ABSTRACT
Objective:To investigate the effects of over-expression of E2F transcription factor 1 (E2F1) on proliferation, invasion, apoptosis and radiosensitivity of glioma cell U251.Methods:Real-time quantitative PCR (qRT-PCR) were used to detect the differential expression of E2F1 mRNA in glioma cells LN18, SW1088, U251 and normal brain glial cells. The stable over-expression of E2F1 plasmid was constructed and transfected into U251 cells. qRT-PCR and Western blot test were used to detect the expression of E2F1, pituitary tumor transforming gene 1(PTTG1), C-Myc, B-cell lymphoma-2 (Bcl-2), Bcl2-associated X (Bax) mRNA and protein expression in the control group and E2F1 over-expression group.U251 cells were divided into control group(no X-ray irradiation), irradiation group(6 Gy dose of X-ray), and irradiation + E2F1 over-expression group(transfected with E2F1 first, then irradiated by 6 Gy of X-ray). Cell proliferation ability was detected by cell counting Kit-8(CCK-8) cell viability detection reagent, and cell invasion and migration ability were detected by Transwell chamber. Apoptosis and cell cycle were detected by flow cytometry.GraphPad Prism 8.0 was used for data analysis.The statistical methods were one-way ANOVA and independent sample t-test. Results:qRT-PCR showed that there was statistical difference in the mRNA levels of E2F1( F=201.92, P<0.05) in different cell lines.The expression levels of E2F1 mRNA in LN18(4.04±0.29), SW1088(3.19±0.16)and U251(4.66±0.20) cells were higher than those in HEB(1.02±0.07)cells ( q=27.00, 19.40, 32.52, all P<0.05). After successfully constructing U251 cells with stable over-expression of E2F1 plasmid, qRT-PCR and Western blot detection results showed that: the mRNA and protein levels of E2F1, PTTG1, C-Myc and Bcl-2 in E2F1 over-expression group were higher than those in control group ( t=77.16, 57.88, 4.63, 51.13, 7.50, 70.85, 8.38, 48.81, all P<0.05). Bax mRNA(0.20±0.01) and protein(0.66±0.01) levels were lower than those in control group((1.00±0.02), (0.94±0.01)), and the differences were statistically significant ( t=1.74, 54.65, both P<0.05). After X-ray irradiation (6 Gy), CCK8 detection results showed: the proliferation ability of the three groups at 24, 48, 72 and 96 h were significantly different ( F=95.41, 187.53, 1 158.49, 7 883.78, all P<0.05). The proliferation capacity of the irradiation group were lower than those of the control group at 24, 48, 72 and 96 h ( q=19.51, 27.20, 66.60, 174.9, all P<0.05). The proliferation capacity of irradiation + E2F1 over-expression group at 24, 48, 72 and 96 h were higher than those of irradiation group ( q=10.63, 10.81, 21.11, 60.90, all P<0.05). Transwell assay results showed that there were significant differences in cell invasion and migration ability among the three groups ( F=315.38, 681.10, both P<0.05). The invasion and migration ability of cells in the irradiation group were lower than those in the control group ( q=35.09, 12.76, both P<0.05), and the invasion and migration ability of cells in the irradiation + E2F1 over-expression group were higher than those in the irradiation group ( q=52.06, 22.81, both P<0.05). Flow cytometry showed that there were significant differences in apoptosis rate and percentage of cells in each cycle among the three groups ( F=667.63, 3 213.30, 3 011.26, 861.98, all P<0.05). The percentage of the apoptosis rate, S phase and G2 phase cells in the irradiation group were higher than those in the control group ( q=51.10, 89.39, 51.82, all P<0.05), while the percentage of G1 phase cells in the irradiation group was lower than that in the control group ( q=141.2, P<0.05). The apoptosis rate and percentage of S phase and G2 phase cells in the irradiation + E2F1 over-expression group were lower than those in the irradiation group ( q=18.87, 41.42, 29.31, all P<0.05), while the number of G1 phase cells in the irradiation + E2F1 over-expression group was lower than that in the irradiation group ( q=70.73, P<0.05). Conclusion:Over-expression of E2F1 can reduce the radiosensitivity of glioma U251 cells by regulating the expression of mRNA and protein of genes related to cell cycle and apoptosis, and E2F1 may be involved in the radioresistance of glioma cells.
ABSTRACT
OBJECTIVE:To study the mechanism of histone deacetylase inhibitor RGFP109 in reversing resistance of glioma U251 cells. METHODS:TR/U251 cells resistance to temozolomide(TMZ)was extrablished. The test was divided into normal con-trol group,TMZ group(40 μmol/L)and TMZ(40 μmol/L)+RGFP109(0-120 μmol/L)different concentrations groups. After 24 h of adding into related medicines,CCK-8 was used to detect the cell survival rate and calculate the half inhibitory concentration (IC50). TUNEL and Annexin V/PI were used to detect the cell apoptosis in normal control group,TMZ group and TMZ+RGFP109 (42μmol/L)group. Immunoblotting was used to detect the O6-methyl guanine-DNA methyltransferase(MGMT),Survivin,B lym-phoma 2(Bcl-2),B lymphoma xL(Bcl-xL)protein expression;and gel migration test was used to detect the p65 acetylation level and its binding capacity with κB-DNA. RESULTS:Compared with normal control group,cell survival rate in TMZ+RGFP109 dif-ferent concentrations groups was obviously decreased (P<0.05),showing a concentration-dependent manner. When the RGFP109 concentration was 42 μmol/L,the sensitivity of TMZ to TR/U251 cells was the same with U251 cells. Compared with normal con-trol group,MGMT,Survivin,Bcl-2,Bcl-xL protein expressions in cells of TMZ groups were enhanced(P<0.01);p65 acetyla-tion level had no obvious changes,while the binding capacity of p65 and κB-DNA was strengthened (P<0.01). Compared with TMZ group,MGMT,Survivin,Bcl-2,Bcl-xL protein expressions in cells of TMZ groups were weakened(P<0.01);p65 acetyla-tion level was enhanced (P<0.01);and the binding capacity of p65 and κB-DNA was weakened (P<0.01). CONCLUSIONS:RGFP109 can reverse the resistance of U251 cells to TMZ by down-regulating the anti-apoptotic protein expressions adjusted by transcription factorκB(NF-κB)and weakening the binding of p65 andκB-DNA.
ABSTRACT
OBJECTIVE:To evaluate the effects of 3 kinds of serum containing blood-activating and stasis-eliminating TCM compound formulas on aggressive behavior,Janus kinase (JAK)/signal transduction and transcriptional activator (STAT) pathway of glioma U251 cells. METHODS:Rats were randomly divided into normal saline group (5 mL/kg),Taohong Siwu decoction group(5.7 g/kg),Xuefu Zhuyu decoction(8.5 g/kg)and Didang decoction(2.8 g/kg),calculated by crude drug,intragastrically administrated once a day,for 10 d. 10% drug-containing serum culture medium was prepared after 2 h of last administration. After 10% drug-containing serum culture medium intervening U251 cells for 1 week,Transwell method was conducted to detect the cell invasion rate, Western blot was adopted to detect the metal matrix protease 2 (MMP-2), MMP-9, phosphorylated JAK2 (p-JAK2),phosphorylated STAT3(p-STAT3)protein expression;and real-time fluorescence quantitative polymerase chain reaction method was used to detect MMP-2,MMP-9 mRNA expression. RESULTS:Compared with blank serum,Xuefu Zhuyu decoction drug-containing serum can reduce cell invasion rate (P0.05). CON-CLUSIONS:In the 3 kinds of blood-activating and stasis-eliminating TCM compound formulas,Xuefu Zhuyu decoction shows sig-nificant invasive effect on inhibiting U251 cells;the mechanism may be related to inhibiting the activation of JAK2/STAT3 signal pathway and decreasing MMP-2,MMP-9 gene and protein expressions.
ABSTRACT
AIM:To investigate whether Notch1 changes stemness and chemotherapeutic sensitivity in human glioma U251 cells.METHODS: The lentiviral vectors, which expressed Notch1-shRNA or Notch1 intracellular domain ( NICD) , were transfected into U251 cells .Western blot and immunofluorescence staining were applied to monitor the va-lidity of the cells, down-regulation of Notch1 expression or over-expression of NICD.The proportion of CD133 +cells was analyzed by flow cytometry.The expression of nestin and GFAP was identified by immunofluorescence staining.The forma-tion rate of tumor cell spheres and the implanted tumor growth in SCID mice were observed.MTT assay was performed to e-valuate the chemotherapeutic sensitivity to VM-26 and BCNU of the cells with different treatments.RESULTS:Stemness was significantly enhanced in the cells over-expressing NICD.For example, the proportion of CD133 +cells was increased, the expression of nestin was up-regulated, the expression of GFAP was down-regulated, and the formation rate of tumor cell spheres and implanted tumor growth were increased.The chemotherapeutic sensitivity to VM-26 and BCNU of the cells was decreased.In the cells with Notch1 gene down-regulation by RNAi, the stemness was inhibited and chemotherapeutic sensi-tivity was increased.CONCLUSION:Notch1, which leads to the change of stemness and chemotherapeutic sensitivity in human glioma U251 cells, is likely to be a potential molecular target for treatment of glioma.
ABSTRACT
0.05);While the differences of the levels of p53,Bcl2,Bax between all D_1+D_2 groups and sham-irradiated group were significant(P