Role Of CHOP-dependent Endoplasmic Reticulum Stress Signaling Pathway In Triptolide Induced A375 Cell's Apoptosis

BackgroundMalignant melanoma has been recognized to be one of the most lethal and aggressive malignancies in human.Although there is a variety of treatment,the cure rate is still unsatisfactorily.Thus,there is an urgent need to find new treatments for melanoma patients.Triptolide,a immunosuppressive agent,has been shown to induce human malanoma A375 cells to death.Studies have shown that the anti-tumor mechanism could via different signaling pathways including endoplasmic reticulum stress mediated apoptosis.Endoplasmic reticulum stress mediated apoptosis is a new apoptosis signaling pathway.C/-EBP homologous protein(CHOP)is commonly used as a marker of endoplasmic reticulum(ER)stress.It is considered as initiation of ER stress-induced apoptosis.Our study are aim to research the role of CHOP-dependent endoplasmic reticulum stress mediated apoptosis in triptolide induced A375 cell's death.Objective1.To estimate the effect of triptolide on malignant melanoma A375 cells,and to observe whether triptolide can cause the occurrence of endoplasmic reticulum stress.2.Assessing the role of endoplasmic reticulum stress in triptolide-induced melanoma apoptosis.Providing a new choice for the treatment of melanoma.MethodsHuman malignant melanoma A3 75 cells were pretreated with different triptolide concentration(OnM?12.5 nM?25 nM?50 nM?100 nM?200 nM)for 24h.Then using optical microscope observing the change of cell's morphological,the inhibition of cell proliferation was measured by using CCK8 assay,the apoptosis was evaluated by flow cytometric of Annexin/PI staning,WesternBlot was used to measure the protein expression of GRP78?p-PERK?PERK and CHOP,and detected GRP78 after triptolide for 48h and 72h,qPCR was used to detect the mRNA expression of GRP78?PERK?CHOP.Results1.Cultured A375 cells with triptolide for 24h,finding the amount of A375 cells decreasing with increasing triptolide concentration.The cell's shape changed to long and thin.And prolonging the cultured time,cell morphology became circular or approaching rounded.2.Compared with the control group,12.5nM?25nM?50nM?10WnM?200nM triptolide for 24h,48h,72h inhibited the proliferation of A375 cells in a time-and dose-dependent manner(p<0.05),with the IC50 of 308nM?83nM?55nM at 24,48,72h,respectively.3.After different concentration of triptolide effecting A375 cells for 24h,the opoptosis rate increased in a dose-dependent manner(p<0.05).4.WesternBlot showed that triptolide can induced the protein expression of ERS markes,including GRP78?p-PERK?PERK and CHOP.All the protein level upregulated with dose going higher,(p<0.05).5.Prolonged treatment with triptolide in A375 cells could inhibits expression of GRP78.6.qPCR showed that triptolide could increase the expression on level of GRP78?PERK and CHOP mRNA in a dose-dependent manner,(p<0.05).Conclusion1.Triptolide can induce the endoplasmic reticulum stress of A375 cells.2.Triptolide activated the CHOP-dependent ER stress in A375 cells,which might exhibit a critical function in triptolide-induced apoptosis in human melanoma.