Experimental Study On The Inhibitory Effect Of Natural Plant Compound Alantolactone On Glioblastoma

Objective:The malignant degree of glioblastoma(GBM)is high and the prognosis is very poor.Alantolactone(ATL)is a natural plant compound of hemiterpene lactones,which has stable metabolism and few toxic and side effects in vivo and has potential anti-tumor activity.The first part of the experiment was to investigate the effect of ATL on the proliferation and growth of GBM and to explore the possible molecular mechanisms.It also verified whether ATL could penetrate the blood brain barrier.The second part was to study the effect of ATL on the migration,invasion and apoptosis of GBM,and to explore the molecular mechanism and regulatory target of ATL.Methods:In the first part ofexperiment,the cell viability of human neural tumor cells U87,U251,U118,SH-SY5 Y and SVG p12 of normal human glial cells were detected by MTT assay.After ATL treatment of U87 and U251 cells,cell proliferation was detected by cell colony formation assay,cell cycle was detected by flow cytometry,and Western Blot was used to detect the expression of key proteins regulating cell cycle progression.In the further study of molecular mechanism,the nuclear localization and interaction between transcriptional co-activator p300 and NF-κB p50/p65 and their binding to COX-2 promoter were analyzed by Western blotting assay,PCR,confocal immunofluorescence,DNA-protein binding by streptavidin-agarose pulldown assay and chromatin immunoprecipitation assays after treatment with ATL.In the study of the target of ATL,we used in vitro kinase experiment and molecular docking method to verify the interaction between ATL and Ikkβ.In in vivo experimental studies,allogeneic nude mice were used to study the effects of ATL on tumor growth in vivo.The effect of ATL on the expression of key proteins in molecular mechanism was detected by immunohistochemistry.In addition,we detected whether ATL could penetrate the bloodbrain barrier.We injected ATL into the abdominal cavity of rats,and detected the content of ATL in the cerebrospinal fluid by liquid chromatography-mass spectrometry.In the second part ofexperiment,scratching experiments and Transwell chamber experiments were used to detect the migration and invasion of human glioblastoma U87 and U251 cells treated with different concentrations of ATL.In the study of molecular mechanism,the protein expression of MMP-2 and MMP-9,the activation of Cofilin and the ratio of G-actin and F-actin were studied by Western Blot and confocal immunofluorescence.On the other hand,flow cytometry was used to detect the number of apoptotic cells in U87 and U251 cells treated with different concentrations of ATL.In the molecular mechanism studies,we investigated the translocation of mitochondria from Cofilin and Actin,the release of cytochrome C,and the expression of key proteins in Caspase cascade by Western Blot,confocal immunofluorescence and protein co-immunoprecipitation.In the study of ATL targeting the LIMK enzyme that regulates Cofilin,we used the method of pretreatment of specific inhibitor LIMKi to detect the changes of migration and invasion ability and apoptosis respectively.In vivo study,Western Blot and immunohistochemistry were used to detect the effect of ATL on the expression of p-cofilin and p-LIMK1/2 protein in nude mice bearing tumor.Results:Part one: ATL can significantly inhibit the proliferation and growth of GBM cells.Its possible molecular mechanism is that ATL acts on the ATP binding site of Ikkβ and restricts its phosphorylation,thereby inhibiting the nuclear transposition of NF-κB,hindering NF-κB binding to COX-2 promoter region and P300 recruitment,affecting COX-2 gene expression,and inhibiting the growth and proliferation of GBM.ATL can also reduce the protein expression of Cyclin D1 and CDK4,and block the cycle cycle of GBM cells in G0/G1 phase.In vivo experiments also demonstrated that ATL could significantly reduce the protein expression level of COX-2 and p-p65 in the allogeneic tumor bearing tissue.In addition,the detection of cerebrospinal fluid showed that ATL was able to penetrate the bloodbrain barrier.Part two: On the one hand,ATL can significantly inhibit the migration and invasion ability of GBM cells.The molecular mechanism may be that ATL can activate Cofilin activity and up-regulate the ratio of G-actin and F-actin.On the other hand,ATL can significantly induce the apoptosis of GBM cells.The molecular mechanism may be ATL can activate Cofilin activity,prompting Cofilin and G-actin co-translocation to the mitochondria,mediating the release of cytochrome C to the cytoplasm,thereby initiating Caspase cascade signaling pathway to induce apoptosis.In the regulation of target research,we found that ATL activates Cofilin by targeting inhibition of LIMK enzyme activity.In vivo experiments,we found ATL can significantly down-regulate the expression of p-cofilin and p-LIMK1/2 in allogeneic tumor-bearing tissues.Conclusions:In the first part of the experiment,ATL was found to exert anti-GBM cell proliferation and growth effects through targeted inhibition of Ikkβ enzyme activity,interference with NF-κB / COX-2-mediated signaling pathway,and arrest of cell cycle.In addition,through living experiments,it is proved that ATL can penetrate the blood brain barrier,which provides potential for the application of ATL in the treatment of nervous system diseases.In the second part of the experiment,we found that ATL cansuppress the migration and invasion of GBM and induce the apoptosis of GBM by interfering with the LIMK/cofilin signaling pathway.The results of this study indicate that ATL has potential application in the anti-GBM migration and invasion and induction of apoptosis.In conclusion,combining these two parts of the experimental results,it is confirmed that there are many ways and targets to play the role of anti glioblastoma,providing powerful experimental data for its clinical transformation and application.