The Mechanism Study Of Anti-glioma Effect Of Jineol By Regulating EGFR And Downstream Signaling Pathways

ObjectiveBrain glioma is one of the most common malignant tumors in the brain,which has the characteristics of high fatality rate and high recurrence rate.Infiltration and growth in brain parenchyma is a remarkable feature of glioma,which makes it almost impossible to remove the lesion surgically.Epidermal growth factor receptor(EGFR)gene is located on chromosome 7p12,and its encoded protein is a transmembrane receptor tyrosine kinase(EGFR/HER1/ERBB1).The extracellular domain of EGFR combined with amphiregulin(AR),EGF or TGF-α can promote the formation of EGFR dimer,promote the phosphorylation of tyrosine kinase in cells,and activate the downstream signal pathway in cells,thus regulating the biological processes of cell growth,proliferation,invasion,metastasis and apoptosis.At present,it is known that the abnormal activation of EGFR is closely related to the occurrence and development of tumors,and EGFR gene is highly expressed in human glioma,which is negatively correlated with the prognosis of patients.Jineol is a monomer extracted from centipede,which has been shown to inhibit the growth and promote apoptosis of various tumor cells,but its mechanism is unclear.In this study,human glioma cell line U87 was used to evaluate the anti-tumor activity of Jineol and explore its mechanism,so as to provide theoretical basis for the treatment of glioma with centipede,and also provide reference for the anti-tumor research of monomer compounds derived from traditional Chinese medicine.Methods1.the effect of Jineol on the viability of glioma U87 cells: after treated with jineol of10,20 and 40 μM,CCK8 method was used to detect the survival rate of glioma u87 cells at 24 h,48 h and 72 h.2.Effect of Jineol on migration and invasion of glioma U87 cells: After treated with Jineol at 10,20 and 40 μ M,scratch test was used to detect the effect of Jineol on migration and invasion of U87 cells.3.Effect of Jineol on cell cycle distribution and apoptosis of glioma U87 cells: After treated with Jineol of 10,20 and 40 μ M,PI staining and Annexin V-FITC/PI double staining were used to stain and mark the cells in each group,and then the cells in each group were detected by flow cytometry.Flow Jo software was used to analyze and count the ratio of cell cycle number and Annexin V-PI double positive cells.4.Effects of 4 Jineol on EGFR and its downstream signaling pathway in glioma U87cells: After treated with 10,20 and 40 μM Jineol,RT-PCR and protein Western blot were used to measure the m RNA level and protein relative expression of EGFR in each treatment group.The relative expressions of p-ERK,p-AKT,PCNA,Cyclin D1,Bcl-2,Bax,PARP,Caspase-3,P53 and PTEN protein were determined by Western blot.Results1.CCK8 experiment showed that compared with Control group,the survival rate of glioma U87 cells treated with 10,20 and 40μM Jineol decreased significantly(all p <0.001),and it was dose-dependent with the increase of Jineol concentration.2.The results of scratch test and invasion test showed that compared with Control group,the number and migration distance of transmembrane cells in Transwell chamber in three experimental groups were significantly reduced(P < 0.001),and showed dose dependence.3.The results of flow cytometry showed that Jineol increased the proportion of cells in G1 phase and decreased the proportion of cells in S phase in U87 cells in a dose-dependent manner(P < 0.001)4.Compared with Control group,Jineol significantly decreased EGFR m RNA level,p-EGFR/EGFR level and p-AKT,p-ERK,PCNA,Cyclin-D1,Bcl-2,PARP and Caspase-3protein expression levels in a dose-dependent manner(all p < 0.05),and increased Bax,P53 and PTEN protein expression levels.ConclusionThis study confirmed that Jineol can significantly inhibit the proliferation,invasion and migration of brain glioma cells in vitro and promote apoptosis,mainly by inhibiting the activation of EGFR pathway and affecting the downstream signal pathway to play an anti-glioma role.