glioblastoma multiforme ( gbm ) is one of the most common primary malignant brain tumors in adults . surgical excision combined with radiotherapy and chemotherapy is the choice of treatment ; however , the overall prognosis remains poor , and the median survival rate of gbm patients is less than 18 months [ 3 - 5 ] . currently , gbm is treated with surgical resection with concurrent radiotherapy and daily temozolomide ( tmz , 75 mg / m ) . tmz is an oral agent used for the treatment of gbm and melanoma ; however , its therapeutic action depends on its ability to alkylate / methylate dna . because this methylation induces dna damage and triggers the death of tumor cells , overexpression of a dna repair enzyme known as o-6-methylguanine - dna methyltransferase ( mgmt ) can diminish the therapeutic efficacy of tmz . other mechanisms including deregulation of cellular enzymes and membrane transporting proteins , genomic aberrations , and altered susceptibility to apoptosis may also be responsible for the higher incidence of chemo - resistance in gbm patients . mitogen - activated protein kinase phosphatases ( mkps ) belong to the family of dual - specificity protein phosphatases , which dephosphorylate both tyrosine and serine / threonine residues of mitogen - activated protein kinases ( mapks ) . mkps are subdivided into three distinct groups based on gene structure , sequence similarity , substrate specificity , and subcellular localization . mkp-1 is localized to the nucleus , and has a high specificity to mapks including p38 mapk and c - jun n - terminal kinase ( jnk ) , but a relatively low specificity to extracellular signal - regulated kinase . mkp-1 is induced by various chemotherapeutics and has been shown to inhibit drug - induced jnk activation and subsequent apoptotic cell death in breast cancers . reduction of mkp-1 expression levels using small interfering rna ( sirna ) increases chemosensitivity and jnk activity in lung and ovarian cancers . in this study , we investigated whether highly expressed mkp-1 in human gbm cells is responsible for the chemo - resistance observed in these cells . rabbit polyclonal antibodies against jnk , phospho - jnk , caspase-3 , and cleaved caspase-3 were purchased from cell signaling technologies ( beverly , ma ) . cisplatin , cyclophosphoamide , doxorubicin , epirubicin , etoposide , 5-fluorouracil , gemcitabine , irinotecan , mitomycin c , and vincristine were purchased from sigma - aldrich ( st . louis , mo ) . rabbit polyclonal anti - human mkp-1 and mkp-2 antibody were purchased from santa cruz biotechnology ( santa cruz , ca ) . jnk inhibitor ( sp600125 ) was purchased from calbiochem ( la jolla , ca ) . human gbm cell lines ( u251-mg and ln215-mg ) were maintained at 37 under an atmosphere of 5% co2 and 95% air in dulbecco 's modified eagle 's medium ( welgene , seoul , korea ) supplemented with 10% fetal bovine serum ( gibco , gaithersburg , md ) , 100 u / ml penicillin , 100 g / ml streptomycin , and minimum essential medium ( mem ) nonessential amino acids ( invitrogen , carlsbad , ca ) . u373-mg and crt - mg were maintained at 37 under an atmosphere of 5% co2 and 95% air in rpmi 1640 medium ( welgene ) supplemented with 10% fetal bovine serum ( gibco ) , 100 u / ml penicillin , 100 g / ml streptomycin , and mem nonessential amino acids ( invitrogen ) . sirna for mkp-1 ( 5'-cucagugugugauccgguu-3 ' ) and a random sequence ( control , 5'-ccuacgccaauuucgu-3 ' ) sirna were synthesized by bioneer ( daejeon , korea ) and used for transfection , which was performed using lipofectamine 2000 ( invitrogen ) according to the manufacturer 's protocols . for transient transfection , cells were plated at 510 cells per well in six - well plates or 610 cells per well in 60 mm dishes . the next day , cells were transfected with mkp-1 sirna oligonucleotides or control sirna using lipofectamine 2000 ( invitrogen ) . after 48 hours of transfection , cells were treated in the presence or absence of various anti - cancer drugs . transfection efficiency was then assessed using both quantitative real - time polymerase chain reaction ( qrt - pcr ) and western blotting of mkp-1 . total rna was isolated using an rneasy mini kit ( qiagen , hilden , germany ) . complementary dna ( cdna ) synthesis was performed with the superscript first - strand synthesis system ( invitrogen ) according to the manufacturer 's instructions , and qrt - pcr was performed using a lightcycler faststart dna kit ( roche applied science , mannheim , germany ) with the lightcycler 2.0 system . the mkp-1 primer sequences were 5'-cctgtccactccacgaacagt-3 ' for the forward primer and 5'-gctgggagaggtcgtaatgg-3 ' for the reverse primer . cell viability was determined using wst-1 reagent ( nalgene , rochester , ny ) with a microplate reader ( bio - rad , richmond , ca ) to measure the absorbance at 450 nm . cell death was confirmed by staining with 100 nmol / l tetramethylrhodamine ethyl ester ( sigma , taufkirchen , germany ) . a total of 10,000 cells were analyzed using an lsrii flow cytometer ( bd bioscience , san jose , ca ) . cell lysates were electrophoresed through 10% or 15% polyacrylamide gels and then transferred to polyvinylidene difluoride membranes ( millipore , bedford , ma ) . the membranes were subsequently probed with the antibodies listed above , after which bound antibody was detected using the eclplus reagent ( amersham pharmacia biotech , little chalfont , uk ) according to the manufacturer 's protocols . a microarray dataset was obtained from gene expression omnibus ( geo ) series ( gse ) , and gse i d gse2223 ; probes 5382 , 40027 , and 40883 were used for mkp-1 . because the probe intensities were recorded on a log2 scale , the intensities were converted to a log10 scale [ 2^(probe intensity recorded in the dataset ) ] , and averaged to give the final mkp-1 expression level . statistical analyses of two independent samples were performed using a student 's t - test . groups were compared by one - way anova with tukey 's post - hoc test , which determines the significance of differences between means . a p < 0.05 was considered to be significant . rabbit polyclonal antibodies against jnk , phospho - jnk , caspase-3 , and cleaved caspase-3 were purchased from cell signaling technologies ( beverly , ma ) . cisplatin , cyclophosphoamide , doxorubicin , epirubicin , etoposide , 5-fluorouracil , gemcitabine , irinotecan , mitomycin c , and vincristine were purchased from sigma - aldrich ( st . louis , mo ) . rabbit polyclonal anti - human mkp-1 and mkp-2 antibody were purchased from santa cruz biotechnology ( santa cruz , ca ) . jnk inhibitor ( sp600125 ) was purchased from calbiochem ( la jolla , ca ) . human gbm cell lines ( u251-mg and ln215-mg ) were maintained at 37 under an atmosphere of 5% co2 and 95% air in dulbecco 's modified eagle 's medium ( welgene , seoul , korea ) supplemented with 10% fetal bovine serum ( gibco , gaithersburg , md ) , 100 u / ml penicillin , 100 g / ml streptomycin , and minimum essential medium ( mem ) nonessential amino acids ( invitrogen , carlsbad , ca ) . u373-mg and crt - mg were maintained at 37 under an atmosphere of 5% co2 and 95% air in rpmi 1640 medium ( welgene ) supplemented with 10% fetal bovine serum ( gibco ) , 100 u / ml penicillin , 100 g / ml streptomycin , and mem nonessential amino acids ( invitrogen ) . sirna for mkp-1 ( 5'-cucagugugugauccgguu-3 ' ) and a random sequence ( control , 5'-ccuacgccaauuucgu-3 ' ) sirna were synthesized by bioneer ( daejeon , korea ) and used for transfection , which was performed using lipofectamine 2000 ( invitrogen ) according to the manufacturer 's protocols . for transient transfection , cells were plated at 510 cells per well in six - well plates or 610 cells per well in 60 mm dishes . the next day , cells were transfected with mkp-1 sirna oligonucleotides or control sirna using lipofectamine 2000 ( invitrogen ) . after 48 hours of transfection , cells were treated in the presence or absence of various anti - cancer drugs . transfection efficiency was then assessed using both quantitative real - time polymerase chain reaction ( qrt - pcr ) and western blotting of mkp-1 . total rna was isolated using an rneasy mini kit ( qiagen , hilden , germany ) . complementary dna ( cdna ) synthesis was performed with the superscript first - strand synthesis system ( invitrogen ) according to the manufacturer 's instructions , and qrt - pcr was performed using a lightcycler faststart dna kit ( roche applied science , mannheim , germany ) with the lightcycler 2.0 system . the mkp-1 primer sequences were 5'-cctgtccactccacgaacagt-3 ' for the forward primer and 5'-gctgggagaggtcgtaatgg-3 ' for the reverse primer . cell viability was determined using wst-1 reagent ( nalgene , rochester , ny ) with a microplate reader ( bio - rad , richmond , ca ) to measure the absorbance at 450 nm . cell death was confirmed by staining with 100 nmol / l tetramethylrhodamine ethyl ester ( sigma , taufkirchen , germany ) . a total of 10,000 cells were analyzed using an lsrii flow cytometer ( bd bioscience , san jose , ca ) . cell lysates were electrophoresed through 10% or 15% polyacrylamide gels and then transferred to polyvinylidene difluoride membranes ( millipore , bedford , ma ) . the membranes were subsequently probed with the antibodies listed above , after which bound antibody was detected using the eclplus reagent ( amersham pharmacia biotech , little chalfont , uk ) according to the manufacturer 's protocols . a microarray dataset was obtained from gene expression omnibus ( geo ) series ( gse ) , and gse i d gse2223 ; probes 5382 , 40027 , and 40883 were used for mkp-1 . because the probe intensities were recorded on a log2 scale , the intensities were converted to a log10 scale [ 2^(probe intensity recorded in the dataset ) ] , and averaged to give the final mkp-1 expression level . statistical analyses of two independent samples were performed using a student 's t - test . groups were compared by one - way anova with tukey 's post - hoc test , which determines the significance of differences between means . a p < 0.05 was considered to be significant . analysis of the cdna microarray data provided by the geo databases ( gse i d gse2223 ) revealed that the mkp-1 mrna expression levels were higher in brain tumor samples than normal samples , and that the amount of the increase was correlated with the grade of the tumor . specifically , mkp-1 levels were only higher in gbm samples ( grade iv ) when compared to normal brain samples ( fig . we tested whether mkp-1 was also highly expressed in human gbm cell lines using immunoblot analysis ( fig . when compared to other cell types and human primary cultured astrocytes , mkp-1 was highly expressed in human gbm cell lines in the absence of any external stimulation . because mkp-2 has a similar intracellular localization and substrate specificity as mkp-1 , we examined the mkp-2 protein levels in the same cell type . interestingly , there were no differences in the expression levels of mkp-2 between various tumor cell types . however , in two of the cell lines ( crt - mg and hepg2 ) , mkp-2 was expressed at relatively higher levels than in the other cell types ( fig . induced expression of mkp-1 has been identified as one of the cancer resistance mechanisms in human breast , lung , and ovarian cancers . therefore , we tested whether highly expressed mkp-1 plays a role in the high incidence of chemo - resistance in gbms . to accomplish this , cells were transiently transfected with control or mkp-1-specific sirna and then tested for drug - induced cytotoxicity . the knockdown efficiency of mkp-1 sirna was confirmed by qrt - pcr and western blot analysis ( fig . the reduction of mkp-1 expression significantly enhanced drug - induced cytotoxicity in u251-mg cells for all of the drugs tested except doxorubicin , epirubicin , and cisplatin ( fig . although the differences were not statistically significant , there were considerable increases in the cytotoxicity of these drugs against cells transfected with mkp-1 sirna . similar results were obtained using ln215 cells ; however , mkp-1 knockdown did not significantly increase vincristine - induced cell death ( data not shown ) . because the effect of mkp-1 knockdown was most pronounced with irinotecan- and etoposide - induced cytotoxicity , we further examined the effect of mkp-1 sirna transfection on drug - induced cell death under these conditions . twenty four hours after transfection with control or mkp-1 sirnas , cells were incubated in the presence or absence of varying doses of irinotecan or etoposide ( fig . these results clearly indicate that mkp-1 may act as a mechanism of chemo - resistance in human gbm cells . anti - cancer drugs are thought to kill cancer cells via sustained activation of the jnk pathway . thus , we tested whether the jnk pathway is also involved in mkp-1-mediated resistance to chemotherapeutic agents . treatment with irinotecan or etoposide increased the phosphorylation of jnk , and this increase was dramatically enhanced by transient transfection with mkp-1 sirna ( fig . 4a ) . cleavage of caspase-3 into active fragments was also induced by treatment with irinotecan or etoposide , while mkp-1 knockdown markedly increased the cleavage of procaspase-3 . consistent with these results , the decrease in viability induced by concomitant treatment with irinotecan and mkp-1 sirna was reversed by pre - incubation with a jnk inhibitor , sp600125 ( fig . 4b and c ) . analysis of the cdna microarray data provided by the geo databases ( gse i d gse2223 ) revealed that the mkp-1 mrna expression levels were higher in brain tumor samples than normal samples , and that the amount of the increase was correlated with the grade of the tumor . specifically , mkp-1 levels were only higher in gbm samples ( grade iv ) when compared to normal brain samples ( fig . we tested whether mkp-1 was also highly expressed in human gbm cell lines using immunoblot analysis ( fig . when compared to other cell types and human primary cultured astrocytes , mkp-1 was highly expressed in human gbm cell lines in the absence of any external stimulation . because mkp-2 has a similar intracellular localization and substrate specificity as mkp-1 , we examined the mkp-2 protein levels in the same cell type . interestingly , there were no differences in the expression levels of mkp-2 between various tumor cell types . however , in two of the cell lines ( crt - mg and hepg2 ) , mkp-2 was expressed at relatively higher levels than in the other cell types ( fig . induced expression of mkp-1 has been identified as one of the cancer resistance mechanisms in human breast , lung , and ovarian cancers . therefore , we tested whether highly expressed mkp-1 plays a role in the high incidence of chemo - resistance in gbms . to accomplish this , cells were transiently transfected with control or mkp-1-specific sirna and then tested for drug - induced cytotoxicity . the knockdown efficiency of mkp-1 sirna was confirmed by qrt - pcr and western blot analysis ( fig . the reduction of mkp-1 expression significantly enhanced drug - induced cytotoxicity in u251-mg cells for all of the drugs tested except doxorubicin , epirubicin , and cisplatin ( fig . 2c ) . although the differences were not statistically significant , there were considerable increases in the cytotoxicity of these drugs against cells transfected with mkp-1 sirna . similar results were obtained using ln215 cells ; however , mkp-1 knockdown did not significantly increase vincristine - induced cell death ( data not shown ) . because the effect of mkp-1 knockdown was most pronounced with irinotecan- and etoposide - induced cytotoxicity , we further examined the effect of mkp-1 sirna transfection on drug - induced cell death under these conditions . twenty four hours after transfection with control or mkp-1 sirnas , cells were incubated in the presence or absence of varying doses of irinotecan or etoposide ( fig . these results clearly indicate that mkp-1 may act as a mechanism of chemo - resistance in human gbm cells . anti - cancer drugs are thought to kill cancer cells via sustained activation of the jnk pathway . thus , we tested whether the jnk pathway is also involved in mkp-1-mediated resistance to chemotherapeutic agents . treatment with irinotecan or etoposide increased the phosphorylation of jnk , and this increase was dramatically enhanced by transient transfection with mkp-1 sirna ( fig . 4a ) . cleavage of caspase-3 into active fragments was also induced by treatment with irinotecan or etoposide , while mkp-1 knockdown markedly increased the cleavage of procaspase-3 . consistent with these results , the decrease in viability induced by concomitant treatment with irinotecan and mkp-1 sirna was reversed by pre - incubation with a jnk inhibitor , sp600125 ( fig . 4b and c ) . we found that mkp-1 is highly expressed in human gbms and transformed gbm cell lines in the absence of external stimulation . we also found that high levels of mkp-1 are responsible for drug - resistance in a jnk - dependent manner . it has been reported that mkp-1 can be induced by various chemotherapeutic agents and can act as an anti - apoptotic mechanism in certain types of human malignancies . in contrast , our results clearly indicate that mkp-1 is constitutively expressed at relatively high levels in human gbm cells and confers resistance to a variety of chemotherapeutic agents in a jnk - dependent manner . due to diverse stimulations triggered by the tumor microenvironment , expression patterns of mkp-1 vary according to tumor types . in human breast cancer , the expression level of mkp-1 is constitutive in tissue surrounding the tumor and greater in tumor lesions . low - grade human colon cancer showed robust mkp-1 expression , but mkp-1 was down regulated in high - grade tumors . as shown in fig . 1 , mkp-1 expression is higher in glioblastoma cell lines than in human primary astrocytes . these results imply that mkp-1 is highly correlated with tumor progression and contributes to subsequent drug resistance in glioblastoma . while multiple mechanisms for multi - drug resistance ( mdr ) have been proposed , there are only a few considered to be primarily responsible for mdr in many human malignancies . first , cancer cells decrease the uptake of water - soluble drugs by downregulating specific transporters . second , mdr can arise from mechanisms involving the increased activity of dna damage repair enzymes , reduced apoptosis , and changes in the cell cycle and drug metabolism . third , the induction of energy - dependent efflux of hydrophobic drugs can cause mdr via increased diffusion through the plasma membrane . unlike previous studies of mdr that address drug acceptability , we investigated the cell signaling associated with mdr . because jnk and p38 mapk are largely responsible for drug - induced apoptosis and are effective downstream targets of mkp-1 , we considered mkp-1 as a potential mechanism for mdr . we observed increased drug sensitivity after mkp-1 knockdown in human gbm cells , found the effect of this knockdown to be most pronounced in irinotecan- and etoposide - induced cytotoxicity . irinotecan , in combination with bevacizumab was tested in a phase 2 clinical study for recurrent glioblastoma . although combination therapy provides impressive response rates , the mechanisms responsible for the clinical benefits in gbm patients remain unclear . etoposide is also being studied in a phase 2 clinical trial and is commonly used in combination with carboplatin in recurrent gbm patients . mkp-1 is highly expressed in the glioblastoma in the basal state , but not in normal brain cells . overexpressed mkp-1 increases the resistance to anti - cancer drugs through inhibition of jnk phosphorylation . therefore , mkp-1 is an attractive target for overcoming drug resistance in this highly refractory malignancy .
purposecurrent treatment of glioblastoma after surgery consists of a combination of fractionated radiotherapy and temozolomide . 
 however , it is difficult to completely remove glioblastoma because it has uncertain boundaries with surrounding tissues . 
 moreover , combination therapy is not always successful because glioblastoma has diverse resistances . to overcome these limitations 
 , we examined the combined effects of chemotherapy and knockdown of mitogen - activated protein kinase phosphatase-1 ( mkp-1).materials and methodswe used ten different anti - cancer drugs ( cisplatin , cyclophosphoamide , doxorubicin , epirubicin , etoposide , 5-fluorouracil , gemcitabine , irinotecan , mitomycin c , and vincristine ) to treat glioblastoma multiforme ( gbm ) cells . 
 knockdown of mkp-1 was performed using sirna and lipofectamine . 
 the basal level of mkp-1 in gbm was analyzed based on cdna microarray data obtained from the gene expression omnibus ( geo ) databases.resultsanti-cancer drug - induced cell death was significantly enhanced by knockdown of mkp-1 , and this effect was most prominent in cells treated with irinotecan and etoposide . 
 treatment with these two drugs led to significantly increased phosphorylation of c - jun n - terminal kinase ( jnk ) in a time - dependent manner , while pharmacological inhibition of jnk partially inhibited drug - induced cell death . 
 knockdown of mkp-1 also enhanced drug - induced phosphorylation of jnk.conclusionincreased mkp-1 expression levels could be the cause of the high resistance to conventional chemotherapeutics in human gbm . 
 therefore , mkp-1 is an attractive target for overcoming drug resistance in this highly refractory malignancy .