Manganese superoxide dismutase (Mn-SOD) is a mitochondrial enzyme that dismutates two superoxide radicals into hydrogen peroxide and molecular oxygen. This enzyme is crucial for the defence against cellular reactive oxygen species (ROS), functioning as an essential anti-oxidant enzyme protecting critical targets from superoxide modification. Diclofenac is a non-steroidal antiinflammatory drug (NSAID) frequently used as an analgesic and in the treatment of rheumatic diseases; more recently, a number of experimental and clinical studies suggested its possible usage as an anticancer agent. Many reports have shown that diclofenac, as well as other NSAIDs, induce apoptosis in a variety of cell lines such as hepatic, gastric and renal, thus influencing their cellular redox state. On the other hand, a few data are available regarding the effects of these drugs on neuronal cells.Here we investigate diclofenac-induced apoptosis in the neuroblastoma cell line SH-SY5Y and the possible involvement of Mn-SOD in this process. Flow cytometric analysis of SH-SY5Y cells treated with diclofenac revealed a time- and dose-dependent increase of apoptotic nuclei. Moreover, the treatment of SH-SY5Y with diclofenac induces an increase in cellular ROS levels, as measured by oxidation-sensitive fluorescence probes. To evaluate the involvement of Mn-SOD in the cytotoxic effect induced by diclofenac, both protein level and enzyme activity have been evaluated in protein extracts obtained from SH-SY5Y cells grown in the absence or in the presence of diclofenac. Western blotting analysis showed that diclofenac decreases the levels of Mn-SOD; concomitantly, its enzymatic activity is reduced, as measured by a colorimetric assay on non-denaturing polyacrylamide gels. However, diclofenac does not affect the mRNA levels of Mn-SOD, as determined by RT-PCR experiments. When SH-SY5Y cells were cultured in the presence of a recombinant thioredoxin from the hyperthermophilic archaeon Sulfolobus solfataricus, a marked attenuation of the diclofenac-induced apoptosis was observed, together with an increase of the Mn-SOD levels. Furthermore, diclofenac induces a reduction of the mitochondrial membrane potential and a release of cytocrome c from mitochondria. These data suggest that mitochondria are involved in the diclofenac-induced apoptosis in SH-SY5Y neuroblastoma cell line and point to a possible role of Mn-SOD in this process.
Involvement of mitochondrial Mn-SOD in the defence of diclofenac-induced apoptosis in the SH-SY5Y neuroblastoma cell line / DE VENDITTIS, Emmanuele; Zappelli, C; Cecere, Francesca; Iuliano, A; Castellano, Immacolata; Grimaldi, P; Masullo, Mariorosario; Ruocco, MARIA ROSARIA. - In: THE FEBS JOURNAL. - ISSN 1742-464X. - 276 (Suppl 1):(2009), pp. 194-194.
Involvement of mitochondrial Mn-SOD in the defence of diclofenac-induced apoptosis in the SH-SY5Y neuroblastoma cell line
DE VENDITTIS, EMMANUELE;CECERE, FRANCESCA;CASTELLANO, IMMACOLATA;MASULLO, MARIOROSARIO;RUOCCO, MARIA ROSARIA
2009
Abstract
Manganese superoxide dismutase (Mn-SOD) is a mitochondrial enzyme that dismutates two superoxide radicals into hydrogen peroxide and molecular oxygen. This enzyme is crucial for the defence against cellular reactive oxygen species (ROS), functioning as an essential anti-oxidant enzyme protecting critical targets from superoxide modification. Diclofenac is a non-steroidal antiinflammatory drug (NSAID) frequently used as an analgesic and in the treatment of rheumatic diseases; more recently, a number of experimental and clinical studies suggested its possible usage as an anticancer agent. Many reports have shown that diclofenac, as well as other NSAIDs, induce apoptosis in a variety of cell lines such as hepatic, gastric and renal, thus influencing their cellular redox state. On the other hand, a few data are available regarding the effects of these drugs on neuronal cells.Here we investigate diclofenac-induced apoptosis in the neuroblastoma cell line SH-SY5Y and the possible involvement of Mn-SOD in this process. Flow cytometric analysis of SH-SY5Y cells treated with diclofenac revealed a time- and dose-dependent increase of apoptotic nuclei. Moreover, the treatment of SH-SY5Y with diclofenac induces an increase in cellular ROS levels, as measured by oxidation-sensitive fluorescence probes. To evaluate the involvement of Mn-SOD in the cytotoxic effect induced by diclofenac, both protein level and enzyme activity have been evaluated in protein extracts obtained from SH-SY5Y cells grown in the absence or in the presence of diclofenac. Western blotting analysis showed that diclofenac decreases the levels of Mn-SOD; concomitantly, its enzymatic activity is reduced, as measured by a colorimetric assay on non-denaturing polyacrylamide gels. However, diclofenac does not affect the mRNA levels of Mn-SOD, as determined by RT-PCR experiments. When SH-SY5Y cells were cultured in the presence of a recombinant thioredoxin from the hyperthermophilic archaeon Sulfolobus solfataricus, a marked attenuation of the diclofenac-induced apoptosis was observed, together with an increase of the Mn-SOD levels. Furthermore, diclofenac induces a reduction of the mitochondrial membrane potential and a release of cytocrome c from mitochondria. These data suggest that mitochondria are involved in the diclofenac-induced apoptosis in SH-SY5Y neuroblastoma cell line and point to a possible role of Mn-SOD in this process.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.