RESUMO
Interference with telomerase and telomere maintenance is emerging as an attractive target for anticancer therapies. Ligand-induced stabilization of G-quadruplex formation by the telomeric DNA single-stranded 3' overhang inhibits telomerase from catalyzing telomeric DNA synthesis and from capping telomeric ends. We report here the effects of a 3,6,9-trisubstituted acridine compound, BRACO-19, on telomerase function in vitro and in vivo. The biological activity of BRACO-19 was evaluated in the human uterus carcinoma cell line UXF1138L, which has very short telomeres (2.7 kb). In vitro, nuclear human telomerase reverse transcriptase (hTERT) expression was drastically decreased after 24 hours, induction of cellular senescence and complete cessation of growth was seen after 15 days, paralleled by telomere shortening of ca. 0.4 kb. In vivo, BRACO-19 was highly active as a single agent against early-stage (68 mm(3)) tumors in a s.c. growing xenograft model established from UXF1138L cells, if given chronically at 2 mg per kg per day i.p. BRACO-19 produced growth inhibition of 96% compared with controls accompanied by partial regressions (P < 0.018). Immunostaining of xenograft tissues showed that this response was paralleled by loss of nuclear hTERT protein expression and an increase in atypical mitoses indicative of telomere dysfunction. Cytoplasmic hTERT expression and its colocalization with ubiquitin was observed suggesting that hTERT is bound to ubiquitin and targeted for enhanced degradation upon BRACO-19 treatment. This is in accord with a model of induced displacement of telomerase from the telomere. The in vitro and in vivo data presented here is consistent with the G-quadruplex binding ligand BRACO-19 producing an anticancer effect by inhibiting the capping and catalytic functions of telomerase.
Assuntos
Acridinas/farmacologia , DNA/metabolismo , Telomerase/antagonistas & inibidores , Telômero/efeitos dos fármacos , Neoplasias Uterinas/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , DNA/efeitos dos fármacos , DNA/genética , DNA de Cadeia Simples/efeitos dos fármacos , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA , Feminino , Quadruplex G , Guanina/metabolismo , Humanos , Camundongos , Camundongos Nus , Telomerase/biossíntese , Telomerase/metabolismo , Telômero/genética , Telômero/metabolismo , Ubiquitina/metabolismo , Neoplasias Uterinas/enzimologia , Neoplasias Uterinas/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Methionine dependence is a unique feature of cancer cells characterized by growth and cell cycle arrest (typically in S and G2/M) under conditions of methionine depletion. Following replenishment of media with methionine, the cell cycle blockade is reversible and during this recovery period, cells may become more susceptible to the action of cell cycle specific drugs. The response of a panel of methionine dependent (HTC, Phi-1, PC3 and 3T3) cells to vinblastine and paclitaxel was compared to methionine independent Hs-27 cells under conditions of methionine depletion (M-H+; methionine depleted media supplemented with homocysteine) and starvation (M-H-; media without methionine or homocysteine). All cell lines were significantly more resistant to both agents under M-H+ and M-H- conditions compared to controls under normal culture conditions [M+H-]; however, the magnitude of resistance was reduced in the methionine independent Hs-27 cells. During recovery from methionine depletion and starvation, the response of the methionine dependent cells to vinblastine and paclitaxel was significantly enhanced compared to controls. Although the activity of vinblastine on the Hs-27 cell line was comparable to controls, these methionine independent cells became significantly more resistant to paclitaxel during recovery studies (IC50 = 2.13 +/- 0.5 microM) compared to control cultures (IC50 = 0.13 +/- 0.15 microM). Whilst the mechanism responsible for this remains uncertain, the increased activity of paclitaxel against methionine dependent cells in conjunction with the decreased activity against Hs-27 cells suggests that methionine depletion strategies may enhance the therapeutic index of paclitaxel.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Metionina/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Paclitaxel/uso terapêutico , Animais , Linhagem Celular Tumoral , Combinação de Medicamentos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos , Ratos , Vimblastina/uso terapêuticoRESUMO
Novel 2-(4-aminophenyl)benzothiazoles (e.g., compounds 1 and 2) possess highly selective, potent antitumor properties in vitro and in vivo. Elucidation of the mechanism of action of this structurally simple class of compounds has occurred in parallel with selection of a candidate clinical agent. Antitumor benzothiazoles induce and are biotransformed by cytochrome P 450 1A1 to putative active, as well as inactive metabolites. Metabolic inactivation of the molecule has been thwarted by isosteric replacement of hydrogen with fluorine atoms at positions around the benzothiazole nucleus. Amino acid conjugation to the exocyclic primary amine function of 2-(4-aminophenyl)benzothiazoles has been used to overcome limitations posed by drug lipophilicity. Water soluble, chemically stable prodrugs rapidly and quantitatively revert to their parent amine in mice, rats, and dogs in vivo. Plasma concentrations of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (2) regenerated from the lysylamide prodrug (2b), sufficient to elicit cytocidal activity against ZR-75-1 and T47D human mammary carcinoma cell lines persist > 6 h. The growth of breast (MCF-7) and ovarian (IGROV-1) xenograft tumors is significantly retarded by 2b. Manageable toxic side effects are reported from preclinically efficacious doses of 2b. Cytochrome P 450 1A1 protein expression, selectively induced in sensitive carcinoma cells, was detected in MCF-7 and IGROV-1 tumors 24 h after treatment of mice with 2b (20 mg/kg). The lysyl amide prodrug of 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole is potentially suitable for clinical evaluation.
Assuntos
Aminoácidos/metabolismo , Compostos de Anilina/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Ovarianas/tratamento farmacológico , Tiazóis/uso terapêutico , Compostos de Anilina/farmacocinética , Animais , Antineoplásicos/farmacocinética , Benzotiazóis , Western Blotting , Divisão Celular/efeitos dos fármacos , Citocromo P-450 CYP1A1/metabolismo , Cães , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Masculino , Neoplasias Mamárias Experimentais/enzimologia , Camundongos , Camundongos Nus , Neoplasias Ovarianas/enzimologia , Pró-Fármacos , Ratos , Ratos Sprague-Dawley , Tiazóis/farmacocinética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The cytochrome P450 family of enzymes is involved in the Phase I metabolism of a wide variety of compounds. Although generally involved with detoxification, overexpression of one family member, cytochrome P450 1B1 (CYP1B1), has been associated with human epithelial tumors. As such, CYP1B1 was hypothesized to be a novel target for the development of anticancer therapies. We investigated expression of CYP1B1 protein in 61 human colorectal adenocarcinomas and compared this to that observed in 14 histologically normal human large bowel samples removed from patients undergoing surgery for large bowel tumors. Although we confirmed that CYP1B1 was expressed at high levels in human colorectal tumor epithelia, we also found that CYP1B1 was not absent from normal colonic epithelia but was expressed at low levels. The expression of CYP1B1 in colon tumors does not correlate with tumor stage or degree of lymph node invasion in this study. Furthermore, in addition to expression in colon epithelia, CYP1B1 is also observed in blood vessels within the colon. As with the epithelia, levels of CYP1B1 were higher in tumor vasculature than that of the normal colon. Although these observations greatly support the development of CYP1B1 targeted anticancer therapies, they also indicate the caution that should be observed when developing such drugs.
Assuntos
Adenocarcinoma/enzimologia , Hidrocarboneto de Aril Hidroxilases/biossíntese , Neoplasias do Colo/enzimologia , Adenocarcinoma/patologia , Western Blotting , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Neoplasias Colorretais/patologia , Citocromo P-450 CYP1B1 , Células Epiteliais/citologia , Epitélio/patologia , Humanos , Immunoblotting , Imuno-Histoquímica , Metástase Linfática , Músculo Liso/metabolismoRESUMO
Methionine dependence is a feature unique to cancer cells, exhibited as inability to grow in a methionine-depleted environment supplemented with homocysteine, the immediate metabolic precursor of methionine. This study explores the effect of methionine depletion and homocysteine supplementation on the viability, sulfur amino acid metabolism and cell-cycle kinetics of normal and cancer cells, as well as their ability to recover from the treatments. An array of cells including hepatomas (HTC, Phi-1), prostate adenocarcinomas (PC-3) and transformed (3T3) and normal (HS-27) fibroblasts, has been used aiming to evaluate the importance of tissue specificity. All cell lines proliferated well in methionine-complete media (M+H-), whilst only the normal fibroblasts HS-27 grew in methionine-depleted homocysteine-supplemented media (M-H+). None of the tested cell lines were able to grow in media without methionine or homocysteine (M-H-). HTC was the only cell line that did not recover from the M-H+ treatment whilst PC-3 did not recover from the M-H- treatment. Methionine and homocysteine depletion (M-H+ and M-H-) were found to induce arrest at different phases of the cell cycle, depending on the cell line: the methionine-dependent HTC, PC-3 and 3T3 arrested at the S and G2/M phase, whilst Phi-1 and the methionine-independent HS-27 accumulated in the G1 phase. The cell-cycle kinetics showed that the observed blockades were reversible. The information resulting from these studies is important for not only the behavior of cancer cells, but also for appreciating the potential of developing cancer therapies based on methionine-depletion strategies.
Assuntos
Homocisteína/farmacologia , Metionina/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Camundongos , Ratos , Enxofre/químicaRESUMO
A panel of tumour models used extensively for in vivo evaluation of new drugs was characterised for their p53 status. Basal p53 protein levels were measured by immunodetection on both formalin-fixed tumour tissue and from protein extracts of fresh tumours. High levels of nuclear-specific staining, indicative of p53 mutation, was seen in 15/25 tumours, with the remainder showing intermittent or no staining. The functional status of p53 cDNA from these tumours was assayed within the functional analysis of separated alleles in yeast (F.A.S.A.Y.) reporter system. The cDNA from those tumours with high levels of p53 protein showed 14/15 failing to activate the reporter gene. The cDNA from tumours with low or non-detectable p53 levels showed 8/10 with wild-type p53. Tumours were grown subcutaneously in mice (n=10). Each mouse was given maximum tolerated doses for either doxorubicin, 5-fluorouracil or cisplatin. Tumour volumes were measured daily, alongside untreated controls. The specific growth delay values for each tumour were separated into two groups, those with functional p53 (wild-type) and those without (mutant and null status). The Mann-Whitney U test was performed on the groups of data, to evaluate differences in their response on the basis of p53 status. Cisplatin was moderately active against tumours with wild-type and mutant p53 genes with no significant difference seen between both groups. However, a significant difference in specific growth delay was seen between the two groups when treated with doxorubicin or 5-fluorouracil (P=0.05), indicating a role for p53 protein in modulating the in vivo efficacy of these agents.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Proteína Supressora de Tumor p53/análise , Animais , Western Blotting , Cisplatino/uso terapêutico , Doxorrubicina/uso terapêutico , Feminino , Fluoruracila/uso terapêutico , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos , Camundongos Nus , Mutação , Neoplasias/metabolismo , Neoplasias/patologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transformação Genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Historically, the process of developing new anticancer agents was largely empirical. Today, because of improvements in our knowledge of the molecular processes involved in the development of cancer, the process of developing new agents is becoming more rational. Researchers from Cancer Research UK, the European Organisation for Research and Treatment of Cancer and the National Cancer Institute have shown that, by undertaking a pharmacological approach to the selection of potential anticancer agents, both meaningful antitumour data and an 80% reduction in animal usage can be obtained. It has also been demonstrated that a new pharmacological tool, the "hollow fibre system", in which tumour cells are grown in biocompatible fibres which are implanted into mice, can be used to produce meaningful antitumour data with pharmacodynamic endpoints. By increasing the amount of data that can be obtained from a single animal and opening up the possibility of eliminating the need for untreated control animals, the hollow fibre system has the potential to make a significant contribution to both reduction and refinement.
Assuntos
Alternativas ao Uso de Animais , Antineoplásicos/farmacologia , Técnicas de Cultura de Células/veterinária , Desenho de Fármacos , Farmacologia/métodos , Animais , Técnicas de Cultura de Células/métodos , Doxorrubicina/farmacocinética , CamundongosAssuntos
Antineoplásicos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Proteínas de Neoplasias/antagonistas & inibidores , Telomerase/antagonistas & inibidores , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Divisão Celular/efeitos dos fármacos , Desenho de Fármacos , Inibidores Enzimáticos/efeitos adversos , Inibidores Enzimáticos/farmacologia , Inibidores do Crescimento/efeitos adversos , Inibidores do Crescimento/farmacologia , Inibidores do Crescimento/uso terapêutico , Humanos , RNA/antagonistas & inibidores , Telômero/efeitos dos fármacosRESUMO
PURPOSE: The quinone based bioreductive drug apaziquone (EO9) failed to demonstrate efficacy in previous phase II studies following intravenous administration. We determined the dose of apaziquone that can be safely administered intravesically and explored its activity for superficial bladder transitional cell carcinoma. MATERIALS AND METHODS: Six patients with multifocal, Ta/T1 and G1/G2 transitional cell carcinoma of the bladder received escalating doses of apaziquone formulated as EOquintrade mark (0.5 mg/40 ml up to 16 mg/40 ml) weekly for 6 weeks. A further 6 patients received weekly apaziquone at the highest nontoxic dose established. Pharmacokinetic parameters were determined in urine and blood, and the pharmacodynamic markers NQO1 (reduced nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase-1) and glucose transporter 1 were also characterized. Efficacy was determined against a marker lesion. RESULTS: Local toxicity (grades 2 and 3 dysuria, and hematuria) was observed at doses of 8 mg/40 ml and above but 4 mg/40 ml was well tolerated with no systemic or local side effects. Apaziquone in urine increased linearly with the dose but no apaziquone was detected in plasma. In 8 of 12 patients complete macroscopic and histological disappearance of the marker lesion occurred. A correlation between response and NQO1 and/or glucose transporter 1 expression could not be established. CONCLUSIONS: Intravesical administration of 4 mg/40 ml apaziquone was well tolerated and had ablative activity against superficial bladder cancer marker lesions.