The Effect And Mechanism Of Temozolomide With Amlexanox On Human Glioblastoma Cells

Background and Purpose: Temozolomide,as a first-line chemotherapeutic agent for glioblastoma multiforme,can increase survival of patients with glioblastoma.However,because of the rapid formation of drug resistance,it is difficult to improve the prognosis of patients suffering from glioma.Thus,a more effective administration is urgently needed for glioma.Based on this background,this study aimed to explore the effect and mechanism of temozolomide combined with a small molecule inhibitor,amlexanox on glioma cells.Methods: This study was divided into two parts: in vivo and in vitro,with classical human glioblastoma cell line U87 and primary glioma cells as experimental subjects.In vitro,we mainly used CCK-8 and colony formation assays to test cell proliferation ability,flow cytometry to exam apoptosis,cell wound scratch and Transwell assays to detect cell invasion and migration ability,Western-Blot to detect changes of IKBKE and proteins related to AKT pathway;in vivo,we mainly conducted the construction of models of intracranial and subcutaneous tumors in nude mice to test the effects of tomozolomide combined with amlexanox on tumor growth,and immunohistochemical staining assay was used to detect the changes in the expression of the following proteins: IKBKE、p-AKT、p-AMPK、p-m TOR.Results: CCK-8 results showed that emozolomide combined amlexanox significantly inhibited the proliferation of U87 and primary glioma cells compared with treatment using single drug(temozolomide or amlexanox),and the combined agents(TMZ and amlexanox)significantly inhibited the formation of cell clone;The results of cell wound scratch and Transwell migration assays showed that temozolomide combined with amlexanox could significantly inhibit healing area and number of cells penetrated;Transwell invasion assay showed that treatment with combined drugs significantly reduced the number of cells penetrating out of the chamber;The results of cell flow cytometry showed that temozolomide combined amlexanox significantly increased the number of apoptotic cells(PI+ 、Annexin-V+ 、 and PI-、Annexin-V+)than other groups did;Western-Blot assay showed that amelexanox inhibited IKBKE activity,temozolomide enhanced AMPK、AKT activity and decreased m TOR activity,and temozolomide combined amlexanox significantly inhibited AKT and m TOR activity;The results in vivo showed that temozolomide combined with amlexanox could significantly inhibit the growth of subcutaneous and intracranial tumors in nude mice compared with treatment in the control group and TMZ group,and prolong the survival time of animal models with intracranial tumors.Immunohistochemical staining assay showed that expression of p-AMPK、p-AKT in tumor samples increased and expression of p-m TOR decreased in temozolomide group,and expression of p-AKT、p-m TOR obviously decreased incombined group.Conclusion: Tomozolomide combined with amlexanox can not only inhibit the ability of proliferation,migration and invasion for glioma cells,but also promote apoptosis.It may involve in the mechanism that amlexanox reverse temozolomide-induced AKT activation by inhibiting IKBKE activity,and then reduce the resistance of glioma cells to temozolomide.This provides a new idea for clinical application of temozolomide combined with small molecule inhibitors in the treatment of glioblastoma.