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ABSTRACT OBJECTIVE To investigate the effects and mechanism of ginsenoside Rh2 on the proliferation and apoptosis in human glioma U87 and U251 cells. METHODS Using human glioma U87 and U251 cells as subjects， the proliferation and apoptosis， as well as the expression of histone deacetylase 1（HDAC1） protein and apoptosis-related proteins [B cell lymphoma-2（Bcl-2）， Bcl-2-associated X protein （Bax） and cleaved caspase-3] were detected after being treated with different concentrations of ginsenoside Rh2. RESULTS The concentrations of 10，20，30，40，50，60，70，80 μmol/L ginsenoside Rh2 could generally significantly increase the proliferation inhibition rate of U87 and U251 cells （P＜0.05 or P＜0.01）， and the half inhibitory concentrations of this component after 48 hours of action were 51.34 and 55.84 μmol/L， respectively；30，50 μmol/L ginsenoside Rh2 could increase the total apoptotic rate of both types of cells， reduced the protein expressions of HDAC1 and Bcl-2， and increased the protein expressions of Bax and cleaved caspase-3 significantly （P＜0.05 or P＜0.01）. CONCLUSIONS Ginsenoside Rh2 has a significant inhibitory effect on the proliferation of glioma cells and promotes the apoptosis of cells， which may be through reducing the expression of HDAC1 protein and activating the Bcl-2 family protein-mediated apoptosis pathway.
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OBJECTIVE@#To explore the effect of atorvastatin (AVT) on biological behaviors and the miR-146a/PI3K/Akt signaling pathway in human glioma cells.@*METHODS@#Human glioma U251 cells were treated with 8.0 μmol/L AVT or transfected with a miR-146a inhibitor or a negative control fragment (miR-146a NC) prior to AVT treatment. RT-PCR was used to detect miR-146a expression in the cells, and the changes in cell proliferation rate, apoptosis, cell invasion and migration were detected using MTT assay, flow cytometry, and Transwell assay. Western blotting was performed to detect the changes in cellular expressions of proteins in the PI3K/Akt signaling pathway.@*RESULTS@#AVT treatment for 48 h resulted in significantly increased miR-146a expression and cell apoptosis (P < 0.01) and obviously lowered the cell proliferation rate, invasion index, migration index, and expressions of p-PI3K and p-Akt protein in U251 cells (P < 0.01). Compared with AVT treatment alone, transfection with miR-146a inhibitor prior to AVT treatment significantly reduced miR-146a expression and cell apoptosis (P < 0.01), increased the cell proliferation rate, promoted cell invasion and migration, and enhanced the expressions of p-PI3K and p-Akt proteins in the cells (P < 0.01); these effects were not observed following transfection with miR-146a NC group (P>0.05).@*CONCLUSION@#AVT can inhibit the proliferation, invasion and migration and promote apoptosis of human glioma cells possibly by up-regulating miR-146a expression and inhibiting the PI3K/Akt signaling pathway.
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Humans , Apoptosis , Atorvastatin/pharmacology , Cell Line, Tumor , Cell Proliferation , Glioma/pathology , MicroRNAs/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal TransductionABSTRACT
Objective To investigate the inhibitory effect and mechanism of temozolomide on migration and invasion of U251 human glioma cells enhanced by plumbagin. Methods CCK-8 method was used to study the effects of plumbagin, temozolomide and plumbagin+temozolomide on the proliferation of glioma U251 cells. Cell scratch test was used to detect the migration of U251 cells in the control (DMSO), plumbagin, temozolomide and plumbagin+temozolomide groups for 48h. Transwell assay was used to detect the effect of the combination therapy on the invasion of U251 cells. Western blot was used to detect the relative expression levels of E-cadherin in three groups. Results CCK-8 showed that the proliferation inhibition rate of U251 cells treated with plumbagin (1.25 μmol/L) combined with temozolomide (200 μmol/L) for 48h was 75.69%, significantly higher than that treated with plumbagin alone (P=0.012) or temozolomide alone (P=0.034). Cell scratch assay showed that the combination of plumbagin and temozolomide could significantly enhance the inhibition effect of temozolomide on the migration of U251 cells (P=0.023). Transwell assay showed that the invasion ability of U251 cells was significantly decreased after the combination therapy (P < 0.05). The protein expression of E-cadherin in the combination group was significantly higher than those in plumbagin or temozolomide groups (P < 0.05). Conclusion Plumbagin combined with temozolomide can inhibit the migration and invasion of glioma cells and enhance the sensitivity of glioma cells to temozolomide. And the effect is achieved by the protein expression of E-cadherin.
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Aim To prepare curcumin-PLGA nanoparticles [curcumin poly(lactic-co-glycolic acid) nanoparticles, Cur-PLGA-NPs ] and evaluate their effects on glioma cells in vitro. Methods C6 cells and U251 cells were subcultured and randomly divided into control group, curcumin group and curcumin-PLGA group. The morphology of Cur-PLGA-NPs was observed by inverted fluorescence microscope and transmission electron microscopy. The particle size of Cur-PLGA-NPs was detected by Malvem particle size potentiometer. The encapsulation efficiency and drug loading rate of Cur-PLGA-NPs were detected by a multi-function microplate reader. The uptake of Cur-PLGA-NPs by C6 cells and U251 cells was observed by fluorescence microscopy. The effect of Cur-PLGA-NPs on the viability of C6 cells and U251 cells was detected by CCK-8. The effect of Cur-PLGA-NPs on apoptosis was assessed by flow cytometry. Apoptosis was detected by Hoechst staining. Results The Cur-PLGA-NPs had an average particle size of (284. 6 ± 9. 0) nm and were spherical. The encapsulation efficiency was (70.712 ±2.615)%, and the drug loading rate was (2.828 ±0.105) %. Compared with control group and curcumin group, C6 cells and U251 cells significantly increased the uptake of Cur-PLGA-NPs (P < 0. 05). Compared with control group and curcumin group, the cell viability of C6 cells and U251 cells in curcumin-PLGA group significantly decreased (P < 0. 05). Compared with control group and curcumin group, the Cur-PLGA-NPs induced apoptosis significantly in C6 cells and U251 cells(P <0. 05). Conclusion Compared with curcumin, Cur-PLGA-NPs can enhance the uptake of drugs by tumor cells, promoting tumor cell apoptosis.
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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.
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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.
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Objective: To investigate the effect of solanine on growth and proliferation of U251 cells. Methods: U251 cells were cultured with different concentration of solanine together. CCK-8 method, wound healing assay, Transwell method, Hochest assay, flow cytometry screening and Western blotting were used to detect inhibitory rate, migration and invasive rates, apoptosis rate and expression level of cell apoptosis-related proteins. Results: CCK-8 assay showed the median inhibition concentration (IC50) of 48 h was 20.05 μg/μL. It was effective that solanine in the concentration range of 5-35 μg/μL could inhibit the growth and proliferation of U251 cells, and played a role in a dose-dependent manner (P < 0.05). Compared with control group, experimental groups can not only inhibit U251 cell migration and invasion in a dose-dependent manner (P < 0.05), but also appear some significant apoptosis characteristics with a concentration dependence. Western blotting assayed that the expression of Bax protein was upregulated, whereas Bcl-2 protein expression was downregulated in experimental groups compared with control group and these changes were dose-dependent. Conclusion: Solanine can inhibit growth and proliferation of U251 cells in effective concentration and in a dose-dependent manner. Solanine can inhibit the invasion and migration of U251 cells. Solanine can induce the apoptosis of U251 cells, and inhibit the proliferation and growth of U251 cells via regulating the expression of Bax and Bcl-2 proteins and affecting Bcl-2/Bax ratio.
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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.
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AIM:TostudythechangeofradiosensitivityofU251cellsaftertreatedwithsodiumdichloroacetate ( DCA) and further to explore the possible mechanism .METHODS: The U251 cells were divided into 4 groups: control group, DCA group, X-ray irradiation without DCA pretreatment ( IR) group and X-ray irradiation with DCA pretreatment ( DIR) group.MTT assay was applied to determine the cell viability .The intracellular reactive oxygen species ( ROS) were detected by DHE fluorescence .The expression level of Bcl-2 was assessed by Western blot .The percentage of apoptosis of cells was determined by flow cytometry .RESULTS:No difference between control group and DCA group in cell viability (P>0.05) was observed.However, the cell viability of both IR group and DIR group was markedly reduced compared with control group ( P<0.05).Furthermore, the viability of DIR group was significantly decreased compared with IR group ( P<0.05 ) .The percentage of ROS positive cells was obviously increased in DIR group compared with IR group (P<0.05).The expression level of Bcl-2 was sharply decreased in DIR group (P<0.05) and the percentage of apoptosis of cells was significantly elevated ( P<0.05) in DIR group compared with IR group .CONCLUSION:The better antitu-mor effect was obtained by improving the radiosensitivity through pretreating the cells with DCA , and the possible mecha-nism was down-regulation of the Bcl-2 expression by developing the intracellular ROS .
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Objective: To investigate the malignant phenotype of human glioma cells by microRNA-7 (miR-7) silencing epidermal growth factor receptor (EGFR)/ phosphatidylinositol kinase-3 (PI3K) pathway. Methods: The human U251 glioma cells were transfected with pri-miR-7. The expression of miR-7 was analyzed by real-time fluorogenic quantitative-PCR (RFQ-PCR). The expressions of epidermal growth factor receptor (EGFR), PI3K and AKT2 proteins were detected by immunocytochemistry and Western blotting. The cell growth curves were drawn, and the cell cycle distribution induced by miR-7 was determined by flow cytometry (FCM). The cell migration ability and tumorigenicity were detected by Transwell chamber assay and soft agar colony assay, respectively. Results: The result of RFQ-PCR showed that the expression level of miR-7 was up-regulated in human glioma cells transfected with pri-miR-7. The expression levels of EGFR, PI3K and AKT2 proteins were down-regulated in glioma U251 cells transfected with pri-miR-7 (P < 0.05). The cell proliferation rate was slowed down, and the proportion of S phase cells was reduced. The abilities of cell migration and soft agar colony formation of U251 cells transfected with pri-miR-7 were significantly reduced (P < 0.05). Conclusion: MiR-7 transfection can effectively silence the expressions of key members of EGFR/PI3K pathway and reverse the malignant phenotype of U251 cells and which is expected to become a new choice of glioma gene therapy. Copyright© 2011 by TUMOR.
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AIM: To evaluate the effectiveness of small interference RNA on inhibiting bcl-2 gene expression. METHODS: With Ambion's software and kit, we designed and synthesized siRNA targeted bcl-2, which were transfected into astrocytoma cell line U251 with lipofectamine. The non-transfected cells and treatment with antisense drug G-3139 were taken as controls. MTT was used to detect the inhibitory rate of cell growth. Flow cytometric method was used to detect the change in cycle of the cells. The inhibitory effect of siRNA on mRNA level was detected by RT-PCR and on protein level was by immunohistochemical method. RESULTS: For the living rate of cell, siRNA 2, 3, 5, 6 groups were significantly lower than siRNA 1, 4 groups, lipofectamine and control group at 24, 48, and 96 h. siRNA 1-6 groups only had statistic difference with antisense group at 24, 48 h. As for PCR and immunohistochemical method, the expression of bcl-2 on siRNA 2, 3, 5, 6 groups were significant lower than other groups. The results of flow cytomytric method showed the cells transfected with siRNA 1-6 and antisense were blocked at S stage. CONCLUSION: siRNA inhibited bcl-2 gene expression more than 50%.