RESUMO
The NADPH oxidase (NOX) is a significant determinant for the expression and activity of γ-glutamyltransferase (GGT), which is frequently upregulated after increased levels of reactive oxygen species (ROS) and oxidative stress. Earlier studies on human colon carcinoma HT-29 cells have shown that treatment with phorbol 12-myristate 13-acetate (PMA) activates NOX thus increasing the intracellular level of ROS and upregulating GGT. Another important source of cellular ROS is the mitochondria, and treatment with the mitochondria uncoupler carbonylcyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP) results in increased ROS levels. The present study shows that when HT-29 cells were simultaneously treated with both agents, a significant and synergic increase in intracellular ROS was detected. NOX activity contributed at least 50 % of this increase as inhibiting NOX activity with apocynin or downregulating the NOX activity using siRNA against p22 phox reduced the synergic ROS production. The combined FCCP and PMA treatment also provoked highly increased GGT mRNA levels after 24 h whereas only minor and delayed increases in GGT protein and enzyme activity levels were detected. The results strongly indicate that ROS production by both mitochondria and NOX is involved in the regulation of GGT expression in colon carcinoma cells.
Assuntos
Neoplasias do Colo/metabolismo , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Desacopladores/farmacologia , gama-Glutamiltransferase/biossíntese , Acetofenonas/farmacologia , Benzopiranos/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Linhagem Celular Tumoral , Combinação de Medicamentos , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Células HT29 , Humanos , Mitocôndrias/metabolismo , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , Oxirredução , Estresse Oxidativo , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Interferente Pequeno , Acetato de Tetradecanoilforbol/farmacologia , gama-Glutamiltransferase/genéticaRESUMO
Nostoc sp. strain KVJ20 was isolated from the symbiotic organs of the liverwort Blasia pusilla This cyanobacterium has been shown to have broad symbiotic competence, and bacterial extracts have inhibitory effects on cancer cell lines and microbes. An array of genes for the production of secondary metabolites is present.
RESUMO
Gamma-glutamyltransferase (GGT) plays a central role in the homeostasis of the antioxidant glutathione (GSH). The expression of GGT has been shown to be upregulated after oxidative stress, but the signalling pathways implicated remain poorly characterized. The results here show that acute exposure of CC531 cells to oxidative stress resulted in activation of Ras and augmented GGT enzyme activity, both at the transcriptional and at the translation level. Moreover, an involvement of the GGT promoter II was detected after RT-PCR and transient transfection studies. Ectopic expression of activated Ras, but not dominant negative Ras, also resulted in increased GGT promoter II transcriptional activity, an effect that was attenuated by over-expression of dominant negative mutants of Akt, p38 MAPK and MEK1. Addition of specific inhibitors of these kinases during oxidative stress diminished the activation of GGT. In conclusion, oxidative stress-induced activation of GGT involves Ras and several downstream signalling pathways.