JWA Differentially Mediates Cisplatin And TRAIL Induced Apoptosis In Human Gastric Cancer Cells

Gastric cancer is the third most common malignancy in China,with a median 5-year survival of only 20%,it becomes a major cause of both morbidity and mortality.Cisplatin,one of the most widely used and effective anticancer agents,targets the DNA by inducing DNA adducts and crosslinks,leading to single and double-strand breaks,activating the DNA damage response,resulting in apoptotic cell death.Cisplatin is the treatment backbone in a variety of cancers including gastric cancer,but intrinsic and acquired resistance impairs its effectiveness to a high degree.Cisplatin resistant cancer cells can also acquire cross-resistant to other Chemotherapy drugs,such as 5-fluorouracil,Doxorubicin,As2O3.The mechanism of drug resistance was attributed to the barrier of apoptosis mediated by dysregulated signaling pathway,such as p53,PI3K/Akt.Therefore,development of novel agents that reactivate apoptosis in drug resistant cancer cells has emerged as an important therapeutic strategy.TRAIL(also known as Apo2L)is a death ligand that belongs to the TNF superfamily of cytokines.Just like other family members,TRAIL is expressed as a type II membrane protein but it also exists in a soluble form.It mediates its apoptotic effects by binding to its death receptors as a homotrimer.Several TRAIL receptors have been discovered to date,including TRAIL-R1,TRAIL-R2,TRAIL-R3 and TRAIL-R4.TRAIL-R1 and TRAIL-R2 are death receptors that are commonly known as DR4 and DR5 respectively,and both of these receptors transduce TRAIL mediated death signals to the intracellular apoptotic machinery.TRAIL-R3 and TRAIL-4 are designated as antiapoptotic decoy receptors that antagonize TRAIL-induced apoptosis.TRAIL triggers apoptosis in cancers,irrespective of p53 status and appears to have a therapeutic index in preclinical studies.The JWA gene,also known as ARL6ip5,was initially cloned from human tracheal bronchial epithelial cells after treatment with all-trans retinoic acid.Subsequent studies indicated that JWA promotes As2O3-induced apoptosis in HeLa and MCF-7 cells via both ROS and mitochondria linked signal pathway and p38 MAPK linked tubulin polymerization.Moreover,we also have demonstrated that JWA regulated cisplatin induced DNA damage-apoptosis.The cisplatin resistant gastric cancer BGC823/DDP cells were produced from their parent BGC823 cells.Our data have showed that The cisplatin resistant gastric cancer cells BGC823/DDP is more sensitive to TRAIL than the cisplatin sensitive gastric cancer cells BGC823.In line with this,the cisplatin resistant gastric cancer cells SGC7901/DDP is more sensitive to TRAIL than the cisplatin sensitive gastric cancer cells SGC7901 as well.We further determined how JWA affected the expressions of DR4,Bcl-2 and Mcl-1 to regulate Cisplatin and TRAIL induced apoptosis.Objective:The objectives of the present study were to investigate the role of JWA in TRAIL induced apoptosis of gastric cancer cells and elucidate the underlying mechanisms of how does JWA differentially mediates cisplatin and TRAIL induced apoptosis in human gastric cancer cells.Methods:The cisplatin resistant subline BGC823/DDP and SGC7901/DDP were induced previously.Compared TRAIL induced apoptosis in cisplatin resistant gastric cancer cells BGC823/DDP and cisplatin sensitive gastric cancer cells BGC823.To identify how JWA affected the expressions of DR4,Bcl-2 and Mcl-1 to regulate Cisplatin and TRAIL induced apoptosis.Result:1.The cisplatin resistant gastric cancer cells BGC823/DDP is more sensitive to TRAIL than the cisplatin sensitive gastric cancer cells BGC823.The CCK-8 assay and Western blotting showed that much more survival of BGC823/DDP cells than BGC823 cells by treated with cisplatin and As2O3.However,it was surprising that the BGC823/DDP cells were more sensitive to TRAIL than the BGC823 cells.In line with this,the cisplatin resistant gastric cancer cells SGC7901/DDP is more sensitive to TRAIL than the cisplatin sensitive gastric cancer cells SGC7901.2.Overexpression of DR4 leads the BGC823/DDP cells more sensitive to TRAIL induced apoptosis.Here,data showed that only DR4 was significantly up-regulated in BGC823/DDP cells compared with BGC823 cells.In line with the pro-apoptotic role of DR4,increased levels of cleaved PARP-1 were observed by treated cells with 100,150 ng/ml of TRAIL for 8h in DR4 overexpressed BGC823 cells.In addition,reduced level of cleaved PARP-1 was observed by treated with 60,80ng/ml of TRAIL for 8h in DR4 knockdown BGC823/DDP cells.3.JWA inhibits TRAIL-induced apoptosis by negatively regulating DR4.Based on the opposite expression patterns between JWA and DR4 in cisplatin sensitive and resistant gastric cancer cells,we postulated that if DR4 was negatively regulated by JWA.Indeed,the DR4 was significantly increased by knockdown of JWA in BGC823 cells.As a result,TRAIL induced more apoptosis in JWA knockdown BGC823 cells.In contrast,overexpression JWA in BGC823/DDP cells led to an obviously decrease of DR4,and in parallel,TRAIL induced less apoptosis in JWA overexpressed BGC823/DDP cells.4.JWA inhibits DR4 expression by promoting its ubiquitination.When the BGC823/DDP cells were transfected with flag-JWA or control plasmid,then treated with CHX for 0,2,4,6,8 h,the degradation of DR4 in the JWA overexpression BGC823/DDP cells was faster than that of control.Immunoprecipitation assay further showed that the degradation DR4 was increased in the JWA overexpressed BGC823/DDP cells.5.JWA via inhibits Bcl-2 or activates Mcl-1 differentially control cisplatin and TRAIL induced apoptosis.We found that Bcl-2 was up-regulated,whereas,for our surprise,the anti-apoptotic protein Mcl-1 was down-regulated in BGC823/DDP cells.We further determined how JWA affected the expressions of Bcl-2 and Mcl-1 in cisplatin sensitive BGC823 cells.As a result,the levels of Bcl-2,but not Mcl-1 were down-regulated in JWA overexpressed BGC823 cells,and the down-regulated Bcl-2 in these cells led to higher levels of cleaved caspase-3 when the cells were treated with 0.8 ?g/ml of cisplatin;Interestingly,the levels of Mcl-1,but not Bcl-2 in these cells were activated by treatment w:ith 150 ng/ml of TRAIL,and the activated Mcl-1 led to reduced levels of cleaved caspase-3.In contrast,in JWA knockdown BGC823 cells,the levels of Bcl-2,but not Mcl-1 were up-regulated,and with less levels of cleaved caspase-3 by treated with 0.8 ?g/ml of cisplatin;However,the levels of Mcl-1,but not Bcl-2 were inhibited and with higher levels of cleaved caspase-3 by treatment with 150 ng/ml of TRAIL in these cells.Conclusion:Our results indicated that JWA as an effective switch via different targets regulates cisplatin or TRAIL induced apoptosis in gastric cancer cells,respectively.We speculate that JWA is a potential biomarker for determine patients who may benefit either from adjuvant chemotherapy with platinum-based regimen or TRAIL in gastric cancer.Further evaluation of JWA in prospective clinical studies is warranted.