Silencing XBP1 Expression Enhances The Sensitivity Of Human Osteosarcoma HOS Cells To MPPA-PDT

Objective: To investigate the role of X-box binding protein 1(XBP1)in the treatment of human osteosarcoma HOS cells by pyropheophorbide-αmethyl ester-mediated photodynamic therapy(MPPα-PDT),and to explore the possible mechanism.Methods: HOS cells were treated by MPPα-PDT for 6,12 and 24 h,and the expression levels of inositol-requiring enzyme 1 alpha(IRE1α)-XBP1 pathway-related protein IRE1α and XBP1 were detected by Western blotting.The specific siRNA targeting XBP1 gene was transfected into HOS cells by lipofection,and treated by MPPα-PDT.The cells were divided into the blank group,siRNA-negative control(NC)group,siRNA-XBP1 group,MPPα-PDT group and MPPα-PDT+siRNA-XBP1 group.The expressions of XBP1 mRNA and protein were detected by realtime fluorescent quantitative PCR and Western blotting,respectively.The proliferation of HOS cells was detected by CCK-8 assay.The apoptotic rate was analyzed by flow cytometry(FCM).The expression levels of Cleaved-caspase 3 and Cleaved-poly ADP-ribose polymerase(Cleaved-PARP)were determined by Western blotting.The intracellular level of reactive oxygen species(ROS)was detected by DCFH-DA probe staining,then the expression levels of Catalase and superoxide dismutase 1(SOD1)proteins were detected by Western blotting.Results: The expression levels of IRE1α-XBP1 pathway-related proteins IRE1α and XBP1 were increased after the treatment of MPPα-PDT(both P < 0.05).siRNA-XBP1 inhibited the expression levels of XBP1 mRNA and protein(both P < 0.01).Silencing siRNA-XBP1 expression inhibited the proliferation activity of HOS cells(P < 0.01),up-regulated the apoptosis rate and the expression level of apoptosis-related protein Cleaved-caspase 3(both P < 0.05),and down-regulated the expression levels of antioxidant enzyme-related proteins Catalase and SOD1(both P <0.05).Compared with the MPPα-PDT group,the proliferation activity of HOS cells treated with MPPα-PDT+siRNA-XBP1 was decreased(P <0.01),the apoptosis rate and the expression levels of Cleaved-caspase 3 and Cleaved-PARP were increased(all P < 0.05),the intracellular ROS level was up-regulated(P < 0.01),and the expression levels of Catalase and SOD1 were decreased(both P < 0.01).Conclusion: MPPα-PDT can induce the activation of IRE1α-XBP1 pathway in HOS cells.Silencing XBP1 can inhibit the proliferation activity of HOS cells,up-regulate the apoptosis rate,and increase the sensitivity of HOS cells to MPPα-PDT.The mechanism may be related to theup-regulation of intracellular ROS level and the down-regulation of anti-oxidation molecules.