The Role Of Luteolin In Glioblastoma And Its Mechanisms

Glioblastoma multiforme(GBM)is the most common and lethal malignant primary brain tumor.Even patients take the most aggressive treatment,they are challenged by poor feedback to surgery,vulnerable relapse and short survival time,which result from the high degree of proliferation,invasion and resistance to chemotherapy and radiotherapy.Therefore,new therapeutic strategies should be developed to inhibit the recurrence of cancer and improve the prognosis of GBM patients,luteolin is a common flavonoid that exists in many types of plants.What’s more,luteolin was shown to be therapeutically active against several solid tumors in vitro and in vivo.In lung cancer cells,the luteolin can inhibit the expression of Nrf2 effectively and selectively and inhibit the growth of solid tumor in nude mice and improve the sensitivity to chemotherapy.The insulin-like growth factor 1(IGF-1)is overexpressed in several tumors,which can regulate the apoptosis of cancer cells.In addition,the abnormal activation of PI3K/AKT/mTOR signaling pathway plays a vital role in tumorigenesis and caner development.A lot of researches have shown this pathway is inextricably linked with the biological behavior of cancer cell,such as invasion,migration,apoptosis and chemotherapy resistance.So in our study,we used U251MG and U87MG human glioblastoma cell lines as in vitro model and the nude mice for in vivo to study the anti-GBM effect of luteolin.We examined the effects of luteolin on invasion and migration,apoptosis in glioblastoma both in vitro and in vivo and in U251MG temozolomide-resistant cell line.What’s more,we studied the role of IGF-1R/PI3K/AKT/mTOR signaling pathway in luteolin-induced biological behavior changes in glioblastoma.This thesis in divided into five parts:Part ⅠThe effect of luteolin on the migration of glioblastoma cell lines.Objective:To explore whether luteolin could inhibit the migration of glioblastoma cell lines.Methods:Glioblastoma cells were seeded into 96-well plates and treated with luteolin at different concentrations(0.5,10,20.40,80μmol/L)for 24h,and the cell proliferation was tested by CCK-8 assay.We used scratch-induced migration assay to detect the effects of luteolin on the migration of U251MG and U87MG cell lines.The protein levels of MMP-2,MMP-9,TIMP-1,TIMP-2 were analyzed in U251MG and U87MG treated with luteolin(0,5,10,20μmol/L)for 24h using western blot.We also tested the protein levels of EMT associated proteins,vimentin,β-catenin,N-cadherin and E-cadherin.We used the rhodamine-conjugated phalloidin to test the cytoskeleton.Results:We choose a concentration range of luteolin lower than 40μM and 24h to determine the related effects on glioblastoma cells.The wound healing assay showed that luteolin significantly decreased the migration ability of glioblastoma cells in concentration-and time-dependent manners.In addition,luteolin significantly decreased the protein levels of MMP-2 and MMP-9,while up-regulated the expression of TIMP-1 and TIMP-2 in a concentration-dependent manner.What’s more,The treatment with luteolin was accompanied by extreme alterations in cell morphology,which attributed to reorganization of the actin cytoskeleton.Conclusion:luteolin could inhibit glioblastoma cell migration.Part ⅡThe effect of luteolin on the apoptosis in glioblastoma cells.Objective:To test the effect of luteolin on apoptosis in glioblastoma cells.Methods:Establish glioblastoma U251MG and U87MG cell line models and primary endothelial cell model.Cells were seeded into 96-well plates and treated with luteolin at different concentrations(0.5,10,20,40,80μmol/L)for 24h,and the cellproliferation was tested by CCK-8 assay.Then we determined cell apoptosis by flow cytometry followed by Annexin V/PI staining after treated with different concentrations for 24h.We also tested cell apoptosis of glioblastoma and xenograft tumor by TUNEL staining.We tested the level of cleaved-caspase-3 by immunofluorescence and western blot.Results:Luteolin could induce cell apoptosis in glioblastoma cell lines and up-regulate the expression of cleaved-caspase-3.Luteolin treatment led to a notable inhibition of tumor growth without obvious affects on body weight and activation of cleaved-caspase-3.Conclusion:Luteolin could induce apoptosis of glioblastoma cells and inhibit the growth of glioblastoma in vivo.Part ⅢThe role of endoplasmic reticulum stress on the pro-apoptosis effect of luteolin.Objective:To explain the role of endoplasmic reticulum stress on the pro-apoptosis effect of luteolin.Methods:We tested the ROS level of glioblastoma cells after the treatment of luteolin by fluorescence microscopy.The protein levels of ROS associated proteins Nrf2,NQO-1 and HO-1 were analyzed in U251MG and U87MG treated with luteolin(0,40,80μM)for 24h using western blot.We tested endoplasmic reticulum stress proteins p-PERK,p-eIF2α,ATF4,CHOP and Cleaved-caspase-12 level after treating with different concentrations of luteolin(0,5,10,20,40,80μmol/L)for 24h and 40μmol/L at different times(0,1,3,6,12,24h).In addition,cells were exposed to 40μM of luteolin for 24h,and we used JC-1 staining to test the mitochondrial membrane potential.We tested the level of cytochrome c and Bcl-2,Bax by immunofluorescence and western blot.We also tested protein level of ATF4,CHOP and Cleaved-caspase-12 of xenograft tumor by western blot.Results:Luteolin could induce cell ROS level in glioblastoma cell lines,and reduce the protein level of Nrf2,NQO-1 and HO-1.What’s more,the protein level of p-PERK,p-eIF2α were highest after 40μM treatment for 6h,while the level of CHOP,Cleaved-caspase-12 and Cleaved-capase-3 were highest at 24h.In addition,the protein level of ERS-associated protein were up-regulated in a concentration-dependent manner in vitro and in vivo.The mitochondrial membrane potential and anti-apoptosis protein Bcl-2 decreased,while the express of cytochrome c and pro-apoptosis Bax increased.Conclusion:Luteolin-induced ROS increases ER stress and mitochondrial dysfunction,which contributes to luteolin lethality in glioblastoma cells.Part ⅣThe role of p-IGF-1Rβ/PI3K/AKT/mTOR signaling pathway in luteolin-induced changes of glioblastoma biological behavior.Objective:To explore the role of p-IGF-1Rβ/PI3K/AKT/mTOR signaling pathway on anti-glioblastoma effect of luteolin.Methods:Cells were exposed to different concentrations of luteolin for 24h,and the expression levels of p-IGF-1Rβ,p-AKT,AKT,p-mTOR,mTOR were detected by western blot.Cells were treated with 100ng/ml IGF-1 after treatment with 20μM or 40μM luteolin,then we tested the expression of MMP-2,MMP-9,TIMP-1,TIMP-2,E-cadherin,Vimentin and Cleaved-caspase-2 by western blot.Results:Luteolin treatment obviously decreased phosphorylation of IGF-1Rβ,AKT.mTOR in concentration manners.Cells were treated with 100ng/ml IGF-1 after treatment with 20μM or 40μM luteolin,the increased expression of TIMP-1,TIMP-2,E-cadherin and Cleaved-caspase-3 were decreased,while the inhibited expression of MMP-2,MMP-9,Vimentin were up-regulated.Conclusion:Luteolin induced apoptosis and inhibition of migration in glioblastoma cells were partly via the p-IGF-1Rβ/PI3K/AKT/mTOR signaling pathway.Part ⅤThe effect of luteolin on U251MG temozolomide resistance cell line.Objective:To test the effect of luteolin on apoptosis in established glioblastoma temozolomide resistance cells and rescue the sensitivity to temozolomide.Methods:We observed the shape of U251MG and U251MG-TR cells by inverted microscope,and tested the IC-50 to temozolomide.The mRNA and protein level of multidrug resistance-associated protein 3(MRP-3)were determined by QRT-PCR and western blot.Cells were seeded into 96-well plates and treated with luteolin at different concentrations(0,5,10,20,40,80μmol/L)for 24h,and the cell proliferation was tested by CCK-8 assay.Then we determined cell apoptosis by flow cytometry followed by Annexin V/PI staining after treating with different concentrations for 24h.We tested the protein level of Cleaved-caspase-3,Bcl-2 and Bax by western blot.The protein level of Nrf2 was tested by western blot and immunofluorescence,and the mRNA and protein level of Nrf2,NQO-1 and HO-1 were determined by QRT-PCR and western blot.The cell viability of U251MG-TR was tested by CCK-8 after knock-down the Nrf2 by lentivirus.CCK-8 assay was used to test the effect on U251MG-TR cell with treatment both luteolin and temozolomide,and the pro-apoptosis effect was tested by flow cytometry.The protein level of Cleaved-caspase-3 and Bax were tested by western blot.Results:The U251MG-TR cell had characteristic changes,more larger and growth into group.What’s more,the MRP-3 mRNA and protein level of U251MG-TR increased.The resistance index of U251MG-TR cell was about 9.luteolin could inhibit the growth of U251MG-TR.which was more sensitive than U251MG cell.luteolin could induce the apoptosis of U251MG-TR,and up-regulate the protein level of Cleaved-caspase-3 and Bax and decrease the level of Bcl-2.In addition,the mRNA and protein level of Nrf2,NQO-1 and HO-1 were up-regulated in U251MG-TR cell.When we knock-down the expression of Nrf2,the viability of U251MG-TR-Nrf2i was higher than that of U251MG-TR after the same concentration of luteolin.At last,luteolin treatment could rescue the sensitivity to temozolomide of U251MG-TR.Conclusion:Luteolin.could induce apoptosis of U251MG temozolomide resistance cell line and rescue the sensitivity to temozolomide.SummaryOur results provided evidence that luteolin inhibited the migration glioblastoma cells via p-IGF-1Rβ/PI3K/AKT/mTOR signaling pathway,and induced apoptosis by prolonged activated endoplasmic reticulum stress in vitro and in vivo.What’s more the U251MG-TR cell was sensitive to luteolin,which might associated with up-regulated Nrf2.In addition,luteolin could rescue the sensitivity to temozolomide of U251MG-TR cell line.These results make luteolin an attractive therapeutic agent for developing alternative treatment protocols,and for combining with other chemotherapy drugs to overcome resistance and achieve better outcomes.