Experimental Studies On Mechanisms Of Nicardipine Combined With Temozolomide To Enhance Chemotherapy Sensitivity Against Glioma Stem Cells

Glioma is the most common primary intracranial malignant tumor,accounting for about 80%of all primary brain malignancies.The overall treatment outcome of glioma is still unsatisfactory,and it is highly susceptible to recurrence even after the existing standardized treatments(surgery,radiotherapy,chemotherapy,and TTFields therapy).The median overall survival(OS)of glioblastoma multiforme(GBM)for patients is only about 15 months,causing great suffering and financial burden to the patient’s family and society.In recent years,significant advances have been made in surgery,radiotherapy,and chemotherapy of glioma.Over the past two decades,intensive researches on the properties of glioblastoma stem cells(GSCs)has confirmed their indispensable roles in tumor formation,maintenance,treatment resistance,and recurrence in glioma.Therefore,targeting on GSCs will offer a new potential strategy against glioma and improve patients’ prognosis.Part I:Analysis of chemotherapeutic resistance of GSCs derived from mesenchymal and proneural GBMObjective:To analyze the biological phenotypes and chemo-resistance-related properties of GSCs derived from different molecular subtypes of GBM,for the purpose of providing a preliminary experimental references for relevant intervention strategies.Methords:Glioma stem cell lines(GSC3,GSC11.GSC23.GSC91,GBM12)derived from mesenchymal(MES)and proneural(PN)GBM patients were cultured under neural stem cell culture conditions.Tumor stem cell spheroid clone formation,cell cycle assay and Western blot were applied to detect biological characters os GSCs and the drug resistance-related proteins expression of GSCs of different molecular subtypes.The expression levels of ATP-binding cassette superfamily G member 2(ABCG2),alkyl purine-DNA-N-glycosylase(APNG),and O6-methylguanine-DNA methyltransferase(MGMT)were measured by Western blot.Then cytotoxic effect assay was performed,TMZ alone,or ABCG2 blocker(nicardipine,Nic)alone,or TMZ combined with Nic was applied to different molecular subtypes of GSCs,respectively.The difference in killing efficiency of TMZ,nic,or combined treatment group(TMZ+Nic)on GSCs.Results:(1)GSCs grew in a sphere-like pattern in neural stem cell culture conditions,MES-type GSCs had a heterogeneous cell sphere pattern,with GSC91 and GBM12 growing in suspension in vitro as irregular sphere-like cell clusters,GSC3 growing like clusters and densely adherent to the bottom of the culture flask,and pre-neural type(PN)GSCs(GSC23,GSC11)showing a more regular sphere-like cell clusters(ii)All five glioma stem cells were grown in suspension.All five stem cell strains expressed the drug resistance-associated protein ABCG2,and the two PN-type GSCs expressed higher levels of APNG than the MES-type GSCs.The expression level of APNG was higher in two PN-type GSCs than in MES-type GSCs,whereas MGMT was only expressed in GSC23(PN-type).(iii)The combination of TMZ and Nic further inhibited the cell viability as well as the self-renewal capacity of GSCs,compared to TMZ or Nic alone,while the percentage of G2 phase cell population was significantly increased in GSCs exposed to the combined drug group environment,indicating cell cycle arrest in the G2/M phase.Conclusion:Glioma stem cells expressed high levels of multiple drug resistance-associated proteins,and Nic in combination with TMZ significantly enhanced the inhibition effect of glioma stem cells,reduced their proliferative activity,especially in PN-type GSCs as well.Part Ⅱ:Experimental studies on reversal of temozolomide resistance phenotype of PN-type GSC and related mechanismsObjective:To analyze the TMZ resistance-related phenotypes of PN-type GSCs,which are key cells for chemoresistance,and explore the molecular mechanism of drug resistance mediated by ABCG2,for the purpose of providing a valuable experimental basis for targeting the TMZ-related transporter proteins of PN-type GSCs to reverse their drug resistance.Methods:The inhibitory effects of TMZ on PN-type GSCs was analyzed.TMZ alone,TMZ in combination with Nic on GSCs proliferation ability were further examined and compared using the CCK8 assay.The effect of TMZ alone and TMZ in combination with Nic on GSCs apoptosis was examined by flow cytometry.Western blot was performed to detect the changes of ABCG2 protein expression in PN-type GSCs after Nic intervention.Flow cytometry was used to detect ABCG2 activity of PN-type GSCs after TMZ alone,or combined with Nic treatment.Results:The CCK8 assay showed that the combination of nicardipine with temozolomide significantly reduced the survival of PN-type GSCs cells in a time-and concentration-dependent manner.A flow cytometric assay showed a significant pro-apoptotic effect of TMZ plus Nic combination on PN-type GSCs.3D invasion assay showed a synergistic effect of temozolomide and nicardipine on the inhibition of invasion distance of GSCs spheres.Nicardipine down-regulated the expression level of ABCG2 protein in PN-type GSCs and inhibited the protein activity of ABCG2 as well.Conclusion:Nicardipine exerted synergistic effects with temozolomide by participating in the regulation of ABCG2 protein expression levels and protein activity in PN-type GSCs,promoting apoptosis and inhibiting proliferation and spheroid invasion of PN-type GSCs,thus providing a preliminary experimental basis for targeting the GSCs chemo-resistance-associated protein ABCG2.