The Study Of Anti-tumor Effect Of Thiabendazole And Its Functional Mechanism For Targeting MCM2 And NUSAP1 Proteins In Glioblastoma Treatment

BackgroundGlioblastoma multiforme(GBM)is the most common and aggressive primary brain malignant tumor in adults.Surgical resection is the first choice,and postoperative local radiotherapy and chemotherapy are required.Chemotherapeutic agents currently commonly used are temozolomide,an oral DNA alkylating agent,which in combination with postoperative radiotherapy increased survival from 12.1 months to 14.6 months.However,at least 50%of patients with GBM are insensitive to treatment with temozolomide.The tumor biology of GBM makes the disease resistant to treatment.First of all,GBM tumor margins are infiltrated into normal brain tissue,making complete surgical resection of the tumor an unsolvable difficulty.Secondly,due to the existence of the blood-brain barrier(BBB)in the brain,most chemotherapeutic drugs cannot penetrate into the brain sufficiently to play an effective therapeutic role.Third,due to GBM’s high cellular,genetic heterogeneity,therapies targeting key molecular pathways are often ineffective.Therefore,new effective drugs and therapeutic targets are urgently needed for the treatment of GBM.Drug reuse has become a widely accepted strategy for cancer treatment,in which drugs with clear efficacy and good safety for a particular disease are used to treat other diseases in order to identify new therapies or achieve better therapeutic effects.Drugs that are known to be relatively safe in humans can be quickly tested in clinical trials,especially in cancer diseases where treatment options are few.For example,flubendazole and mebendazole,compounds in the benzimidazole carbamate family,are approved for use as deworming agents.In recent years,studies have shown that these drugs have anti-tumor properties against a variety of tumors,including human gliomas.For example,flubendazole has been shown to inhibit glioma proliferation in previous studies.Mebendazole was shown to be cytotoxic to gliomas in both homogenic and heterogenic glioma mouse models,significantly prolongating mean survival.Mebendazole has also been used in phase 1 clinical trials,showing long-term safety and acceptable toxicity at doses up to 200 mg/kg.Thiabendazole(TBZ;2-(thiazol-4-yl)benzimidazole)has been used to treat human intestinal parasites for more than 50 years.TBZ can inhibit Candida albicans,penicillium,help treat psoriasis,prevent the formation of aflatoxin in plant feed,and has no carcinogenic properties,and does not affect animal fertility.A previous study showed that TBZ reduced the growth of fibrosarcoma in humans.Therefore,TBZ,as a member of the non-toxic benzimidazole compound family,has attracted our attention for its anticancer potential.GBM is similar to other highly malignant solid tumors in that its cells show abnormal proliferation and cell cycle,which is related to the mal-regulated expression of cell cycle regulating proteins.Therefore,based on the biological characteristics of GBM,radiotherapy and chemotherapy as therapeutic targets have been established.Mitotic disorder refers to abnormal mitosis caused by various stimuli.Mitosis eventually leads to cell death by inducing abnormal mitosis.In vitro,benzimidazole have been reported to induce mitosis,polyploid,aneuploid,up-regulation of autophagy,and death through apoptosis or necrosis.In this study,we investigated the anticancer effects of TBZ on GBM cells in vitro and in vivo and their molecular mechanisms.TBZ can induce G2/M phase arrest of GBM cell cycle,inhibit proliferation and inhibit invasion.We sequenced RNA from TBZ-treated tumor cells to identify differentially expressed genes,and found that mini-chromosome maintenance protein 2(MCM2)is an important transcription factor regulated TBZ.These results support the reuse of TBZ as a possible treatment for GBM.Part I Thiabendazole targeting MCM2 inhibits glioblastoma cell proliferation and invasionObjectivesGlioblastoma cell lines were treated with thiabendazole(TBZ)on in vitro cell models and in vivo orthotopic xenograft model.The inhibitory effect of thiabendazole on glioblastoma cells was verified by observing the changes of tumor behavior in glioblastoma cell lines and detecting the level changes of intracellular molecular expression.Methods1.In vitro culture of human glioblastoma cell lines.2.Cell viability of glioblastoma cell lines and changes after treatment were detected by CCK-8(cell counting kit 8).The proliferation of glioblastoma was detected by clonal formation assay.Cell replication and division of glioblastoma cell line was detected by EdU reagent.3.Glioblastoma cells were planted in trans-well chamber to observe the changes in the number of cells passing through after treatment,and evaluate the effect of thiabendazole on the invasion ability of glioblastoma cells.After the 3D tumor spheres were constructed,the range of cells invaded from the tumor spheres in the matrix glue was observed to determine the effect of thiabendazole on the invasion ability of glioblastoma cells.Tumor spheres were co-cultured with brain organoids to observe the degree and speed of tumor cells’ invasion into brain organoids,and to detect the changes of glioblastoma invasion ability before and after treatment with thiabendazole.4.The important proteins related to cell proliferation(PCNA),cell cycle(Cyclin B1,Cyclin B2,CDK1)and tumor cell invasion(ZEB1,N-cadherin,MMP2)after thiabendazole treatment were detected by western blot technology to verify the effect of drugs on the proliferation and invasion of glioblastoma.5.After 2 days of thiabendazole treatment,cells were sequenced for further analysis of intracellular RNA expression changes under thiabendazole treatment.Bioinformatics analysis was conducted on genes with different expressions.Pathways and molecules with significant expression differences were found through GO and KEGG enrichment analysis.Key proteins that frequently interact with other proteins were screened through protein interaction network analysis,and potential targets of thiabendazole on glioblastoma were finally searched.6.Small interfering RNA transfection technology was used to act on the target molecule,mini-chromosome maintenance complex component 2(MCM2),and reduce its protein expression.The effects of target proteins on cell proliferation and invasion in glioblastoma cell lines were detected.7.Build target protein expression of the virus,and infection of glioblastoma cell line,establish stable MCM2 expression of cell line,further verify the MCM2 in glioblastoma cell lines,and testing in the role of thiamethoxam benzene of azole,after MCM2 expression,glioblastoma cell line proliferation,invasion suppression is mitigated.8.P3 cells were xenografted into the brain of nude mice,and the nude mice were given thiabendazole daily by intraperitoneal injection to observe the effect of the drug on glioblastoma in vivo.Results1.Thiabendazole can clearly inhibit the proliferation of glioblastoma cell lines(P3,U251,LN229,A172,U118MG)in vitro experiments,and its inhibitory effect becomes stronger with the increase of concentration.The IC50 of normal human astrocyte(NHA)cells was higher than that of tumor cells and showed some resistance to the toxic effects of thiabendazole.Thiabendazole inhibited the proliferation of P3 and U251 cells in a quantitative and timedependent manner,and induced G2/M phase arrest of P3 and U251 cells,down-regulated the expression of proteins related to proliferation and cell cycle regulation,and inhibited the proliferation of P3 and U251 cells.2.According to the results of Trans-well invasion assay and THREE-DIMENSIONAL invasion ball assay,thiabendazole inhibited the invasion of glioblastoma cell lines P3 and U251,and the expression of invasion-related proteins was down-regulated,and the inhibition effect of high concentration treatment group(TBZ 300μM)was more significant.The results showed that thiabendazole could inhibit the degree and speed of invasion of P3 tumor spheres into brain organoids.3.According to the RNA sequencing results of P3 and U251 cell lines in the dosed and untreated groups,bioinformatics analysis was conducted on the data of differentially expressed genes in glioblastoma,and it was found that the expression differences of cell cycle and mitosis related genes were the most obvious.In P3 and U251 cell lines,MCM2,UHRF1 and MCM5 were among the top 10 genes with down-regulated expression levels,among which MCM2 protein expression level was most significantly down-regulated by protein imprinting.4.After MCM2 down-regulation,P3 and U251 cells showed decreased proliferation,G2/M phase arrest,and down-regulation of proliferation and cell-cycle-related protein expression levels.Moreover,the invasion ability of GBM cells was weakened and the expression of invasion-related proteins was down-regulated.5.After MCM2 overexpression,proliferation ability of P3 and U251 cells was enhanced,G2/M phase inhibition of thiabendazole was weakened,and invasion inhibition induced by thiabendazole was alleviated.The expression of proteins related to cell cycle,cell proliferation and cell invasion increased.6.After thiabendazole treatment,the proliferation of intracranial tumor cells in nude mice was inhibited,the survival time of nude mice was prolonged,and the expression level of MCM2 in tumor tissues was decreased.7.MCM2 is correlated with NUSAP1,and thiabendazole can inhibit the expression of NUSAP1 in GBM cells.ConclusionsThiabendazole can inhibit the proliferation and invasion of glioblastoma cell lines P3 and U251.According to the analysis of RNA sequencing results,MCM2 is an important therapeutic target of thiabendazole.Therefore,this drug offers a choice to glioblastoma addjuvant treatment strategy.Part Ⅱ NUSAP1 knockdown inhibits cell proliferation and invasion by down-regulating TOP2A gene level in human glioblastomaObjectivesTo study the expression and function of Nucleolar and Spindle Associated Protein 1(NUSAP1)in glioma,and its effect on patient survival.Further research on downstream regulatory genes related to NUSAP1 will provide new therapeutic targets for glioma.Methods1.NUSAP1 expression and patient survival were analyzed in TCGA,CGGA and REMBRANT databases.2.Immunohistochemical technique and protein immunoprecipitation method were used to detect NUSAP1 expression in glioblastoma tissue samples collected by our hospital.3.EdU reagent to detect cell replication and proliferation.4.Trans-well invasion assay was used to detect changes in cell invasion.5.Apoptosis was detected by flow cytometry using PI and Annexin V double fluorescence staining.6.The protein expression levels of NUSAP1 and TOP2A were down-regulated by small interfering RNA transfection technique,and the role of their proteins in glioblastoma cell lines was detected.7.Downstream regulatory genes of NUSAP1 were identified by RNA sequencing and bioinformatics analysis.8.U251 cells were xenografted into the brain of nude mice to observe the proliferation of glioblastoma in vivo after the down-regulation of NUSAP1 expression.Results1.NUSAP1 expression is up-regulated in high-grade gliomas and negatively correlated with the prognosis of patients with gliomas.The expression level of NUSAP1 in glioma samples collected from our hospital increased with the increase of tumor grade.NUSAP1 was highly expressed in glioblastoma cell lines U251,T98 and P3.2.After NUSAP1 was down-regulated,the results of EdU assay showed that the proliferation of glioblastoma cells was reduced,and the results of Trans-well assay showed that the number of invading cells of glioblastoma was reduced.Apoptosis was promoted by PI and Annexin-V double fluorescence staining.3.Analysis of RNA sequencing results showed that eight genes(MT1F,S1PR1,IGFN1,UTP14C,TOP2A,KRT6A,ANO1,UFSP1)were down-regulated after NUSAP1 was downregulated.The expression of TOP2A was significantly different,and there was a potential interaction relationship with NUSAP1 in protein interaction network analysis.Moreover,the down-regulation of NUSAP1 significantly inhibited the protein expression level of TOP2A in glioblastoma cells.4.The down-regulation effect of TOP2A expression was similar to that of NUSAP1 expression,which significantly inhibited the proliferation and invasion of glioblastoma cells and induced apoptosis.5.Down-regulation of NUSAP1 in vivo can inhibit the expression of TOP2A and inhibit the growth and invasion of glioma.ConclusionsAfter NUSAP1 is knocked down,the proliferation and invasion ability of glioblastoma cells is decreased and apoptosis is promoted.NUSAP1 inhibits the proliferation and invasion of glioblastoma cells and induces glioblastoma cells apoptosis by down-regulating the expression of TOP2A gene.This study provides new insights into the mechanism of glioblastoma inhibition mediated by NUSAP1 silencing and demonstrates the therapeutic feasibility of NUSAP1 in malignant brain tumors.