| Aim: To clarify how melatonin sensitized human hepatocellular carcinoma(HCC)Hep G2 cells via inhibition of cyclooxygenase-2(COX-2)expression to endoplasmic reticulum(ER)stress-induced apoptosis and indentify the molecular mechanisms.Methods:We detected the expression of COX-2,activating transcription factor 6(ATF-6),inositol-requiring enzyme 1(IRE-1),protein kinase RNA-like endoplasmic reticulum kinase(PERK)in HCC tissues by using immunohistochemical staining.We next evaluated the effect of four concentrations of melatonin on the three unfolded protein response(UPR)pathways by Western blot.To investigate the underlying mechanisms of which pathway is associated with the expression of COX-2 under the condition of ER stress,we used RNA interference to knockdown the m RNA in all three UPR pathways.Then we observed the changes in COX-2 m RNA and protein levels by q RT-PCR and Western blot.We also evaluated the expression of COX-2,C/EBP homologous protein10(CHOP),B-cell lymphoma-2(Bcl-2)and Bcl-2-associaed X protein(Bax)by Western blot.To further determine whether melatonin influences ER stress-induced apoptosis,FACS analysis and TUNEL staining were performed in Hep G2 cells.Results:1.The association of three UPR pathways in HCC with the expression of COX-2: We have evaluated and analyzed the expression of COX-2,ATF-6,IRE-1 and PERK in HCC tissues by using immunohistochemical staining.Among the tissues with positive COX-2 expression,the positive rate of ATF-6,IRE-1 and PERK was 87%,60% and60%,respectively.Among the tissues with negative COX-2 expression,the negative rate of ATF-6,IRE-1 and PERK was 29%,55% and 53%,respectively.In conclusion,the expression of COX-2 was correlated with the expression of ATF-6(P=0.011).There was no association between COX-2 and IRE-1(P=0.086).We also found no association between COX-2 and PERK(P=0.134).2.The effects of different concentrations ofmelatonin on the three UPR pathways in Hep G2 cells: To meet this requirement,Hep G2 cells were first pretreated withtunicamycin for 8 h;then,we treated the cells with four concentrations of melatonin(10-9,10-7,10-5,10-3mmol/L)for 24 h.We evaluated the expression of proteins in the three pathways by Western blot.We found that melatonin at a concentration of both 10-3and 10-5mmol/L could obviously downregulate the expression of ATF-6,IRE-1 and PERK.We next chosen the concentration of 10-5mmol/L for further study.3.We have evaluated the interference efficiency of si RNA using Western blot: We have transfected the three homologous sequences of si RNA for ATF-6,IRE-1 and PERK to Hep G2 cells individually.The total protein was extracted 24 h after transfection.The expression of ATF-6,IRE-1 and PERK was detected using Western blot.We have obersved that the expression of ATF-6 was inhibited obviously by transfected with si-ATF-6 1#.Furthermore,the expression of IRE-1 was Inhibited obviously by transfected with si-IRE13# and the expression of PERK was Inhibited obviously by transfected with si-PERK 3#.4.We have investigated the association of different UPR pathways with the expression of COX-2 under the condition of ER stress.Hep G2 cells first pretreated with tunicamycin(3μmol/L)for 8h;then,we used si RNA to knockdown the m RNA in all three UPR pathways and observed the changes in COX-2 m RNA and protein levels by q RT-PCR and Western blot.The results revealed that the expression of COX-2 was downregulated in the group transfected with si-ATF-6,compared with the control(P<0.01).However,there was no significant change for the expression of COX-2 when transfected with si-IRE-1 or si-PERK.5.We have further determined the effect of si-ATF6 on Hep G2 cells.Hep G2 cells first pretreated with tunicamycin(3μmol/L)for 8 h;then,we transfected si-ATF-6 to Hep G2 cells for 24 h.Apoptosis assays were performed using an Annexin-V FITC apoptosis kit and TUNEL staining.The apoptotic cell ratio was increased in the group transfected with si-ATF-6,as compared with the control group(P<0.01).6.We have identified the pathway of which melatonin induced Hep G2 cell apoptosis under ER stress.Hep G2 cells first pretreated with tunicamycin(3μmol/L)for 8 h.Then,we have evaluated the expression of COX-2,CHOP,Bcl-2 and Bax by Western blot in four groups: blank control group,TM group,melatonin group(treated with melatonin at the concentration of 10-5mmol/L for 24h)and si-ATF-6 group(transfected with si-ATF-6 for 6h).The expression of CHOP and Bax was upregulated in the melatonin group and si-ATF-6 group.Meanwhile,both the expression of Bcl-2 and Bcl-2/Bax ratio were downregulated in the melatonin group and si-ATF-6 group(P<0.01).7.We have compared the effects of melatonin and si-ATF6 on Hep G2 cell apoptosis under ER-stress.Hep G2 cells first pretreated with tunicamycin(3μmol/L)for 8 h.Then,we have determined cell apoptosis by using an Annexin-V FITC apoptosis kit and TUNEL staining in four groups: blank control group,TM group,melatonin group(treated with melatonin at the concentration of 10-5mmol/L for 24h)and si-ATF-6 group(transfected with si-ATF6 for 6h).Compared with blank control group,ER-stress could induce cancer cell apoptosis mildly(P<0.05).Nevertheless,both treated with si-ATF-6and melatonin resulted in a dramatic increase in the number of apoptotic Hep G2 cells compared with TM group(P<0.01).Conclusions:1.The expression of COX-2 was correlated with the expression of ATF-6.Suppression of ATF-6 could downregulate the expression of COX-2,which suggest that overexpression of COX-2 may be associated with the of ATF-6 pathway.2.Supression of ATF6 resulted in a dramatic increase in the number of apoptotic Hep G2 cells.ATF-6 may take part in the process associated with resistance to ER stress–mediated apoptosis.3.Under the tunicamycin-induced ER stress,melatonin can inhibit the expression COX-2 by downregulating one of the UPR pathways,ATF-6,which increases the apoptosis of Hep G2 cells via CHOP and Bcl-2/Bax pathway. |
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