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     

Histamine H1 Receptor Activation Stimulates Mitogenesis in Human Astrocytoma U373 MG Cells

In human astrocytoma U373 MG cells that express histamine H₁ receptors (180 ± 6fmol/mg protein) but not H₂ or H₃ receptors, histamine stimulated mitogenesis as assessed by [³H]-thymidine incorporation (173 ± 2% of basal; EC₅₀, 2.5 ± 0.4M). The effect of 100M histamine was fully blocked by the selective H₁ antagonist mepyramine (1M) and was markedly reduced (93 ± 4% inhibition) by the phospholipase C inhibitor U73122 (10M).The activator of protein kinase C (PKC) phorbol 12-tetradecanoyl-13-acetate (TPA, 100nM) stimulated [³H]-thymidine incorporation (270 ± 8% of basal), and this response was not additive with that to 100M histamine. The incorporation of [³H]-thymidine induced by 100M histamine was partially reduced by the PKC inhibitor Ro 31-8220 (57 ± 7% inhibition at 300nM) and by the compound PD 098,059 (30M, 62 ± 14% inhibition), an inhibitor of the mitogen-activated kinase (MAPK) kinases MEK1/MEK2.These results show that histamine H₁receptor activation stimulates the proliferation of human astrocytoma U373 MG cells. The action of histamine appears to be partially mediated by PKC stimulation and MAPK activation.     

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.     

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.     

Comparison of the bioavailability of uridine in mice after either oral or parenteral administration

Summary We compared the bioavailability of uridine (Urd) (350 and 3500 mg/kg) administered either as a single SC injection or by gavage, in male CD8F₁ mice. Plasma samples were analyzed for Urd and uracil (Ura) using high-pressure liquid chromatography. After Urd (3500 mg/kg, SC), plasma Urd levels peaked at 4900 M and then declined to pretreatment levels (< 10 M) within 6h. Plasma Ura concentrations peaked at 1400 M and then declined initially more slowly than Urd. After Urd (3500 mg/kg, PO) plasma levels of Urd were fairly constant (range 33–82 M) for up to 8 h and had returned to pretreatment levels at 16 h. Plasma Ura concentrations paralleled Urd, but were approxximately ten-fold higher. Areas under the concentration-time curve for Urd showed that the bioavailability of Urd after PO administration was 7% of that after SC administration. After Urd (350 mg/kg, SC) Urd levels peaked at 210 M returning to pretreatment levels within 2 h. Plasma Ura levels reached a peak with 300 M and then declined initially more slowly than those of Urd. After Urd (350 mg/kg, PO) plasma Urd levels were not perturbed, although Ura levels peaked at 50 M after which they declined and could no longer be detected at 4 h. These data indicate that (a) the bioavailability of Urd (350 or 3500 mg/kg) was lower when given PO than when it was administered by SC injection; and (b) Urd (3500 mg/kg) PO resulted in prolonged and relatively constant plasma Urd levels compared with Urd (3500 mg/kg) SC. These results suggest that Urd PO should be compared with parenterally administered Urd in attempts to increase the therapeutic index of 5-fluorouracil and of antimetabolite inhibitors of de novo pyrimidine biosynthesis.     

Inhibition of cell proliferation and glutathione S-transferase by ascorbyl esters and interferon in mouse glioma

Summary Mouse glioma-26 (G-26) cell line established in this laboratory was used in the study. Thein vitro effect of ascorbyl esters, viz., ascorbyl-palmitate (As-P), -stearate (As-S) and mouse interferon-/ (MulFN-/) on the glioma cell viability, proliferation and glutathione S-transferase (GST) activity was investigated. Cell viability and proliferation were examined by colorimetric MTT assay and [³H]-thymidine incorporation, respectively. Incubation (24 h) of G-26 cells with As-S, As-P or MulFN-/, resulted in a dose dependent decrease in cell viability (IC₅₀=125M As-S; 175M As-P and 3.6×10⁴ U/ml MulFN-/) and proliferation (IC₅₀=157M As-S; 185M As-P and 3.6×10⁴ U/ml MulFN-/). A combined exposure to 175 M As-S and 800 U/ml of MulFN-/ resulted in a greater than an additive effect on cell viability and proliferation. The inhibition of cell proliferation/viability by interferon was species specific and was observed only with homologous MulFN-/, but not with human interferon- lymphoblastoid or human interferon-. Ascorbyl esters inhibited cytosolic GST activity (1–50=15.0 M As-S and 28.5 M As-P) towards 1-chloro-2,4-dinitrobenzene in a dose dependent manner. The apparent Ki values for affinity purified GST, deduced from Dixon plots were 0.95 M and 2.0 M for As-S and As-P, respectively. Significant inhibition of GST was also observed in the cytosol isolated from G-26 cells exposed to 300 M As-S or 800 U/ml MulFN-/.     

High-dose chemotherapy with autologous stem cell rescue for children with high risk and recurrent medulloblastoma and supratentorial primitive neuroectodermal tumors

Current treatment for high risk and recurrent medulloblastoma (MB) and supratentorial primitive neuroectodermal tumors (stPNET) has a very poor prognosis in children. High dose chemotherapy (HDCT) and autologous stem cell rescue have improved survival rates. We present 19 patients (thirteen classified in the high risk group and six patients with recurrent disease) that received HDCT and autologous stem cell rescue.In the high risk group [Med Pediatr Oncol 38 (2002) 83], all patients underwent neurosurgical debulking. Standard chemotherapy was prescribed in 10 patients. Radiotherapy was given to 4 patients (all older than 4years old). In the recurrence disease group [Childs Nerv Syst 15 (1999) 498], five patients underwent surgery. Radiotherapy was given to those who were not previously irradiated. The HDCT in twelve patients consisted of busulfan 4mg/kg/day, orally over 4days in 6-hourly divided doses and melphalan at a dose of 140mg/m²/day by intravenous infusion over 5min on day –1. Three patients additionally received thiotepa 250mg/m²/day intravenously over 2days and four patients additionally received topotecan 2mg/m²/day over 5days by intravenous infusion over 30min. The other seven patients received busulfan and thiotepa at the same doses.Patients stem cells were mobilized with granulocyte colony-stimulating factor at a dose of 12g/kg twice daily subcutaneously for four consecutive days. Cryopreserved peripheral blood progenitor cells were re-infused 48h after completion of chemotherapy. With a median follow-up of 34months (range 5–93) eight complete responses and one partial response were observed. Three patients died of treatment-related toxicities (15%). The 2 year event-free survival was 37.67±14% in all patients and 57±15% for the high risk group.Therefore we conclude that HDCT may improve survival rates in patients with high risk/recurrent MB and stPNET despite treatment toxicity.     

Role of Ceramide During Cisplatin-induced Apoptosis in C6 Glioma Cells

Cisplatin is commonly used for the treatment of malignant brain tumors. However, the mechanisms of cell death by cisplatin are not fully understood. Therefore, the present study was designed to elucidate the apoptotic signaling pathway(s) activated by cisplatin in a C6 rat glioma cell line. C6 cells were treated with various concentrations of cisplatin (0.2–10g/ml) for 24–72h. At 10g/ml cisplatin, over 90% of the cells became dead at 72h. Apoptotic death was confirmed by condensation and fragmentation of nuclei, and DNA laddering. Even in cells treated with 1.5g/ml cisplatin, typical apoptotic cells were observed at 72h. The intracellular level of ceramide, measured Escherichia coli diacylglycerol kinase markedly increased during 24–72h after the addition of 10g/ml cisplatin. The activity of caspase-3(-like) proteases increased and reached a peak at 48h. Inhibitors of caspases reduced the number of apoptotic cells. Pretreatment of C6 cells with glutathione or N-acetyl-cysteine, which are known to block the activation of neutral magnesium-dependent sphingomyelinase, inhibited ceramide formation, leading to suppression of both activation of caspase-3(-like) proteases and apoptosis by cisplatin. In contrast, pretreatment of the cells with N-oleoylethanolamine (OE), a ceramidase inhibitor, potentiated apoptosis induced by cisplatin. Furthermore, OE enhanced sensitivity of the cisplatin-resistant cells to cisplatin. These results suggest that ceramide is closely implicated in apoptosis of glioma cells by cisplatin through activation of caspase-3(-like) proteases.     

Prevention of rat mammary carcinoma utilizing leuprolide as an equivalent to oophorectomy

A clinical trial is currently under way to examine the effectiveness of leuprolide as a breast cancer chemopreventive agent and contraceptive. This trial, as well as similar proposed studies, is based on the assumption that leuprolide is as effective as surgical castration in preventing the onset of mammary tumors; however, this has not been well documented in the DMBA animal model. We directly compared leuprolide and oophorectomy in this model and examined a combined therapy of leuprolide/bromocriptine. Twenty-seven day old female Sprague-Dawley rats were randomly allocated into one of eight groups. All rats received a 20-mg dose of DMBA at the age of 55 days. Group 1 (n=10), no treatment; Group 2 (n=9), leuprolide (100g/kg/day) for eight weeks beginning four weeks prior to DMBA; Group 3 (n=10), oophorectomy four weeks prior to DMBA with replacement estrogen beginning four weeks following DMBA. Estrogen replacement was achieved with a 0.05-mg estradiol tablet releasing 0.833g/day over a 60-day period. Group 4 (n=10), leuprolide (100g/kg/day) initiated two weeks prior to DMBA and continuing for two weeks following DMBA; Group 5 (n=9), oophorectomy two weeks prior to DMBA with 0.05mg of estradiol in depot form, releasing 0.833g/day, beginning four weeks following DMBA and continuing until week 16 of the study; Group 6 (n=10), leuprolide (100g/kg/day) beginning two weeks prior to DMBA and continuing for the duration of the experiment; Group 7 (n=10), leuprolide (100g/kg/day) for eight weeks beginning two weeks prior to DMBA; Group 8 (n=9), leuprolide (100g/kg/day) and bromocriptine (83g/day) for eight weeks beginning two weeks prior to DMBA. At nineteen weeks (15 weeks post DMBA), animals were sacrificed and autopsies performed. One hundred percent of untreated animals developed tumors. No animals undergoing oophorectomy four weeks prior to DMBA or receiving leuprolide four weeks prior to and simultaneously with DMBA developed tumors. In animals pretreated two weeks prior to DMBA with leuprolide or oophorectomy, each group had one animal with tumor development. No tumors developed in the animals receiving ongoing injections of leuprolide. However, one tumor developed in those receiving leuprolide for the first eight weeks beginning two weeks prior to DMBA administration. One animal receiving both leuprolide and bromocriptine developed one tumor. We conclude that chemical oophorectomy (with leuprolide) is as effective as surgical oophorectomy in inhibiting DMBA induced carcinogenesis.     

Intrathecal Chemotherapy with MX2 for Treating Glioma Dissemination in vivo

We examined whether the intrathecal MX2 chemotherapy for treating dissemination of malignant glioma would be a feasible therapy. In the toxicity study, physiological and histological neurotoxicity was not observed in the rats treated with less than 100g/kg of MX2 administered intracisternally. But physiological side effects were observed in the treatment group of more than 200g/kg and histological brain toxicity was in the treatment group of more than 1000g/kg. Dissemination models were induced in rats by intracisternal inoculation of C6 glioma cells. The median survival times of the rats treated with 100g/kg of intrathecal MX2 on day 1, 3, or 7 after tumor inoculation were prolonged by 52.4% (p=0.0006), 31.5% (p=0.0007), and 7.1% (p=0.0180), respectively, compared to that of untreated control animals. Intrathecal MX2 treatment also cured 33.6% of rats in the treatment group. These findings suggested that there was a possibility that intrathecal MX2 would be a safe and effective method for treating dissemination of malignant glioma.     

Reduced formation of lesions in the DNA of a multidrug-resistant L1210 subline selected for teniposide resistance

Summary Earlier studies have suggested that higher cellular levels of teniposide (VM-26) are required for the inhibition of growth in L1210/VM-26 sublines than in parental L1210 cells [8]. On the basis of this observation, we hypothesized that resistance to VM-26, which is partly attributed to multidrug resistance, also resulted in reduced formation of DNA lesions by the drug. In confirmation of this hypothesis, equitoxic concentrations of VM-26 produced fewer breaks in the DNA of LIa5M cells, the prototype L1210/VM-26 subline, than in that of L1210 cells. Previously, potassium cyanide (KCN) and verapamil were shown to increase the levels of VM-26 in LIa5M but not L1210 cells. These agents also selectively increased the formation of breaks in the DNA of LIa5M but not L1210 cells. The DNA unknotting assay with phage P4 DNA indicated equivalent DNA type II topoisomerase activity in nuclear extracts of LIa5M and L1210 cells. The factor that reduced the formation of breaks in cellular LIa5M DNA by VM-26 provided less protection against equitoxic levels of doxorubicin, to which LIa5M cells are crossresistant.This work was supported by Research Project Grant CA 39426 and Cancer Center Support (Core) Grant CA 21765 from the National Cancer Institute and by the American Lebanese Syrian Associated Charities     

Human tissue distribution of platinum after cis-diamminedichloroplatinum

Summary Using X-ray fluorescence spectrometry, platinum concentrations were determined in autopsy tissue samples from 12 patients who had received cis-diamminedichloroplatinum (DDP) 20–120 mg/m² up to 6 months antemortem. Tissue platinum concentrations were highest in liver (0.5–3.7 g/g wet weight), prostate (1.6–3.6 g/g), and kidney (0.4–2.9 g/g), somewhat lower in bladder, muscle, testicle, pancreas, and spleen, and lowest in bowel, adrenal, heart, lung, cerebrum, and cerebellum, Platinum concentrations in tumors were generally somewhat lower than the concentration in the organ in which the tumor was located, with the exception of intracerebral tumors. Different metastatic sites in the same patient had substantially different platinum concentrations and hepatic metasutases had the highest concentrations. Intra-arterial administration of drug may augment tissue concentrations of platinum. In a patient undergoing therapeutic abortion 4 days after treatment, the platinum concentration was 0.5 g/g in the placenta and 0.3 g/g in the fetus. The data suggest that for in vitro sensitivity testing, DDP concentrations of 7 g/ml should be used.     

Intrathecal Busulfan Treatment of Human Neoplastic Meningitis in Athymic Nude Rats

The current study was designed to evaluate the toxicity and activity of Spartaject Busulfan, a microcrystalline preparation of busulfan, following its intrathecal administration into a nude rat model of human neoplastic meningitis. Animals were treated through permanent indwelling subarachnoid catheters. Human glioma D-456 MG growing in the subarachnoid space was treated with 8.1µmol of intrathecal Spartaject Busulfan. Single-dose therapy was also subsequently compared with 4 doses of 8.1 and 2.0µmol busulfan, respectively, against D-456 MG neoplastic meningitis. Additional experiments evaluated a saline control versus 8.1µmol×1, 6.2µmol×4 and 4.1µmol×4, respectively, against D-456 MG. A single dose of 8.1µmol of intrathecal Spartaject Busulfan resulted in an increase in median survival of 61.7% compared with the saline control. In experiment 2, all busulfan treatments showed increases in median survival of 142.8% (8.1µmol×1), 52.3% (2.0µmol×4), and 23% (8.1µmol×4) (p<0.001 for all groups) compared with the saline control. These results suggest that a narrow therapeutic dose range for both toxicity and activity has been defined for intrathecal busulfan in the treatment of human neoplastic meningitis in athymic nude rats. Although busulfan has only limited activity against solid tumors, the high doses achievable in the CSF following intrathecal administration coupled with the steep dose–response relationships of alkylating agents, provide rationale for further evaluation of this agent.     

High-performance liquid chromatographic procedures for the analysis of carboplatin in human plasma and urine

Summary Specific, sensitive and reproducible high-performance liquid chromatographic procedures were developed for the quantitative analysis of carboplatin in human plasma and urine. Plasma and urine were ultrafiltered with an Amicon Centrifree^TM micropartition system, and samples were injected onto a LiChrosorb^TM diol column. The mobile phase was CH₃CN/H₂O (92:8, v/v) for plasma and CH₃CN/0.015% H₃PO₄ (89:11, v/v) for urine. The effluents were monitored at 229 nm. Carboplatin eluted by 10 min. The detector response was linear from 0.5 (plasma) or 5 (urine) to 500 g/ml. The lower limit of quantification was 1.0 g/ml plasma and 5.0 g/ml urine. Constitutents in plasma and urine, and possible degradation products (cyclobutane mono- and dicarboxylic acids) did not interfere. Within-day precision was less than 4% for plasma and 9% and 4% for urine concentrations of 40 and 401 g/ml, respectively. Within-day accuracy was 96% or greater for both matrices. Carboplatin was not bound to the Centrifree^TM membrane and recovery was 94% for plasma and 96% for urine. The storage stability of carboplatin in water, plasma, plasma ultrafiltrate, and urine and the extent of binding to human plasma proteins were evaluated. The percentage of carboplatin reversibly bound to plasma proteins was minimal (10%) over a range of 1–50 g/ml. In human plasma at 37 °C the drug was stable for about 2 h, but then degraded with a half-life of 32 h. Carboplatin had limited stability in water, plasma, and urine stored at-25 °C. Biological samples, therefore, should be stored frozen and analyzed within a week of collection to obtain valid results.     

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     

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.     

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.     

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).     

18b-甘草次酸诱导人乳腺癌细胞凋亡及与细胞内Ca2+释放的关系

Objective: To study the effects of 18-glycyrrhetinic acid (GA) on proliferation inhibition, apop- totic induction, and the relationship between GA-induced apoptosis and intracellular Ca²⁺ concentration in human breast carcinoma (MCF-7) cells. Methods: After MCF-7 cells were treated with GA at the concentrations from 50 mol/L to 250 mol/L for 24 h, cell viability of proliferation was assessed by MTT assay. After the cells were treated with 100 mol/L, 150 mol/L, and 200 mol/L GA for 24 h, the rates of cell apoptosis were examined by terminal deoxynucleotide transferase mediated dUTP nick-end-labeling method and flow cytometry with Annexin V/propidium iodide fluorescent stain. After the cells treated with 150 mol/L GA for 24 h, intracellular Ca²⁺ concentration was measured by Fure-2 fluorescein load method. Results: After the cells were treated with GA at the concentrations from 100 mol/L to 250 mol/L, the rates of proliferative inhibition were increased significantly (P<0.05 and P<0.01) in a dose- dependent fashion. IC50 of the proliferation inhibition was 234.33 mol/L. Treated with 100 mol/L, 150 mol/L, and 200 mol/L, the rates of cell apoptosis were increased significantly (P<0.01). Intracellular Ca²⁺ concentration after treatment with GA was higher evidently than that of control (P<0.05). Conclusion: 18-glycyrrhetinic acid has the effects of the proliferation inhibition and the apoptotic induction on MCF-7 cells. The rise of intracellular Ca²⁺ level may be depended on apoptosis induced by GA in MCF-7 cells.     

A limited sampling model for the pharmacokinetics of etoposide given orally

A limited sampling model of etoposide after oral administration to estimate the area under the plasma concentration-time curve from 0 to 24 h (AUC) by determination of the drug plasma levels at only two time points was developed by a multiple regression analysis on a training data set of 15 patients receiving oral doses ranging from 54 to 90 mg/m². The equation describing the model is AUC (g ml^–1 h)=5.183 (g ml^–1 h)+1.193 (h)×C_1h (g/ml)+8.439 (h)×C_4h (g/ml) (R ²=0.93,P=0.0001), whereC _1h andC _4h represent the plasma etoposide concentrations at 1 and 4 h, respectively. The model was validated prospectively on a test data set of 13 patients receiving oral doses ranging from 52 to 87 mg/m² and, additionally, on a data set of 7 patients receiving oral doses ranging between 176 and 200 mg/m², investigated in a previous study. Validation on both test data sets gave a relative mean predictive error of 0.1% and a relative root mean square error of 15.8% and 16.7%, respectively. The present study shows that it is possible to obtain a good estimate of the plasma AUC after oral administration of etoposide using a two-time-point sampling model. The model can be used to monitor the etoposide AUC in patients receiving chronic oral treatment.