| Glioblastoma(GBM)is most malignant tumor in the central nervous system,which is often primary and more common in adults.It also has the highest intracranial incidence,with about 80% of all the malignant tumors in the brain.Glial cells are mostly the sources of it.GBM is a malignant tumor with high fatality rate,high disability rate,and poor prognosis.Although we have taken a variety of treatment methods,among which the main treatment is through the surgery and supplemented by chemotherapy,radiation therapy and biological treatment methods.Besides,with the improvement of neuroimaging and neurosurgical techniques,the status quo has been improved.However,the treatment of malignant glioma is still not elusive.Temozolomide(TMZ)is a chemotherapy drug,belonging to the oral two generation of alkylating agent.It is effective for the treatment of various kinds of solid tumors and has become the first-line drug therapy for glioma chemotherapy at present.TMZ concurrent radiotherapy combined with adjuvant chemotherapy is the standard treatment for newly diagnosed gliomas.But even so,the effect of chemotherapy on gliomas remains limited.The reasons are as belowed: First,it is possible that the presence of blood-brain barrier affects the distribution and absorption of drugs in the brain.Second,there may be the presence of drug resistance genes.Therefore,the loss of TMZ sensitivity,drug resistance and the subsequent tumor progression or recurrence are still the main reasons for the failure of TMZ chemotherapy.Among the related genes of glioma intrinsic or acquired drug-resistance,O6-methylguanine-DNA-methyltransferase(MGMT)is one of the most important genes in the related research which can repair the DNA damage caused by alkylating agents.This results in a reduction in DNA damage caused by TMZ,BCNU and other chemotherapeutic agents which leads to drug resistance.Methylation promoter has been widely recognized as the main mechanism for silencing MGMT gene by epigenetic modification,thereby enhancing the sensitivity of tumor to alkylating agents.Other studies have also shown that methylation of MGMT promoter is closely related to the status of IDH and genome-wide epigenetic variation(G-CIMP phenotype).Finding the related gene or mechanism that can regulate the expression of MGMT,and thus further enhance the effectiveness of TMZ therapy,has become a hot spot.NDRG2 is a candidate tumor suppressor gene,which was discovered by subtractive hybridization in 1999.In many issues,it is associated with tumor development,such as lung cancer,colon cancer,breast cancer,gastric cancer and the others.In the malignant glioma cell line,the expression of NDRG2 was also decreased,and the exogenous overexpression of NDRG2 in vitro could inhibit the proliferation of glioma cells.And the downregulation of NDRG2 was also found in meningioma.NDRG2 promoter methylation was found to be highly correlated with the decrease in NDRG2 transcript levels.In addition to hyper-methylation of the NDRG2 promoter,NDRG2 also regulates the growth of glioma by up regulating histone acetylation.The expression level of NDRG2 is negatively correlated with the pathological grading of brain tumor,and positively correlated with the survival rate of patients with neuroastrocytoma.The relationship between NDRG2 and MGMT is unclear at present,and rarely reported in related literature.So our research can provide new directions and strategies for enhancing the sensitivity of TMZ in the treatment of glioma,exploring the new synergistic factors associated with it,enhancing the therapeutic effect and improving the prognosis of the patients.Based on the previous findings,we sought to investigate whether the NDRG2 gene played a role in TMZ chemotherapy in patients with gliomas,whether it was associated with MGMT,the role it played,and the related mechanism.In order to resolve these scientific issues,we conducted the following studies.Objective:1.To determine the correlation between NDRG2 and the efficacy of TMZ chemotherapy in patients with glioma.2.To explore the factors that NDRG2 increased the sensitivity of glioma to TMZ chemotherapy drugs.3.To analysis of molecular signaling pathways regulating MGMT by NDRG2.4.To elucidate the relationship between NDRG2 and MGMT in TMZ resistant glioma cells.Methods and results:1.NDRG2 enhanced the sensitivity of TMZ to glioma(1)Construct NDRG2 overexpressing and control cherry cell line We have successfully constructed U87-NDRG2 cell,U87-Cherry cell,A172-NDRG2 cell,A172-Cherry cell,T98G-NDRG2 cell,T98G-Cherry cell,U251-NDRG2 cell,U251-Cherry cell using p Lenti6.3/V5-DEST lentiviral expression vector.At the same time Real-Time PCR was performed to detect the expression of NDRG2 m RNA and Western Blot was performed to detect the expression of NDRG2 protein.(2)NDRG2 promotes the treatment sensitivity of TMZ to glioma Previous studies showed that NDRG2 can inhibit the multiplication phenomenon of glioma cells.So,what's the trend after using TMZ? Is there any other change? Does the overexpression of NDRG2 increase the cytotoxicity of TMZ to glioma? We evaluated it from measuring the cell growth,killing curves,the cell cycle and apoptosis indexes etc.We detected the MTT index to observe the cell viability trend.The results showed that in the U87 cells,with the treatment of TMZ for 24 h,the relative cell viability decreased with the increase of TMZ concentrations.But this phenomenon was limited to the concentration range of 0-400 ?mol/L.Within the concentration range of 400-800umol/L,the curve started to flatten,which indicated that after reaching a certain level,the concentration was no longer the main factor affecting the viability of cells.We also observed that the relative cell viability of the NDRG2 group was lower than that of the cherry group at the same time.After the treatment of TMZ(400 ?mol/L)for 5 days,the MTT detection showed that the relative viability of NDRG2 overexpression group was lower than that of Cherry cells.After the treatment of TMZ for 24 h,we found that in U87 cells,the apoptotic rate of NDRG2 overexpression group was significantly higher than that of Cherry group,both in the drugs treatment group and in the control group without drugs.And the rate of apoptosis in the treatment group was significantly higher than that in the control group.The same trend was observed in T98 G cells.Then we gave different concentrations of TMZ to U87 cells.Within a certain range,we found that the rate of apoptosis increased with the increase of the concentration of the drug.The apoptosis rate in NDRG2 overexpression group was higher than that in Cherry group.In the previous work,we found that overexpression of NDRG2 could affect the cell cycle by flow cytometry.After the addition of TMZ,the analysis of the cell cycle distribution showed that the G1 phase rate increased in the NDRG2 over expression group,compared the control group,but the addition of drugs and drug concentrations did not show significant effects on the cycle.This part of the experiment indicated that NDRG2 can increase the sensitivity of TMZ to glioma,decrease the cell proliferation ability,and increase the apoptosis rate.2.NDRG2 enhances the efficacy of TMZ by blocking MGMT upregulation(1)The expression of NDRG2 and MGMT in different glioma cells In the first part,we found that under the treatment of TMZ,overexpression of NDRG2 could significantly improve the cytotoxicity of TMZ on cells,inhibit the growth of glioma,and promote apoptosis of glioma cells.What is the underlying mechanism? As we allknow,MGMT is a gene closely related to the treatment of glioma with drug resistance and resistance to TMZ.It can repair DNA damage caused by TMZ.In normal human and animal tissues,MGMT acts as a protective agent which can avoid alkylating damage and prevent cancer from occurring.In the tumor tissue,it can also avoid alkylating drugs such as chemotherapy injury,which will results in drug resistance.Therefore,we assume whether NDRG2 regulates the effect of TMZ on glioma cells by regulating the expression of MGMT?Therefore,we performed related experiments to observe the relationship between the change of expression of MGMT and NDRG2 after the treatment of TMZ on brain glioma cells.We observed the background expression of NDRG2 and MGMT in U87,T98 G,U251 and A172 cell lines.The results showed that the expression of endogenous MGMT was remarkable in T98 G cells,but very low,even invisible in U87,U251,and A172 cell lines.(2)MRNA and protein expression after the treatment of TMZ on U87,T98 G,U251 and A172 cell lines separately In U87 cells,the expression of MGMT increased after the treatment of TMZ on cherry group,and the expression of MGMT in NDRG2 group did increased slightly after the treatment of TMZ.However,after the treatment of TMZ,we obviously observed that the expression of MGMT in NDRG2 group was lower than that in Cherry group.After the treatment of TMZ on A172,U251 and T98 G cells,we also found that the expression of MGMT in NDRG2 group was lower than that in control group.Therefore,we think that NDRG2 may be associated with the upregulation of MGMT expression in gliomas under TMZ administration.3.Study on the mechanism of NDRG2 regulating MGMT(1)Observation on the regulation of MGMT by NDRG2 at the level of methylation In previous studies,we have proved that there is a correlation between NDRG2 and MGMT.Then we focus on the mechanism between NDRG2 and MGMT.Previous studies have shown that methylation of the MGMT promoter is an important cause of decreased expression of MGMT.Therefore,we first investigated whether NDRG2 enhanced the evel of MGMT methylation,thus reducing the activity of MGMT expression and increasing the sensitivity of glioma to TMZ.NDRG2 may also inhibit the development of tumor cells by inhibiting the PI3K/AKT pathway.Some literatures have reported that NF-?B may be the main factor in the process of MGMT mediated alkylating drug resistance.Therefore,we explored the mediated pathway of NDRG2 regulating MGMT from these aspects.We treated cells with the methyltransferase inhibitor GSK343,which can able to compete with SAM and selectively inhibit EZH2,a histone lysine methyltransferase inhibitor.High expressions of EZH2 or mutations in EZH2 have been found in many cancers,suggesting that EZH2 is a possible target for cancer treatment.In the absence of TMZ,the expression of MGMT was up-regulated by GSK343;with the addition of TMZ,the MGMT increased significantly in the control and NDRG2 group.However,there was no significant difference between the two groups when GSK343 was added.This result showed that NDRG2 might not work by GSK343 methylation.5-AZA belongs to the DNA methyltransferase inhibitor.In the presence of TMZ,if NDRG2 decline the expression of MGMT by methylated MGMT,the downward trend in MGMT should be reduced after using methylation inhibitors.However,the result was not like this,suggesting that it might not work through this pathway(2)Regulation observation of NDRG2 in AKT pathway and MGMT Previous investigators have found that NDRG2 can inhibit the development of tumor cells by inhibiting the AKT pathway.We established NDRG2 overexpression group and corresponding control group respectively in U87 and T98 G cells to observe the expression of AKT and p-AKT protein.The results showed there were no significant difference.(3)Regulation observation of NDRG2 in NF-?B pathway on MGMT In U87 and T98 G cells,it could be observed that in Cherry group,MGMT increased significantly after TMZ addition,and in NDRG2 group,the expression of MGMT also increased after TMZ addition.Comparing the Cherry group with NDRG2 group with the treatment of TMZ,we saw that the MGMT in the NDRG2 group decreased obviously,with p-p65(the phosphorylated subunit of NF-?B)the similar trend. TNF-? can stimulate the expression of NF-?B.In U87 and T98 G cells,it could be observed that the expression of MGMT was significantly higher than that before adding TNF-?.JSH-23 belongs to the NF-?B transcriptional activity inhibitor.In T98 G cells,it could be observed that the expression of MGMT was significantly lower than that before adding JSH-23,and it is basically consistent with the trend of p-p65.This result suggested that MGMT might be regulated positively by NF-?B to some extent.4.Expression of NDRG2 and MGMT in TMZ resistant cells Comparing the growth curves between U251 resistant cells and parental cells,we found that with the advance of time,the proliferation ability of drug resistant cell U251 was obviously higher than that of parental cells,suggesting that the drug-resistant cells proliferated faster and became more malignant.Under the treatment of TMZ,with the increase of the concentration of TMZ drugs,the killing efficiency showed obvious difference between U251 resistant cells and parental cells.The resistant cell group was significantly lower than that of the parental group.It showed that drug-resistant cells were more resistant to TMZ,and the therapeutic effect was worse.The expression of MGMT in U251 resistant cells was obviously increased,and the expression of NDRG2 was decreased.After administration of TMZ,the expression of MGMT in the NDRG2 group was decreased in the resistant group,which also confirmed that NDRG2 achieved the enhancing drug sensitivity in U251 resistant cells by the regulation of MGMT.Conclusion:Through the above research contents and results,we found that NDRG2 might prevent MGMT upregulation after the treatment of TMZ to enhance the sensitivity of TMZ in the treatment of glioma.This process might be achieved by NDRG2 inhibiting the NF-?B pathway and then blocking the upregulation of MGMT.We have not found that this process was related to the AKT pathway or depend on MGMT methylation. |
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