| Background:Glioma of the brain is the most frequent primary brain tumor,characterized by its intense proliferation ability and its highly invasive growth.Surgery is usually not able to reach desired outcomes,and so postoperative adjunct treatment is frequently necessary in conjunction with radiotherapy and chemotherapy.Temozolomide is a lipophilic imidazoltetrazine alkylating agent,which is currently used as the first-line treatment of postoperative chemotherapy for glioma.However,postoperative patients combined with radiotherapy and chemotherapy still have a very high recurrence rate and mortality,which is closely related to the drug resistance of glioma.LDHA,a major enzyme involved in glycolysis,interacts with pyruvate to create lactic acid,thereby stimulating glucose absorption and enhancing lactic acid production within tumor cells..To satisfy the cell’s need for energy.Studies have demonstrated that LDHA is a critical player in the onset,progression,and prognosis of a variety of cancers,additionally exhibiting sensitivity to temozolomide.Thus,exploring LDHA expression and its reaction to temozolomide in glioma cells is of immense research interest.Objective:In this study,glioma patient samples with different pathological grades were compared,and the expression levels of LDHA,MGMT and drug-resistant related proteins in the samples were analyzed,the relationship between LDHA and glycolysis was discussed,and the changes of intracellular reactive oxygen species content were compared.The drug resistance of LDHA to human glioma cells and its mechanism were further discussed.Methods:1.Western-Blot method was used to compare the expression of LDHA in 8patients with glioma of different pathological grades2.The effects of temozolomide at different doses and for different time on LDHA content in glioma were determined by Western-Blot.3.Human U87 and U251 cells were used for the experiment.Groups of cells were separately treated with SiRNA targeted to LDHA,as well as with non-functional SiRNA of the negative control.4.The experimental,control,and blank groups were split into those that were administered TMZ and those that were not,with the concentration of the treatment being 200μmol/L.The contents of P-GP,MRP-1 and MGMT in cytoplasm and PKM2,HKII,PFKP,NOX1 and NOX4 in cytoplasm of each group were detected by Western-Blot.5.The MTT,ROS,and DHE assays were conducted on the six groups of cells to measure the viability of tumor cells,as well as the alteration in reactive oxygen species levels.Results:1.LDHA expression in gliomas has been linked to the severity of the tumor,with higher expression levels correlating to an increase in malignancy.2.The expression of LDHA in glioma tissue increased with the increase of TMZ duration and concentration.3.Following the introduction of knocked out LDHA,the expression of TMZpromoted drug-resistant proteins such as P-GP,MGMT and MRP-1 markedly dropped in the experimental group when compared to the control and null groups.4.After LDHA was knocked out,the expressions of PKM2,HKII,PFKP and other proteins in cells were decreased to a certain extent.5.MTT results showed that the killing effect of TMZ on glioma cells was significantly enhanced after LDHA knockout.6.The results of ROS and DHE fluorescence experiments significantly reduced LDHA and increased the content of intracellular reactive oxygen species.7.Inhibition of LDHA can improve the expression of NOX1 and NOX4,and promote the generation of hydrogen peroxide.Conclusion:1.LDHA expression levels in glioma rose in correlation with the tumor’s malignancy.2.The expression of LDHA was higher in temozolomid-resistant glioma cells.3.LDHA gene knockout can reduce the resistance of glioma cells to TMZ.4.LDHA knockout can enhance the killing effect of temozolomide by inhibiting glycolysis and increasing the level of intracellular reactive oxygen species. |
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