ABSTRACT
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.
ABSTRACT
Objective: To investigate the regulatory effects of resveratrol (Res) on the epithelial to mesenchymal transition (EMT), migration and invasion abilities of the glioma U87 cells, and to clarify the inhibitory effect of Res on EMT of the cells. Methods: The glioma U87 cells were divided into control group (no treatment), EMT group (EMT was induced with TGF-β1) and Res treatment group (EMT was treated with Res). The expression levels of mesenchymal markers (N-cadherin, β-catenin, Vimentin), EMT-inducing transcription factors (Snail, Slug, Twistl), matrix metalloproteinase (MMP)-2 and MMP-9 in the glioma U87 cells were measured by Western blotting method. Scratch-healing assay and Trans well assay were performed to examine the cell migration and invasion abilities. Results: Compared with control group, the protein expression levels of mesenchymal markers (N-cadherin, β-catenin, Vimentin), EMT-inducing transcription factors (Snail, Slug, Twistl), and MMP-2 and MMP-9 in EMT group were significantly increased (P<0. 05). Compared with EMT group, the expression levels of above proteins in Res treatment group were markedly down-regulated (P<0. 05 or P <0.01); the effect in 40 μmol · L1 Res treatment group was more obvious than that in 20 μmol · L-1 Res treatment group. The migration and invasion rates of the U87 cells in EMT group were obviously elevated compared with control group (P<0. 01), and the migration and invasion rates of the glioma U87 cells in Res treatment group were significantly lower than those in EMT group (P<0. 01). Conclusion: Res can inhibit EMT of the glioma U87 cells and reduce the migration and invasion abilities of the cells.
ABSTRACT
Objective@#To investigate the effects of formyl peptide receptor 2 (FPR2) silencing on the proliferation, migration and invasion of human glioma U87 cells and its possible mechanisms.@*Methods@#The expression of FPR2 was detected in normal glial cells, glioma cells, normal brain tissues and glioma tissues using Western blot and immunohistochemistry staining. A synthesized siRNA duplex was employed to inhibit FPR2 in human glioma cells (U87). The knockdown efficiency was evaluated by real-time reverse transcription-polymerase chain reaction (RT-qPCR) and Western blot. MTT, transwell assays and flow cytometry analyses were used to determine the cell proliferation, migration, invasion and apoptotic rates of U87 cells, respectively. Mice xenograft experiments were used to observe the effect of FPR2 silencing on the tumorigenesis of U87 cells in vito. Western blot and enzyme-linked immunosorbent assays were performed to detect the expression and release of cell cycle and migration-related proteins.@*Results@#The expression of FPR2 was significantly higher in glioma cell lines and glioma tissues than that in normal glial cells and brain tissues. Compared with blank control and negative control, FPR2 mRNA and protein levels in siRNA group were significantly downregulated. The cell proliferation inhibitory rates in FPR2 siRNA group were (23.1±5.1)%, (39.6±5.6)% and (44.4±6.7)% at 24 h, 48 h and 72 h, respectively, which were significantly increased than those in negative control group [(3.2±0.6)%, (5.7±0.8)% and (7.9±0.9)%, respectively; P<0.05]. The apoptosis rate in FPR2 siRNA group was (17.4±2.1)%, which was significantly elevated than that in the negative control group with (5.4±0.5)% and blank control group with (3.8±0.3)% (all P<0.05). In addition, the numbers of migrated cells were 108.7±9.5 in FPR2 siRNA group, which was significantly lower than that in blank control group 312.9±17.5 and negative control group (304.4±15.7, all P<0.05). Likewise, the numbers of invaded cells were 19.3±3.2 in FPR2 siRNA group, which was significantly lower than that in blank control group 106.9±8.5 and negative control group (102.4±7.4, all P<0.05). Moreover, the growth of FPR2 siRNA transfected U87 cells in vivo was remarkably decreased comparing with the negative group (P<0.05). Furthermore, the expression of cyclin D1 and VEGF in FPR2 silencing U87 cells was suppressed mainly through β-catenin signaling pathway.@*Conclusions@#FPR2 silencing by siRNA can inhibit the growth, migration and invasion ability, but promote the apoptosis of U87 cells. The possible mechanisms might be associated with the inhibitory expression of cyclin D1 and VEGF.
ABSTRACT
ABSTRACT:Objective To investigate the anti-metastatic effect of allicin on glioma cell line U87 and related mechanisms.Methods In this study,we employed MTT assay to test the anti-proliferative effect of allicin. Transwell assay was used to test the anti-metastatic ability of allicin.Real-time PCR and Western blotting were employed to test the effect of allicin on the expressions of matrix metalloproteinase-2 (MMP-2 ) and matrix metalloproteinase-9 (MMP-9).Western blotting was employed to test the phosphorylated level of p38.Results Allicin could significantly inhibit the proliferation and invasion of U87 cells (concentration>8 μg/mL,P <0.05). Meanwhile allicin (concentration<8μg/mL)could inhibit the invasion of U87 cells.After treatment with allicin for 24 hours,the expressions of MMP-2 and MMP-9 were decreased significantly (P < 0.05 ).Moreover,allicin treatment decreased the phosphorylated level of p38 obviously (P < 0.05 ).Conclusion Allicin inhibits the invasion and migration of glioma cell line U87 by reducing the expressions of MMP-2 and MMP-9 via suppressing the activity of p38 signal pathway,suggesting that allicin is a potential therapeutic agent for glioma.
ABSTRACT
Objective To identify the role of microRNA-17(miR-17)in human glioma U87 cells invasion which may regulate expression of matriv metalloproteinase(MMP)-2.Methods U87 cells were cultured in vitro,while changes in cellular morphology were observed by phase contrast microscope.The miR-17 which might regulate the expression of MMP-2 was predicted by bioinformatics and identified using dual luciferase report system.Expressions of miR-17 and MMP-2 were determined using real-time PCR and Western blot after transfection of miR-17 mimics.The invasion of U87 cells was detected in vitro by Transwell chamber.Results Expression of MMP-2 was positive by immunofluorescence cytochemistry. Using dual luciferase reporter system,miR-17 could inhibit the expression of MMP-2 by binding to its mRNA 3′UTR. Results of real-time PCR and Western blot showed that over-expression of miR-17 down-regulated expression of MMP-2. The invasion of U87 cells was suppressed by over-expression of miR-17.Conclusion MiR-17 may negatively regulate expression of MMP-2 in human glioma U87 cells and inhibit cell invasion.
ABSTRACT
OBJECTIVE Toobservetheeffectoftemozolomide(TMZ)incombinationwithtetran-drine(TET)on cell viability,colony formation,migration and cell apoptosis of human glioblastoma U87 cells.METHODS TheviabilityofU87cellstreatedwithTET(8-64μmol·L-1),TMZ(50-400 μmol·L-1 )and TMZ combined with TET (3.2,6.4 μmol·L-1 )was detected by cytotoxicity assays with Cell Counting Kit-8 (CCK-8),the colony formation was detected by Giemsa staining,cell migration ability was detected by Transwell migration assay,cell apoptosis was assayed by flow cytometry using Annexin Ⅴ /PI double staining,and the expression of apoptosis-related proteins expression was detec-tedbyWesternblotting.RESULTS ThedataofCCK-8showedthatTET(r=0.903,P<0.05)orTMZ (r=0.995,P<0.05)could inhibit U87 cell viability alone in a concentration-dependent manner.The cell viability inhibition rate of U87 cells by TMZ co mbined with TET was higher than by TMZ or TET alone. Data showed that the effect of TMZ combined with TET was additive.TMZ 100 μmol·L-1 inhibited U87 cell colony formation and migration ablility compared with normal control.The inhibition rate of U87 cells by TMZ 100 μmol·L-1 combined with TET (3.2 and 6.4 μmol·L-1 )was more significant than by TMZ alone (P<0.05).Compared with TMZ alone,TMZ combined with TET (3.2 and 6.4 μmol·L-1 )signifi-cantly down-regulated the expression of anti-apoptotic protein Bcl-XL,but significantly up-regulated the expression of cleaved caspase 3 protein and cleaved poly(ADP-ribose)polymerase.CONCLUSION TET combined with TMZ can inhibit U87 cell viability,colony formation and migration by activating caspase-dependent apoptotic pathway,resulting in apoptosis.
ABSTRACT
Agents that elevate cellular cAMP are known to inhibit the activation of phospholipase D (PLD). We investigated whether PLD can be phosphorylated by cAMP-dependent protein kinase (PKA) and PKA-mediated phosphorylation affects the interaction between PLD and RhoA, a membrane regulator of PLD. PLD1, but not PLD2 was found to be phosphorylated in vivo by the treatment of dibutyryl cAMP (dbcAMP) and in vitro by PKA. PKA inhibitor (KT5720) abolished the dbcAMP-induced phosphorylation of PLD1, but dibutyryl cGMP (dbcGMP) failed to phosphorylate PLD1. The association between PLD1 and Val14RhoA in an immunoprecipitation assay was abolished by both dbcAMP and dbcGMP. Moreover, RhoA but not PLD1 was dissociated from the membrane to the cytosolic fraction in dbcAMP-treated cells. These results suggest that both PLD1 and RhoA are phosphorylated by PKA and the interaction between PLD1 and RhoA is inhibited by the phosphorylation of RhoA rather than by the phosphorylation of PLD1.