Baicalein Inhibits MGMT Expression In Glioblastoma By Modulating Wnt/β-catenin Pathway

Background and objectiveGlioblastoma is an intracranial primary astrocytoma with high malignancy and poor prognosis.Temozolomide,as the current first-line postoperative chemotherapeutic drug,has little effect on MGMT-positive glioblastomas.This clinical status is closely related to the temozolomide resistance induced by MGMT positive expression.The study is to investigate the inhibitory effect of baicalein in vitro on the expression of MGMT protein in human glioblastoma T98 G and GBM-X cells and the related signaling pathways.MethodsWe have found 18 kinds of Chinese medicine monomers that may inhibit the growth of tumor and affect the expression of MGMT in glioblastoma cells through literature review.Western blot was used to preliminarily screen for the Chinese medicine monomer,baicalein,which has the most significant inhibition of MGMT in glioblastoma cells.We have selected MGMT highly positive experimental cells,T98 G and GBM-X cells,from the purchased cell lines and the cultured primary cells.The expression of MGMT protein and related signaling pathway proteins after 72 hours of baicalein treatment were detected by Western blot and the localization of MGMT protein in glioblastoma cells was observed by immunofluorescence technique.The transcription level of MGMT mRNA was detected by reverse transcription polymerase chain reaction.Then we detected the cytotoxicity of baicalein,temozolomide and combined use of two drugs by CCK-8 test to explore the possibility of baicalein and temozolomide combination therapy.ResultsUnder normoxia/hypoxia condition,the expression of T98 G cells MGMT protein in the baicalein treatment group(0.757±0.058,0.714±0.049)was significantly lower than that of the control group(1.000±0.101,1.000±0.072)(P<0.05,P<0.01)and compared with the control group(1.000±0.031,1.000±0.082),the expression of MGMT protein in GBM-X cells in the experimental group(0.843±0.027,0.697±0.052)decreased significantly(P<0.01,P<0.01).As compared to control group,the expression level of TCF1/TCF7,LEF1 and Survivin proteins reduced significantly after baicalein treatment in T98 G and GBM-X cells.The results of immunofluorescence showed that MGMT protein was mainly expressed in the nucleus of glioblastoma cells and baicalein did not change the intracellular localization of MGMT protein.There was no significant difference via CCK-8 test in T98 G and GBM-X cells between the temozolomide group and the control group after 24 h,48h and 72 h.However,with the difference statistically significant,the value of CCK-8 in baicalein and temozolomide combination group was significantly lower than that of the control group.ConclusionMGMT protein is mainly expressed in the nucleus of glioblastoma cells.Baicalein can stably inhibit the expression of MGMT protein but does not change the intracellular localization of MGMT protein.Temozolomide has no obvious killing effects on MGMT-positive glioblastoma cells,but combined baicalein could reverse temozolomide resistance of glioblastoma cells and significantly enhance the cytotoxic effects of temozolomide.Although baicalein inhibits MGMT protein expression,it does not affect MGMT mRNA expression level.The decrease of MGMT protein expression which is closely related to the abnormal expression of Wnt/β-catenin /MGMT/Survivin signaling pathway is related to the decrease of its upstream TCF/LEF protein and could lead to the decrease of apoptosis-related Survivin protein expression.