A Study On The Mechanisms Of Myeloid Cell Leukemia 1 Glutathionylation-enhanced Chemosensitivity Of Gallbladder Cancer Cells To Cisplatin

Gallbladder cancer(GBC)is highly malignant with a low response rate to chemotherapy because GBC cells are drug resistant.GBC side population(SP)cells are considered equivalent to cancer stem cells in biological behaviors and show stronger drug resistance abilities.We previously found that an increase of reactive oxygen species(ROS)could enhance the efficacy of cisplatin(CDDP)via drug resistance-associated protein downregulation.But how the high levels of ROS decreased drug resistance-associated protein expression was unclear.The recent study showed that myeloid cell leukemia 1(Mcl-1)was an anti-apoptotic protein.Glutathionylation,one kind of oxidative modifications,regulated Mcl-1 stability and made tumor cells resistant to chemotherapy.These data suggested that an increase of ROS could possibly overcome CDDP resistance via glutathionylation-dependent Mcl-1 downregulation in GBC cells.Now the relationship between Mcl-1 and CDDP resistance and the effect of glutathionylation on Mcl-1 protein in GBC cells and SP cells are unclear.Our study will investigate the role of Mcl-1 protein in CDDP resistance,the mechanisms that glutathionylation regulates Mcl-1 expression in GBC cells and SP cells and try to prove that ?-phenylethyl isothiocyanate(PEITC)can enhance the sensitivity of GBC cells to CDDP via Mcl-1 glutathionylation.In addition,recent studies revealed that glutathionylation of Signal transducer and activator of transcription 3(STAT3),a modification that may exert regulatory function in STAT3 signaling.And cell survival genes products,including Mcl-1 are regulated by STAT3.We also try to prove that STAT3 glutathionylation can inhibit Mcl-1 protein production and overcome CDDP resistance in GBC cells.The first part:A study on the role of Mcl-1 protein in CDDPresistance in GBC cellsObjective:To investigate the role of Mcl-1 protein in CDDP resistance in GBC cells and SP cells.Methods:The expression of Mcl-1,Bcl-2 and Bcl-xl in CDDP-treated GBC-SD cells was determined by western blot.The expression of Mcl-1 in GBC SP and non-SP cells was determined by western blot.The diversity of Mcl-1 expression in human GBC tissues and chronic cholecystitis tissues was assayed by immunohistochemistry.After GBC-SD cells were transfected with siRNA oligonucleotides specific to Mcl-1 and then treated with CDDP for 24hrs,apoptotic rate was monitored by flowcytometry.Results:1.CDDP increased Mcl-1 expression in GBC cells,but did not increase Bcl-2 or Bcl-xl expression.2.Mcl-1 expression was increased in GBC SP cells,compared to its expression in non-SP cells.3.Mcl-1 expression was found in 71.4%human GBC tissues.In contrast,Mcl-1 expression was found in 26.7%chronic cholecystitis tissues.4.Knocking down expression of Mcl-1 increased CDDP-induced apoptosis in GBC cells.Conclusions:GBC cells show CDDP resistance and SP cells show stronger drug resistance abilities due,in part,to the anti-apoptotic protein Mcl-1.The second part:A study on the mechanisms of PEITC-enhanced chemosensitivity of gallbladder cancer cells to cisplatinObjective:To investigate the mechanisms by which PEITC can enhance the cytotoxicity of CDDP in GBC cells,SP cells and in vivo.Methods:Human GBC GBC-SD cells,cholangiocarcinoma RBE cells and GBC SP cells were treated with CDDP,PEITC or a CDDP/PEITC combination.Then the cell viability was assayed by MTT,the apoptotic rate was monitored by flowcytometry,caspase 3 activity was measured by the kit,and Mcl-1 expression was determined by western blot.GBC-SD cells were transplanted into 28 nude mice.When the tumor size was approximately 50 mm3,the mice were sorted into four equal groups(n=7 mice per group).The tumor-bearing mice of the treatment groups were intraperitoneally administered with PEITC,CDDP or PEITC/CDDP twice a week.The control group received an equal volume of solvent control.The mice were sacrificed after 10 days.The mice body weight,tumor weight and tumor volumes were measured.Mcl-1 expression in tumor tissues was determined by western blot.Results:1.PEITC could enhance the cytotoxicity of CDDP in GBC cells,SP cells and cholangiocarcinoma cells.2.PEITC could enhance the sensitivity of xenografted tumors to CDDP without obvious toxic effects.3.PEITC could decrease Mcl-1 expression and the PEITC/CDDP treatment significantly decreased Mcl-1 expression compared to CDDP alone in GBC cells,SP cells and xenografted tumors.Conclusions:PEITC can enhance the cytotoxicity of CDDP via Mcl-1 downregulation in GBC cells,SP cells and in vivo.The third part:A study on the mechanisms of Mcl-1 glutathionylation-enhanced chemosensitivity of gallbladder cancer cells to cisplatinObjective:To investigate whether PEITC can regulate Mcl-1 glutathionylation and the mechanisms that glutathionylation regulates Mcl-1 stability in GBC cells.Methods:GBC-SD cells were treated with PEITC.Then Mcl-1 mRNA was assayed by quantitative real time PCR,cellular GSH level and GSH/GSSG ratio were measured by the kit,Mcl-1 expression was determined by western blot,cellular glutathionylation of Mcl-1 was determined by immunoprecipitation.Site-directed mutagenesis was used to confirm the Mcl-1 glutathionylation sites.After GBC-SD cells were transfected with the Flag-Mcl-1 mutant which was not glutathionylated and then treated with PEITC,Flag-Mcl-1 expression was detected by western blot.Results:1.PEITC decreased Mcl-1 expression via proteasomal degradation in GBC cells.2.PEITC induced proteasomal degradation of Mcl-1 through depletion of GSH and decrease of GSH/GSSG ratio.3.PEITC increased the glutathionylated Mcl-1.4.Cys16 and Cys286 are Mcl-1 glutathionylation sites.5.The mutation of the two cysteine sites for glutathionylation of Mcl-1 to serines resulted in resistance of Mcl-1 protein to PEITC-mediated degradation.Conclusions:1.PEITC can deplete GSH,decrease GSH/GSSG ratio and increase the glutathionylated Mcl-1,making Mcl-1 more susceptible to proteasomal degradation.2.Cys16 and Cys286 are Mcl-1 glutathionylation sites.The fourth part:A study on the mechanisms of STAT3 glutathionylation-enhanced chemosensitivity of gallbladder cancer cells to cisplatinObjective:To investigate the mechanisms that STAT3 glutathionylation regulates Mcl-1 protein production and enhances CDDP-induced apoptosis in GBC cells.Methods:GBC GBC-SD cells were treated with L-Buthionine-sulfoximine(BSO).Then cellular GSH level and GSH/GSSG ratio were measured by the kit,Mcl-1 and STAT3 expression were determined by western blot,cellular glutathionylation of STAT3 was determined by immunoprecipitation.GBC GBC-SD cells were treated with CDDP,BSO or a CDDP/BSO combination.Then the cell viability was assayed by MTT,the apoptotic rate was monitored by flowcytometry,and caspase 3 activity was measured by the kit.Results:1.BSO inhibited STAT3 activation and downregulated Mcl-1 expression in GBC cells.2.BSO significantly depleted GSH and decreased GSH/GSSG ratio.3.BSO induced the S-glutathionylation of STAT3.4.BSO enhanced CDDP-induced apoptosis in GBC cells.Conclusions:BSO can inhibit STAT3 activation and Mcl-1 protein production via STAT3 glutathionylation and enhance the chemosensitivity of GBC cells to CDDP.In conclusion,these results identified that GBC cells show CDDP resistance and SP cells show stronger drug resistance abilities due,in part,to the anti-apoptotic protein Mcl-1,and found a novel mechanism of Mcl-1 glutathionylation-regulated stability that the glutathionylated Mcl-1 is more susceptible to proteasomal degradation.Our study first certified that Cys16 and Cys286 are Mcl-1 glutathionylation sites.Because PEITC can enhance the cytotoxicity of CDDP via glutathionylation-dependent degradation of Mcl-1 in GBC cells,SP cells and in vivo without obvious toxic effects,the combinative therapeutic strategy may develop a clinical promising approach to treat GBC.In addition,we also found that BSO can inhibit Mcl-1 protein production via STAT3 glutathionylation and enhance the chemosensitivity of GBC cells to CDDP.