| ObjectiveGlioma is the most common primary intracranial malignant tumor,accounting for 80%of intracranial tumors,among which glioblastoma(GBM)accounts for 60%of adult intracranial tumors.The prognosis of glioma patients is very poor due to the high proliferation,invasion and clonal heterogeneity of glioma cells.At present,the classic treatment mode of glioma is surgical resection within the maximum safe range,supplemented with temozolomide(TMZ)chemotherapy and radiotherapy.The median overall survival time of glioma patients receiving systematic treatment is 14-15 months,while the survival time of patients with no treatment is only 3-4 months.After clinical diagnosis,most glioma patients lose the opportunity of surgery due to the large tumor volume,obvious edema,complex location and injury of multiple brain functional areas.At this time,temozolomide(TMZ)chemotherapy and radiotherapy often become the main choice of treatment.Temozolomide,as a first-line chemotherapy drug(alkylating agent)for glioma,presents a cytotoxic effect by inducing base mismatch,DNA single strand/double strand breaks and cycle arrest in glioma cells.Although temozolomide can prolong the survival time and improve the prognosis and life quality of patients with glioma,with the increase of drug dosage and time,the proportion of glioma patients with resistance to temozolomide is also significantly increased,which is also an important reason for the rapid progress of patients with glioma(especially glioblastoma)in clinical late stage.Chemotherapy resistance of glioma is still a huge challenge for neurosurgery.Recently,there are more and more studies on plant herbs in the field of anti-tumor.As a commonly used clinical medicine in traditional Chinese medicine,saffron has the effects of hemostasis,pain elimination,nourishing essence,cooling blood and detoxification.Saffron acid is the main active component of saffron.Nowadays,more and more researches have proved that saffron acid can induce apoptosis of a variety of tumor cells(cervical cancer,breast cancer,colon cancer,etc.)and play an anti-cancer role in tumorigenesis.In addition,the effects of crocetin on tumor chemosensitivity and anti-inflammation has also been reported.At present,there is rare study of crocetin in gliomagenesis.In view of its characteristics of promoting apoptosis and enhancing chemoresistance in a variety of tumors,and considering its own characteristics of high blood brain barrier(BBB)permeability,this study aims to explore the role of crocetin in the malignant progression of glioma and its molecular mechanism in reducing temozolomide resistance.MethodsResearch Part Ⅰ:Effect of crocetin on proliferation,apoptosis and malignant phenotype of glioma cellsU251/LN229 glioma cell lines and HA 1800 normal human astrocyte line were stimulated by a series of different concentrations of crocetin,CCK8 assay was used to detect the cell viability of glioma cells and normal astrocytes stimulated by crocetin at different concentrations,and fitted the cell optical density(OD)of each concentration group on 48h to determine the half maximal inhibitory concentration(IC50)of crocetin in glioma cells and normal astrocytes.The concentration of crocetin used in this study was determined by IC50 value.200μMol/L、400μMol/L crocetin were used to stimulate U251/LN229 glioma cells,colony formation assay and Transwell assay were performed to vertify the effects of different concentrations of crocetin on the proliferation and invasion ability of glioma cells.Flow cytometry was carried to exam the effect of different concentrations of crocetin on apoptosis of glioma cells.Western blotting was used to detect the expression changes of N-cadherin,E-cadherin and Vimentin protein(EMT markers)levels,so as to verify the changes of epithelial mesenchymal transformation(EMT)process of glioma cells under different concentrations of crocetin.Research Part Ⅱ:Explore the synergistic anti-tumor mechanism of crocetin and temozolomide on gliomagenesis via FOS-DUSP1 signal axisBased on the combine bioinformatic analysis via GEO and TCMSP database,FOS,the drug target of crocetin,was screened,and the molecular docking was performed by SailVina to verify the crocetin and its drug target.Bioinformatics analysis of the correlation between FOS and its downstream related gene DUSP1 were performed in different glioma-related microarrays(GSE4290,GSE16011,GSE109857).The expression of FOSmRNA and DUSP1 mRNA in multiple glioma cell lines was verified by qRT-PCR,and the correlation between them was analyzed.FOS overexpression and FOS knockdown glioma cell lines were constructed.Western blotting was used to verify the changes of downstream gene DUSP1 expression after FOS pertubation.U251/LN229 glioma cells were stimulated by20μMol/L、400μMol/L crocetin,qRT-PCR,western blotting,and cellular immunofluorescence(IF)assays were performed to detect the changes of FOS-DUSP1 signal axis at mRNA and protein levels.Knocking down or overexpress FOS in glioma cells respectively,and then give 200μMo1/L、300μMol/L、400μMol/L crocetin stimulation,CCK8 experiment verified the change of crocetin toxicity on glioma cells after FOS disturbance.FOS overexpressed U251/LN229 cells were stimulated by 400μMol/L temozolomide,then,CCK8 assay was carried to exam the influence of FOS expression changes on the sensitivity of glioma cells to TMZ;Cellular immunofluorescence assay was performed to verify the changes of γ-H2AX(markers of DNA damage)expression.Flow cytometry was used to verify the changes of cellular apoptosis rate of after receiving the combine strategy(crocetin combined with temozolomide).Subsequently,Western blot was used to verify the changes of FOS-DUSP1 signal axis under different therapeutic strategy.Part Ⅲ:Crocetin down-regulates FOS-DUSP1 axis to alleviate the TMZ induced-chemotherapeutic resistance in glioma cellsThe TMZ related resistance genes FOS/DUSP1 were identified by differential gene analysis based on the temozolomide-resistance microarrays in glioma.Knocking down DUSP1 in FOS empty vector cell lines and FOS overexpression cells lines respectively,meanwhile,a series of concentration of TMZ were used to stimulate glioma cells above,then,CCK8 experiment was performed to explore the effect of FOS-DUSP1 signal axis on the resistance to TMZ toxicity in glioma cells.Colony formation assay further verified the cell viability under the above treatment.Inducing culture glioma drug resistant cell line(Insensitive_TMZ200-U251/LN229)under 200μMol/L TMZ concentration,then,IC50 and drug resistance index were measured.Western blotting was used to detect the change of FOS/DUSP1 expression in TMZ drug resistant cell lines.400μMol/L TMZ and DMSO treatment were given to TMZ200-U251/LN229 cell line,RT-qPCR and immunofluorescence assay verified whether the up-regulation of FOS-DUSP1 axis was stable in drug-resistant glioma cell lines.Flow cytometry assay was carried to detect the apoptosis rate in TMZ resistant glioma cells(Insensitive_TMZ200-U251/LN229)after receiving 200μMol/L Crocetin combined with the 400μMol/L temozolomide.The subcutaneous tumorigenesis experiment in nude mice further verified the synergistic effect of crocetin+TMZ strategy and its effect on FOS-DUSP1 signal axis in vivo.Part Ⅳ Expression of FOS/DUSP1 in patients with glioma and its clinical prognostic significance:Bioinformatic analysisBased on bioinformatics analysis,the expression of FOS/DUSP1 mRNA in glioma samples of different grades was demonstrated.Collect 70 glioma samples of different grades and 12 normal brain tissue samples.Immunohistochemical staining assay and western blotting assay were used to verify the expression of FOS/DUSP1 in glioma samples of different grades.Based on TCGA/CCGA/CCLE database,FOS/DUSP1 related Kaplan-Meier survival analysis were performed to predict the prognosis of patients with glioma.Results:Part Ⅰ:Crocetin inhibited the malignant phenotype of glioma and epithelial mesenchymal transformation(EMT)process1.The results of CCK8 experiment illustrated that the inhibitory effect of crocetin on the proliferation of U251/LN229 glioma cells presented concentration dependent.The IC50 fitting curve showed that the IC50 of normal astrocyte cells was significantly higher than that of glioma cells,indicating that the toxicity of crocetin on normal astrocyte cells is relatively small in 200-400μmol/L concentration.2.The results of colony formation and Transwell assays showed that the cell proliferation and migration ability of glioma cells decreased significantly with the increase of crocetin concentration,indicating that crocetin can inhibit the gliomagenesis in a concentration dependent manner.3.The results of western blotting showed that crocetin could up-regulate the expression of epithelial marker(E-cadherin)and down regulate the expression of mesenchymal markers(N-cadherin and Vimentin)in glioma cells,indicating that crocetin could inhibit the epithelial mesenchymal transformation process of glioma cells in a concentration dependent manner.4.The results of flow cytometry showed that with the increase of crocetin concentration,the apoptosis rate of glioma cells increased significantly.Part Ⅱ:Crocetin inhibits growth and sensitizes glioma to temozolomide(TMZ)through down-regulating the FOS-DUSP1 axis1.The results of network pharmacology/bioinformatics analysis indicate that FOS may be the drug target gene of crocetin in glioma cells,and the downstream gene DUSP1 is significantly positively correlated with FOS expression.2.The results of cell transfection and western blotting assays showed that DUSP1 was the target of FOS downstream,and the expression of DUSP1 and DUSP1 was positively correlated.The cytotoxic effect of crocetin on glioma cells can significantly decrese the expression of FOS/DUSP1 protein.The results of CCK8 assay results demonstrated that crocetin significantly attenuated the proliferation in FOS overexpression glioma cell lines.3.The sensitivity of FOS overexpressing glioma cell lines to temozolomide was decreased when the cells were stimulated with temozolomide at different concentration gradients,and the results of western blotting assay and immunofluorescence assay also showed that temozolomide induced y-H2AX(DNA double strand damage markers)was significantly down-regulated in FOS overexpression cell lines.4.The results of flow cytometry analysis and western blotting assays showed that crocetin combined with temozolomide could present a synergistic cytotoxicity on glioma cells by down-regulation of FOS-DUSP1 signal axis,and promote further apoptosis of glioma cells.Part Ⅲ:Crocetin alleviates the chemotherapy tolerance of glioma cells induced by TMZ by down-regulation of FOS-DUSP1 signal axis1.Bioinformatics analysis showed that FOS/DUSP1 mRNA expression was significantly up-regulated in glioma TMZ drug resistant cell lines.2.CCK8 and colony formation experiments showed that knockdown of DUSP1 in FOS overexpression glioma cell lines decreased the resistance to TMZ and the proliferation ability,indicating that DUSP1 was a key effector molecule of FOS-induced chemotherapy tolerance of TMZ.3.The results of qRT-PCR and western blotting showed that FOS/DUSP1 expression was significantly up-regulated at mRNA and protein levels in the induced TMZ drug-resistant glioma cell line.4.TMZ drug-resistant glioma cell line were cultured with DMSO and 200μMol/L TMZ respectively,after 10 days of culture,the results of qRT-PCR and immunofluorescence assays showed that FOS-DUSP1 signal axis was up-regulated in drug-resistant glioma and stably expressed with time.5.The results of flow cytometric analysis showed that the combination of crocetin and temozolomide could further increase the percentage of apoptosis of TMZ-insensitive glioma cells,and reduce the TMZ chemotherapy resistance of glioma cells to some extent.6.The results of xenograft glioma model in nude mice illustrated that the inhibitory effect of crocetin+temozolomide treatment on the growth of subcutaneous glioma was significantly enhanced compared with the single drug treatment.The results of western blotting showed that the expression of FOS-DUSP1 signal axis increased after only TMZ therapeutic strategy,and the Crocetin+TMZ combination therapy could sensitize glioma to temozolomide by down-regulation of FOS-DUSP1 axis.Part Ⅳ:FOS/DUSP1 is up-regulated in glioma patients,and is related to the poor prognosis of glioma patients1.In GEO database,bioinformatics analysis results showed that FOSmRNA/DUSP1 mRNA expression in glioma was significantly increased.2.The results of western blotting assay and immunohistochemical staining showed that the expression of FOS/DUSP1 in glioma tissues was higher than that in normal brain tissues,and was up-regulated with the increase of glioma WHO grade.3.Based on the expression of FOS/DUSP1 in TCGA/CGGA public database,Kaplan-Meier survival analysis showed that the survival time of glioma patients with high FOS/DUSP1 expression was significantly shortened.Conclusion:1.Crocetin can inhibit the malignant phenotype of glioma cells,promote apoptosis of glioma cells,and reverse the epithelial mesenchymal transformation process.2.Crocetin combined with temozolomide has synergistic cytotoxicity on glioma cells by down-regulation of FOS-DUSP1 signal axis.3.Crocetin can reduce the TMZ-chemoresistance of glioma cells to to some extent via inhibiting of FOS-DUSP1 signal axis,and further promote the apoptosis of TMZ-resistant glioma cells.4.FOS/DUSP1 were overexpressed in glioma tissues,and the glioma patients with high expression of FOS/DUSP1 indicates poor prognosis. |
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