RESUMO
BACKGROUND: The aim of this study was to evaluate the radiosensitising effect of gemcitabine, in terms of cell-cycle progression, induction of apoptosis, and to investigate the molecular events regulating apoptosis. METHODS: Tumour cells were treated with gemcitabine, radiation, or the combination. 0-72 h after treatment, cells were collected for cell-cycle analysis and apoptosis determination. Caspase 8 and 9, Bid and tBid expression were determined by western blot. The mitochondrial membrane potential was determined using flow cytometry. An RT(2) Profiler PCR Array for human apoptotic genes was performed after the combination or TRAIL treatment. RESULTS: Gemcitabine and radiation resulted in an early S-phase block immediately after treatment, after which the cells moved synchronously through the cell cycle. When cell-cycle distribution returned to pre-treatment levels, an increased induction of apoptosis was observed with activation of caspase 8 and 9 and a reduction of the mitochondrial membrane potential. Gene expression after treatment with radiosensitising conditions was comparable with expression after the TRAIL treatment. CONCLUSION: A role for the cell-cycle perturbations and the induction of apoptosis could be attributed to the radiosensitising effect of gemcitabine. Apoptosis induction was comparable with the apoptotic pathway observed after the TRAIL treatment, that is the involvement of the extrinsic apoptosis pathway.
Assuntos
Apoptose/efeitos dos fármacos , Desoxicitidina/análogos & derivados , Radiossensibilizantes/farmacologia , Apoptose/fisiologia , Apoptose/efeitos da radiação , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/efeitos dos fármacos , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/efeitos da radiação , Western Blotting , Caspase 8/efeitos dos fármacos , Caspase 8/metabolismo , Caspase 8/efeitos da radiação , Caspase 9/efeitos dos fármacos , Caspase 9/metabolismo , Caspase 9/efeitos da radiação , Linhagem Celular Tumoral , Desoxicitidina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/efeitos da radiação , Citometria de Fluxo , Humanos , Marcação In Situ das Extremidades Cortadas , Potencial da Membrana Mitocondrial , Reação em Cadeia da Polimerase , GencitabinaRESUMO
Programmed cell death (PCD) is a genetically controlled cell death that is regulated during development and activated in response to environmental stresses or pathogen infection. The degree of conservation of PCD across kingdoms and phylum is not yet clear; however, whereas caspases are proteases that act as key components of animal apoptosis, plants have no orthologous caspase sequences in their genomes. The discovery of plant and fungi metacaspases as proteases most closely related to animal caspases led to the hypothesis that metacaspases are the functional homologues of animal caspases in these organisms. Arabidopsis thaliana has nine metacaspase genes, and so far it is unknown which members of the family if any are involved in the regulation of PCD. We show here that metacaspase-8 (AtMC8) is a member of the gene family strongly up-regulated by oxidative stresses caused by UVC, H(2)O(2), or methyl viologen. This up-regulation was dependent of RCD1, a mediator of the oxidative stress response. Recombinant metacaspase-8 cleaved after arginine, had a pH optimum of 8, and complemented the H(2)O(2) no-death phenotype of a yeast metacaspase knock-out. Overexpressing AtMC8 up-regulated PCD induced by UVC or H(2)O(2), and knocking out AtMC8 reduced cell death triggered by UVC and H(2)O(2) in protoplasts. Knock-out seeds and seedlings had an increased tolerance to the herbicide methyl viologen. We suggest that metacaspase-8 is part of an evolutionary conserved PCD pathway activated by oxidative stress.
Assuntos
Apoptose/fisiologia , Proteínas de Arabidopsis/genética , Caspase 8/genética , Cisteína Endopeptidases/genética , Peróxido de Hidrogênio/farmacologia , Raios Ultravioleta , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Arabidopsis , Proteínas de Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/efeitos da radiação , Caspase 8/efeitos dos fármacos , Caspase 8/efeitos da radiação , Morte Celular , Cisteína Endopeptidases/deficiência , Cisteína Endopeptidases/efeitos dos fármacos , Cisteína Endopeptidases/efeitos da radiação , Primers do DNA , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Estresse Oxidativo , Plantas Geneticamente Modificadas/metabolismo , Protoplastos/efeitos dos fármacos , Protoplastos/fisiologia , Protoplastos/efeitos da radiação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Hyperthermia is a useful adjunct in cancer therapy as it can increase the effectiveness and decrease the toxicity of currently available cancer treatments such as chemotherapy and radiation. In the present study, we investigated whether 41 degrees C hyperthermia (mild HT) for 20 min can enhance macrosphelide (MS5)-induced apoptosis in human lymphoma U937 cells. Our results revealed that, compared with MS5 (5 microM) and mild HT alone, the combined treatment exhibited significant enhancement in apoptosis at 6 h, which was evaluated by observing morphological changes and DNA fragmentation. Marked increase in the reactive oxygen species (ROS) generation was observed immediately after the combined treatment. Significant increase in Fas externalization, caspase-8 and caspase-3 activation, and loss of mitochondrial membrane potential (MMP) was found after the combined treatment compared with MS5 and mild HT alone. Moreover, this combination can also alter the expression of apoptosis-related proteins as evident by the cleavage of Bid and down-regulation of Bcl-2 while no change in the expression of Bax was observed. Furthermore, an immediate rise in the intracellular calcium ion ([Ca(2+)]i) concentration was observed after the combined treatment, which continuously increased in a time-dependent manner. In addition, mild HT treatment alone also increases [Ca(2+)]i concentration without inducing apoptosis. Our data indicate that early increase in ROS generation is mainly responsible for the enhancement of apoptosis after the combined treatment.