A Study Of Licochalcone-A Sensitizing Human Esophageal Carcinoma Cells To TRAIL-Mediated Apoptosis

Esophageal carcinoma is a highly malignant tumor of human digestive system,occurring in esophageal epithelial tissue.At present our country is one of the highest mortality rates about esophageal carcinoma in the world with the fifth incidence and the fourth mortality rate,which are far more than the world average.Squamous carcinoma and the adenocarcinoma are two main types of esophagus carcinoma that squamous cell carcinomas occupied 90%.The incidence rate of esophageal squamous cell carcinomas was declining while the esophageal adenocarcinoma was gradually improving during the past 30 years.The mechanism of esophageal carcinoma pathogenesis was identified in DNA,RNA and protein levels,while the definite pathogenesis was still unclear.With the development of immune molecular targeted therapy,it is urgent to explored new drugs and treatment for esophageal carcinoma patients.Licorice chalconeA(L-A)is a kind of flavonoids that existing in all kinds of edible plants with extensive pharmacological properties,such as anti-carcinoma,anti-oxidation and anti-inflammatory activity.MTT assay,Western Blot,and RT-qPCR were used to identify the molecular mechanism of L-A though treating Eca109 and TE1 by combining TRAIL and L-A.As is shown in the results,L-A sensitizes TRAIL-induced cytotoxicity in esophageal carcinoma cells.Eca109 and TE1 cells were found to be resistant to TRAIL-induced apoptosis at different concentrations.The apoptosis of ECS cells is dosed dependent of combination of L-A and TRAI.HEK293 did not show any apoptosis with the same treatments.L-A potentiated TRAIL-induced apoptosis in Eca109 cellsAnnexinV/PI assay was employed to detect the TRAIL induced apoptosis by TRAIL combined with L-A.As shown in the results that the apoptosis of Eca109 was significantly higher with combination of TRAIL and L-A than TRAIL treating alone(40.1%versus 23.1%).It is reported that DR4/DR5 induced apoptosis by TRAIL and caspase was the key mechanism.TRAIL induced apoptosis is mainly executed by cell death receptor pathway initiating from Caspase-8 activation,and ultimately activate the effector Caspase-3.In our study,L-A at 5?M or TRAIL at 50ng/mL alone did not cause obvious change in Caspase 3 and PARP,while the combination treatment with TRAIL and L-A led to the complete cleavage of Caspase-3 and PARP.Furthermore,we examined whether the sequence of treatment would affect the cell death.It showed it was no obvious difference of the sensitization among the three ways that L-A pre-treatment,post-treatment and pari-treatment.We then used pan Caspase inhibitor z-VAD-fink to confirm the role of the Caspase cascade in apoptosis induced by TRAIL and L-A.The inhibitor completely blocked Caspase-8 and Caspase-3 activation,and PARP cleavage in cells treated with TRAIL and L-A.Therefore,it is believed that L-A and TRAIL have synergistic effect on Eca109 and TE1 cells death and L-A sensitizes TRAIL-induced apoptosis mainly by the DR4/DR5 pathway.The inactivation of Akt contributes to sensitized cell apoptosisAkt play an important role in both cell survival and the inhibition of apoptosis.Inhibition of the phosphorylated-Akt enhances the sensitivity of TRAIL-mediated apoptosis in various human carcinomas.Data show that the reduction of Akt protein level is mediated by L-A in dose dependent way.To determine whether L-A could inactivate Akt,Eca109 cells were treated with L-A at various time points,and cellular protein extracts were prepared to analyze for phosphorylated-Akt(Ser473,Thr308)and total Akt by immunoblot assays.The level of phosphorylated-Akt at Ser473 not at Thr308 was down-regulated by low-dose L-A in time-dependent manner,whereas the level of total Akt was remained unchanged after treatment.Results also showed that L-A can attenuate Akt activation in cells treated with TRAIL.Moreover,in control group most cells underwent apoptosis after TRAIL and L-A treatment,and Akt-transfected cells remained alive.The dates indicated that L-A sensitized TRAIL-mediated apoptosis by inhibiting Akt activation.Down-regulation of XIAP contributes to sensitiza-tion effect of L-A on TRAIL-induced apoptosisIn TRAIL-induced apoptosis,XIAP is a critical attenuator via inhibition of the Caspase cascade.There was also report demonstrate that XIAP is a physiological substrate of Akt.In order to identify the molecuL-Ar mechanisms which may be involved in the sensitization activity of L-A,we examined whether co-treatment of TRAIL with L-A can influence the protein levels of XIAP proteins.Results showed that significantly decrease of XIAP in cells treated with TRAIL and L-A in a time-and dose-dependent way.To confirm the role of Akt in XIAP stability,we further tested the effect of wortmannin,a known Akt inhibitor,on XIAP protein level in cells treated with TRAIL.The effect of L-A on Akt phosphorylation was comparable with that of wortmannin.Data show low-dose L-A inhibited Akt phosphorylation on Ser473,without altering total Akt levels,which is simillar to the effect of wortmannin.This effect could be abolished by transfecting the cells with wild-type Akt plasmid.XIAP protein show a significant decline in non-transfected cells while was stable in Akttransfected cells.These data indicate that XIAP as a substrate of Akt involved in the sensitization of L-A on TRAIL-mediated apoptosis.L-A promotes proteasomal degradation of XIAPTo elucidate the moleculer of L-Ar mechanism,we first measured the XIAP mRNA level using qRT-PCR.Either TRAIL,L-A,or their combined treatment did not alter the XIAP mRNA level up to 24 h,indicating that the XIAP is mainly regulated post-transcriptionally.Thus we tested the effects of proteasome inhibitor on XIAP protein level.MG132 completely abolished the XIAP down-regulation induced by TRAIL and L-A.The results showed that TRAIL and L-A promote XIAP proteasomal degradation.L-A enhance the sensitivity of the esophageal carcinoma cells on the cytotoxicity induced by TRAIL though inhibiting the activation of Akt protein,degrading XIAP protein.In conclusion,we found that L-A enhance TRAIL-induced apoptosis in Eca109 and TE1 cells,Our results provide an important insight for understanding the mol-ecular mechanism of the anticarcinoma effect of luteolin.It may therefore be possible that luteolin could widen the therapeutic window,allowing carcinoma cells killing while protecting normal cells.Because of the importance of Akt and XIAP in oncogenesis,these findings will be helpful to recognize significance of L-A in antitumor strategies.The combination of TRAIL and L-A might be a novel therapeutic strategy for esophageal carcinoma patients who fail to respond to standard chemotherapy.