Elemene Enhances The Efficacy Of Gefitinib On Glioblastoma Multiforme Cells Through The Inhibition Of The EGFR Signaling Pathway

Background and ObjectiveIntracranial tumor,also referred to as brain tumors,mostly originated in the intracranial primary tumor tissue,a few for other malignant tumor tissues,such as liver cancer,lung cancer,breast cancer metastasis or invade into the cavity formed by the secondary tumors.Glioblastoma multiforme(GBM)which accounts for 30%to 40%of primary malignant tumors of the central nervous system is the most frequently occurring malignant intracranial tumors in adults,with aggressive,invasive,angiogenic,and destructive features.It is recognized as diffuse astrocytoma grade Ⅳ by World Health Organization.The current standard therapy includes surgery of maximal safe resection and then followed by radiotherapy and chemotherapy.Due to the special areas of glioblastoma multiforme agents,rapid proliferation ability,extensive invasive growth and highly invasive,it makes hard for surgical microscope nerve surgery removed completely.At the same time,the therapeutic method of radiotherapy and chemotherapy is not sensitive to GBM cells and the survival estimates for GBM are very poor.With the development of medical biology,molecular target therapy specificity for cell receptors has caused the extensive concern.Gefitinib(brand name Iressa),a specific small molecule inhibitor of EGFR,has been shown to possess exert therapeutic effect on these highly aggressive brain tumors via inhibiting proliferation and inducing apoptosis of tumor cells and it is reported that EGFR gene is generally amplified and/or overexpressed in high-grade glioblastoma multiforme.However,multiple studies have demonstrated that as a single agent for the therapy of human glioblastoma multiforme,gefitinib is limited to its frequent drug resistance and the serious cytotoxicity in clinical trials.Elemene,a novel anticancer agent,is the major active component extracted from the traditional Chinese medicinal plant Curcuma wenyujin.The significant effect of elemene is mainly caused by its ability to pass through the blood-brain barrier(BBB)and reverse the resistance of glioblastoma multiforme to other drugs.In the present study,we will further study to the synergy therapy of gefitinib and elemene in human GBM cells with low toxicity and few side-effects.MethodsThis study selected the important role of high malignant degree of glioblastoma cell line.Two different GBM cell lines U251 and U87-MG from human were cultured in vitro.The treatment set up blank control,gefitinib alone,elemene alone and the combination treatment group for this experiment.On the functional study we used MTT and colony forming assay to evaluate the proliferation and survival of GBM cells.The mechanism experiment research found that the expression of the proteins was analyzed by western blot.And the other related change conditions of marker proteins in GBM cells by Immunofluorescence and transmission electron microscopy.Results(1)The treatment determined by MTT and clone formation experiments shown:the combination index Cl value with elemene and gefitinib two drugs:the value of U251 is 0.53 and U87-MG is 0.78.Elemene increases the inhibitory effects of gefitinib on the proliferation and survival of U251 and U87-MG(2)Western blot experiments shown:elemene enhances the sensitivity of U251 and U87-MG to gefitinib via down regulating the activation of EGFR,AKT and ERK signaling.(3)Western blot experiments shown:Elemene enhances the induction effects of gefitinib on the apoptosis of U251 and U87-MG through down-regulating caspase-3,PARP.(4)Immunofluorescence and electron experiment shown:the combination treatment of elemene and gefitinib producing more autophagosome.Elemene increases the occurrence of autophagy induced by gefitinib in U251 and U87-MG(5)Effects of elemene and gefitinib combined on autophagy related protein Beclinel in U251.ConclusionIn this study,we first time discussed that a combination therapy regimen with the traditional Chinese medicine anti-cancer drugs elemene and the molecular targeted drugs gefitinib.We found that elemene not only increased the inhibitory effects of gefitinib on the proliferation and survival of GBM cells through the inhibition of the EGFR signaling pathway but also induced more distinct apoptosis and autophagy in the GBM cells.Above all,elemene could enhance the chemosensitivity of GBM cells to gefitinib and it might be one potential adjuvant to enhance the effect of EGFR inhibitor and reduce the resistance of gefitinib in GBM.