Study On The Damage Effects Of Radiation Or Temozolomide Combined With Rabeprazole On Glioma Cells

Research Background:Glioma is the most common intracranial tumor originating from the central nervous system.Glioma can occur at any age.The median survival period is about 15months.At present,the commonly used treatment of glioma includes surgical resection,radiotherapy,chemotherapy,etc.However,in an effort to protect the normal brain tissue,it is difficult to guarantee that the tumor tissue is removed after surgery completely.Owing to the heterogeneity and inherent drug resistance of the blood-brain barrier（BBB）,Besides,chemotherapy drugs are prone to lead to many side effects.Thus,in terms of chemotherapy drugs,it is difficult to achieve good therapeutic effects.Radiotherapy could only kill radio-sensitive cells instead of curing glioma.Moreover,radiotherapy can not kill glioma cells that are infiltrating and still exist exceeding the normal radiation range.In addition,glioma cells easily generate radiation resistance,resulting in glioma recurrence.Therefore,it is urgent to find methods to enhance the injurious effect of radiation and chemotherapy.Clinically,Rabeprazole is one of the second-generation proton pump inhibitors（PPIs）used for treating gastric ulcers.Recently,it has been found the role of enhancing the sensitivity of chemotherapy in the treatment of lung cancer.However,whether Rabeprazole can treat glioma is still unknown.Early growth response factor-1（EGR1）is a kind of transcription factor,mainly involved in the process of tissue damage and fibrosis,closely related to the progression and metastasis of cancer.It may participate in the proliferation,migration,invasion,and other processes of cancers.At present,MGMT is the only methyltransferase that can repair the damage of O⁶methylguanine.It also plays an virtually important role in averting gene mutation,cell death,and tumorigenesis caused by alkylating agents.The high expression of MGMT may be the main cause of drug resistance in glioma.Some studies have illustrated that there is a significant correlation between the expression of EGR1 and the methylation of the MGMT promoter.But the specific mechanism has not been elucidated.Moreover,whether Rabeprazole can further enhance the radiation and chemotherapy damage by regulating EGR1/MGMT and suppressing the progression of brain glioma remains to be further explored.Research purposes:Rabeprazole combined with radiation or a chemotherapeutic drug（such as Temozolomide）was used to observe whether Rabeprazole could enhance the damaging effect of radiation and chemotherapy on glioma cells（U251 and U87）;To explore whether there is a targeted regulatory relationship between early growth response factor EGR1 and MGMT gene;To clarify whether Rabeprazole can further regulate the expression of MGMT by acting on EGR1,thus enhancing the damaging effect of radiation and chemotherapy drugs on glioma cells.Research methods:1.The GEPIA bio-informatics website was used for analyzing the expression of the EGR1 gene in various tumors and adjacent tissues;2.Analyzing the relationship between the expression of the EGR1 and MGMT gene and the survival period of patients with glioma through the CGGA database;3.The effect of different concentrations of Rabeprazole,Rabeprazole combined with radiation,and combined with a chemotherapeutic drug（Temozolomide）on the proliferation of glioma cells was detected by the CCK8 test kit;4.The effect of different concentrations of Rabeprazole on the clonogenic ability of glioma cells was detected by clonogenic assay;5.The effects of different concentrations of Rabeprazole on the migration and invasion of glioma cells were observed by the Scratch test and Transwell test;6.Flow cytometry was used to detect the effects of different concentrations of Rabeprazole,Rabeprazole combined with radiation,Rabeprazole combined with the chemotherapeutic drug Temozolomide on apoptosis and cell cycle of glioma cells;7.The EGR1 overexpression plasmid was transfected into cells by lentivirus transfection to establish a stable cell line;8.The CHIP-q PCR experiment was used to verify the targeted regulatory relationship between transcription factor EGR1 and MGMT Promoter.Research result:1.The effect of the expression of EGR1 and MGMT in tumor tissue on tumor progressionThe expression of EGR1 and MGMT in various tumors and adjacent tissues were analyzed by the GEPIA database.The results showed that the expression of EGR1 and MGMT in various tumor tissues was higher than that in adjacent tissues.GEPIA database analyzed the correlation between the expression of the EGR1 gene,MGMT gene,and the survival time of patients.The results showed that the expression of the EGR1 gene and MGMT gene was negatively correlated with the survival of tumor patients.Patients with relatively low expression of the EGR1 gene and MGMT gene have higher survival rates and longer survival periods than patients with relatively high expression of the EGR1 gene and MGMT gene.2.The effect of Rabeprazole on the proliferation and clonogenesis of glioma cellsThe CCK8 test was used to detect the effect of Rabeprazole on cell proliferation activity.The results showed that the proliferation rate of U251 cells and U87 cells decreased significantly with the increased Rabeprazole concentration.The proliferative activity of U251 and U87 cells decreased significantly in pace with time.At the same concentration of Rabeprazole,the proliferation activity of U251 cells was significantly lower than that of U87 cells,suggesting that U251 cells were more sensitive to Rabeprazole.The effect of Rabeprazole on the clonogenic ability of U251and U87 cells was detected by clonogenic assay.The results showed that with the increased Rabeprazole concentration,the number of U251 cells and U87 clones gradually decreased.3.The effect of Rabeprazole on migration and invasion of glioma cellsWith the increase in Rabeprazole concentration,the migration distance of U251and U87 cells gradually decreased,suggesting that Rabeprazole has an increased inhibitory effect on the migration ability of U251 and U87 cells.When Rabeprazole was treated for 12 hours,the migration distance of U251 cells was not obvious.When the treatment time was 24 hours,the migration distance of U251 cells was significantly different.With the increase of Rabeprazole concentration,the number of cells penetrating into the lower chamber gradually decreases.When the concentration of Rabeprazole is 200μM,the number of cells penetrating into the lower chamber decreased significantly（P<0.001;P<0.001）;When the concentration of Rabeprazole is 300μM,the inhibition of invasion is more obvious（P<0.001;P<0.001）.4.The effect of Rabeprazole on apoptosis and cycle of glioma cellsThe apoptosis and cell cycle of U251 and U87 cells were detected by flow cytometry with different concentrations of Rabeprazole.When the treatment concentration is 200-400μM,the apoptosis rate of U251 cells was significantly higher than that of the control group（P<0.01;P<0.05;P<0.001）.Only when the treatment concentration is 400μM,compared with the control group,the apoptosis rate of U87 cells changed significantly when treated with Rabeprazole（P<0.05）.When the concentration of Rabeprazole is 100 and 200μM,compared with the control group,while the percentage of U251 cells in the S phase increased significantly（P<0.01）.When the concentration of Rabeprazole is 300μM,compared with the control group,the percentage of the U251 cell in the G₀/G₁phase increased significantly（P<0.01）.When the action concentration of Rabeprazole is 100μM,compared with the control group,the percentage of the U87 cell in the G₀/G₁phase increased significantly.When the action concentration of Rabeprazole is 300μM,compared with the control group,while the percentage of the U87 cell in the G₀/G₁phase was significantly higher.5.Effect of Rabeprazole combined with radiotherapy and chemotherapy on glioma cells injuryThe results of CCK8 test showed that the cell proliferation activity of Rabeprazole combined with radiation or Temozolomide was significantly lower than that of Rabeprazole alone,radiation alone or Temozolomide alone（P<0.01）;Flow cytometry analysis showed that the apoptosis rate of Rabeprazole combined with radiation or Temozolomide was significantly higher than that of Rabeprazole alone,radiation alone or Temozolomide alone(P<0.01_{（Rabe+Radiation）,U251};P<0.01_{（Rabe+Radiation,U87）});P<0.01_{（Rabe+TMZ,U251）};P<0.01_{（Rabe+TMZ,U87）}).It is suggested that Rabeprazole combined with radiotherapy and chemotherapy can significantly enhance the damage to glioma cells.The percentage of S phase cells in U251 cells treated with Rabeprazole and radiation alone was significantly higher than that treated with radiation alone（P<0.001）.Compared with Temozolomide alone,the percentage of G₂/M cells in U251 cells treated with Rabeprazole and Temozolomide increased significantly（P<0.01）.The percentage of G₂/M phase cells in U87 cells treated with Rabeprazole and radiation alone was significantly higher than that treated with radiation alone（P<0.01）.Compared with the treatment with Temozolomide alone,the percentage of S phase cells in U87 cells treated with Rabeprazole and Temozolomide was significantly higher（P<0.01）.It was suggested that Rabeprazole combined with radiation or Temozolomide could cause S phase block and G₂/M phase block in U251cells,respectively;Rabeprazole combined with radiation or Temozolomide caused G₂/M phase block and S phase block in U87 cells,respectively.6.Effect of Rabeprazole combined with radiation and Temozolomide on the expression of EGR1 and MGMT in glioma cellsThe results of PCR and Western blot showed that the m RNA of EGR1 and MGMT genes in cells treated with Rabeprazole combined with radiation or Temozolomide(P<0.01_{（EGR1,U251）};P<0.001_{（MGMT,U251）};P<0.001_{（EGR1,U251）;}P<0.01_{（MGMT,U87）})and protein（P<0.01）were significantly decreased.It is suggested that Rabeprazole may affect the damage of radiation or Temozolomide to glioma cells by acting on EGR1/MGMT.7.The targeted regulation between EGR1 and MGMTThe PCR test confirmed that the binding ability of EGR1 and MGMT promoter region is strong,suggesting that EGR1 could bind to the promoter of MGMT.Analysis conclusions:1.Rabeprazole can reduce the proliferative activity,clonogenic ability of U251and U87 cell;inhibit the migration and invasion of U251 and U87 cell,promote the apoptosis of U251 and U87 cells;low concentration of rabeprazole promotes S phase delay in U251 cells;high concentration of rabeprazole promoted U251 cells to undergo G₀/G₁phase arrest;low concentration rabeprazole induced G₀/G₁phase arrest in U87 cells;high concentration of rabeprazole promotes G₂/M phase arrest in U251cells;2.Radiation or Temozolomide combined with Rabeprazole can further inhibit the proliferation activity of U251 and U87 cells;further promote the apoptosis of U251;Rabeprazole combined with radiation induced S phase delay and G₀/G₁phase arrest in U251 and U87 cells,respectively;Rabeprazole combined with Temozolomide induced G₀/G₁phase arrest and S phase delay in U251 and U87 cells,respectively;3.EGR1 can combine with the promoter of MGMT gene to promote the expression of MGMT;Rabeprazole may inhibit the damage of glioma cells by EGR1/MGMT enhanced radiation or Temozolomide.