Studies Examining The Synergy Between GLA And TMZ Against Glioblastoma Cells And The Potential Mechanisms

Objective:The aim of this study is to preliminarily investigate the synergy between Glaucocalyxin A(GLA)and Temozolomide(TMZ)and the possible regulatory mechanism via using human glioma cancer cell lines U251,U87 and SGH44 cells as model.Methods:CCK8 and colony formation assay were used to detect the activity of cell proliferation;Annexin V/PI was used to detect the cytotoxicity of GLA/TMZ;hoechst 33258 staining and flow cytometry were used to detect cell apoptosis;Cell cycle was detected by flow cytometry;western blot and qPCR were used to detect the expression of apoptosis and cycle related genes and proteins;the effect and mechanism of GLA/TMZ combined therapy on tumor in vivo were investigated in animal experiments.Results:GLA could inhibit the proliferation of glioma cells alone,while low concentration of GLA has no obvious inhibitory effect on glioma cells.2 μM GLA combined with TMZ of different concentrations significantly inhibited cell proliferation and increased cell mortality in a time-dose dependent manner.The combined effect of 0.1 mM TMZ and 2 μM GLA induced cell apoptosis,the expression of apoptotic bodies increased,the nucleus was broken in the combined treatment group,and the cells were obviously blocked in G2/M phase.Western blot results showed that the expressions of apoptosis-related proteins caspase 8,caspase 9 and bax were increased,while the expression of anti-apoptotic protein bcl-2 was decreased.The expression of cell cycle-related proteins cyclin B and cdc25A were decreased.The results of qPCR were consistent with the results of western blot.These results indicated that the synergy between GLA and TMZ was achieved through the regulation of cell cycle-related genes and proteins.In vivo,the tumor volume was significantly reduced in the GLA/TMZ combined treatment group,and the expression of cyclin B and cdc25A was significantly decreased by western blot,which was consistent with the results of in vitro experiments.Conclusion:GLA/TMZ could synergistically treat glioma cells,and GLA could significantly increase the chemosensitivity of TMZ and decrease the concentration of TMZ.This process is mainly related to the regulation of cyclin B and cdc25A.