. Combinations of deoxycoformycin (l g/ml) and either adenosine or deoxyadenosine gave growth inhibition in all three systems. Deoxyadenosine was the most toxic in all the systems, the LD₅₀ values being 20–25 M. The LD₅₀ values for adenosine were 45–55 M. There was no evidence of selective toxicity by adenosine or deoxyadenosine with these three colony assay systems. In the T-lymphocyte colony system deoxyadenosine appeared to be toxic to both the inducer/helper and the suppressor/cytotoxic T-lymphocyte subpopulations.     

Interactions of vinblastine and vincristine with methotrexate transport in isolated rat hepatocytes

The accumulation of methotrexate (MTX) in the presence of vinblastine (VBL) and vincristine (VCR) was studied in isolated rat hepatocytes. In accordance with our recent study on vindesine (VDS), we found VBL and VCR to reduce net MTX accumulation significantly at 15 min after MTX addition. Drug concentrations of 100 M VBL and 500 M VCR led to 67% and 82% reductions in intracellular MTX, respectively. Since there was only a slight inhibition of MTX efflux by 100 M VBL, the accumulation data demonstrate that the major effect of VBL is on MTX influx. Dixon-plot analyses are suggestive of competitive inhibition of the MTX influx, yielding inhibition constants (K _i values) of 55 M for VBL and 110 M for VCR. Since theK _i values correspond grossly to plasma levels obtained in humans shortly after the infusion of therapeutic doses of the vinca alkaloids studied herein, the interaction with MTX uptake could serve to diminish the toxicity of MTX to nonmalignant cells.This study was supported financially by the Norwegian Cancer Society     

Development of alkylating agent-resistant human tumor cell lines

Summary Survival curves and dose escalation studies of four representative human tumor cell lines exposed to the various alkylating agents are presented. With HN2, at a level of one log of cell kill there was a fivefold range in drug concentration required to achieve this degree of cell kill among the cell lines, from 4.5 M for the SL6 lung adenocarcinoma to 22 M for the SW2 small-cell lung carcinoma. Four logs of SCC-25 squamous carcinoma cells were killed by 100 M CDDP; however, there was only about one log of SL6 cells killed by 500 M CDDP. To kill one log of G3361 melanoma cells required 380 M 4-HC and to kil one log of SCC-25 cells required 24 M, approximately a 16-fold difference. The curves for cell kill by L-PAM appeared to be biphasic, with a break at about 100 M. There was about a threefold range in drug concentration required to achieve one log of cell kill with L-PAM, from 60 M in the SCC-25 cell line to 18 M in the SW2 line. To kill one log of SCC-25 cells required 295 M BCNU and to kill one log of SW2 cell required 120 M, about a 2.5-fold difference. The range of maximally tolerated HN2 concentrations were from 1200M for the SL6 cell line, 48 times the initial concentration, to 300 M for the SCC-25 line, 16 times the initial concentration. The G3361 line tolerated the highest level of CDDP, 1900 M, 48 times the initial concentration. The SCC-25 line, on the other hand, tolerated only 600 M, 30 times the initial concentration. The SL6 cell line maximally tolerated 36 times the initial concentration of 4-HC (1450 M), whereas the SCC-25 cell line tolerated only 18 times the initial concentration (720 M). The G3361 melanoma tolerated 1555 M, 30 times the initial concentration of L-PAM, and the SCC-25 cell line tolerated 700 M, 14 times the initial concentration. The SL6 cell line tolerated the highest concentration of BCNU, 4200 M, 24 times the initial concentration. The SCC-25 cell line tolerated 1450 M, 8 times the initial concentration. In all cases, the SCC-25 cell line was least able to tolerate exposure to increasing concentrations of alkylating agents. The SL6 and G3361 cell lines showed the greatest tolerance for increasing concentrations of alkylating agents. With maximal selection pressure, in terms of intensity and duration, 5-to 15-fold resistance at best could be produced to these alkylating agents. This contrasts with other drugs, indicates that alkylating agents are more like X-rays, and has implications for high-dose clinical treatments. The importance of these findings to the clinical treatment of cancer is discussed.Abbreviations NH2 Nitrogen mustard (mustragen) - CDDP cis-diamminedichloroplatinum (II) (cisplatin) - BCNU N,N-bis(2-chloroethyl)-N-nitrosourea - L-PAM L-phenylalanine mustard (melphalan) - 4-HC 4-hydroperoxycyclophosphamide - DMEM Dulbecco's modified Eagle's medium - FBS fetal bovine serum - PBS phosphate-buffered saline This work was supported by NCI grant #5PO1-CA38493 and a grant from the Mathers Foundation     

The cytotoxicity of sarcosinamide chloroethylnitrosourea (SarCNU) and BCNU in primary gliomas and glioma cell lines: analysis of data in reference to theoretical peak plasma concentrations in man

Summary The cytotoxicity of a new compound, sarcosinamide chloroethylnitrosourea (SarCNU), was compared with that of the clinically available bis-chloroethylnitrosourea (BCNU) in 13 primary human gliomas and in 3 human glioma cell lines using the Human Tumor Cloning Assay (HTCA). At concentrations 16 g/ml, SarCNU reduced the growth to 30% of control in 11 of 13 primary gliomas. At similar concentrations, BCNU produced a comparable cytotoxic effect in 6 out of 13 specimens. At concentrations 16 g/ml, BCNU reduced colony growth to 30% of control in all three glioma cell lines and SarCNU produced the same effect in only one glioma cell line. A recently described statistical model [10], which employs the LD₅₀ dose of new agents in mice, was used to estimate the achievable peak plasma concentration (PPC) of SarCNU. The calculated PPC for SarCNU was found to be 14.8 g/ml compared with 2 g/ml for BCNU. A reevaluation of the cytotoxic activities of SarCNU and BCNU at concentrations approximating their respective PPCs revealed that SarCNU reduced the growth to 30% of control in one cell line at a concentration below its PPC. In contrast, BCNU exhibited similar toxicity in each cell line only at concentrations exceeding its PPC of 2 g/ml. In the case of the primary gliomas, SarCNU was active (30% of control) in ten tumors at concentrations 14.8 g/ml, whereas BCNU was active in only one glioma at a concentration 2 g/ml. The results suggest that SarCNU should be more active than BCNU against human gliomas, provided that the statistical model used has correctly estimated the PPC of SarCNU.     

1-β-d-arabinofuranosylcytosine-, mitoxantrone-, and paclitaxel-induced apoptosis in HL-60 cells: improved method for detection of internucleosomal DNA fragmentation

We investigated the ability of different doses and durations of exposure to the chemotherapeutic drugs 1--d-arabinofuranosylcytosine (Ara-C), mitoxantrone (MTN), and paclitaxel (taxol, TXL) to induce internucleosomal DNA fragmentation and apoptosis in human acute myeloid leukemia (AML) HL-60 cells in suspension culture. At clinically achievable concentrations, all three drugs have been shown to induced apoptosis in HL-60 cells. An improved method was developed for the isolation of pure genomic DNA and the detection of drug-induced intergenomic DNA and the detection of drug-induced internucleosomal DNA fragmentation in <1.0 g of DNA sample by agarose gel electrophoresis. Morphologic evidence for apoptosis was determined by light microscopy following Wright staining, and cell viability was assessed by trypan blue dye exclusion. Internucleosomal DNA fragmentation was observed following exposure to 1.0 M Ara-C for 4 h, which increased with 10 and 50 M Ara-C. Incubation with 100 M Ara-C produced internucleosomal DNA fragmentation starting at 3 h, which increased with longer periods of exposure to Ara-C. Utilizing a schedule of 1-h exposure followed by 3-h suspension in drug-free medium, 0.25 M MTN was found to initiate DNA fragmentation, which increased with exposure to 1.0 and 5.0 M MTN. However, identical treatment with higher concentrations of MTN resulted in random DNA degradation. Alternatively, continuous exposure to 1.0 M MTN for 3 h was necessary to initiate internucleosomal DNA fragmentation. This increased with exposure intervals of up to 6 h. Exposure to TXL concentrations as low as 0.01 M for 24 h caused internucleosomal DNA fragmentation, which increased with dose escalation (0.05, 0.1, 0.5, and 1.0 M) of TXL. Although continuous exposure to 1.0 M TXL for a period as short as 8 h produced internucleosomal DNA fragmentation, this increased significantly with longer exposure intervals. In general there appears to be a threshold concentration and duration of exposure below which non of these three drugs activates endonucleolytic internucleosomal DNA fragmentation and apoptosis. This threshold is lower for the DNA-interactive drugs MTN and Ara-C but higher for the non-DNA-interactive drug TXL. Higher doses or prolonged treatments with the drugs produce random DNA fragmentation associated with necrotic cell death. These in vitro results may further improve our understanding of the antileukemic cytotoxic effects of these drugs, which may enable a more rational design of drug regimens for optimal treatment of AML.     

Phase I/II trial of high dose mitoxantrone in metastatic breast cancer: the M.D. Anderson Cancer Center experience

Anthracyclines are among the most active agents in metastatic breast cancer. Mitoxantrone demonstrated a different toxicity profile when compared to doxorubicin. We performed a phase I/II study of singleagent highdose mitoxantrone therapy for advanced breast cancer. Nineteen patients who had a diagnosis of metastatic breast cancer received treatment at the M.D. Anderson Cancer Center between June 1986 and December 1987. The patients received escalating doses of mitoxantrone until a maximum tolerated dose (MTD), defined as grade 3 or 4 nonhematologic toxicity or infection, was obtained. The starting dose of 25 mg/m², given by short intravenous infusion, was escalated by 25% in each fivepatient cohort if each patient in the previous cohort tolerated the initial course and 2 or fewer patients reached the MTD. The median cumulative dose of mitoxantrone was 93 mg/m² (range, 25–205) and the maximum single dose was 39mg/m². Myelosuppression was the dose limiting toxicity. The median duration of granulocyte count 250/l was 5–7 days. Four patients (22%) had infections that required hospitalization, 3 patients (17%) had cardiac toxicity. One patient (6%) achieved a complete response, and 3 (17%) had a partial response, with an overall response rate of 22.3%. No apparent doseresponse relationship was observed in our study. The mitoxantrone dosage recommended for phase II studies is 25mg/m² every 3–4 weeks. We conclude that highdose mitoxantrone therapy for metastatic breast cancer was relatively well tolerated but was not associated with a higher response rate than that of standard dose mitoxantrone.     

Micromolar concentrations of 2-methoxyestradiol kill glioma cells by an apoptotic mechanism,without destroying their microtubule cytoskeleton

The purpose of this study was to investigate the potential effects of 2-methoxyestradiol, a natural mammalian steroid, in glioma cells, since antiproliferative effects of this compound had been shown earlier in several leukemia and carcinoma cell lines. The effects of 0.2, 2 and 20M concentrations of 2-methoxyestradiol were measured in three malignant human glioma cell lines (U87MG, U138MG, LN405) and one malignant rat glioma cell line (RG-2) using a microtiter-tetrazolium (MTT) assay. In all cell lines, a significant reduction of the viable cell number by more then 75% occurred ( P < 0.05) for concentrations of 2 and 20M 2-methoxyestradiol after 6days. A concentration of 0.2M had smaller effects (10–40% cell reduction), which were significant in two of the cell lines tested. The apoptotic nature of cell death was further analyzed in U87MG and RG-2 cells. Caspase-3 activity was significantly induced to levels between 3.4- and 23-fold after 4days for the two higher 2-methoxyestradiol concentrations (P < 0.05). In the cell line RG-2 nuclear fragmentation was visible in many nuclei, following stains with Hoechst H33258. A round cell morphology occurred in most treated cells, which was not accompanied by a complete destruction of the microtubule network, as it can be observed with other microtubule targeting drugs. K. Chamaon: These authors contributed equally to the work.J. Stojek: These authors contributed equally to the work.     

Tamoxifen Increases Photodynamic Therapeutic Response of U87 and U25ln Human Glioma Cells

We tested the hypothesis that Tamoxifen (TMX), an inhibitor of protein kinase C (PKC), augments the cytotoxicity of photodynamic therapy (PDT) treatment of human (U87) and (U25ln) glioma cells. U87 and U251n glioma cells were plated and treated with PDT using Photofrin as the sensitizer. Cells were treated with Photofrin at various doses and with various optical (632nm) irradiation intensities 24h later. Cells were also treated with Photofrin at a fixed dose alone and with various doses of Tamoxifen and subjected to laser treatment 24h later. Tumor response was tested using the (3-94,5-dimethyl-2-yl)-2,5-diphenyl-tetrazolium (MTT) method. Total toxicity of U87 cells was achieved with PDT at all doses of Photofrin (1, 2.5, 5, 10g/ml) with irradiation densities equal to or greater than 200mJ/cm². Using an irradiation intensity of 100mJ/cm², U87 and U251n cells were killed in a Photofrin dose-dependent manner. Significant cytotoxicity was detected with Photofrin doses of 5g/ml (p < 0.05) and 10g/ml (p < 0.001). Tamoxifen at a dose of 500g/ml and higher, significantly increased the toxicity of the PDT response with 5g/ml Photofrin and 100mJ/cm² (p < 0.05). In summary, our data demonstrate that Tamoxifen significantly enhances the Photofrin PDT activity of U87 and U251n human glioma cells.     

Studies on the myelosuppressive activity of doxorubicin entrapped in liposomes

Summary The myelosuppressive activity of doxorubicin encapsulated in liposomes of differing lipid composition and size was quantified in mice by measurement of changes in spleen weight, peripheral white blood cells (WBC), and bone marrow nucleated cells. Following i. v. administration of free doxorubicin at a dose of 20 mg/kg, a 90% reduction in marrow cellularity was observed on day 3. The marrow nucleated cell count was similar to control values by day 7. Administration of an equivalent dose of doxorubicin that was encapsulated in large (diameter, 1.0 m) egg phosphatidylcholine/cholesterol (EPC/Chol)(molar ratio, 5545) liposomes induced an 80% reduction in bone marrow cellularity that lasted for periods of >7 days. Similar results were obtained following administration of large (1.0 m) liposomal doxorubicin systems formulated with distearoylphosphatidylcholine/cholesterol (DSPC/Chol) (molar ratio 5545). In contrast, liposomal doxorubicin prepared using small (diameter, 0.1 m) DSPC/Chol liposomes induced only a 40% reduction (day 3) in bone marrow cellularity, which returned to control values by day 7. Other indicators of doxorubicin-mediated myelosuppressive activity (spleen weight loss and peripheral leukopenia) correlated well with changes observed in marrow cellularity. An exception to this, however, was observed in animals treated with small (0.1-m) DSPC/Chol liposomal doxorubicin, which displayed peripheral leukopenia for periods of >14 days. This extended leukopenia was not observed following administration of small (0.1-m) EPC/Chol liposomal doxorubicin. Marrow-associated liposomal lipid and doxorubicin were quantified to determine if the extent of doxorubicin-mediated myeloid toxicity could be correlated to changes in biodistribution of the entrapped drug. It was demonstrated that 10–20 times more doxorubicin is delivered to the bone marrow when the drug is given encapsulated in largeliposomes than when it is associated with small liposomes. These data are useful in defining characteristics of liposomal preparations that modulate the myelosupressive behaviour of entrapped antineoplastic agents.Abbreviations MLV multilamellar vesicle - LuV large unilamellar vesicle - SUV small unilamellar vesicle - EPC egg phosphatidylcholine - DPPC dipalmitoylphosphatidylcholine - DSPC distearoylphosphatidylcholine - Chol cholesterol - RES reticuloendothelial system - QELS quasielastic light scattering - FACS fluorescence-activated cell sorting - WBC white blood cell - HBSS HEPES buffered saline solution     

Measurement of in vivo glucose transport from blood to tissue of experimentally-induced glioma in rat brain

Summary In experimentally-induced F98 glioma of rat brain, regional blood flow and glucose transfer were assessed by means of double tracer autoradiography to measure Michaelis-Menten constants for the determination of unidirectional glucose transport across the blood-tumor and blood-brain barrier. In brain regions opposite the tumor hemisphere, the maximal glucose transport rate constant, T_m, ranged from 1.41 ± 0.12 to 3.22 ± 0.29 mol/g/min and the half saturation transport constant of glucose, K_t, varied from 2.78 ± 0.83 to 5.6 ± 1.94 mol/ml (estimate ± standard error of the estimate) yielding a normoglycemic unidirectional glucose inward transport which ranged from 1.24 ± 0.24 to 1.97 ± 0.13 mol/g/min (mean ± standard deviation). In the tumor periphery, the T_m and the Kt values were 3.64 ± 0.56 mol/g/ml and 7.32 ± 2.12 mol/min, and in the tumor center, 1.77 ± 0.25 mol/ml and 2.76 ± 1.13 mol/min, respectively. The unidirectional glucose influx of tumor periphery and center in normoglycemia was 1.98 ± 0.22 and 1.34 ± 0.16 mol/g/min, respectively. Despite comparable unidirectional glucose influxes, however, glucose metabolism of tumor tissue located in the periphery (0.83 ± 0.12 mol/g/min) and the center (0.41 ± 0.10 mol/g/min) of the tumor mass exceeded that of normal gray matter by about 68% and 100% which indicates uncoupling between glucose transport and phosphorylation in experimentally-induced F98 glioma of rat brain.     

Levels of water-soluble antioxidants in astrocytoma and in adjacent tumor-free tissue

Summary The aim of the present study was to investigate the oxidative status in astrocytoma. Samples of brain tissue from the centre to the periphery of the tumor were obtained from 11 astrocytoma patients undergoing computer tomography-guided stereotaxic operation, who had been previously treated with the corticosteroid dexamethasone. Part of the sample was investigated histologically for clarification of tumor type, and the presence of neoplastic and non-neoplastic tissue and necrosis. The rest was used for the quantification of the antioxidants ascorbic acid, uric acid, glutathione and cysteine by high performance liquid chromatography, and for quantification of DNA. Levels of antioxidants were calculated as g/g fresh tissue and mol/g DNA, a parameter related to cell content. There was significantly more DNA in neoplastic samples than in nonneoplastic ones, indicating increased cell density. Uric acid (g/g fresh tissue) was significantly increased in neoplastic compared with non-neoplastic tissue, and levels were even higher in necrotic tissue. There were no significant differences between neoplastic and non-neoplastic tissue levels of ascorbic acid, glutathione or cysteine, expressed as g/g fresh tissue. However, when levels of these three compounds were expressed as mol/g DNA, i.e. taking into account the higher cell density, ascorbic acid, glutathione and cysteine were significantly reduced in neoplastic samples compared with non-neoplastic ones. Results thus show that there are differences between the antioxidant levels in astrocytoma and non-neoplastic tissue, providing additional support for the hypothesis that free radicals play a role in tumor growth.     

Pharmacokinetics of (glycolato-0,0′)-diammine platinum (II), a new platinum derivative,in comparison with cisplatin and carboplatin

Summary The pharmacokinetics of (glycolato-0,0)-diammine platinum (II) (254-S; NSC 375101D), one of the new platinum analogues, was examined in a phase I study of this drug and compared with that of cisplatin and carboplatin. All drugs were given in short-term (30-min) i.v. drip infusions; the doses of 254-S, cisplatin, and carboplatin were 100, 80, and 450 mg/m², respectively. Platinum concentrations in whole plasma, plasma ultrafiltrate, and urine were determined by atomic absorption spectrometry. After the infusion, the plasma concentration of total platinum for the three agents decayed biphasically. Ultrafilterable platinum in plasma decreased in a biexponential mode after infusions of 254-S and carboplatin, whereas the free platinum of cisplatin showed a monoexponential disappearance. The peak plasma concentrations and AUC for free platinum were 5.31 g/ml and 959 g/min per ml for 254-S, 3.09 g/ml and 208 g/min per ml for cisplatin, and 19.90 g/ml and 3446 g/min per ml for carboplatin, respectively. The mean ratio of plasma ultrafilterable platinum to total platinum were calculated, and the results showed that the protein-binding abilities of 254-S and carboplatin were almost identical. More than 50% of the 254-S was excreted in the urine within the first 480 min after its administration. Thrombocytopenia was reported as a dose-limiting toxicity for both 254-S and carboplatin. This similarity in side effects may mainly be due to the comparable pharmacokinetic behavior of these two platinum compounds.     

Chemopreventive and adjuvant therapeutic potential of pomegranate (Punica granatum) for human breast cancer

Fresh organically grown pomegranates (Punica granatum L.) of the Wonderful cultivar were processed into three components: fermented juice, aqueous pericarp extract and cold-pressed or supercritical CO₂-extracted seed oil. Exposure to additional solvents yielded polyphenol-rich fractions (polyphenols) from each of the three components. Their actions, and of the crude whole oil and crude fermented and unfermented juice concentrate, were assessed in vitro for possible chemopreventive or adjuvant therapeutic potential in human breast cancer. The ability to effect a blockade of endogenous active estrogen biosynthesis was shown by polyphenols from fermented juice, pericarp, and oil, which inhibited aromatase activity by 60–80%. Fermented juice and pericarp polyphenols, and whole seed oil, inhibited 17--hydroxysteroid dehydrogenase Type 1 from 34 to 79%, at concentrations ranging from 100 to 1,000g/ml according to seed oilfermented juice polyphenols>pericarp polyphenols. In a yeast estrogen screen (YES) lyophilized fresh pomegranate juice effected a 55% inhibition of the estrogenic activity of 17--estradiol; whereas the lyophilized juice by itself displayed only minimal estrogenic action. Inhibition of cell lines by fermented juice and pericarp polyphenols was according to estrogen-dependent (MCF-7)estrogen- independent (MB-MDA-231)>normal human breast epithelial cells (MCF-10A). In both MCF-7 and MB-MDA-231 cells, fermented pomegranate juice polyphenols consistently showed about twice the anti-proliferative effect as fresh pomegranate juice polyphenols. Pomegranate seed oil effected 90% inhibition of proliferation of MCF-7 at 100g/ml medium, 75% inhibition of invasion of MCF-7 across a Matrigel membrane at 10g/ml, and 54% apoptosis in MDA-MB-435 estrogen receptor negative metastatic human breast cancer cells at 50g/ml. In a %% murine mammary gland organ culture, fermented juice polyphenols effected 47% inhibition of cancerous lesion formation induced by the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). The findings suggest that clinical trials to further assess chemopreventive and adjuvant therapeutic applications of pomegranate in human breast cancer may be warranted.     

In Vitro and in Vivo Inhibition of SK-N-MC Neuroblastoma Growth Using Cyclic nucleotide Phosphodiesterase Inhibitors

The effect of cyclic nucleotide phosphodiesterase (PDE) inhibitors Zaprinast and DC-TA-46 has been tested on SK-N-MC neuroblastoma growth. Antiproliferative activity of the tested drugs was assayed both in vitro and in the xenograft model of nude mice. In clonal density experiments, the IC₅₀ value was 3.3M for Zaprinast and 1.9M for DC-TA-46, while 7.5M BCNU alkylating agent was required to obtain the same effect. SK-N-MC cells xenografted in the nude mouse showed that the administration of Zaprinast and DC-TA-46 caused a significant 50% decrease of the tumour weight. These data demonstrate that PDE inhibitors may be useful for at least reducing tumour growth; they may be of interest for further evaluation as alternative molecules in the design of multiple agent protocols for neuroblastoma treatment.     

Taxol and taxotere in bladder cancer: in vitro activity and urine stability

In this study the antimicrotubular agents taxol, taxotere, and vinblastine were compared for their ability to inhibit the clonal growth of human bladder tumor cell lines using a soft-agar clonogenic assay. The stability of taxol and taxotere was evaluated by high-performance liquid chromatography over a range of pH in human urine. Both taxol and taxotere were shown to maximally inhibit the clonal growth of human bladder cell lines within 1 h of drug incubation. The most active agent in the panel of tumor lines was taxotere, with 6 of 12 lines being sensitive to the agent at 0.01 M and all cell lines being sensitive at 0.1 M. Taxol was active in 1 of 12 lines at 0.01 M and in 11 of 12 at 0.1 M. Only 2 of 12 cell lines were sensitive to vinblastine over the 0.01- to 0.1-M dose range. Taxol and taxotere were found to be stable in human urine for 4 h over a pH range of 5–7. At least 85% of both drugs were present during this period of drug incubation. Our findings suggest that both taxol and taxotere may be clinically useful agents for systemic and intravesical use in bladder cancer.This work was supported in part by the Veterans Administration Research Service     

Pharmacokinetics of methotrexate and 7-hydroxy-methotrexate in plasma and bone marrow of children receiving low-dose oral methotrexate

Summary The absorption, distribution, and elimination kinetics of low-dose p.o. methotrexate (MTX) were repeatedly studied in 19 children during maintenance treatment of childhood acute lymphoblastic leukemia. Plasma concentrations, urinary elimination, and bone marrow concentrations of MTX and 7-hydroxymethotrexate (7-OH-MTX) were monitored during 24 h following a routime p.o. dose (30 mg/m²) using high-pressure liquid chromatography. Significant interindividual variability was found in time to peak concentration (30–180 min), peak concentration (0.41–2.77 M), and to a lesser extent the half-lives (t_1/2: 32.8–86.1 min; t_1/2: 43.6–350.0 min; t_1/2 absorption: 25.2–60.3 min) and plasma area under the concentration-time curve from zero to infinity (195.6–818.5 M.min). Significant amounts of 7-OH-MTX were detected in plasma, with a mean area under the concentration-time curve from zero to infinity of 208 M.min compared with 365.6 M.min for MTX. High concentrations of 7-OH-MTX were present in bone marrow 24 h after oral MTX (15/19 patients) and were at least five fold those in plasma and three fold the concentration of MTX in bone marrow. In four patients occasionally neither MTX nor metabolite could be detected. Repeated examination of these pharmacokinetic parameters in plasma and bone marrow showed that the intraindividual variability was small.This study was supported by the Netherlands Cancer Foundation Koningin Wilhelmina Fonds     

Irofulven Induces Apoptosis in Breast Cancer Cells Regardless of Caspase-3 Status*

Caspase-3 deficiency can limit the efficiency of pro-apoptotic anticancer treatments. Irofulven (hydroxymethylacylfulvene, HMAF, MGI 114, NSC 683863) is an antitumor drug, currently in a Phase III and multiple Phase II trials, which can differentiate between tumor and normal cells in apoptosis induction. This study investigated whether apoptosis induced by irofulven requires caspase-3. Irofulven action was compared in breast cancer cells differing in caspase-3 status: deficient MCF-7 cells and proficient MDA-MB-231 cells and in normal human mammary epithelial cells, HMEC. Irofulven induces significant, concentration and time-dependent apoptotic DNA fragmentation in breast cancer cell lines, regardless of caspase-3 status. After 12, 24 and 48h incubation at 1M irofulven ( 3×GI₅₀), fragmented DNA comprised 3.7, 14.1 and 34.6% and 8.4, 12.6 and 20.3% of total DNA in MCF-7 and MDA-MB-231 cells, respectively. Cell viability (trypan blue exclusion) remained largely unaffected during the first 24h but decreased markedly after 48h, indicating secondary necrosis. Net losses in cell numbers were apparent at 48h. Normal HMEC cells were refractory to 1M drug with only 3–9% fragmented DNA after 12–48h, although apoptosis was observed at drug levels >3M. The broad-spectrum caspase inhibitor Z-VAD-fmk inhibited irofulven-induced apoptosis of all cell lines at 20M with nearly complete abrogation of apoptosis at 100M. Irofulven treatment resulted in marginal caspase-3 processing in MDA-MB-231 and HMEC cells. These results indicate that whereas the caspase cascade mediates irofulven- induced apoptosis, caspase-3 is dispensable (supported by NIH CA70091 and CA78706).     

Deoxypyrimidine-induced inhibition of the cytokinetic effects of 1-β-d-arabinofuranosyluracil

Summary Ara-U-induced S-phase accumulation and the interaction between high concentrations of ara-U (HiCAU) and ara-C were investigated in L1210 leukemia cells in vitro. Treatment of exponentially growing L1210 murine leukemia cells with ara-U (200–1000 m) for 48 h caused a dose-dependent accumulation of cells in the S-phase. The extent of this ara-U-induced S-phase accumulation correlated with ara-U incorporation into DNA and with increases of up to 172% and 464% in the specific activities of deoxycytidine kinase and thymidine kinase, respectively, over control values. Metabolism of 1 m ara-C following the exposure of cells to ara-U (1mm) resulted in 4.5 pmol ara-C DNA/mg protein vs 2.1 pmol/mg protein in control cells. Although 48-h exposure of cells to 200 and 400 m ara-U is not cytotoxic, it enhances the cytotoxicity of ara-C (10–100 m) 4- to 10-fold. Ara-U-induced S-phase accumulation is inhibited by deoxypyrimidine nucleosides but not by pyrimidine or deoxypurine nucleosides. Some of the ara-U and ara-C concentrations used in this study are achievable in clinical practice, and ara-U/ara-C interactions may explain in part the unique therapeutic utility of high-dose ara-C.Abbreviations ara-C 1--d-arabinofuranosylcytosine - ara-U 1--d-arabinofuranosyluracil - ara-CTP 1--d-arabinofuranosylcytidine triphosphate - HiDAC high-dose ara-C - HiCAU high concentrations of ara-U - dCTP deoxycytidine triphosphate - HiDAU high-dose ara-U - FiTC Fluoroisothiocyanate - dUDP deoxyuridine diphosphate - dUTP deoxyuridine triphosphate - dTTP thymidine triphosphate - BrdUrd bromodeoxyuridine - dCyd kinase deoxycytidine kinase Supported in part by grant CH-35H from the American Cancer Society, by Public Health Service grant CA-12197 from the National Cancer Institute, National Institutes of Health, and by the Gaston Cancer Society     

Systemic treatment with murine recombinant interleukin-1β inhibits the growth and progression of malignant glioma in the rat

We investigated the effects of daily subcutaneous (SC) injections of 100, 200, or 400 g/kg murine recombinant interleukin-1 (rIL-1) or its excipient on normal Fischer 344 rats and ones harboring a malignant RT-2 glioma. The tumor model has a predictable course with animals dying on days 14–17 following an intracerebral inoculation of 10⁴ RT-2 glioma cells. Treatments with (rIL-1) or excipient began on day seven post-tumor inoculation and continued for 7 days. We observed no significant effect on core body temperatures although there was a significant (p < 0.05) decrease in body weight in all (rIL-1) treated animals. When tumor-bearing animals became moribund, they received an intraperitoneal injection of bromodeoxyuridine (BUdr) and were sacrificed two hours later. Blood samples were obtained prior to their sacrifice by transcardiac perfusion with a buffered aldehyde solution. Recombinant IL-4ß affected blood differentials; causing neutrophilia, lymphopenia, and slight thrombocythemia. The BUdr labeling index of glioma cells did not significantly differ between treatment groups, although tumors differed histologically at the time of necropsy. Tumors of rIL-1 treated animals had more extensive necrosis and a greater degree of leukocyte infiltration. Survival studies were conducted in which rats were given continuous daily SC injections of (rIL-1) until day of death. Overall survival between the two groups differed significantly in studies using 100 g/kg/d (p < 0.05); (rIL-1ß) treated rats had a mean survival time of 22 (± 3.0) days while excipient controls had a mean survival time of 17 (± 0.5) days. Similarly, at a dose of 200 g rIL-1(3/kg/d), mean survival was significantly (p < 0.05) increased as compared to excipient controls (18.75 ± 1.5 vs. 15.25 ± 1.7 days, respectively). Daily injections of 400 g/kg did not significantly increase the survival of glioma bearing animals, possibly as a consequence of fIL-1ß toxicity at this dose.