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<p><b>OBJECTIVE</b>To elucidate how ethanol extract of L. serratum (ELS) could exert anti-migratory effects on glioma with the suppression of nuclear factor kappa B (NF-κB) downstream pathway.</p><p><b>METHODS</b>Cell viability of ELS on C6 glioma was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Nitric oxide (NO) assay and 2',7'-dichlorofluorescin diacetate (DCFH-DA) assay were applied to measure NO production and reactive oxygen species (ROS) generation on lipopolysaccharide (LPS)-induced C6 glioma cells. NF-κB, mitogen-activated protein kinase (MAPK), inducible nictric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein were determined by Western blot. Wound healing assay was used to investigate the inhibitory effect of ELS on fetal bovine serum (FBS)-induced migration and matrix metalloproteinase (MMP)-9 and -2 activity was examined by zymography.</p><p><b>RESULTS</b>ELS suppressed LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 through inhibiting the expression of chemokine CCL2 (or monocyte chemoattractant protein-1, MCP-1). In addition, ELS inhibited the expression of iNOS, COX-2, and the production of NO by LPS in C6 glioma cells. ELS also significantly decreased serum-induced migration of C6 glioma cells in scratch wound healing in a dose-dependent manner (P<0.01). The activity of MMP-9 and -2 were also significantly attenuated by ELS with LPS treatment (P<0.01).</p><p><b>CONCLUSIONS</b>Our results suggest that downregulation of MMP-9 gene expression might be involved in the anti-migration effect of ELS against LPS-induced C6 glioma cells.</p>
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Objective To clarify the inhibitory effect of Tripterine combined with Cisplatin on C6 glioma ceils and its apoptosis mechanism.Methods C6 glioma cells were treated with Tripterine,Cisplatin,or Tripterine combined with Cisplatin.CCK-8 assay was used to detect the growth inhibition rate in each group.Flow cytometry analysis was used to test the cell apoptosis rate.The expressions of apoptosis-related proteins including Bcl-2,Bax,XIAP,NF-kB were analyzed by ELISA.Results Compared with Tripterine-or Cisplatin-treated group,the inhibition ratio of cell growth in Tripterine and Cisplatin combination group was (69.76±7.28)％,which could significantly inhibit the growth of C6 glioma cells(t=23.78,P＜0.01).The apoptosis rate was significantly higher (47.75±5.63)％ in combination group than in Tripterine or Cisplatin treated group.The results of ELISA showed that the expression of Bax was significantly higher and Bcl-2,XIAP,NF-κB were obviously lower in the combination group than in Tripterine or Cisplatin treated group (t=35.27,P＜ 0.01).Conclusion The combination of Tripterine and Cisplatin significantly increases the inhibition rate on C6 glioma cells through upregulating Bax and inhibiting Bcl,XIAP and NF-kB to induce the apoptosis of C6 glioma cells.
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Aim To investigate the effects of isoliquiri-tigenin(ISL)on C6 glioma cell proliferation and differ-entiation.Methods C6 glioma cells’viability and proliferation were respectively measured by SRB test. Colony formation of C6 glioma cells from different groups was assayed.After culturing the cells from each group,giemsa staining was used to observe cell mor-phology.RT-PCR was applied to detect mRNA expres-sion of GFAP.Western blot was applied to detect the expression of GFAP.Results ISL effectively inhibited the viability of C6 glioma cells when compared with the control group in a concentration-dependent manner (P<0.01).The morphological observation under light mi-croscope showed that:in the control group,most of the undifferentiated C6 cells showed long fusiform and po-lygonal shape.Compared to the control group,the C6 cells treated with ISL revealed alteration in morphology such as astrocytes with smaller smooth,round body and much finer longer,tapering processes.The cloning for-mation rate detection revealed that:the colonies in the control group semerged earlier and were larger than those experimental ones,the cloning formation rate was higher,while almost no effective cells colony emerged in ISL treated groups(P <0.01 ).Western blot and RT-PCR analysis showed that GFAP expression in the ex-perimental groups increased(P <0.01).Conclusion ISL may inhibit the proliferation of C6 glioma cells and induce their differentiation.
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@#Objective To investigate the inhibitory effect of Pingliukang (a prescription) medicated serum on the proliferation of cultured rat C6 glioma cells and influence on cell cycle in vitro. Methods MTT colorimetry were performed to measure the levels of the proliferation of rat C6 glioma cells cultured with 2.5%, 5%, 10% and 20% of Pingliukang medicated serum for 24 h, 48 h and 72 h in vitro. The effect of Pingliukang medicated serum on cell cycle were observed by FCM. Results The proliferation of C6 cell was obviously inhibited by Pingliukang medicated serum with dose-effect relationship. The inhibitory effect of 20% of medicated serum was the strongest. When the C6 glioma cells were treated with 10% and 20% medicated serum for 48 h and 72 h, the cells of S period declined. Conclusion The Pingliukang medicated serum can inhibit the proliferation and block cell cycle of cultured C6 glioma cells.
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Objective To investigate the anti-tumor effect and mechanism of tamoxifen on rat C6 glioma cells. Methods C6 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 3% fetal calf serum (FCS), and treated with tamoxifen of different concentrations, i.e. group A (1.25μmol/L), group B (2.50 μmol/L), group C (5.00 μmol/L), group D (10.00 μmol/L), group E (20.00 μmol/L) and control group (0.00 μmol/L). Morphological changes, MTT assay and 5-bromo-2'-deoxyuriding labeling ratio were assessed. Apoptosis was observed by flow cytometry. Results C6 cells treated with different doses of tamoxifen for 24, 48, and 72 hours became irregular in shape, while cells treated with vehicle grew normally. MTT assay showed that tamoxifen did not suppress C6 cell growth until 72 hours after treatment. Seventy-two hours after treatment, there were significant differences in cell viable rate between group A versus groups C, D and E; so did group B versus group D as well as group E (P<0.05). BrdU incorporation assay indicated significant difference of BrdU labbled index (BrdU LI) among groups A, C, E and control group 48 hours after treatment (P<0.05). And the BrdU LI decreased with the increased concentration of tamoxifen. Flow cytometry (FCM) showed significant difference between treated group and control group at 24, 48, and 72 hours after treatment (P<0.05). Conclusion Tamoxifen significantly suppresses the growth of C6 glioma cells in a time- and dose-dependent manner. The mechanism of tamoxifen suppressing C6 glioma cells may be inhibiting proliferation and inducing apoptosis. Therefore, tamoxifen can be a candidate as a chemotherapy agent for glioma.
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rAdinbitor was cloned from Gloydius blomhoffi brevicaudus in the laboratory. Previous researches had proved that rAdinbitor could inhibit proliferation of C6 glioma cells as well as promote their apoptosis. The molecular mechanism of rAdinbitor’s effects on C6 cells need to be further studied. rAdinbitor was expressed in E. coli BL21/pET23b-adinbitor and purified with Ni Sepharose 6 Fast Flow. The purified protein was confirmed by Western blotting. C6 cells were induced with fibronectin (FN). The effects of rAdinbitor with different concentrations on the expression of FAK, MEK1/2 and Caspase-3 as well as on activity of FAK and ERK1/2 in FN-induced C6 cells were studied by immunoblotting and immunoprecipitation. Results showed that rAdinbitor with different concentrations could obviously reduce the expression of FAK and MEK1/2, increase the expression of Caspase-3, as well as decrease ERK1/2 phosphorylation; besides 10 mg/L rAdinbitor, other concentrations’ rAdinbitor could inhibit FAK phosphorylation obviously. All those effects were dose-dependent. Results indicate that the effects of rAdinbitor on decreasing expression and activity of FAK and inhibiting Ras-MAPK signaling pathway play an important role in suppressing the proliferation of C6. Furthermore, the increase in Caspase-3 expression implies that the increase in apoptosis of C6 cells might be due to the suppression of rAdinbitor on the activity of ILK and PI-3K/Akt pathway.
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To evaluate whether different doses of X ray irradiation can induce apoptosis of rat C6 glioma cells. A rat C6 glioma cell was irradiated with X ray in different doses in vitro. Apoptosis was identified by the classical “ladder” pattern (oligonucleosome sized fragments) in DNA agarose gel electrophoresis. Results: The results showed that significant apoptosis was found in 15, 20 or 30 Gy groups at 4 hours and 1 day after X ray irradiation compared with control, whereas this phenomenon could only be seen vaguely in the 40 Gy group. The latter group showed more necrosis than apoptosis. It suggested that the apoptotic mode of cell death and necrosis may represent the response in this X ray irradiated rat C6 glioma cells in vitro.
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To clarify the effect of extracellular ATP in cultured C6 glioma cells, ATP-induced cytosolic free calcium ((Ca2+)i) mobilization and cell proliferation were investigated. ATP-induced (Ca2+)i increased in a dose-dependent manner (10-7 M apprx 10-3 M). ATP-induced (Ca2+)i increases were slightly slowed in extracellular calcium-free conditions especially in sustained phase. ATP-induced (Ca2+)i increment was also inhibited by the pretreatment of U73122, a phospholipase C (PLC) inhibitor, in a time-dependent manner. Suramin, a putative P2Y receptor antagonist, dose-dependently weakened ATP-induced (Ca2+)i mobilization. Significant increases in cell proliferation were observed at 2, 3, and 4 days after ATP was added. Stimulated cell proliferation was also observed with adenosine at days 2 and 3. This cell proliferation was significantly inhibited by the treatment with suramin. Ionomycin also stimulated cell proliferation in a concentration-dependent manner. In conclusion, we suggest that extracellular ATP stimulates C6 glioma cell proliferation via intracellular free calcium mobilization mediated by purinoceptor.
Subject(s)
Adenosine , Adenosine Triphosphate , Calcium , Cell Proliferation , Cytosol , Glioma , Ionomycin , Receptors, Purinergic , Suramin , Type C PhospholipasesABSTRACT
Rat C6 glioma cells were cultured in vitro,and 10?L C6 cell suspension containing 10g/L agarose and 1?10 6 C6 cells was injected into the right caudate nucleus of rat brains to establish a rat brain tumor model by directional implantation method.General observation and MRI scan were conducted after implantation.Trans aorta paraformaldehyde perfusion for 5 group rats having received implantation was carried out at 10,15,20,25,days and before natural death.The tumor containing samples were prepared histologically by hematoxylin and eosin stains.MRI scan showed that intracerebral growth occurred in 50 implanted rats,with a distant metastasis of 4%.The results indicated that tumor globular intracerebral growth occurred and extracranial growth extension was scarce after implantation.The model was very stable and the implanted rat survival duration is easily determined. The experiment lays the foundation for chemotherapy,radiotherapy and gene therapy of the glioma.