reactive   oxygen species ( ros ) , normally produced during the aerobic metabolism , function as second messengers involved in many cellular functions . on the other hand , when ros level increases because of oxidant treatments and/or defective antioxidant systems , these highly reactive compounds and radicals become dangerous toxic agents .
in fact , ros may cause severe damages to several cellular macromolecules , including proteins , lipids , and dna , thus contributing to the development of many pathological conditions .
indeed several evidences have been reported , indicating that the redox homeostasis is a finely regulated mechanism involved in normal cellular functions and prevention of several stress - associated pathologies .
many drugs have toxic side effects , because they provoke an imbalance of the intracellular ros level . in the past
, cellular death due to a chemical injury was frequently linked to a necrotic process ; now , it is clear that the main effect provoked by several drugs is the programmed cell death .
diclofenac , a nonsteroidal anti - inflammatory drug ( nsaid ) widely used in clinical therapeutics , has cytotoxic effects and induces apoptosis in many cultured cell lines [ 3 , 4 ] .
, many experimental , epidemiologic and clinical studies suggest that nsaids , and in particular the highly selective cyclooxigenase-2 inhibitors , could act as anticancer agents [ 5 , 6 ] .
it has been reported that a combination of a specific nsaid and certain anticancer drugs has potential clinical applications .
for instance , diclofenac potentiates the chemotherapeutic effects of some drugs in neuroblastoma cell lines .
however , little is known about the effect of this nsaid on nervous cell lines , because most of the studies on this compound regard hepatic , gastric , or kidney cells [ 3 , 4 , 9 ] . in particular , ros are involved in the diclofenac - induced apoptosis of cultured gastric cells as well as in nephrotoxicity in vivo [ 9 , 10 ] ; furthermore , an oxidative injury causes the mitochondrial permeability transition in diclofenac - treated hepatocytes .
however , the molecular mechanisms underlying the induction of apoptosis by diclofenac have not been clarified in neuronal cells . in this paper , we have investigated the involvement of mitochondrial dysfunction in the mechanism of diclofenac - induced apoptosis in the neuroblastoma cell line sh - sy5y .
in particular , we have analyzed the role of the manganese superoxide dismutase ( sod2 ) in this process .
sod2 is a key enzyme of the mitochondrial matrix involved in the protection against oxidative stress , which converts the toxic superoxide anions to hydrogen peroxide and molecular oxygen .
numerous reports have demonstrated that sod2 has an essential role in the protection against many apoptotic stimuli [ 1214 ] .
in fact , in mice a partial deficiency of the sod2 gene ( sod2(+/ ) ) increases the sensitivity to apoptosis , whereas its overexpression has an antiapoptotic effect .
in particular , sod2 is involved in the inhibition of the mitochondrial permeability transition after cell treatment with tumor necrosis factor- or ionizing radiations [ 1618 ] , and blocks the fas - mediated apoptosis [ 19 , 20 ] .
our data show that the treatment of the neuroblastoma cell line sh - sy5y with diclofenac induces a decrease in sod2 protein level and an increase of the ros concentration .
this impaired redox balance predisposes the cell to apoptosis through a mechanism involving the mitochondrial pathway .
rpmi 1640 medium , fetal bovine serum ( fbs ) , l - glutamine , penicillin g , streptomycin and   trypsin were purchased from cambrex .
rhodamine 123 ( r123 ) , dichlorofluorescein diacetate ( dcfh - da ) , and propidium iodide were purchased from sigma .
polyclonal antibody against human sod2 was purchased from upstate ; polyclonal antibody against gapdh was obtained from cell signaling ; polyclonal antibody against -tubulin , goat polyclonal antibody against cox-4 , monoclonal antibody against cytochrome c , and each secondary antibody conjugated to horseradish peroxidase were obtained from santa cruz biotechnology .
recombinant thioredoxin a2 from the hyperthermophilic archaeon sulfolobus solfataricus ( sstrx ) was obtained as previously reported .
the human neuroblastoma cell line sh - sy5y was obtained from american type culture collection .
sh - sy5y cells were grown in rpmi 1640 medium supplemented with 10% fbs , 2  mm l - glutamine , 100  iu / ml penicillin g , and 100  g / ml streptomycin , in humidified incubator at 37c under 5%  co2 atmosphere .
they were split and seeded in plates ( 75  cm ) every three days and used for assays during exponential phase of growth .
an increasing concentration of diclofenac was added to cultures , and cells were incubated for different times .
cytotoxicity was quantitatively assessed by measurements of lactate dehydrogenase ( ldh ) activity released in the extracellular fluid from damaged or destroyed cells .
briefly , different aliquots of cell incubation media were added to a 1-ml reaction mixture containing 0.1  m tris - hcl , ph 7.5 , 125  m nadh , and incubated for 15 minutes at 30c .
the reaction started with the addition of 600  m sodium pyruvate and was followed by the decrease in absorbance at 340  nm .
the results were normalized to 100% death caused by cell sonication . to determine the number of apoptotic nuclei in diclofenac - treated cells , 3  10 cells / well were seeded into 96-well plates ;
at the end of each treatment , cell suspensions were centrifuged and pellets were resuspended in a hypotonic lysis solution containing 50  g / ml propidium iodide .
after incubation at 4c for 30 minutes , cells were analysed by flow cytometry to evaluate the presence of nuclei with a dna content lower than the diploid .
the intracellular ros level was detected using the oxidation - sensitive fluorescence probe dcfh - da .
briefly , cells were seeded into 6-well plate ( 3  10 cells / well ) and treated with 150  m diclofenac for different times .
dcfh - da was added in the dark at 10  m final concentration 30 minutes before the end of each incubation ; then cells were collected , washed in 10  mm sodium phosphate , ph 7.2 buffer containing 150  mm nacl ( pbs ) , and finally resuspended in 500  l of pbs for the fluorimetric analysis .
the measurement of the ros levels was realised with a cary eclipse fluorescence spectrophotometer ( varian ) .
excitation and emission wavelengths were 485  nm and 530  nm , respectively ; both excitation and emission slits were set at 10  nm . for the analysis of sod2 mrna expression ,
total rna was extracted from 1  10 cells using a trizol reagent ( invitrogen ) as described by the manufacturer .
the yield and integrity of each rna sample were checked spectrophotometrically at 260  nm and by agarose gel electrophoresis , respectively .
equal amounts of rna ( 24  g ) were subjected to a reverse transcriptase polymerase chain reaction ( rt - pcr ) , using a specific kit ( invitrogen ) and random primers ; three dilutions of cdna were amplified by pcr using taq dna polymerase ( invitrogen ) . for dna amplification of both sod2 and glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) ,
the pcr program was 5 minutes initial denaturation at 95c , 2023 cycles of amplification ( 95c , 1 minute ; 50c , 1 minute ; 72c , 1 minute ) , final extension step at 72c for 10 minutes .
the following primers were used : 5d - tacgtgaacaacctgaacgt-3 ( sense ) and 5d - caagccatgtatctttcagtta ( antisense ) for sod2 ; 5d - caccatcttccaggagcgag-3 ( sense ) and 5d - tcacgccacagtttcccgga-3 ( antisense ) for gapdh .
sod2 pcr products were normalized to the respective intensity of the house - keeping gene gapdh .
briefly , cells were seeded into 6-well plate ( 3  10 cells / well ) , incubated at 37c for 1 hour in the presence of 5  m r123 , washed twice with pbs and placed in fresh complete medium containing 150  m diclofenac .
after different times of drug treatment , the medium was withdrawn , and collected cells were washed twice with pbs .
after detachment with trypsin , cells were harvested in pbs and centrifuged at 4c for 10 minutes .
following aspiration of supernatant , the cellular pellet was resuspended in 500  l of pbs .
the fluorescence of cell - associated r123 was measured in the above - mentioned fluorescence spectrophotometer , using excitation and emission wavelengths of 490  nm and 520  nm , respectively ; both excitation and emission slits were set at 10  nm .
sh - sy5y cells were plated at a density of 3  10 cells / well in 6-well plates , and 150  m diclofenac was added to the cultures . after the drug treatment , cells were harvested , washed with pbs , and then lysed in ice - cold modified ripa buffer ( 50  mm tris - hcl , ph 7.4 , 150  mm nacl , 1% nonidet p-40 , 0.25% sodium deoxycolate , 1  mm na3vo4 ,   and 1  mm naf ) , supplemented with protease inhibitors and incubated for 30 minutes on ice .
the supernatant obtained after centrifugation at 12,000  g for 30 minutes at 4c constituted the total protein extract .
briefly , protein samples were dissolved in sds / reducing loading buffer , run on a 14% sds - page , then transferred to immobilon p membrane ( millipore ) .
the filter was incubated with the specific primary antibody at 4c overnight and with the horseradish peroxidase - linked secondary antibody at room temperature for 1 hour .
membranes were then analyzed by an enhanced chemiluminescence reaction , using super signal west pico kit ( pierce ) according to the manufacturer 's instructions ; signals were visualized by autoradiography .
sh - sy5y cells were plated at a density of 2  10 cells / plate ( 75  cm ) . after treatment with 150  m diclofenac
, cells were harvested , washed in pbs , then resuspended in buffer m ( 5  mm hepes , ph 7.4 , 250  mm mannitol , 0.5  mm egta , 0.1% bsa ) , supplemented with protease inhibitors , and homogenized .
the homogenate was centrifuged at 800  g for 10 minutes at 4c , and the supernatant was then centrifuged at 12,000  g for 30 minutes at 4c .
the resulting pellet ( mitochondrial fraction ) was resuspended in buffer m and the final supernatant represented the cytosolic fraction .
aliquots of both fractions ( cytosolic and mitochondrial ) were used in western blotting analysis for the cytochrome c localization .
sh - sy5y cells were plated at a density of 2  10 cells / plate ( 75  cm ) and , after one - day plating , 150  m diclofenac was added to the cultures .
cells were then collected at different times as previously indicated and then centrifuged at 300  g at 4c for 5 minutes , washed once with pbs , and finally resuspended in 50  mm potassium phoshate , ph 7.8 , containing 1  mm edta .
the cellular suspension was sonicated , centrifuged at 20,000  g at 4c for 30 minutes , and after protein quantitation , the supernatant was used for an activity gel assay . in particular , aliquots of cell extracts ( 50  g ) were fractionated on a 10% polyacrylamide gel and sod activity was evaluated as previously described .
this method is based on the inhibitory effect of sod on the reduction of nitro - blue tetrazolium by the superoxide anions generated by the photochemical reduction of riboflavin ; sod is visualized as a colourless band over a blue background .
all results are presented as histograms and data are the average  sd of at least three independent measurements . in particular ,
data were analyzed by one - way anova , and differences were considered significant when the corresponding p - values were < .05 in the bonferroni 's post - hoc test .
to evaluate whether diclofenac had cytotoxic effects on neuronal cells , the human neuroblastoma cell line sh - sy5y was incubated for 24 hours with increasing concentrations   of diclofenac .
propidium iodide incorporation followed by flow cytometric analysis showed a dose - dependent increase of apoptotic nuclei with sub - diploid dna content ( figure 1(a ) ) .
the cytosolic enzyme lactate dehydrogenase ( ldh ) has been already used as a marker of cytotoxic injury .
indeed , a significant increase of ldh activity is typically found in culture media of cells undergoing a disruption of plasma membrane . to this aim
, we have assayed the ldh activity in the culture media of sh - sy5y cells incubated up to 48 hours with increasing concentrations of diclofenac .
no significant release of the intracellular ldh was observed up to 150  m diclofenac ( not shown ) , which was taken as the maximal concentration of this compound not causing a significant cytotoxic effect ( mntc ) . to analyse the time - dependent increase of apoptosis
, sh - sy5y cells were exposed to 150  m diclofenac and analysed at different times . as shown in figure 1(b ) , the apoptotic process was already evident after 8-hour treatment and progressively increased up to 72 hours .
it is known that nsaids alter the redox state of different cell types through an enhancement of intracellular ros levels [ 911 ] .
therefore , we have decided to investigate whether diclofenac affects the intracellular ros levels also in sh - sy5y cells , using the fluorescent probe dcfh - da .
indeed , when these cells were incubated with diclofenac and dcfh - da at different times , an early increase of the ros level was detected with respect to control cells ; furthermore , ros production progressively continued at least up to 4 hours ( figure 2 ) .
as sod2 is the primary antioxidant enzyme in mitochondria protecting cells from oxidative injuries , we have evaluated the role exerted by this enzyme in sh - sy5y cells in the course of diclofenac treatment . to this aim
, the dose- and time - dependent effects of this drug on sod2 protein levels of sh - sy5y cells were analysed in the experiments reported in figure 3 .
when these neuronal cells were incubated for 24 hours with increasing diclofenac concentrations , a dose - dependent decrease of the sod2 protein level was observed , as evaluated by western blotting using antibodies against human sod2 ( figure 3(a ) ) .
a similar picture emerged when sh - sy5y cells were incubated with 150  m diclofenac and analysed at different times .
the decrease of sod2 levels ( figure 3(b ) ) was evident after 24-hour treatment and increased at 48 hours . to investigate the possibility that the reduction of the sod2 protein levels corresponded also to a decrease in sod activity
, the protein extracts obtained from cells incubated with 150  m diclofenac were analysed at different times using a sod activity gel , which allows the detection of the activity of both mitochondrial sod2 and cytosolic sod1 .
interestingly , the sod2 activity decreased in a time - dependent manner ( figure 4 ) , thus indicating that the reduction of the enzyme level caused a lower efficiency in the defence against the superoxide anions .
vice versa , the activity of sod1 was not affected by the diclofenac treatment ( not shown ) .
we also evaluated whether the diclofenac - induced decrease of sod2 levels / activity depends on a down - regulation of the corresponding mrna . to this aim
, sh - sy5y cells were incubated in the presence or in the absence of 150  m diclofenac for 4 , 8 , and 24 hours . in order to evaluate a possible regulation of the enzyme at transcriptional level , after rna extraction
, a cdna was obtained by reverse transcriptase and used as a template in a pcr realised in the presence of specific oligonucleotides for sod2 .
the data presented in figure 5 indicate that the diclofenac treatment did not significantly alter the mrna levels of sod2 at all times investigated .
a similar picture with proportionally lower signals emerged when dilutions of the cdna template were used in the rt - pcr assay ( not shown ) .
these results , together with the low number of amplification cycles used , confirm that the employed pcr conditions fell in the linear dose - response range .
thioredoxin ( trx ) , a small ( 12  kda ) and ubiquitous protein involved in many cellular functions , is a potent disulphide oxidoreductase controlling the reduced state of intracellular proteins .
the crucial antioxidant power of trx also takes advantage of its ability to cross the cellular membrane ; in fact , evidences have been reported that trx can be released from cells [ 2628 ] and even enter a living cell [ 29 , 30 ] . moreover ,
endogenously produced or exogenously added trx increases the sod2 expression in lung carcinoma   or neuronal cells . to this aim , the effect of trxa2 from the hyperthermophilic archaeon sulfolobus solfataricus ( sstrx ) on the reduction of sod2 levels caused by diclofenac was evaluated ( figure 6 ) . when sh - sy5y cells were treated with 150  m diclofenac , the sod2 protein level was significantly reduced .
the addition of increasing concentrations of sstrx allowed a restoration of the sod2 level , thus counteracting the effect of diclofenac .
in particular , in the presence of 10  m sstrx , the amount of sod2 was even higher compared to untreated cells . in a control experiment ,
the addition of 10  m sstrx did not cause any significant variation of the sod2 level in the absence of diclofenac ( not shown ) .
the possible protective effect of sstrx on the diclofenac - induced apoptosis was also evaluated . as shown in figure 7 , the addition of sstrx partially reverted the programmed cell death provoked by the drug .
in particular , in the presence of 10  m sstrx , the apoptosis of the diclofenac - treated cells was reduced at all times investigated .
loss of the membrane mitochondrial potential occurs as an early event during the apoptosis induced by specific stimuli in some cellular systems . to better evaluate the molecular mechanisms underlying the diclofenac - induced apoptosis in sh - sy5y cells , the efficiency of the mitochondrial function was evaluated through measurements of its membrane potential , using the fluorescent probe r123 .
it is known that this compound crosses the mitochondrial membrane and accumulates into the matrix , only when the transmembrane potential is preserved ; therefore , in case of a loss of the membrane potential , the r123 fluorescence undergoes a significant reduction . as shown in figure 8 , the mitochondrial incorporation of r123 in sh - sy5y cells treated with 150  m diclofenac underwent a significant reduction compared to that measured on untreated cells .
this hypopolarization of the mitochondrial membrane caused by diclofenac became evident after 2-hour treatment and significantly increased with the incubation time .
a deeper insight on the involvement of mitochondria in the diclofenac - induced apoptosis was realised with a western blotting analysis aimed at the evaluation of the cytochrome c release from mitochondria .
in particular , the detection of cytochrome c was carried out on both cytosolic and mitochondrial protein fractions from sh - sy5y cells incubated in the absence or in the presence of 150  m of diclofenac at different times ( figure 9 ) .
interestingly , the cytochrome c was already present after a 8-hour incubation mainly in the cytosolic fraction of diclofenac - treated cells ; moreover , the amount of this mitochondrial marker in the cytosol significantly increased after a 24-hour treatment ( figure 9(a ) ) .
this behaviour was confirmed by the concomitant analysis on the mitochondrial fraction , where a reduction of the cytochrome c level was observed in the diclofenac - treated cells ( figure 9(b ) ) .
these data indicate that cytochrome c is released from the mitochondria , as a consequence of the diclofenac treatment of sh - sy5y cells .
in this paper we describe the effects of diclofenac on cultures of the neuroblastoma cell line sh - sy5y , thus extending to this neuronal cell our knowledge on the possible toxicity of this drug , already reported for gastric , hepatic , or renal cells .
indeed , we present evidence that diclofenac induces apoptosis through a modulation of the mitochondrial function , associated to an alteration of the redox homeostasis . it is known that apoptosis is the primary cell death induced by drugs   and that this process is mainly mediated by the mitochondrial pathway [ 36 , 37 ] . here
we show that diclofenac induces apoptosis of sh - sy5y cells in a time- and concentration - dependent manner , thus suggesting that this compound is somehow toxic even for a neuronal - type cell .
previous pharmacokinetic studies demonstrated that the blood level of diclofenac , on the basis of the therapeutic doses used in patients ( 50150 mg / day ) , ranges between 10 and 30  m .
however , in some circumstances the local concentration of the drug might increase . in particular
, diclofenac concentration increases as a consequence of long - term treatments , overdosing , limited clearance , and so forth , as previously reported [ 3942 ] . for these reasons , in our in vitro studies we have used a diclofenac concentration of 150  m , because this value corresponded to the maximal concentration of this compound not causing cytotoxicity , as determined by cytotoxicity assays on sh - sy5y cells . in the light of the large therapeutic usage of diclofenac , these data are more relevant because of the known ability of this drug to cross the blood - brain barrier .
apoptosis is regulated by many signals and metabolic events , and the cellular redox state plays a critical role in this process .
indeed , several data showed that ros , mainly produced by mitochondria , are involved in the programmed cell death through an induction of the oxidative stress .
in particular , an increased ros level frequently represents a triggering event upstream of the mitochondrial membrane depolarization , cytochrome c release , caspase activation , and nuclear fragmentation .
an alteration of the ros levels was already demonstrated in diclofenac - induced apoptosis in gastric and renal cells [ 9 , 10 ] .
also in our neuronal cell system diclofenac provokes an early and significant increase of the intracellular ros levels .
these results prompted us to evaluate the downstream effects of altered ros levels in the course of diclofenac treatment of sh - sy5y cells . under this concern
, sod2 represents the major antioxidant enzyme in mitochondria , where the intense cellular respiration may produce a large amount of ros .
therefore , an efficient sod2 activity is required to counteract the mitochondrial dysfunction induced by an oxidative stress , which could lead to the programmed cell death usually observed in various disease contexts
. the antiapoptotic role of sod2 is even demonstrated by the resistance to the radiation - induced damage reported for cell lines overexpressing sod2 .
our data on sh - sy5y cells show that diclofenac impairs sod2 functions , thus suggesting that this enzyme is involved in the apoptotic mechanism induced by the drug . in particular , this process is associated to a significant and concomitant reduction of both protein level and enzymatic activity of sod2 , whereas rt - pcr experiments showed that the corresponding mrna levels are not affected by the diclofenac treatment .
therefore , a transcriptional regulation of the sod2 gene by diclofenac could be excluded ; probably , an increased degradation of the protein levels could explain the reduced activity of the enzyme .
the behaviour of sod2 in the diclofenac response was compared with the effects reported for other compounds in various experimental systems .
for instance , stautosporine , a protein kinase inhibitor , did not affect the mrna levels of sod1 and sod2 , but decreased protein and activity levels of both enzymes .
other authors reported that sod2 is degraded by caspases in jurkat t cells , following oligomerization of the fas receptor . on the other hand , in rat
astrocytes the lipopolysaccharide induced an increase of sod2 mrna , but not of the sod2 protein .
these observations confirm the regulation of sod2 functions following a drug treatment ; however , the differences observed among the various experimental systems indicate that the sod2 response is a multifactorial process only poorly clarified .
it is known that the induction of sod2 can be mediated by various macromolecules , such as interleukyn-1 , lipopolysaccarides , and tumor necrosis factor- ; furthermore , thiol - reducing agents can affect sod2 biosynthesis , as demonstrated by the enhanced sod2 expression caused by thioredoxin , a potent disulfide oxidoreductase .
in this paper , the addition of a heterologous thioredoxin to diclofenac - treated cultures of sh - sy5y led to an enhancement of the sod2 levels , as well as to a reduction of the apoptosis .
this result confirms the involvement of sod2 in the apoptotic processes induced by the drug and suggests that an archaeal thioredoxin is active also in human cells .
furthermore , we can speculate on a possible functional interaction between heterologous components of the thioredoxin system , because purified archaeal sstrx was added in its oxidised inactive form ; its conversion to the reduced active form involves a reaction putatively catalysed by the human thioredoxin reductase .
the increased ros level , the low functionality of the mitochondrial antioxidant enzyme sod2 , together with a partial recovery of sod2 properties by a heterelogous thioredoxin , strongly suggest the involvement of mitochondria in the diclofenac - induced apoptosis of sh - sy5y cells .
indeed , an excessive production of ros and a decrease in sod2 levels contribute to the mitochondrial dysfunction .
in particular , after a significant loss of the mithocondrial membrane potential , apoptosis - inducing factors are released from the mitochondria , thus leading to the activation of the caspase cascade , and ultimately to nuclear condensation [ 44 , 45 , 53 , 54 ] .
also in our system , the diclofenac treatment induces an early mitochondrial hypopolarization , correlated to an increase of the intracellular ros levels , both events representing typical features of the onset of mitochondrial apoptosis .
another crucial marker of the intrinsic apoptosis is the cytocrome c release from the mitochondria , as a consequence of membrane depolarization .
our data showed that cytochrome c translocates from the mitochondria to the cytosol during the diclofenac treatment of sh - sy5y cells .
this finding , together with the mitochondrial membrane depolarization , provides a more direct link between mitochondria and diclofenac - induced apoptosis , thus confirming that the programmed cell death in sh - sy5y follows the mitochondrial pathway .
the results obtained in this work could be relevant for a deeper insight on the therapeutic usage of diclofenac , pointing to the oxidative damage related to its cytotoxic effect .
in particular , the involvement of the main antioxidant mitochondrial enzyme in the apoptotic process may suggest the use of sod2 small interfering rna in combination with diclofenac , in order to improve the treatment of cancer , such as neuroblastoma . on the other hand , diclofenac ,
because of its ability to alter the cellular redox state of neuronal cells , could be considered a neurotoxic compound . under this concern , previous studies showed that diclofenac and indomethacin , another nsaid , enhance the effects of some neurotoxins on pc12 cells   and that diclofenac inhibits the proliferation and differentiation of neuronal stem cells .
our results on the protective effects of sstrx open some perspectives on the possible counteraction of the side effects caused by diclofenac .
 diclofenac , a nonsteroidal anti - inflammatory drug , induces apoptosis on the neuroblastoma cell line sh - sy5y through a mitochondrial dysfunction , affecting some antioxidant mechanisms . 
 indeed , the time- and dose - dependent increase of apoptosis is associated to an early enhancement of the reactive oxygen species ( ros ) . 
 mitochondrial superoxide dismutase ( sod2 ) plays a crucial role in the defence against ros , thus protecting against several apoptotic stimuli . 
 diclofenac decreased the protein levels and the enzymatic activity of sod2 , without any significant impairment of the corresponding mrna levels in the sh - sy5y extracts . 
 when cells were incubated with an archaeal exogenous thioredoxin , an attenuation of the diclofenac - induced apoptosis was observed , together with an increase of sod2 protein levels . 
 furthermore , diclofenac impaired the mitochondrial membrane potential , leading to a release of cytochrome c. these data suggest that mitochondria are involved in the diclofenac - induced apoptosis of sh - sy5y cells and point to a possible role of sod2 in this process . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion