RESUMEN
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.
Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Metionina/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Paclitaxel/uso terapéutico , Animales , Línea Celular Tumoral , Combinación de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Ratones , Ratas , Vinblastina/uso terapéuticoRESUMEN
BACKGROUND: We have examined, in this study, the feasibility of determining cellular factors contributing to irinotecan activity in colorectal cancers. Irinotecan is a camptothecin derivative requiting carboxylesterase activation to SN-38, which interacts with its target enzyme, topoisomerase I. MATERIALS AND METHODS: In 9 surgical or biopsy samples of colorectal tumours and corresponding normal tissue, kept in a tumour bank, we evaluated topoisomerase I expression and activity, respectively by Western blotting and DNA relaxation assay, carboxylesterase activity using two different substrates and p53 status by immunohistochenistry. RESULTS: Topoisomerase I expression and activity were significantly correlated, as were the two types of determinations for carboxylesterase activity. Topoisomerase I was significantly more active in tumours than in corresponding normal tissue. The three samples presenting the highest topoisomerase I expression and activity originated from the patients who responded to irinotecan treatment. No such features were apparent for carboxylesterase activity and p53 staining. CONCLUSION: Topoisomerase I expression appeared as the parameter most likely to predict response to irinotecan therapy in the clinical setting.
Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Camptotecina/análogos & derivados , Camptotecina/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Adulto , Anciano , Western Blotting , Carboxilesterasa/metabolismo , Neoplasias Colorrectales/enzimología , ADN-Topoisomerasas de Tipo I/metabolismo , Estudios de Factibilidad , Femenino , Humanos , Inmunohistoquímica , Irinotecán , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Estudios Retrospectivos , Inhibidores de Topoisomerasa I , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
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.
Asunto(s)
Homocisteína/farmacología , Metionina/farmacología , Animales , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular Tumoral , Humanos , Ratones , Ratas , Azufre/químicaRESUMEN
PURPOSE: Irinotecan is a drug of the camptothecin family that has proven activity in advanced colon cancer, with about 20% responses in untreated as well as in 5-fluorouracil-resistant tumors. Irinotecan is considered as a prodrug which needs to be activated to SN-38 by carboxylesterases to become able to interact with its target, topoisomerase I. The work reported here intended to identify the determinants of the cytotoxicity of irinotecan in two human colorectal tumor cell lines, LoVo and HT-29, at the level of the target of the drug and at the level of the availability of the active metabolite to the target. RESULTS: The cytotoxicity of irinotecan and SN-38 markedly differed in the two cell lines: irinotecan IC(50) values were 15.8 microM for LoVo cells and 5.17 microM for HT-29 cells; SN-38 IC(50) values were 8.25 n M for LoVo cells and 4.50 n M for HT-29 cells. Topoisomerase I expression (at the mRNA and the protein levels) and catalytic activity were similar in the two cell lines. Irinotecan induced similar amounts of cleavable complexes at its IC(50) in both cell lines. SN-38 induced a concentration-dependent formation of cleavable complexes, which was not significantly different in the two cell lines. Expression of the carboxylesterase CES1 was higher in HT-29 than in LoVo cells. Expression of the carboxylesterase gene CES2 was comparable in the two cell lines and much higher than CES1 gene expression. Carboxylesterase activity was extremely low using p-nitrophenylacetate as a substrate (1.45 and 1.84 pmol/min per mg proteins) and could not even be detected using irinotecan as a substrate. Cell accumulation of irinotecan was markedly different, reaching consistently higher levels in HT-29 cells than in LoVo cells. CONCLUSIONS: Our results indicate that (1) the cytotoxicity of irinotecan was likely due to the drug itself and not to its metabolite SN-38, and (2) that irinotecan uptake was more predictive of its cytotoxicity than topoisomerase I availability and activity in these two cell lines.