Antisense down-regulation of thymidylate synthase to suppress growth and enhance cytotoxicity of 5-FUdR, 5-FU and Tomudex in HeLa cells. (25/1894)

1. Thymidylate synthase (TS), the key enzyme in de novo synthesis of thymidine, is an important target for antitumour chemotherapy. It was hypothesized that antisense oligonucleotide down-regulation of TS mRNA would decrease TS levels and enhance the cytotoxicity of inhibitors of TS, including the pyrimidine analogues 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine (5-FUdR), and the folate analogue Tomudex (ICI D1694; N-(5-[N-(3, 4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino ]-2-theon yl-L-glutamic acid). 2. 2'-Methoxyethoxylated, phosphorothioated 20-mer oligodeoxynucleotides (ODNs), complementary to various sequences in TS mRNA, were synthesized, along with control oligomers consisting of the same, respective bases in randomized order, against which all the biological effects were compared. Following a 6-h transfection of HeLa cells using polycationic liposome at 3 microg ml(-1), ODN 83 (50 nM), complementary to a region in the 3'-untranslated region of the TS mRNA, decreased TS mRNA levels by approximately 70% within 24 h. ODN 83 also decreased TS enzyme activity, as measured by binding of TS to radiolabelled 5-fluorodeoxyuridine monophosphate. In addition to inhibiting proliferation by up to approximately 40%, ODN 83 enhanced the cytotoxicity of Tomudex or 5-FU, added 1 day following transfection, by 50 - 60%. ODN 83 also enhanced sensitivity to 5-FUdR by 70%, but did not affect the toxicity of cisplatin, chlorambucil, melphalan, doxorubicin, ionizing radiation, paclitaxel, or irinotecan. 3. These data indicate that antisense ODN down-regulation of TS can inhibit human tumour cell proliferation and enhance the efficacy of TS-targeted drugs.  (+info)

Patterns of DNA adduct formation in liver and mammary epithelial cells of rats treated with 7,12-dimethylbenz(a)anthracene, and selective effects of chemopreventive agents. (26/1894)

7,12-Dimethylbenz(a)anthracene (DMBA) is a prototype carcinogen that induces a high yield of mammary tumors in rats after a single feeding. We investigated the induction and chemoprevention of DNA adducts in female Sprague Dawley rats receiving DMBA by gavage according to a variety of treatment schedules. The patterns of 32P-postlabeled DNA adducts in liver and mammary epithelial cells were similar to those produced by the in vitro reaction of metabolically activated DMBA with calf thymus DNA. There was a high and statistically significant correlation between dose of DMBA administered to rats (0, 0.6, 2.4, and 12 mg/kg body weight) and levels of DNA adducts in both types of cells. The regression lines relating DMBA doses to total DNA adduct levels were significantly divergent and crossed at 1.5 mg/kg body weight, indicating that, at lower doses, the formation of DNA adducts is more intense in target mammary cells, whereas at higher doses, DNA adduct levels are more elevated in liver cells, presumably due to the greater metabolic capacity of this organ. When the rats were sacrificed 7 days rather than 2 days after DMBA administration, DNA adduct levels were approximately halved in both liver and mammary cells. The observed patterns can be interpreted based on toxicokinetic factors, local and distant metabolism, removal of DNA adducts by excision repair, and cell proliferation rate. Of three chemopreventive agents given with the diet to rats treated with 12 mg of DMBA, 5,6-benzoflavone (1650 ppm) was the most effective, inhibiting DNA adduct formation in liver and mammary cells by 96.5 and 83.5%, respectively. Feeding of 1,2-dithiole-3-thione (600 ppm) inhibited this biomarker by 68.5 and 50.2%, whereas butyl hydroxyanisole (BHA; 5000 ppm) showed a significant inhibition in the liver (46.5%) but was ineffective in mammary cells (29.0%, not significant). These data correlate nicely with the results of a parallel study in which 5,6-benzoflavone, 1,2-dithiole-3-thione, and BHA inhibited formation of hemoglobin adducts by 80.0, 44.0, and 0%, respectively; the incidence of mammary tumors by 82.4, 47.1, and 5.9%, respectively; and their multiplicity by 92.6, 80.0, and 7.4%, respectively. Therefore, biomarkers of biologically effective dose are highly predictive of the efficacy of chemopreventive agents in the DMBA rat mammary model. The selective inhibition by BHA of DNA adducts in the liver but not in mammary cells is consistent with the finding that this phenolic antioxidant stimulated phase II activities in the liver but not in the mammary gland (L. L. Song et al., manuscript in preparation). In any case, the broad-spectrum inducer 5,6-BF appears to be more effective than the two monofunctional phase II inducers, presumably because an enhanced activation of DMBA to reactive metabolites is coordinated with their blocking, detoxification, and excretion.  (+info)

BN 80933, a dual inhibitor of neuronal nitric oxide synthase and lipid peroxidation: a promising neuroprotective strategy. (27/1894)

Nitric oxide (NO) and reactive oxygen species (ROS) act independently as well as cooperatively to induce neuronal death in acute neurological disorders. Inhibition of neuronal nitric oxide synthase (nNOS) and inhibition of lipid peroxidation induced by ROS have both been proposed as neuroprotective strategies in stroke and trauma. Recently, in our laboratory, the combination of the two strategies was found to be synergistic in reducing neuronal damage. Here, we report that BN 80933 [(S)-N-[4-[4-[(3,4-dihydro-6-hydroxy-2, 5,7, 8-tetramethyl-2H-1-benzopyran-2-yl)carbonyl]-1-piperazinyl]phenyl]-2- thiophenecarboximidamide], a compound that combines potent antioxidant and selective nNOS inhibitory properties in vitro, affords remarkable neuronal protection in vivo. Intravenous administration of BN 80933 significantly reduced brain damage induced by head trauma in mice, global ischemia in gerbils, and transient focal ischemia in rats. Treatment with BN 80933 (0.3-10 mg/kg) significantly reduced infarct volume (>60% protection) and enhanced behavioral recovery in rats subjected to transient (2-h) middle cerebral artery occlusion and 48-h or 7-day reperfusion. Furthermore, treatment with BN 80933 commencing up to 8 h after the onset of ischemia resulted in a significant improvement of neurological outcome. All these results indicate that BN 80933 represents a class of potentially useful therapeutic agents for the treatment of stroke or trauma and possibly neurodegenerative disorders that involve both NO and ROS.  (+info)

Pindolol suppresses serotonergic neuronal activity and does not block the inhibition of serotonergic neurons produced by 8-hydroxy-2-(di-n-propylamino)tetralin in awake cats. (28/1894)

Clinical studies have shown that pindolol can enhance the effects of antidepressant drugs, presumably by acting as an antagonist at somatodendritic 5-hydroxytryptamine (5-HT)(1A) autoreceptors, which regulate the firing rate of central serotonergic neurons. The current study characterized the action of pindolol on the single-unit activity of serotonergic neurons in the dorsal raphe nucleus of freely moving cats. (+/-)-Pindolol produced a dose-dependent inhibition of neuronal activity after i.v. (ED(50) = 0.25 mg/kg) and s.c. (ED(50) = 1.23 mg/kg) administration. The active enantiomer (-)-pindolol (1 mg/kg i.v.) also suppressed neuronal activity (maximal decrease, 88%). Upon p.o. administration, (+/-)-pindolol (10 mg/kg) produced a marked, long-acting suppression of neuronal activity similar to that observed after s.c. administration. In all cases, the reduction in firing rate produced by pindolol was completely reversed by low doses of N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]-ethyl]-N-(2-pyridinyl)cycloh exanecarboxamide (WAY-100635) (0.1 mg/kg i.v. or 0.2 mg/kg s.c.), a selective 5-HT(1A) antagonist. Systemic administration of (-)-tertatolol (1-5 mg/kg i.v.), another beta-adrenoceptor blocker/putative 5-HT(1A) antagonist, had no significant effect on neuronal activity. The ability of i.v. (+/-)-pindolol (0.1-1 mg/kg) to reverse the suppression of serotonergic neuronal activity produced by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (10 microg/kg i.v.), a selective 5-HT(1A) agonist, also was examined. (+/-)-Pindolol had no appreciable effect on the action of 8-OH-DPAT. In contrast, the 5-HT(1A) antagonist drugs WAY-100635 (0.1 mg/kg i.v. ), 4-fluoro-N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl benzamide (0.1 mg/kg, i.v.), N-tert-butyl-3-(4-(2-methoxyphenyl)piperazin-1-yl)-2-phenylprop anamid e [(S)-WAY-100135] (0.5 mg/kg i.v.), and (-)-tertatolol (1-5 mg/kg i. v.) reversed the effect of 8-OH-DPAT to varying degrees. Overall, these results indicate that pindolol acts as an agonist rather than an antagonist at 5-HT(1A) autoreceptors in awake animals.  (+info)

Involvement of bcl-2 and p21waf1 proteins in response of human breast cancer cell clones to Tomudex. (29/1894)

Mechanisms of resistance to Tomudex include increased thymidylate synthase activity, as well as reduced intracellular drug uptake and polyglutamation. However, little is known about other mechanisms of resistance, such as a possible protection against Tomudex-induced apoptosis mediated by bcl-2. We transfected the MDA-MB-435 human breast cancer cell line, which is characterized by a mutated p53 gene, with cDNA of the bcl-2 gene and generated two clones (MDA-bcl4 and MDA-bcl7) characterized by bcl-2 expression twofold and fourfold that observed in the control cell clone (MDAneo). A concomitant overexpression of p21wafl was also detected in the MDA-bcl7 clone. The MDA-bcl4 clone was three times more resistant to a 24-h Tomudex exposure than the MDAneo clone, whereas the MDA-bcl7 clone was as sensitive to Tomudex as the control cell clone. A lower sensitivity of the MDA-bcl4 clone than MDAneo and MDA-bcl7 clones to 5-fluorouracil and gemcitabine was also observed. No significant difference was noted in the susceptibility of clones to fludarabine and methothrexate. Basal levels of thymidylate synthase activity were superimposable in the three clones. Tomudex induced a marked accumulation of cells in the S phase in all the clones. However, an apoptotic hypodiploid DNA peak and the characteristic nuclear morphology of apoptosis were observed only in the MDA-bcl7 clone after exposure to Tomudex. No difference in the treatment-induced modulation of proteins involved in cell cycle progression (cyclin A, cdk2, pRB, E2F-1) and apoptosis (bcl-2, bax) was observed in the three clones. The only exception was that the expression of p21wafl in the MDA-bcl4 clone was inducible at a Tomudex concentration much higher than that required to induce the protein in the other clones. Overall, the results indicate that bcl-2 and p21wafl proteins concur in determining the cellular profile of sensitivity/resistance to Tomudex.  (+info)

A Phase I study of raltitrexed, an antifolate thymidylate synthase inhibitor, in adult patients with advanced solid tumors. (30/1894)

The purpose of this study was to perform a Phase I trial of raltitrexed, a selective inhibitor of thymidylate synthase, and to determine the pharmacokinetic and toxicity profiles as a function of raltitrexed dose. Fifty patients with advanced solid tumors and good performance status were treated with raltitrexed as a 15-min i.v. infusion every 3 weeks, at doses escalating from 0.6 to 4.5 mg/m2. Asthenia, neutropenia, and hepatic toxicity were the most common dose-limiting toxicities in this largely pretreated patient population, but they occurred during the initial cycle in only one of nine patients treated with 4.0 mg/m2 and in two of nine patients treated with 4.5 mg/m2. Only 2 of 13 patients treated with 3.5 mg/m2 ultimately experienced unacceptable toxicity after three and seven cycles, compared with 42 and 56% of patients receiving 4.0 and 4.5 mg/m2 after medians of three and two cycles, respectively. The maximum raltitrexed plasma concentration and the area under the plasma concentration-time curve increased in proportion to dose. Raltitrexed clearance was independent of dose and was associated with the estimated creatinine clearance. Asthenia, neutropenia, and hepatic transaminitis were dose-related and tended to occur more frequently when patients received three or more cycles of therapy. A 3-week treatment interval was feasible in the majority of patients at all doses. Although 4.0 mg/m2 appeared to be a safe starting dose in this pretreated patient population, about half who received two or more courses ultimately experienced dose-limiting toxicity. A dose of 3.5 mg/m2 was well tolerated in most patients.  (+info)

Impact of polyglutamation on sensitivity to raltitrexed and methotrexate in relation to drug-induced inhibition of de novo thymidylate and purine biosynthesis in CCRF-CEM cell lines. (31/1894)

The aim of this study was to investigate the influence of folylpolyglutamyl synthetase (FPGS) activity on the cellular pharmacology of the classical antifolates raltitrexed and methotrexate (MTX) using two human leukemia cell lines, CCRF-CEM and CCRF-CEM:RC2Tomudex. Cell growth inhibition and drug-induced inhibition of de novo thymidylate and purine biosynthesis were used as measures of the cellular effects of the drugs. CCRF-CEM:RC2Tomudex cells had <11% of the FPGS activity of CCRF-CEM cells, whereas MTX uptake and TS activity were equivalent. In CCRF-CEM:RC2Tomudex cells, MTX polyglutamate formation was undetectable after exposure to 1 microM [3H]MTX for 24 h. After exposure to 0.1 microM raltitrexed, levels of total intracellular raltitrexed-derived material in CCRF-CEM:RC2Tomudex cells were 30- to 50-fold lower than in the CCRF-CEM cell line. CCRF-CEM: RC2Tomudex cells were >1000-fold resistant to raltitrexed and 6-fold resistant to lometrexol but sensitive to MTX and nolatrexed when exposed to these antifolates for 96 h. After 6 h of exposure, CCRF-CEM cells retained sensitivity to MTX and raltitrexed but were less sensitive to lometrexol-mediated growth inhibition. In contrast, CCRF-CEM: RC2Tomudex cells were markedly insensitive to raltitrexed, lometrexol, and to a lesser degree, MTX. Simultaneous measurement of de novo thymidylate and purine biosynthesis revealed 90% inhibition of TS activity by 100 nM MTX in both cell lines, whereas inhibition of de novo purine synthesis was only observed in CCRF-CEM cells, and only after exposure to 1000 nM MTX. Ten nM raltitrexed induced >90% inhibition of TS activity in CCRF-CEM cells, whereas in CCRF-CEM:RC2Tomudex cells, there was no evidence of inhibition after exposure to 1000 nM raltitrexed. These studies demonstrate that polyglutamation is a critical determinant of the cellular pharmacology of both raltitrexed and MTX, markedly influencing potency in the case of raltitrexed and locus of action in the case of MTX.  (+info)

A phase II study of Tomudex alternated with methotrexate, 5-fluorouracil, leucovorin in first-line chemotherapy of metastatic colorectal cancer. (32/1894)

PURPOSE: This multicenter phase II study was designed to assess the efficacy of the alternating schedule of tomudex with methotrexate (MTX)/5-fluorouracil (5-FU)/leucovorin (LV) in first-line chemotherapy for metastatic colorectal cancer. PATIENTS AND METHODS: Patients with histologically proven metastatic colorectal cancer and at least one bidimensionally measurable lesion, aged 18-70, with performance status < or = 2, normal baseline biological values, and no prior chemotherapy, were selected. Treatment was tomudex 3 mg/m2 and, after two weeks, MTX, 200 mg/m2 by 30' infusion after hydration with 1500 ml saline solution, followed on day 2 by 5-FU, 600 mg/m2 and leucovorin, orally, 15 mg for six times every 6 hours, beginning 24 hours after MTX. Cycles were repeated every four weeks. Tumor response assessment was performed after three cycles. RESULTS: Thirty-four patients were enrolled in this study, of whom twenty-four had liver metastases, nine local relapse, five lymph node involvement, four lung metastases, and three peritoneal carcinomatosis. Four patients achieved objective responses (one complete and three partial), for an overall response rate of 12% (95% CI: 0%-22%). Twelve patients had stable disease and 18 progressed on therapy. Median survival for all patients was 13 months. Two patients experienced grade 3 WHO neutropenia while hepatotoxicity was reported in 13 patients (6 grade 1, 3 grade 2, 3 grade 3, 1 grade 4), suggesting that this combination could increase hepatic toxicity in comparison to tomudex or MTX/5-FU alone. CONCLUSIONS: Our results suggest that this regimen does not warrant further investigation in advanced colorectal cancer patients, at least not with this schedule and doses.  (+info)