Study On The Mechanism Of HDAC6-p97/VCP Regulating "Ubiquitination-autophagy Pathway" In Reversing Endoplasmic Reticulum Stress Tolerance In Glioblastoma

Objective 1.To investigate the killing effect of histone deacetylase inhibitor Tubastatin A in combination with temozolomide on glioma cell resistant strains;2.To investigate the mechanism of histone deacetylase inhibitor Tubastatin A combined with temozolomide to kill glioma cell resistant strains.Materials and Methods: Construction of four different glioma cell lines resistant strains,previously treated with Tubastatin A,using CCK-8 method,Caspase-3 apoptosis detection method,Hoechst 33342 / PI double staining and Tunel method to detect cell proliferation and apoptosis.The gene expression of histone deacetylase 6-related cell signaling pathway in each cell line and the interaction between the corresponding molecules were detected by protein imprinting experiment and immunoprecipitation assay.The experimental data were analyzed by SPSS19 statistical software.All experiments were performed a minimum of three times.Data are presented as Mean ± SEM.Independent experiments were pooled when the coefficient of variance could be assumed identical.One-way ANOVA was used to assess significant differences for multiple groups,followed by post hoc Bonferroni’s test.P <0.05 was considered statistically significant.Results 1.Western blotting showed that the expression level of GRP78,phosphorylated IRE1α and HDAC6 in tumor tissue was higher than that in normal brain tissue.The expression level of p97 / VCP in tumor tissue was lower than that in normal brain tissue.The expression level of GRP78,phosphorylated IRE1α and HDAC6 in TMZ-resistant strain was higher than that in parent strain.The expression level of p97 / VCPTMZ-resistant strain was lower than that of parent strain.2.Cell viability and apoptosis test showed that CCK-8 method was used to detect the apoptosis of TUB and TMZ in four different glioma cell lines,which was significantly higher than that of TUB alone group.Four kinds of tumor cell lines(P <0.05);U27 group: 11.7μM(P <0.05);U87 group: 17.3μM(P <0.05);U118 group: 8.2μM(P <0.05)<0.05).Caspase-3 activity in four different glioma cell lines,TUB and TMZ combined group of patients with apoptosis was significantly higher than the TUB alone group.Hoechst 33342 / PI double staining method to detect A172 glioma cell line,TUB and TMZ combined group of patients with apoptosis was significantly higher than the TUB alone group.The apoptosis of A172 glioma cell line,TUB and TMZ group was significantly higher than that of TUB group.3.The results of Western blotting showed that the unfolded protein response and endoplasmic reticulum stress-related pathway protein were higher in TUB group than in TUB group,respectively,in glioma cell lines U87 and U118.4.Western blotting and immunoprecipitation showed that in the glioma cell lines U87 and U118,the heat shock response pathway protein in TUB combined with TMZ group was lower than that of TUB alone group.5.Western blotting,immunoprecipitation and immunofluorescence showed that HDAC6-mediated autophagy pathway-associated protein in Tlu and TMZ group were significantly lower than those in TUB alone group in glioma cell lines U87 and U118.The ubiquitin-proteasome pathway-related protein mediated by p97 / VCP in TUB combined with TMZ was more potent than that of TUB alone.Conclusions1.The TMZ resistance of glioma cells is positively correlated with the stress tolerance mechanism of endoplasmic reticulum.2.The endoplasmic reticulum stress tolerance mechanism of glioma cells was associated with intracellular HDAC6-p97 / VCP concentration balance(HDAC6 up-p97 / VCP decline).3.HDAC6 selective inhibitor TUB inhibited the deacetylation and ubiquitin binding capacity of HDAC6,reversing the concentration of intracellular HDAC6-p97 / VCP,thus preventing the endoplasmic reticulum stress tolerance mechanism.4.TUB and TMZ synergistically enhance the killing of TMZ-resistant cells.