Mechanism Of Transforming Growth Factor β1-connective Tissue Growth Factor Signaling Axis In Mediating Glioma Temozolomide Chemoresistance

BackgroundGlioblastoma(GB)is one of the most common primary malignant tumors in central nervous system.Though the surgical-based combination of radiation and chemotherapy treatment has become a comprehensive guide,the prognosis of GB patients has not significantly improved yet.Among these treatments,temozolomide(TMZ),the second-generation oral alkylating agent,is the first-line chemotherapy of malignant glioma.However,during the TMZ clinical application,the inherent or acquired TMZ resistance of glioma seriously restricted the efficacy of TMZ chemotherapy,which is one of the main reason in glioma chemotherapy failure.Connective tissue growth factor(CTGF)is one kind of cysteine-rich secreted peptides and its abnormal expression is associated with tumor drug resistance.However,its specific regulatory mechanisms in glioma TMZ chemoresistance is not yet clear.Based on preliminary research,we will explore in-depth on molecular network of CTGF mediated TMZ chemoresistance and further enrich the molecular regulatory mechanisms of glioma chemoresistanc,which may provide a theoretical basis for elucidating the TMZ chemoresistance mechanisms and clinical reversal.Main research contentObjectiveFirst,to identify the role of CTGF in the process of glioma TMZ resistance.Second,to confirm CTGF-mediated TMZ resistance is through stem-like properties acquisition in glioma.Third,to elucidate the molecular mechanism of TGF-β1 signal activation in CTGF mediated TMZ chemoresistance in glioma.MethodsThe correlation between CTGF expression and glioma resistance was analyzed by immunohistochemistry,qRT-PCR and Western blot.The shRNA interferance or lentivirus overxpression were used to investigate the effect of CTGF expression on TMZ chemoresistance in glioma.The relationship between CTGF and tumor stem properties expression was detected with qRT-PCR,tumorsphere formation,CD44 flow cytometry assays.ELISA,qRT-PCR and Western blot were applied to analyze the molecular mechanism of TGF-β1 signal activation in CTGF mediated TMZ chemoresistance in glioma.Results1)The cell survival assay showed that the IC50 value of TMZ resistant glioma cells was between 1.5-2.0mM with higher drug resistant properties than its parental cells(1.0mM).The qRT-PCR and Western blot analysis revealed that the drug resistant phenotype gene and protein expression of ABCB1,ABCC,BCRP and MGMT were significantly increased in glioma TMZ-resistant cells.2)The microarray analysis and screening of the TMZ resistant cells showed that CTGF was significantly upregulated in glioma TMZ resistant cells,and further clinical glioma tissue sample analysis confirmed that the expression of CTGF was positively correlated to the glioma tumor grades and tumor drug resistance.3)In vitro TMZ treatment promoted CTGF expression in glioma cells with a does and time dependent manner.4)CTGF downregulation inhibited glioma cell cloning ability,improved TMZ chemosensitivity and enhanced TMZ-induced apoptotic C-PARP protein expression.The subcutaneous tumor xenograft model found that CTGF interferance inhibited glioma growth and tumor formation.5)CTGF overexpression enhanced glioma cell clone formation ability and restored the clone formation ability after TMZ pretreatment,promoted glioma TMZ chemoresistance and decreased TMZ-induced apoptosis.In addition,overexpressed CTGF promoted glioma cell growth,downregulated the C-PARP expression and enhanced drug resistance to TMZ chemotherapy in in vivo subcutaneous tumor model.6)CTGF overexpression significantly upregulated the expression of tumor sternness related gene ALDH1,CD44,Nestin and Nanog,and promoted the ability of tumorsphere formation,increased the percentage of CD44+ cells and CD44 protein expression.Furthermore,co-transfected siCD44 inhibited the ability of tumorsphere formation and reversed the CTGF-mediated TMZ chemoresistance in glioma.7)ELISA and qRT-PCR analysis showed and confirmed that TGF-β1 was elevated in glioma cells after TMZ treatment and with a does dependent manner.Immunofluorescence showed that there was co-expression of TGF-β1 and CTGF by TMZ treatment.With the TGF-β1 neutralization or inhibition,the TMZ-induced CTGF elevation was significantly reversed while the addition of recombinant growth factor rTGF-β1 promoted CTGF expression in glioma cells.8)TMZ treatment promoted the phosphorylation of Smad3 and ERK1/2 signals,and the si-Smad3 or si-ERK1/2 downregulated CTGF expression and reversed overexpressed CTGF-mediated TMZ resistance.In addition,in vivo study showed TMZ promoted glioma chemoresistance,which closely related to TGF-β1-Smad3/ERK1/2-CTGF signal pathway activation.Conclusions1)The TMZ resistant cell model and clinical tissue samples confirmed that CTGF expression was closely related to the glioma resistance.2)In vitro TMZ treatment promoted CTGF expression in glioma cells with a does and time dependent manner.CTGF downregulation enhanced TMZ sensitivity and TMZ induced apoptosis while CTGF overexpression promoted TMZ chemoresistance in glioma.The cell apoptotic modulation was involved in CTGF-mediated TMZ chemoresistance in glioma.3)CTGF overexpression promoted tumor sternness properties and targeting tumor stemness properties modulated CTGF-mediated TMZ resistance in glioma.4)TGF-β signaling activation promoted TMZ-induced CTGF upregulation and Smad3 and ERK signaling were involved in the activation of TMZ-induced TGF-p/CTGF axis in glioma TMZ chemoresistance.