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OBJECTIVE To investigate the effects of polyphyllin Ⅵ（PPⅥ） on the proliferation and apoptosis of glioma cells and potential mechanism. METHODS Using human glioma LN229 cells as objects， MTT assay was used to detect the survival rate after treated with different concentrations of PPⅥ [0 （control group）， 1， 2， 4， 8， 16， 32， 64 μmol/L] for different time （24， 48， 72 h）. The clone formation experiments were adopted to detect the number of cell clones and clone formation rate after being treated with different concentrations of PPⅥ [0 （control group）， 2， 4， 8 μmol/L] for 14 days. The flow cytometry and Western blot assay were used to detect the apoptotic rate of cells， the expressions of apoptosis-related protein [B cell lymphoma-2 （Bcl-2）， Bcl-2 associated X protein （Bax）， cleaved caspase-3]， and the expressions of related proteins of Fas/Fas ligand （FasL） death receptor pathway and protein kinase B （Akt）/glycogen synthesis kinase-3β （GSK-3β） pathway after being treated with different concentrations of PPⅥ [0（control group）， 4， 8 μmol/L] for 24 h. RESULTS Compared with the control group， the survival rate of cells， the number of clones and clone formation rate， the protein expression of Bcl-2， and the phosphorylation levels of Akt and GSK-3β protein were decreased significantly in different concentration groups of PPⅥ （P＜0.05 or P＜0.01）. The apoptotic rate， the protein expressions of Bax， cleaved caspase-3， Fas， FasL and cleaved caspase-8 were increased significantly （P＜0.05 or P＜ 0.01）. CONCLUSIONS PPⅥ can inhibit the proliferation and induce the apoptosis of human glioma LN229 cells， which may be related to the activation of the Fas/FasL death receptor pathway and the inhibition of the Akt/GSK-3β 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 effect of hydroxyurea (HU) combined with temozolomide (TMZ) and radiotherapy (RT) on the sensitivity of human glioma U251 cells to chemoradiotherapy (CRT).Methods:Human glioma U251 cell line was cultured in vitro. CCK8 cell assay was used to detect the proliferation activity of U251 cells treated with different concentrations of HU/TMZ under different conditions. Flow cytometry was utilized to detect apoptosis rate and cell cycle distribution of U251 cells. Transwell chamber assay and scratch test were performed to evaluate the changes of cell invasion and migration. The expression levels of apoptosis proteins were determined by Western blot. Colony formation assay was adopted to detect the cell survival fraction . Results:HU concentration at 50μmol/L and below did not affect the proliferation of human glioma U251 cells ( P>0.05). Low-dose HU combined with CRT significantly inhibited cell proliferation ( P<0.05), invasion ( P<0.01) and migration (12h P<0.001, 24h P<0.01), and promoted cell apoptosis ( P<0.01) compared with the use of CRT alone. Application of 50μmol/L HU combined with RT increased the radiosensitivity of cells (SER=1.49), significantly prolonged the cell cycle of S phase and G 2 phase (both P<0.05), considerably up-regulated the expression levels of the apoptosis-associated proteins of Caspase-3 and Bax and significantly down-regulated the expression level of anti-apoptosis protein of Bcl-2(all P<0.001). Conclusions:Compared with CRT, HU combined with CRT can further inhibit the proliferation, invasion and migration of human glioma U251 cells, promote cell apoptosis, increase the radiosensitivity and prolong the cell cycle of S and G 2 phases, thereby enhancing the sensitivity of human glioma U251 cells to CRT.
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BACKGROUND: In recent years, increasing studies have shown that abnormally activated Hedghog signaling pathway is widely involved in the injury repair of systemic multi-tissue organ diseases, but its related role in myocardial fibrosis is still unclear. OBJECTIVE: To study the effect of blocking Hedgehog-Gli signaling pathway on epithelial-mesenchymal transition of myocardial fibroblasts induced by transforming growth factor-β1 (TGF-β1). METHODS: (1) Animal experiment: The model of myocardial infarction in mice was established by left coronary artery ligature. The pathological changes of myocardial tissue were observed by hematoxylin-eosin staining and Masson staining at 3, 7, and 14 days after infarction. The mRNA, protein and spatio-temporal expressions of Gli1 at different time points were detected by RT-PC, western blot and immunofluorescence. The mRNA and protein expression changes of Gli1 before and after adding inhibitor were detected using RT-PCR and immunofluorescence. (2) Cell experiment: The primary cultured myocardial fibroblasts of neonatal Sprague-Dawley rats were cultured by differential adherent method. The cultured myocardial fibroblasts were cultured with 0, 1, 5, and 10 μg/L TGF-β1 intervention. The expression of Gli1 mRNA and protein was detected by RT-PCR and western blot, respectively. Myocardial fibroblasts were induced by TGF-β1 of 10 μg/L for 24 hours, and the spatio-temporal expression of Gli1 protein and expression of epithelial-mesenchymal transition markers were detected by immunofluorescence. Gli1 and Smo in Hedgehog signaling pathway were specifically blocked by GANT61 and Cyclopamine for 24 hours, respectively, and 10 μg/L TGF-β1 was added. The mRNA expression level of Gli1 was detected by RT-PCR, and the spatio-temporal expression of Gli1 and expression of epithelial-mesenchymal transition markers were detected by immunofluorescence. RESULTS AND CONCLUSION: (1) Results of the animal experiment: As the time after infarction prolonged, the mRNA and protein expression levels of Gli1 in the mouse myocardium gradually increased, but the mRNA and protein expressions of Gli1 in the infarcted tissue decreased after addition of the Hedgehog pathway specific blocker. (2) Results of the cell experiment: After stimulation with TGF-β1, the epithelial mesenchyme of myocardial fibroblasts transformed into myofibroblasts (positive for a-SMA). As the intervention dose of TGF-β1 increased, the mRNA and protein expression of Gli1 gradually increased. After specifically blocking Gli1 and Smo in the Hedgehog signaling pathway for 24 hours, the mRNA and protein expression levels of Gli1 were inhibited, accompanied by the changes in the expression of E-Cadherin and Vimentin during epithelial-mesenchymal transition, indicating the existence of epithelial-mesenchymal transition. By the combination of in vivo and in vitro experiments, this study confirmed that the Hedgehog-Gli signal pathway is involved in the occurrence and development of myocardial fibrosis and myocardial fibroblast transdifferentiation.
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OBJECTIVE：To study the anti-tumor effect of artemether （ARM）self-microemulsifying drug delivery system （SMEDDS） on human glioma subcutaneously transplanted model mice. METHODS ：Human glioma cell line SHG 44 was inoculated and passed on to establish subcutaneous transplanted tumor model of nude mice. At the 5th，10th，15th，20th and 25th day after inoculation ，the tumor tissue volume was measured and the growth curve was drawn to confirm the initial stage of rapid tumor proliferation. Thirty nude mice was collected to establish subeutaneously transplanted tumor nude model ，and then divided into control group （normal saline ），ARM suspension group [ 60 mg/（kg·d）]，ARM-SMEDDS low-dose ，medium-dose and high-dose groups [ 10，20，30 mg/（kg·d）] at the initial stage of rapid tumor proliferation. They were given normal saline and relevant solution intragastrically once a day ，for consecutive 30 d. The weight change and general sibuation of mice were recorded. The change of tumor volume was determined and relative tumor proliferation rate was calculated. RESULTS ：The subcutaneously transplanted tumor tissue entered the initial stage of rapid tumor proliferation from the 10th day after transplantation. The general situation was normal ，and there was no obvious abnormal reaction in mice of each group during treatment. Since 10th day of administration，tumor tissue volume of mice in ARM-SMEDDS groups were shortened significantly than control group （P＜0.05）. At 15th day of administration ，tumor volume of mice in ARM-SMEDDS groups were shortened significantly than ARM suspension group（P＜0.05）. After last administration ，relative tumor proliferation rates of mice in ARM-SMEDDS groups were decreased significantly，compared with ARM suspension group （P＜0.05）. CONCLUSIONS ：ARM-SMEDDS show significant inhibitory effect on the proliferation of human glioma ，and are better than suspension with higher dosage.
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Objective:To explore the effects of Bufalin on the growth and proliferation of human glioma cells U251. Methods:Methyl thiazolyl tetrazolium(MTT)assay was used to detect the effect of Bufalin on the proliferation of human glioma cells U251. An in-verted microscope was used to observe the changes of cell number,morphology and activity. AnnexinV/ PI was used to measure the in-duction of cell apoptosis caused by Bufalin. Results:Bufalin at different concentrations(0. 001 - 10. 0μmol·L - 1 )inhibited the pro-liferation of U251 cells in a dose and time-dependent manner. Compared with that of the control group,the apoptosis rate of Bufalin group was increased significantly(P < 0. 01). Conclusion:Bufalin can inhibit the growth and proliferation of U251 cells in a dose and time-dependent manner,and induce the apoptosis of U251 cells.
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Objective The derivative of Gefitinib was used to treat glioma cells in vitro to explore a more effective new drug for the clinical treatment of astrocytoma. Methods (1) Fifteen kinds of gefitinib derivatives, gefitinib and temozolomide were used to treat glioma cells, and the effect of 0, 10, 15, 20, 25 and 30 μmol/L of each kind of drug on cell proliferation was detected by by MTT assay , respectively. (2) To calculate the concentration of IC50 , then select lower IC50 of derivativs combinate gefitinib and temozolomide with 10, 20 and 30 μmol/L to treat cells, then the apoptosis of cells were detected by flow cytometry. Expression of p-EGFR was detected by western–blot assay. Results (1) NO.LPY-5,9,11, but not other derivatives of Gefitinib could effectively inhibit the growth of cells. (2) IC50 of NO.LPY-9 was less than that of the 5th drug, and both of them were lower than those of gefitinib and temozolomide; NO. LPY-11 was excluded. (3) The cell apoptosis of No. LPY-9 was higher than that of gefitinib and temozolomide , respectively. However, No.LPY-9-induced cell apoptosis was significantly higher than that of No. LPY-5-induced cell. (4) Levels of p-EGFR expression in No.LPY-9 and gefitini-induced cells were significantly lower than that in the negative control group. Conclusion No.LPY-9 has asignificant inhibitory effect on glioma cells in vitro , resulting from the inhibition of the ERFR-mediated signaling pathways and induction of cell apoptosis.
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Objective To investigate the effect of knocking down miR-19a on proliferation and apoptosis of human glioma cell line U251 in vitro.Methods U251 cells were cultured routinely.MiR-19a inhibitor was transfected into U251 cells by Lipofectamine 2000.At the same time,the negative control group and blank control group were established.The expression level of miR-19a was detected by RT-PCR.and cell proliferation was analyzed by CCK-8 assay.The changes of cell apoptosis and cycle were monitored by flow cytometry.Results Compared with the negative control group and blank control group,qRT-PCR showed that the expression level of miR-19a was significantly reduced after transfection (F=124.72,P ＜ 0.01).CCK-8 revealed that proliferation ability of miR-19a inhibitor group was significantly suppressed.The cell survival rate in miR-19a inhibitor group after 48 h was (48.27-±8.23) ％,compared with the blank control group (100.00 ％),the difference was statistically significant (t =12.45,P ＜ 0.01).After low-expression of m iR-19a,the G0/G1 phase cells were increased and S phase cells were decreased.The low-expression of miR-19a could induce cell apoptosis.Conclusions Low-expression of miR-19a can inhibit cell proliferation,block G1/S transition and induce apoptosis in human glioma cell line U251.miR-19a may serve as an attractive target of gene therapy for glioblastoma.
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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.
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OBJECTIVE@#To explore the suppressing effect of γ-secretase inhibitor DAPT on proliferation of human glioma cell line SHG-44 in vitro and its mechanism.@*METHODS@#The SHG-44 cell was treated by DAPT with different concentration. The proliferation of cells was detected by MTT assay; cell cycle and TSC of CD133(+) were determined by flow cytometry analysis technique; the key factor in Notch signaling pathway (Notch-1, Delta-1, Hes-1) was measured by reverse transcriptase-polymerase chain reaction and western blotting.@*RESULTS@#DAPT inhibited the growth and proliferation of SHG-44 cells significantly(P<0.05). And the inhibiting effect on SHG-44 cells produced by DAPT showed a dose-dependent manner. DAPT increased the rate of cells in G0/G1 phase of SHG-44 cells, while it decreased the rate of cells in S phase. TSC of CD133(+) was significantly reduced after DAPT treated SHG-44 cells. The expression of protein and mRNA of Notch-1, Delta-1 and Hes-1 were gradually downregulated with the increase of DAPT doses.@*CONCLUSIONS@#DAPT can downregulate these key factor in Notch signaling pathway, reduce the TSC of CD133+ and inhibit the proliferation of SHG-44 cells.
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Humans , Antineoplastic Agents , Pharmacology , Cell Cycle , Cell Line, Tumor , Cell Proliferation , Cell Shape , Dipeptides , Pharmacology , Glioma , Signal TransductionABSTRACT
O astrocitoma grau IV ou glioblastoma multiforme (GBM) é o mais maligno e com prognóstico ruim entre os gliomas. Esse prognóstico sombrio está associado, em parte, à quimiorresistência (QR). Ao lado disso, a classificação atual dos gliomas não consegue responder a heterogeneidade da resposta ao tratamento. Assim, parece existir subtipos de GBM com características distintas. Dessa forma, o objetivo desse trabalho foi caracterizar fenotipicamente uma nova linhagem, ESP12, e, desenvolver um modelo in vitro para a avaliação da QR. Amostras obtidas de glioma humano foram estudadas quanto aos achados característicos de malignidade e subtipadas quanto aos fenótipos proliferativo e pró-neural, imunohistoquimicamente. As culturas obtidas das amostras foram mantidas a 37 ºC em atmosfera com 5% de CO2. A caracterização de ESP12 incluiu: a) subtipagem por imunocitoquímica e por citometria de fluxo; b) investigação de um fenótipo de resistência, através da identificação de células CD133+...
The astrocytoma grade IV also known as glioblastoma multiforme (GBM) is the most malignant and has a poor prognosis among gliomas. This poor prognosis is associated, in part, to chemoresistance (QR). Furthermore, the current classification of gliomas cannot answer the heterogeneity of treatment response. Therefore, it seems to exist GBM subtypes with distinct characteristics. The aim of this study was to characterize phenotypically a new cell line, ESP12, and to develop an in vitro model for the assessment of QR. Human glioma samples were studied by immunohistochemistry for the characteristic findings of malignancy and subtyped as to proliferative and proneural phenotypes. Primary cultures were obtained from samples and maintained at 37 °C in an atmosphere with 5% CO2. The characterization of ESP12 included: a) subtyping by immunocytochemistry and flow cytometry; b) investigation of a resistance phenotype by identifying CD133+...
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Humans , Glioblastoma/mortality , Glioblastoma/pathology , Brain Neoplasms/complications , Brain Neoplasms/diagnosis , Brain Neoplasms/mortality , Brain Neoplasms/pathologyABSTRACT
Background and purpose:Integrinαvβ3 receptor plays an important role in promoting, sustaining and regulating the angiogenesis. It is overexpressed on neovascular endothelial cells and tumor cells. RGD peptide specifically binds to integrinαvβ3, which could evaluate growth status and invasiveness of tumor. This study aimed to investigate the biodistribution in healthy KM mice and micro PET/CT imaging in U87MG tumor-bearing mice of 18F-E[c(RGDfK)2]. Methods: 18F-E[c(RGDfK)2] was produced using an automated synthesis module via a simple one-step 18F-labeling strategy of the precursor 4-NO2-3-TFMBz-E[c(RGDfK)2]. The percentage activity of injection dose per gram of tissue (%ID/g) was calculated at 0.5, 1, 2, 4 h post injection of the probe. Micro PET/CT images of U87MG tumor-bearing nude mice with or without 18F-E[c(RGDfK)2] blocking were acquired at each time point. Results: The labeling efficiency and radiochemical purity of 18F-E[c(RGDfK)2] were 10% and 98%, respectively. 18F-E[c(RGDfK)2] was excreted via renal route, with a high blood clearance. The other organs had background-level activity accumulation. At 1 h, the%ID/g of kidney, liver, intestine, muscle and blood was (1.02±0.16)%ID/g,(0.24±0.06)%ID/g, (0.35±0.03)%ID/g, (0.13±0.03)%ID/g and (0.11±0.03)%ID/g 18F-E[c(RGDfK)2] had initial high tumor uptake [(5.2±0.56)%ID/g] and good tumor-to-background contrast (5.36) at 1 h post injection. Tumor uptake for blocking group was lower than those without blocking, and T/M reduced to 1.57. Conclusion: 18F-E[c(RGDfK)2] appears a promising PET molecular imaging probe targeting integrin αvβ3, with high tumor uptake. It could be suitable for prognosis evaluation of integrin-positive tumor, selection of vascular targeting therapy and therapy effect monitoring.
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To investigate the inhibitory effects of Ginsenoside Rb1 (GRb1) on apoptosis caused by Herpes Simplex Virus-1 (HSV-1) in Human Glioma Cells (U251),U251 cells were infected by HSV-1 at a multiplicity of infection of 5 and GRb1,GRb1+HSV-1,HSV-1 and control groups.MTT and cell apoptosis assays were used to detect the inhibitory effects of GRbl on the apoptosis of U251 cells that caused by HSV-1 infection for various concentrations of drug and virus treatments by MTT assay.We found that in the 400 μg/mL GRbl and 400 μg/mL GRbl+HSV-1 groups,MTT values were higher than control group at all times (P＜0.05).Moreover,the apoptosis rate in the 400 μg/mL GRb1+HSV-1 group was lower than the HSV-1 group (P＜0.05).These results confirmed that,at appropriate concentrations,GRb 1 could inhibit nerve cell apoptosis in HSV-1 infections.
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Objective To provide more convincing evidences and experimental data for exploring vanillin derivative BVAN08,6-bromine-5-hydroxy-4-methoxy-benzaldehyde,as a new anticancer drug,and to investigate the effect on the growth,radiosensitization of human glioma cell line U-251 and the relative mechanism.Methods The effect of BVAN08 on cell proliferation of U-251 and radiosensitivity to 60Co γ-rays (irradiation dose rate 2.3 Gy/min) were analyzed with MTT and colony-forming ability assay.Change in cellular morphology was observed by using light microscope.Change in cell cycle and apoptosis was detected with flow cytometry.The autophagy was observed by using TEM (irradiation dose rate is transmission electron microscope).DNA-PKcs protein level was detected through Western blot analysis.Results BVAN08 exhibited a dose- and time-dependent inhibition on the proliferation of U-251 cells during the concentration range of 10-100 mol/L (t = 1.83-3.07,P ＜ 0.05).IC50 at 48 h and 72 h after administration with BVAN08 were 55.3 and 52.7 mol/L,respectively.Obvious G2/M arrest was induced in U-251 cells after 4 h administration with BVAN08,and reached peak at 12 h.The G2/M population reached 63.3% in U-251 cells after 12 h administration of 60 μmol/L BVAN08 and kept increasing with the time,while both apoptosis and autophagic cell death were induced.The most effective radiosensitization time for BVAN08 treatment was 12 h before irradiation.The enhancement ratio of radiosensitivity was 3.14 for 20 μmol/L of BVAN08 12 h before 2 Gy irradiation.Conclusions BVAN08 can nduce apoptosis as well as autophygic cell death of U-251 cells,and sensitize U-251 cells.The mechanism of its radiosensitizing effect might be associated with the induction of G2/M arrest and inhibition of DNA-PKcs expression.BVAN08 seemed to be a romising radiosensitizing anticancer drug.
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OBJECTIVE: We determined whether the expression of GRIM-19 is correlated with pathologic types and malignant grades in gliomas, and determined the function of GRIM-19 in human gliomas. METHODS: Tumor tissues were isolated and frozen at -80degrees C just after surgery. The tissues consisted of normal brain tissue (4), astrocytomas (2), anaplastic astrocytomas (2), oligodendrogliomas (13), anaplastic oligodendrogliomas (11), and glioblastomas (16). To profile tumor-related genes, we applied RNA differential display using a Genefishing(TM) DEG kit, and validated the tumor-related genes by reverse transcription polymerase chain reaction (RT-PCR). A human glioblastoma cell line (U343MG-A) was used for the GRIM-19 functional studies. The morphologic and cytoskeletal changes were examined via light and confocal microscopy. The migratory and invasive abilities were investigated by the simple scratch technique and Matrigel assay. The antiproliferative activity was determined by thiazolyl blue Tetrazolium bromide (MTT) assay and FACS analysis. RESULTS: Based on RT-PCR analysis, the expression of GRIM-19 was higher in astrocytic tumors than oligodendroglial tumors. The expression of GRIM-19 was higher in high-grade tumors than low-grade tumors or normal brain tissue; glioblastomas showed the highest expression. After transfection of GRIM-19 into U343MG-A, the morphology of the sense-transfection cells became larger and more spindly. The antisense-transfection cells became smaller and rounder compared with wild type U343MG-A. The MTT assay showed that the sense-transfection cells were more sensitive to the combination of interferon-beta and retinoic acid than U343MG-A cells or antisense-transfection cells; the anti-proliferative activity was related to apoptosis. CONCLUSION: GRIM-19 may be one of the gene profiles which regulate cell death via apoptosis in human gliomas.
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Humans , Apoptosis , Astrocytoma , Brain , Cell Death , Cell Line , Collagen , Drug Combinations , Glioblastoma , Glioma , Interferon-beta , Laminin , Light , Microscopy, Confocal , Oligodendroglioma , Polymerase Chain Reaction , Proteoglycans , Reverse Transcription , RNA , Tetrazolium Salts , Thiazoles , Transfection , TretinoinABSTRACT
Objective To investigate the invasion ability and collagenolytic activity of human glioma cells in vitro.Methods Boyden chamber invasion assay was employed to evaluate the cell migration ability of human malignant glioma cell line U87MG in vitro and the effect of conditioned medium of U87MG cells on matrix collagenolytic activity was tested by agar-gelatin gel.Results The number of U87MG glioma cells migrating through the Matrigel-coated membrane was more than that of addition of EDTA or anti-MMP-2 antibody in U87MG glioma cells(P0.05).EDTA or anti-MMP-2 antibody markedly inhibited the number of the migrated cells,the inhibitory rates were 79.2% and 77.1%,respectively. The conditioned medium collected from the U87MG cells showed an increase in the transparent ring area on agar-gelatin gel.Inhibition of MMP-2 enzymatic activity by EDTA or anti-MMP-2 antibody reduced the transparent ring area on agar-gelatin gel.Conclusion Both invasion ability and collagenolytic activity of U87MG cells are depended on MMP-2,suggesting that MMP-2 plays an important role in glioma invasiveness.
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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
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Objective:To study the effects of curcumin on the activity of ATPase and the mechanism of apoptosis in U-251 cell.Methods:U-251 cells were treated with 20,40,80,100?mol/L curcumin for 24 h and the growth inhibition rates of U-251 cells were measured with MTT method.Cell apoptosis was inspected with flow cytometry(FCM).The activities of ATPase were determined by colorimetry method.Results:Curcumin inhibited the proliferation of U-251 cells and induced apoptosis of U-251 cells.level of ATPase in U-251 plasma Membrane was low remarkably.Conclusion:Curcumin induced apoptosis and inhibited proliferation of U-251 cell via inhibition of activation of plasma Membrane ATPase.
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Objective :To construct the small interfering RNA(siRNA) eukaryotic expression vectors of human ClC-2 gene. Methods: According to the program and principles of DEQOR about designing siRNA,two pairs ClC-2 mRNA-targeted hairpin siRNA were devised,and the two pairs complemantary oligonucleotide strands of DNA fragments that encoded the above siRNA were synthesized by chemosynthesis.After annealing of the complementary strands,the DNA fragments were connected to the polyclone sites of plasmid eukaryotic expression vector pSUPER.puro that was cut by restriction endonuclease BglⅡ and HindⅢ,followed by transformation,amplification and plasmid extraction in E.coli,and finally,the two recombinant plasmids were identified by agarose gel electrophoresis by means of cutting with EcoRⅠand HindⅢ and by DNA sequence analysis.The plasmids were transfected transiently into human glioma cell line BT-325 cells by Lipofectamine~(TM)2000.The mRNA expression of ClC-2 gene was detected by(RT-PCR.) Results: The connections between the DNA fragments encoding ClC-2-targeted siRNA and the pSUPER.puro plasmid were correct,as confirmed by agarose gel electrophoresis and DNA sequencing(analysis.) ClC-2 mRNA expression of the BT-325 cells transfected two recombinant vectors was(significantly) decreased.Conclusion: The two RNAi recombinant vectors of human ClC-2 gene were successfully constructed.They were named pSUPER.puro-siClC-21 and pSUPER.puro-siClC-22,respectively.This laid the groundwork for future research about ClC-2 gene affecting invasion and migration of human glioma cells.
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OBJECTIVE: The objective of this study was to determine the photodynamic therapeutic response of U-87 human glioma cell in vitro as well as in the nude rat xenograft model using photofrin as photosensitizer. MATERIAL AND METHOD: U-87 cells were cultured on 96-well culture plates, photofrin(Quadralogic Technologies Inc., Vancouver, Canada) was added into the cell culture medium at concentration of 1ng/ml, 2.5ng/ml, 5ng/ml, 10ng/ml and 20ng/ml. 24 hour after drug treatment, cells were treated with optical(632nm) irradiation of 100mJ/cm2, 200mJ/cm2 and 400mJ/cm2. Photofrin(12.5mg/kg, i.p.) was administered to 28 nude rats containing intracerebral U-87 human glioma as well as 26 normal nude rats. 48 hours after administration, animals were treated with optical irradiation(632nm) of 35J/cm2, 140J/cm2 and 280J/cm2 to exposed tumor and normal brain. The photofrin concen-tration was measured in tumor and normal brain in a separate population of animals. RESULTS: By MTT assay, there was 100% cytotoxicity at any dose of photofrin with optical irradiation of 200mJ/cm2 and 400mJ/cm2. But at the optical irradiation of 100mJ/cm2 cells were killed in dose dependent manner 28.5%, 49.1%, 54.4%, 78.2%, and 84.6% at concentration of 1ng/ml, 2.5ng/ml, 5ng/ml, 10ng/ml and 20ng/ml, respectively. Dose dependent PDT lesions in both tumor and normal brain were observed. In the tumor lesion, only superficial tissue damage was found with optical irradiation of 35J/cm2. However, in the optical irradiation group of 140J/cm2 and 280J/cm2 the volume of lesions was measured of 7.2mm3 and 14.0mm3 for treatment at 140J/cm2 and 280J/cm2, respectively. The U-87 bearing rats showed a photofrin concentration in tumor tissue of 6.53+/-2.16ng/g, 23 times higher than that found in the contralateral hemisphere of 0.28+/-0.15ng/g. CONCLUSION: Our data indicate that the U-87 human glioma in vitro and in the xenografted rats is responsive to PDT. At these doses, a reproducible injury can be delivered to human glioma in this model. Strategies to spare the normal brain collateral damage are being studied.