glutathione s - transferase pi ( gstp ) is a biomarker protein in drug - resistant solid epithelial tumors , including ovarian , breast , liver , pancreatic , lung , and lymphoma . in some cases , gstp can be the most abundant protein in the tumor and , consequently , has the potential to serve as an important drug target [ 24 ] . one therapeutic approach has been to develop prodrugs that are substrates for gstp and become cytotoxic when liberated in cancer cells , yet exhibit diminished activation / toxicity in normal tissue . paba / no ( o-[2,4-dinitro-5-[4-(n - methylamino ) benzoyloxy]phenyl ] 1-(n , n - dimethylamino ) diazen-1-ium-1,2-diolate )   is a gstp - activated prodrug that releases high levels of nitric oxide ( no ) at physiological ph . this reaction results in the formation of a meisenheimer - complex intermediate and subsequently the leaving group of the reaction generates two molecules of no . elevated no levels lead to cytotoxic effects by forming rns / ros intermediates that can alter protein function directly through posttranslational modifications on redox sensitive cysteine residues ( s - nitrosylation , p - sno or s - glutathionylation , p - ssg ) [ 3 , 6 ] . prior studies have shown that protein disulfide isomerase ( pdi ) is a molecular target of paba / no treatment in cancer cells [ 2 , 5 , 79 ] . pdi is the most abundant chaperone / isomerase in the endoplasmic reticulum and plays a pivotal role in protein folding through isomerase and chaperone activity . the active site cysteine residues are s - glutathionylated ( pdi - ssg ) following paba / no treatment . the functional consequences are reduced isomerase activity , accumulation of unfolded / misfolded proteins , and activation of the unfolded protein response ( upr ) . at least one mechanism of action for paba / no can be attributed to upr - induced cell death . pdi shares homology with the estrogen - binding domain of the estrogen receptor alpha ( er ) . despite the kdel sequence present in the c - terminus that retains pdi in the endoplasmic reticulum ( er ) , pdi is also found in non - er locations . with immune - affinity , steroid - affinity and site - specific dna - affinity chromatography , landel et al . have found that in extracts from mcf-7 cells and er-expressing cho ( cho - er ) cells er forms complexes with pdi , hsp70 , and two unidentified proteins . moreover , pdi enhances the binding of purified er to the estrogen response element ( ere ) in a dose - dependent manner , suggesting that this accessory protein may be required for optimal interactions of er with eres [ 12 , 13 ] . the dna - binding domain and hinge domain of er are the minimal structures sufficient for pdi - er interactions . however , information regarding the corresponding domain(s ) of pdi involved in this interaction is not presently available . the presence of estrogen neither is required for pdi - er interaction   nor has any effect on the stoichiometry of pdi association with er . the pdi - er interaction may protect er from oxidation and alter er conformation so that the susceptibility of er to different proteases is altered . the present study evaluates the efficacy of paba / no in er-positive breast cancer cells and interrogates the impact of s - glutathionylation of pdi on its chaperone activity , specifically with er. we hypothesize that s - glutathionylation of pdi not only blunts protein folding but also prevents protein interaction with er , destabilizing the receptor and subsequent er signaling ( see scheme in figure 1 ) . reduced glutathione ( gsh ) , oxidized glutathione ( gssg ) , and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide ( mtt ) reagent , and -estradiol were purchased from sigma ( st . antibodies were purchased from the following sources : anti - pdi ( affinity bioreagents ) ; anti - glutathionylation ( virogen ) , anti - actin ( abcam ) , anti - flag ( sigma ) , anti - er/anti - er ( millipore ) , and anti - c - myc / anti - cyclin d1/anti - p21 ( cell signaling ) . paba / no ( o-[2,4-dinitro-5-[4-(n - methylamino ) benzoyloxy ] phenyl]1-(n , n - dimethylamino ) diazen-1-ium-1,2-diolate ) was provided by dr . larry keefer , chemistry section , laboratory of comparative carcinogenesis , nci at frederick ( md ) . his - tagged recombinant human pdi in the bacterial expression vector pet-28b was provided by dr . lana lee ( university of windsor , ontario ) and expressed as previously described [ 8 , 9 ] . the his - tagged pdi mutant flfl was synthesized using the quikchange site - directed mutagenesis kit ( stratagene ) , in which histidine 55 and 399 were changed to phenylalanines , and lysine 57 and 401 were changed to leucines , simultaneously . purified plasmids were transformed into escherichia coli bl21(de3)plyss strain , which were then induced with 1  mm iptg at 37c for 3  h and pelleted at 6000  rpm for 15  min at 4c . cells were lysed by sonication in lysis buffer ( 20  mm nah2po4 , 500  mm nacl ) . the lysates were centrifuged at 13,000  rpm for 30  min at 4c and the pellets were discarded . the supernatants were incubated with ni - nta agarose beads ( qiagen ) for 1  h at 4c . the beads were washed three times with wash buffer ( 20  mm nah2po4 , 500  mm nacl , and 10  mm imidazole ) . his - pdis were eluted with elution buffer ( 20  mm nah2po4 , 500  mm nacl , and 250  mm imidazole ) . the eluates were dialyzed against dialysis buffer ( 25  mm hepes , ph7.3 , 100  mm nacl , 1  mm edta , and 1  mm dtt ) . full - length human pdi was amplified from a cdna prepared from hl60 cells using the following primers : 5-gacggatccatgctgcgccgcgctctgctg-3and 5-gtcgaattcttacagttcatctttcacagc-3. the resulting pcr fragment was cloned into the bamhi / ecori sites of pcdna3 vector for mammalian expression . a flag tag sequence dykddddk was inserted between amino acids e497 and d498 , upstream of the kdel sequence . the human breast cancer line mcf7 was purchased from the american type culture collection and grown as recommended by the supplier . cells were maintained in mem containing 10% fcs , 100  g / ml streptomycin , 100  units / ml penicillin , and 2  mmol / l l - glutamine at 37c in a 5% co2 incubator . expression plasmids containing flag - tagged pdi and flfl mutant were transfected into mcf7 cells using genjet reagent according the manufacturer 's instruction . cells ( 2  10 per treatment group ) were plated 24  h before drug treatment . to validate that overexpression of pdi ( pdi - oe ) does not lead to altered localization , transfected mcf7 cells ( vector or pdi ) were seeded onto labtek ii chamber slides and allowed to adhere overnight . cells were rinsed 2x with pbs and fixed with 4% paraformaldehyde for 30  min . nonspecific protein binding was blocked using 1% bovine serum albumin ( bsa ) for 30  min . cells were then incubated for 1  h in primary anti - pdi ( vector ) or anti - flag ( pdi - oe ) antibody in pbs . unbound primary antibody was removed by rinsing the cells 3x with pbs containing 0.05% tween-20 . following removal of the secondary antibody , the cells were imaged by fluorescent microscopy ( nikon eclipse e800 , nikon instr . , in . lewisville , tx using nikon ds - u1 software v. 5.03 , photometrics , tucson , az ) . cell pellets were suspended in lysis buffer ( 20  mm tris - hcl , ph 7.5 , 15  mm nacl , 1  mm edta , 1  mm egta , 1% triton x-100 , 2.5  mm sodium pyrophosphate , and 1  mm -glycerophosphate with freshly added protease and phosphatase inhibitors , 5  mm naf and 1  mm na3vo4 ) and incubated for 30  min on ice . lysates were sonicated for 10  sec and centrifuged for 30  min at 10,000  g at 4c . protein concentrations in the supernatant were assayed with the bradford reagent ( bio - rad laboratories , hercules , ca ) using igg as a standard . equivalent amounts of protein were electrophoretically resolved under nonreducing conditions on 10% sds - polyacrylamide gels ( sds - page ) ; unmodified proteins were separated under reducing conditions . proteins were transferred onto nitrocellulose membranes ( bio - rad , hercules , ca ) . nonspecific binding was reduced by incubating the membrane in blocking buffer ( 20  mm tris - hcl , ph 7.5 , 150  mm nacl , 0.1% tween 20 , 1  m protease inhibitors , 5  mm naf , and 1  mm na3vo4 ) containing 10% nonfat dried milk for 1  h. membranes were incubated with the indicated antibody ( blocking buffer containing 5% nonfat dried milk ) at stated dilutions overnight at 4c , washed 3x with pbs for 15  min , and incubated with the appropriate secondary antibody conjugated to horseradish peroxidase for 1  h. the membranes were washed 3 times and developed with enhanced chemiluminescence detection reagents ( bio - rad ) . in relation to actin . the structure of the s - glutathionylation refractory mutant ( pdi - flfl ) was evaluated and compared to the wild type ( pdi - wt ) by protein tryptophan fluorescence scanning [ 8 , 16 ] . the scan was recorded on an f 2500 spectrofluorometer ( hitachi ) using 10  10  40  mm quartz cuvette , excitation and emission slits were 2.5 and 5.0  nm , respectively . the excitation wavelength was 295  nm to minimize an effect of protein tyrosines and phenylalanines . quenching experiments were performed as follows : 1  m of pdi - wt or pdi - flfl in 20  mm pb , ph = 7.4 was titrated with 05000  nm cscl at room temperature under constant stirring . = 295  nm ) were recorded and integrated using felix32 standard software ( pti , nj ) . the enzyme activities of pdi - wt and pdi - flfl were monitored using the turbidimetric assay of insulin disulphide reduction . briefly , 0.25  m purified human pdi was added to a solution of insulin ( 0250  m ) and gsh ( 500  m ) in a 0.1  m k2hpo4 buffer containing 2  mm edta ( ph 7.0 ) . dtt has been shown to completely reduce the disulfide bonds and is used as a positive control . the increase in turbidity was monitored (  = 630 ) at 30  s intervals for 30  min . mean values and s.e . were computed for each group , n = 3 . 10,000 cells were seeded in 96-well plates in 50  l medium . increasing drug concentrations of paba / no were added to a final volume of 100  l and maintained in drug for 72  h. following drug exposure , cell viability was assayed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide ( mtt ) conversion assay . experimental data presented represent the mean value  sd for at least three independent experiments . data were analyzed for statistically significant differences between groups with student 's t - test using sigmastat 3.5 ( systat software inc . prior studies have shown that paba / no induces dose - dependent cell death that is concurrent with s - glutathionylation and activation of the upr in ovarian and leukemia cancer cells . using mcf7 cells , we confirmed the presence of a dose- ( figure 2(a ) ) and time - dependent inverse relationship of p - ssg relative to cell death ( figure 2 ) . under nonstressed conditions , pdi coimmunoprecipitates with er , figure 3(a ) . however , paba / no - induced nitrosative stress - induced s - glutathionylation of pdi abrogates the interaction within 30  min following drug treatment ( figures 3(b ) and 3(c ) ) . chaperone functions of pdi are considered to be protective of er when exposed to either oxidative or nitrosative damage . in fact , protein levels of er were diminished 81  2% following treatment with paba / no ( figure 4(a ) ) . to determine if er levels were decreased as a consequence of proteosomal degradation , mcf7 cells were pretreated with proteosomal inhibitors ( mg132 and ps341 ) prior to subsequent paba / no exposures . proteosomal inhibition blunted paba / no - induced er degradation ( figure 4(c ) ) . to validate that pdi levels are critical to er stability , mcf7 cells were transfected with empty vector or with recombinant pdi to generate overexpressing cells . figure 5(a ) shows fluorescent images confirming that the sub - cellular localization of transfected , overexpressed pdi ( right panel ) is consistent with native pdi ( left panel ) . comparative analysis of vector or pdi - transfected cells treated with paba / no ( figure 5(b ) ) suggested that overexpression of pdi stabilized intracellular er levels . previous studies have shown that estrogen stimulation can lead to activation of a range of proteins involved in cell proliferation . we evaluated a cadre of such response proteins following -estradiol  paba / no treatments ( figure 6 ) . paba / no treatment appeared to counteract the expected estrogenic effects , leading to downregulation of c - myc , cyclin d1 , and p21 . such data are consistent with the antiproliferative and growth arrest effects of paba / no . s - glutathionylation invariably occurs at cysteine residues found in a motif that exists in a low pka environment . using site - directed mutagenesis we generated an s - glutathionylation refractory mutant , transfected it into host cells ( pdi - flfl ) , and evaluated its secondary and tertiary structure compared to pdi and pdi - ssg [ 8 , 9 ] . the trypophanyl fluorescence scans of pdi - wt and pdi - flfl have overlapping spectra ( p < 0.05 ) , indicating the proteins are folded similarly ( figure 7(a ) ) . further validation that protein folding of pdi - flfl was equivalent to pdi - wt was based upon the data showing quenching of the protein trp fluorescence with cscl ( figure 7(b ) ) . there was no statistical difference between the pdi - wt protein and the s - glutathionylation refractory mutant ( p < 0.05 ) . measurement of isomerase activity showed pdi and pdi - flfl were essentially equivalent ( figure 7(c ) ) . pdi was s - glutathionylated in a dose - dependent manner , however pdi - flfl was refractory ( figure 7(d ) ) . pdi - wt and pdi - flfl were transfected into mcf7 cells and treated with paba / no to induce s - glutathionylation and activate upr - induced cell death . following immunoprecipitation , pdi - wt was s - glutathionylated as detected by western blot analysis . however , pdi - flfl was not modified ( figure 7(e ) ) . s - glutathionylation of pdi is the trigger for er-pdi complex disruption and activation of the upr . consequently , the toxic effects of paba / no were diminished in mcf7 cells transfected with the s - glutathionylation - resistant mutant , pdi - flfl ( figure 7(f ) ) . targeting er signaling is an important and successful treatment strategy for most breast cancer patients that are er positive . in the preclinical evaluation of paba / no , pdi was identified as a molecular target of the drug [ 2 , 5 , 8 , 1820 ] . specifically , pdi is s - glutathionylated on cysteine residues within the catalytic domains following nitrosative stress , thereby blunting isomerase activity [ 8 , 9 ] . pdi has homology with the estrogen - binding domain of er and acts as a molecular chaperone that is required for er-mediated gene regulation . in the present study we evaluated whether s - glutathionylation of pdi also impacts chaperone function . our data suggest that s - glutathionylation of pdi leads to disruption of the pdi : er complex , proteosomal degradation of transcription factor , and diminished er mediated gene regulation . pdi is organized into five domains ( a , b , b , a , and c ) . there are two active sites in the catalytic domains a and a , each having two conserved cysteine residues that cycle between oxidized ( disulfide ) and reduced ( dithiol ) states to facilitate the folding and correct disulfide bond formation of its protein substrates . the crystal structure of yeast pdi suggests that the a , b , b , and a domains form a twisted u shape with the catalytic domains facing each other and an internal hydrophobic surface that interacts with substrate proteins . residues 101144 of mature pdi have significant similarity ( 29.5% identity ) with residues 350392 of er. moreover , residues 163211 of mature pdi are homologous with residues 304349 of er. it is noteworthy that pdi segment 101144 comprises the c - terminal part of the a domain , a short interdomain linker , and n - terminal part of the b domain , whereas segment 163211 is exclusively located in the b domain . it would seem plausible that estrogen binding may cause a conformational change that interferes with the functional coordination between the catalytic domain and substrate - binding region , with the consequence that pdi enzymatic activity is suppressed . accordingly , the apparent kd value of pdi for estrogens such as -estradiol has been determined to be 1.52  m . the catalytic activity of pdi ( measured by insulin degradation or reactivation of randomly oxidized rnase ) is inhibited by estradiol in vitro , with an ic50 of approximately 100  nm . as an estrogen - binding protein , pdi can serve to modulate the intracellular estrogen reservoir and slow down estrogen metabolic disposition . moreover , under certain conditions , pdi - bound estrogen can be released and augment the transcriptional activity of er . s - glutathionylation of proteins generally occurs on cysteine residues with a low pka . using site - directed mutagenesis we generated a mutant pdi that is s - glutathionylation refractory with isomerase activity equivalent to native pdi . transfection of pdi - flfl into breast cancer cells resulted in diminished paba / no - induced toxicity as a result of s - glutathionylation refractory pdi . hence the triggers for abolishing the er-pdi complex and activation of the upr were blunted . rnai technology has been used to knock down pdi in mcf-7 cells but interpretation of results has not always been consistent . for example , fu et al . demonstrated that pdi knockdown is associated with downregulation of er , whereas schultz - norton et al . reported the exact opposite finding that decreased expression of pdi leads to an increase in er protein levels . both groups have used at least two different rnai sequences , thus a precise understanding of the relationship between pdi and er stability remains undefined . the results from the present study demonstrate that pdi levels do have a direct impact on er stability . moreover , changes in redox homeostasis induced by nitrosative / oxidative stress cause s - glutathionylation of pdi and mediate cell death through activation of the upr and abrogation of er stability and signaling .
s - glutathionylation of cysteine residues within target proteins is a posttranslational modification that alters structure and function . 
 we have shown that s - glutathionylation of protein disulfide isomerase ( pdi ) disrupts protein folding and leads to the activation of the unfolded protein response ( upr ) . 
 pdi is a molecular chaperone for estrogen receptor alpha ( er ) . 
 our present data show in breast cancer cells that s - glutathionylation of pdi interferes with its chaperone activity and abolishes its capacity to form a complex with er. such drug treatment also reverses estradiol - induced upregulation of c - myc , cyclind1 , and p21cip , gene products involved in cell proliferation . 
 expression of an s - glutathionylation refractory pdi mutant diminishes the toxic effects of paba / no . 
 thus , redox regulation of pdi causes its s - glutathionylation , thereby mediating cell death through activation of the upr and abrogation of er stability and signaling .