| Background:Glioma is the most common and aggressive tumor in brain and other central nervous system.It is characterized by high incidence rate,high recurrence rate,high malignancy,high invasiveness,difficult treatment,poor clinical prognosis and high mortality.Temozolomide(TMZ)is a first-line chemotherapy agent for the treatment of glioma patients.TMZ can inhibit the proliferation and induce apoptosis of glioma cells by alkylating the DNA of glioma cells,which can effectively play an important role in improving the prognosis of glioma patients.However,many glioma patients develop intrinsic or acquired resistance to TMZ,which is the reason for the failure of TMZ treatment in glioma patients,and TMZ resistance significantly shortens the progression free survival(PFS)and overall survival(OS)of these patients.Therefore,there is an urgent need to develop treatment strategies for TMZ resistant glioma patients.O6-methylguanine-DNA methyltransferase(MGMT),which can repair the DNA damage caused by TMZ treatment,is one of the main reasons why gliomas develop resistance to temozolomide.Chlorogenic acid(CGA)is an active polyphenol extracted from plants,which has anti-tumor effects against a variety of tumor types,including glioma.It can reverse the drug resistance of multidrug-resistant lymphoma and enhance the cytotoxicity of Adriamycin in osteosarcoma.However,its direct anti-tumor effect against glioma and whether it improves chemotherapy sensitivity has not been reported yet.Therefore,further exploration of the role and mechanism of chlorogenic acid on glioma and key regulatory molecules/proteins is expected to lay a foundation for the pathogenesis of brain glioma and brand-new therapeutic strategies.Objective:This study intends to explore whether chlorogenic acid has a direct therapeutic effect on glioma cells and whether chlorogenic acid has a synergistic effect with temozolomide treatment.This study proposed a new hypothesis that chlorogenic acid may enhance the chemosensitivity of glioma cells to temozolomide by regulating the expression of MGMT,and further studied the key pathways and protein molecules involved.Method:In this study,bioinformatics was used to analyze the comprehensive role of MGMT in gliomas.The effect of CGA on the viability of glioma cells was detected by CCK-8 assay.The effect of CGA on the proliferation of glioma cells was observed by cell colony formation assay.The effect of CGA on the migration and invasion of glioma cells was evaluated by Transwell assay and scratch assay.The effects of CGA on cell cycle and apoptosis of glioma cells were analyzed by flow cytometry.The changes of MGMT transcription levels in glioma cells were analyzed by Reverse transcriptionq PCR(RT-q PCR).The effects of MGMT and β-catenin on CGA-induced glioma cell death and reduction of temozolomide resistance were studied by cell transfection method.The expression levels of related protein molecules in glioma cells and human glioma subcutaneous transplantation model tissues in nude mice were detected by Western Blot.The expression levels of related protein molecules in human glioma subcutaneous transplantation model tissues in nude mice were observed by immunohistochemical staining.The role of CGA in vivo experiment was verified by establishing human glioma subcutaneous transplantation model in nude mice.Results:1.Bioinformatics analysis found that the high expression of MGMT was negatively correlated with the prognosis of glioma patients,and there were many highly correlated pathways affecting glioma treatment that was worth studying.2.The results of CCK-8 assay showed that CGA could effectively reduce the viability of glioma cells,and had little effect on the viability of normal cells.The results of cell colony formation assay showed that CGA could inhibit the proliferation of glioma cells.Transwell assay and scratch assay showed that CGA could inhibit migration and invasion in glioma cells.The results of flow cytometry showed that CGA could induce apoptosis and cell cycle arrest of glioma cells.Western Blot showed that the expression of Cleaved PARP-1,Cleaved Caspase-3 and Bax proteins increased in glioma cells under the action of CGA.At the same time,the expression of Bcl-2 and Cyclin B1 protein decreased.3.The results of RT-q PCR and Western Blot showed that CGA can inhibit the MGMT transcription levels and expression levels in glioma cells.At the same time,the combined application of CGA and TMZ has a synergistic effect in glioma cells.Overexpression of MGMT in glioma cells can reverse the synergistic effect of CGA and TMZ in glioma cells.Gene silencing β-catenin can inhibit the expression of MGMT m RNA and protein expression in glioma cells,and enhance the chemotherapeutic sensitivity of glioma to TMZ.4.CGA can inhibit the expression of p-GSK-3β and β-catenin protein in glioma cells,decrease MGMT expression,reduce cell proliferation activity,induce apoptosis and enhance the chemotherapeutic sensitivity of glioma to TMZ.GSK-3β Inhibitor(CHIR-99021)or overexpression of β-catenin can reverse the effect of CGA above.5.CGA can upregulate the expression of PTEN protein in glioma cells,inhibit the expression of p-AKT,p-GSK-3β,β-catenin and MGMT protein,reduce cell proliferation activity,induce apoptosis and enhance the chemotherapeutic sensitivity of glioma to TMZ.PTEN inhibitor(SF1670)can reverse the effect of CGA above.6.In vivo,CGA can inhibit tumor growth and reduce the expression of p-AKT,pGSK-3β,β-catenin and MGMT protein in human glioma subcutaneous transplantation model in nude mice.Compared to CGA or TMZ alone,the combination of CGA and TMZ significantly reduced tumor volume in human glioma subcutaneous transplantation model in nude mice.Conclusion:1.Chlorogenic acid can inhibit the proliferation of glioma cells in vivo and in vitro and enhance the chemotherapeutic sensitivity of glioma to TMZ.2.Chlorogenic acid enhances the chemotherapeutic sensitivity of glioma to TMZ by inhibiting the MGMT transcription levels and expression levels in glioma cells.3.Chlorogenic acid regulates MGMT expression by inhibiting Wnt/β-catenin pathway.4.Chlorogenic acid inhibits PI3K/AKT pathway by upregulating PTEN,further affecting Wnt/β-catenin pathway. |
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