The Role And Molecular Mechanism Of JWA Involved In As₂O₃ Induced Apoptosis In HeLa And MCF-7 Cells

Arsenic trioxide(As₂O₃),emerging as a standard therapy for both newly diagnosed and refractory acute promyelocytic leukemia(APL), induces apoptosis of APL cell as the main mechanism of antineoplastic properties of APL.More recently,studies revealed that apoptosis inducing properties of As₂O₃ were not restricted to APL,but also in different types of tumor cells' viability,including other types of leukocythemia cell,hepatocellular carcinoma cell,prostatic carcinoma cell,renal carcinoma cell,esophageal carcinoma cell,uterine cervix cancer cell,bladder carcinoma cell and such solid tumor cell.The mechanisms of As₂O₃ to induce cellular apoptosis are probably much more complicated than we understood.In APL cells,As₂O₃ induces apoptosis mainly through the mechanisms that result in the degradation of PML-RARαand the covalent linkage between SUMO-1 and PML. However,the precise mechanism of As₂O₃-induced apoptosis in solid cancers has yet to be clarified.As₂O₃ was shown to(1)generate reactive oxygen species(ROS),(2)activate MAPK,suppress Bcl-2 family expression,(3)disrupt mitochondrial membrane potential(Δψm)and release cytochrome c,(4)and increase the activities of caspase-3,-8 and -9,resulting in apoptosis.As₂O₃ apparently affects numerous intracellular signal transduction pathways and produces alterations in cellular functions.These actions of As₂O₃ may result in the induction of apoptosis and depending on cell type,exposure concentrations,and time.JWA(GenBank:AF070523.1,1998),a newly identified ATRA-responsive gene,was primarily cloned from human tracheal bronchial epithelial cells by Zhou J,et al.Several cis-elements were found within the identified 3,000 bp of its promoter region,such as phorbol ester response element(TRE),hemin response element(HRE),heat shock response element(hRE),stress response element(SRE),and the retinoic acid response element(ARE)semi-site and so on.In addition,two potential PKC phosphorylation sites(SDR-SLR)were recognized in JWA coding region.The accumulating data have shown that JWA is a novel structurally microtubule associated protein(MAP),not only related to differentiation and/or apoptosis inducers(e.g.TPA,ATRA,4HPR,and As₂O₃)mediated leukemia cells differentiation and apoptosis,but actively responsive to stress stimulations(e.g.heat shock,oxidative stress). However,the underlying molecular mechanism of JWA gene and involved signal pathways in the regulation of cellular apoptosis is still unknown.In this study,we chose As₂O₃ as the apoptosis inducing chemotherapeutic agent,to explore whether JWA is involved in As₂O₃ induced apoptosis and the underlying molecular mechanisms in human cervical cancer cell line HeLa and human breast cancer cell line MCF-7 as cell culture models.1.As₂O₃ induces apoptosis and accompanied with enhancement of JWA expressions in HeLa and MCF-7 cellsThe Hoechst 33258 staining assay,TUNEL and Annexin V-PE flow cytometry assays showed that As₂O induces apoptosis in both HeLa and MCF-7 tumor cells in a dose-dependent manner.However,the two cell lines indicated differential sensitivity to As₂O₃ exposure.For HeLa cells, treatment with 5μM As₂O₃ for 48 h induced about 100%apoptosis; however,induced only 25%apoptosis in MCF-7 cells.Western blot analysis showed an enhanced JWA expressions in both HeLa and MCF-7 cells and also with a dose-dependent increasing manner.These data suggest JWA may be involved in As₂O₃ induced apoptosis.2.JWA is required for As₂O₃-inducing apoptosisTo evaluate whether increased JWA expression is required for As₂O₃ inducing apoptosis in both HeLa and MCF-7 cells,we established stable JWA deficient HeLa and MCF-7 cells.Interestingly,these JWA deficient cells showed significant resistance to As₂O₃ treatment when compared with their vector controlled cells;In contrast of this,the MTT assay demonstrated significantly increased viabilities in both asJWA-HeLa and asJWA-MCF-7 cells compared with their vector controls after As₂O₃ treatment.When expression of JWA protein was rescued by transiently transfected JWA cDNA pEGFP vector(sense vector)into JWA deficient cells,As₂O₃ induced apoptosis were rose to the level of the vector control after treatement with As₂O₃ for 48h.These results indicating that JWA protein may act as a pro-apoptotic molecule in the process of apoptosis induced by As₂O₃.3.JWA enhances As₂O₃ induced apoptosis through mitochondrial pathwayHow does JWA play its pro-apoptotic role in the pathway of As₂O₃ triggered cell apoptosis? Genetic and biochemical evidences indicate that apoptosis proceeds in two major cell death pathways:an intrinsic pathway that involves mitochondrial membrane permeabilization and release of several apoptogenic factors,followed by caspase-9 activation; and an extrinsic apoptotic signaling pathway that occurs via caspase-8 activation.To determine whether caspases signal pathways were involved in As₂O₃ triggered and JWA dependent cell apoptosis,studies examined expressions of caspase-8 and caspase-9 and their potential cleavages. Data showed that As₂O₃ treatment(5μM for 24 hr)significantly triggered caspase-9 but not caspase-8 cleavage,this phenomenon was obviously attenuated in JWA deficient cells.Further,when expression of JWA protein was rescued by transiently transfected JWA cDNA pEGFP vector (sense vector)into JWA deficient cells,the cleavage of caspase-9 was increased to the level of vector control after treatment with As₂O₃ for 24h. Suggesting that JWA exerted its pro-apoptotic effect mostly through the intrinsic pathway of As₂O₃ triggered cell apoptosis.To testify whether caspase-9 activation regulated by JWA may be due to the changes of mitochondrial membrane potential(Δψm),we further performed FACS analysis to detect the loss ofΔψm.Interestingly,results revealed that asJWA-HeLa cells treatment with As₂O₃(5μM for 24 hr)were still maintained theirΔψm in contrast of markedly losingΔψm in control cells. It was suggested that loss of mitochondria membrane potential in the presence of JWA expression may be linked to caspase-9 activation.4.As₂O₃ generated ROS induces JWA expressionROS are known to affect mitochondria membrane potential,therefore trigger a series of mitochondria-associated events including cell apoptosis. In addition,ROS plays a key component role underlying the As₂O₃ generated-effects.It is possible that the role of JWA involved in As₂O₃ induced apoptosis was mediated by As₂O₃ generated ROS.To test this hypothesis,DCFH-DA staining assay was used to detect intracellular content of ROS induced by As₂O₃ treatment.In this cell culture model, catalase was used to remove intracellular H₂O₂.As a result,removing of intracellular H₂O₂ by addition of catalase resulted in reduced apoptosis and JWA expression.These evidences indicated that H₂O₂ might act as a metabolite of As₂O₃ via activating JWA and then induce apoptosis.5.Molecular mechanisms underlying the effect of JWA on As₂O₃ induced apoptosisMAPK signal pathways to be responsible for mitochondria mediated cell apoptosis.The Bcl-2 family proteins are also known to be important for apoptosis-regulating molecules that modulate mitochondrial dependent signal pathways.To further investigate the role of JWA on the pathway of As₂O₃ induced apoptosis,we tested whether the role of JWA was associated with MAPK or Bcl-2 family proteins.We observed the effects of JWA on As₂O₃(0,5μM for 24h)induced expression or phosphorylation of MAPK and Bcl-2/Bad molecules.The results showed the As₂O₃ activated phosphorylations of MEK,ERK1/2 and JNK were almost completely blocked by knock-down JWA expression;although the total expressions(unphosphorylation level)of these signal molecules were unaffected and the Bad phosphorylation was further enhanced in As₂O₃ treated asJWA-HeLa cells.In summary,the results presented here indicate that(1)As₂O₃ generated H₂O₂ via inducing JWA expression and then triggered a series of signal transduction events to induce apoptosis in HeLa and MCF-7 cells.(2)JWA is required for As₂O₃ induced activation of MEK-ERK and JNK which further closely linked to changes of mitochondrial membrane potential and the activation of caspase-9;these cascade events play a central role for As₂O₃ inducing apoptosis.(3)As₂O₃ induced Bad activation was also contributed to apoptosis and regulated by JWA.JWA may exert its pro-apoptotic effect on As₂O₃ induced apoptosis through the following pathway:As₂O₃→H₂O₂→JWA→MAPK family(MEK-ERK, JNK)→Bcl-2 family(Bad,Bax)→mitochondrial membrane potential→cytochrome c→caspase-9→apoptosis.