XCT Regulates Redox Status To Effect The Sensitivity Of Chemotherapy In Diffuse Large B Cell Lymphoma

Background and Objectives:Diffuse large B-cell lymphoma(DLBCL),characterized by a high degree of heterogeneity in immunophenotype,pathogenetics,and clinical response,is the most common type of non-Hodgkin lymphoma.Based on gene expression profiling,DLBCL can be sub-divided into 3 distinct subtypes:germinal center B cell like(GCB),activated B-cell like(ABC),and type 3,with different cell-of-origin(COO)and survival outcome.The addition of rituximab to the traditional CHOP regmin has markedly improved response rates in DLBCL over the last decade.Still,approximately 40%of DLBCL patients will relapse within a short period of time and eventually succumb to the disease.Treatement response is the worst among ABC-DLBCLs,where the 5-year overall survival rate for is only about 50%.These clinical observation highlights the need for improved prognostic models that can guide risk-justified treatment selection and development of new treatment.Doxorubicin,as the most important cytotoxic anticancer drug in CHOP(cyclophosphamide,vincristine,doxorubicin(Dox),and prednisone)chemotherapy,had been used in DLBCL for more than 30 years.The following mechanisms are believed to contribute to the cytotoxic effect of Dox in cancer cells:1)intercalation into DNA,2)topoisomerase II inhibition,3)generation of free radicals leading to oxidative stress.The anticancer mechanisms of Dox may differ different cancers,and in the different subtypes of the same cancer.Unpublished results from our laboratory(Yun et al.)showed that Dox-induced cytotoxicity in GCB-DLBCLs is dependent on DNA damage response;however,in ABC-DLBCLs Dox mainly triggers oxidative cell death rather than DNA damage response.Reactive oxygen species(ROS)are chemically reactive molecules containing oxygen,including oxygen ions and peroxides.Mitochondrial respiration is a major source of ROS in most mammalian cells.In some cancer cells,that are not depend on mitochondrial respiration for energy production,ROS generation likely results both from cell signaling-stimulated increase in NADPH oxidase,cyclooxygenases and lipoxygenases.Macrophages and neutrophils,as tumor-infiltrating inflammatory cells,can generate ROS as a result of NADPH oxidase activation.Chemo-and radiation therapy also induce ROS generation.Anticancer drugs,including the anthracyclines(such as Dox)and platinum coordination complexes,are known to exert part of their anticancer effects through ROS generation.Because in ABC-DLBCLs the main mechnanisam of Dox-induced cytoroxicity relies on increased ROS.We hepothesise that pharmacolopical ROS insult may improve the Dox sensitivity in ABC-DLBCL.xCT(SLC7A11)is the light-chain subunit of System xc(-).Together with the heavy-chain subunit(CD98hc or SLC3A2),they constitute of System xc(-).As a Na+-independent transporter,xCT mediates the exchange of extracellular cystine(the predominant form of cysteine in plasma,extracellular body fluids and cell culture medium)for intracellular glutamate.The availability of cysteine is rate limiting for GSH synthesis,with the activity of xCT therefore being essential for the GSH-dependent antioxidant system.It has been reported that xCT expression can promote the proliferation,metastasis and drug resistence in different solid tumors and chronic lymphocytic leukemia(CLL).However the role of xCT in DLBCL therapeutic response has not been explored.In particular,it is unknown whether xCT can privide Dox resistance in ABC-DLBCLs by upregulating antioxidant capacity.In summary,in this project we will evaluate xCT expression in different DLBCL cell lines anddetermine whether,and if so how,xCT inhibition affects the response of ABC-DLBCLs to Dox-containging therapy.Methods:1.Expression pattern of xCT and CD44 in different DLBCL cell linesExponential growth phase ABC-DLBCL(HBL1,Ly3-S,Ly3-R,SuDHL2,Riva,U2932,Pfeiffer,TMD8,and Ly10),GCB-DLBCL(SuDHL-6,SuDHL-5,Val,Karpass-422,Ly1)and Type-3 DLBCL cells were used to analyze the expression of xCT and CD44.Q-PCR was used to detect the mRNA expression of CD44 and xCT.Western blot and Flow cytometry were used to detect CD44 and xCT expression,respectively.The expression of CD44v8-10 was studied by RT-PCR.2.The effect of xCT inhibition on cell proliferation and the underlying mechanism.xCT was interefered by xCT siRNA transient transfection in 2 ABC-DLBCL cell lilnes(SuDHL2 and Riva).Following confirmation of knockdown effect,cells were exposed to Dox.The viability of different treatment groups.Ongoing apoptosis was followed by FCM analyzed after staining with SYTOX Green and Annexin V.ROS was measured by with H2DA-DCF staining followed by FCM.p38 MAPK/p-p38 MAPK,JNK/p-JNK,Bax,Bim,Mcl-1,Bcl-2,Caspase 3 and PARP were detected by Western Blot.Following the treatment with xCT inhibitor SASP,cell viability,ROS generation,and status of stress and apoptosis regulators were analyzed as described above.3.SASP synergized the sensitivity of Dox in ABC-DLBCL cell linesAfter pretreatment with xCT inhibitor SASP and Dox,Riva and SuDHL2 cell lines were exposed to Dox.Cell viability,ROS generation,and status of stress and apoptosis regulators were analyzed as described above.SuDHL2 and Riva Cells pretreated by glutathione ethyl ester(GEE)and p38 inhibitor were exposed to SASP/Dox combination and analyzed as described above.4.Statistical analysisDistributions of variables between the different groups were carried out by t-test or one way-ANOVA.Levene test was used to evaluate the homogeneity of variance.LSD method was used to analyze the difference among groups after one way ANOVA.Otherwise,F test(Welch)and Dunnett’s T3 test were used to evaluate the difference among the groups.A value P<0.05 was considered to indicate statistical significance.Statistical analysis was done using the Statistical Package of Social Sciences version 13.0 for Windows.Results:1.xCT mRNA can be detected in all DLBCL cell lines by qRT-PCR.The levels of xCT mRNA in ABC-DLBCL cell lines are higher than those in GCB-DLBCL cell lines(t = 2.583,P = 0.041).xCT expression showed no difference in ABC-DLBCL and GCB-DLBCL cell lines detected by Western Blot(t = 0.328,P = 0.750).CD44v8-10 can be only detected in Pfeiffer cell line.It may imply that CD44v8-10 may not a major factor regulating the stabilize of xCT.2.xCTi siRNA-transfected in Riva and SuDHL2 cell lines did not show reduction of xCT protein even after 96 hours.xCT has a very long half-life in Riva,SuDHL2,and Ly3-S cell lines.SASP,the xCT inhibitor,could inhibit the proliferation of DLBCL cell lines at a high concentration.However it promoted cell growth at a low concertration.GSH level decreased(P<0.001,<0.001)and ROS level increased(P<0.001,<0.001)in Riva and SuDHL2 cell lines 24 hours after SASP treatment.After 12 hours and 24 hours of treated with SASP,oxidative stress signaling proteins p-p38 and p-JNK increased.The apoptosis related proteins Cleaved PARP,Bax,Bim are increased while Mcl-1 and Bcl-2 decreased.3.Combined SASP and Dox treatment inhited the proliferation of Riva(F=141.039,P= 0.001)and SuDHL2(F=145.039,P-= 0.030),induced apoptosis in Riva(F=1131.469,P<0.001)and SuDHL2(F=3332.651,P<0.001),and increased ROS levels in Riva(F=153.281,P<0.001)and SuDHL2(F= 157.407,P<0.001)cells.The combination further increased the expression of p-p38 and p-JNK,Cleaved PARP,Bax,Bim,and decreased the expression of Mcl-1,Bcl-2 compared to Dox treatment alone.GEE attenuated ROS accumatation(P<0.001,<0.001),suppressed apoptosis(P<0.001,<0.001),and antagonized caspase,p38,and JNK activation by SASP/Dox combination in Riva and SuDHL2 cell lines.p38 inhibition restored viability and suppressed apoptosis triggered by SASP/Dox combination in Riva and SuDHL2 cell lines.Conclusions:1.xCT mRNA and protein can be detected in all DLBCL cell lines analyzed.High levels of xCT mRNA were prodominatly detected in ABC-DLBCL cell lines;however xCT protein expression subtype difference.CD44 mRNA and protein were predominately expressed in ABC-DLBCL cell lines.CD44v8-10 only can be dected in the Pfeiffer cell lines.It seemed that xCT protein may not regulated by CD44v8-10.2.xCT is resistant to siRNA mediated knock-down and has a very long half-life in ABC-DLBCL cell lines.Sulfasalazine(SASP),the xCT inhibitor,decreased the GSH level and increased ROS accumulation as well as apoptosis in ABC-DLBCL cell lines.At the same time,the expression of p-p38 and p-JNK,cleaved PARP,Bax,Bim increased and the expression of Mcl-land Bcl-2 decreased.3.SASP/Dox combination induced ROS,increased the expression of p-p38 and p-JNK,cleaved PARP,Bax,Bim and decreased the expression of Mcl-13 Bcl-2.SASP synergized with Dox the sensitivity of Dox in ABC-DLBCLs in increasing apoptosis and inhibiting survival of ABC-DLBCL cell lines.The cytotoxic effect of SASP/Dox combination can be reversed by treatment with GEE or p38 inhibitor.