Ethanol Extract of Lycopodium serratum Thunb. Attenuates Lipopolysaccharide-Induced C6 Glioma Cells Migration via Matrix Metalloproteinase-9 Expression / 中国结合医学杂志

<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>

Extracellular diffusion quantified by magnetic resonance imaging during rat C6 glioma cell progression

Solution reflux and edema hamper the convection-enhanced delivery of the standard treatment for glioma. Therefore, a real-time magnetic resonance imaging (MRI) method was developed to monitor the dosing process, but a quantitative analysis of local diffusion and clearance parameters has not been assessed. The objective of this study was to compare diffusion into the extracellular space (ECS) at different stages of rat C6 gliomas, and analyze the effects of the extracellular matrix (ECM) on the diffusion process. At 10 and 20 days, after successful glioma modeling, gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) was introduced into the ECS of rat C6 gliomas. Diffusion parameters and half-life of the reagent were then detected using MRI, and quantified according to the mathematical model of diffusion. The main ECM components [chondroitin sulfate proteoglycans (CSPGs), collagen IV, and tenascin C] were detected by immunohistochemical and immunoblot analyses. In 20-day gliomas, Gd-DTPA diffused more slowly and derived higher tortuosity, with lower clearance rate and longer half-life compared to 10-day gliomas. The increased glioma ECM was associated with different diffusion and clearance parameters in 20-day rat gliomas compared to 10-day gliomas. ECS parameters were altered with C6 glioma progression from increased ECM content. Our study might help better understand the glioma microenvironment and provide benefits for interstitial drug delivery to treat brain gliomas.

Inhibitory effects of lapachol on rat C6 glioma in vitro and in vivo by targeting DNA topoisomeraseⅠ and topoisomeraseⅡ / 中国药理学与毒理学杂志

OBJECTIVE Lapachol is a natural naphthoquinone compound that possesses extensive biological activities. The aim of this study is to investigate the inhibitory effects of lapachol on rat C6 glioma both in vitro and in vivo, as well as the potential mechanisms. METHODS The antitumor effect of lapachol was firstly evaluated in the C6 glioma model in Wistar rats. The effects of lapachol on C6 cell proliferation, apoptosis and DNA damage were detected by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)/ phenazinemethosulfate (PMS) assay, hoechst 33358 staining, annexinⅤ-FITC/PI staining, and comet assay. Effects of lapachol on topoisomerase I (TOP I) and topoi?somerase Ⅱ (TOP Ⅱ) activities were detected by TOP Ⅰ and TOP Ⅱ mediated supercoiled pBR322 DNA relaxation assays and molecular docking. TOPⅠ and TOPⅡ expression levels in C6 cells were also determined. RESULTS High dose lapachol showed significant inhibitory effect on the C6 glioma in Wistar rats (P<0.05). It was showed that lapachol could inhibit proliferation, induce apoptosis and DNA damage of C6 cells in dose dependent manners. Lapachol could inhibit the activities of both TOPⅠ and Ⅱ. Lapachol-TOPⅠ showed relatively stronger interaction than that of lapachol-TOPⅡ in molecular docking study. Also, lapachol could inhibit TOPⅡ expression levels, but not TOPⅠ expression levels. CONCLUSION These results showed that lapachol could significantly inhibit C6 glioma both in vivo and in vitro, which might be related with inhibiting TOPⅠ and TOPⅡ activities, as well as TOPⅡ expression.

The investigation of the effect of Tripterine combined with Cisplatin on the apoptosis of C6 glioma cells / 中华老年医学杂志

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.

Establishment of mice model of C6 glioma stem cells and its evaluation / 中国药理学通报

Aim To establish ICR animal model with C6 glioma stem cells, to provide the ideal model for the further study of gli-oma stem cells in brain glioma model. Methods C6 glioma stem cell was cultured in vitro by suspension,and was identified with Nestin antibody. C6 stem cells of ICR mouse glioma model were used to investigate survival state and tumor volume in mice after the operation. HE staining and CD133 immunohistochemi-cal study were adopted to investigate the postoperative pathologi-cal changes in mice. Results The expression of Nestin was 96. 01% in C6 glioma stem cells, and Nestin was highly ex-pressed in the cultured C6 glioma stem cells. Mice were inocula-ted with tumor after loss of appetite, weight, behavior and slow, sluggish reaction. Tumor volume at day 21 after modeling was (9. 77 ± 6. 58) mm3 . After HE staining, the model showed the invasive growth, tumor cell shrinkage and derangement. Immu-nohistochemical CD133 staining revealed that tumor cytoplasm color was brown. Conclusion Glioma model can be established based on glioma stem cells into a high rate of tumor, the tumor cycle is short, which can be used as an ideal model for glioma.

Experimental study of effect of Xiao Chai Hu Tang on C6 glioma / 中国免疫学杂志

Objective:To investigate the inhibitory effect and mechanism of different doses of Xiao Chai Hu Tang on C6 glioma cells cultured in vitro. Methods:C6 glioma cells were inoculated in 96 holes,24 holes and 6 holes,each culture plate was divided into 4 groups:control group, low dose group, middle dose group and high dose group, when the cells covered the bottom of culture plate 80%-90%,began adding,cultured for 24 hours after the ter mination of training. Cell proliferation activity,cell viability,protein content and protein positive expression intensity of VEGF and ESM-1,cell apoptosis in early and late stage were detected by CCK-8,in vivo staining,ELISA, immunocytochemistry and flow cytometry. Results: CCK-8 assay showed that the growth of C6 glioma cells was inhibited by low,medium and high dose group;ELISA and immunocytochemistry showed that the expression of VEGF and ESM-1 was lower in the lower, middle and high dose groups, and the levels of protein expression and protein levels were decreased. The flow cytometry showed that the low dose of small radix,middle and high dose group could promote the cell apoptosis. Inverted microscope ob-servation showed that with the increase of dose,the number of cells increased gradually,and the number of dead cells increased,and all kinds of detection methods showed that the inhibition effect increased with dose and dose dependence. The difference between the two groups was statistically significant(P<0. 01). Conclusion:The growth of C6 glioma cells was significantly inhibited by Xiao Chai Hu Tang. It may play a role in inhibiting tumor growth by down regulating ESM-1 and VEGF protein level and promoting cell apoptosis.

Effect of temozolomide combined with curcumin on the apoptosis of the C6 glioma cells / 实用医学杂志

Objective To study the temozolomide combined with curcumin on the inhibitory effect and apoptosis of the C6 glioma cells. Methods The C6 glioma cells were treated with temozomide in combination with curcumin. The anti proliferation effect of liposomes on the C6 glioma cells was investigated by using the method of sulforhodamine B (SRB). Flow cytometry was used to detect apoptosis of the C6 glioma cells. Confocal laser scan-ning microscope was used to observe apoptosis and location in the C6 glioma cells. Results The results of SRB as-say showed that temozolomide in combination with curcumin inhibition rate were (91.22 ± 0.51)%in 48 h of the C6 glioma cells; Flow cytometry showed that the apoptosis rate were (33.15 ± 0.79)% with temozolomide (5 μmol/L) in combination with curcumin (10 μmol/L). Laser scanning confocal scanning microscope indicated that the apop-tosis of in the C6 glioma cells treated with temozolomide in combination with curcumin was more than that of free drug. Conclusion The temozolomide in combination of curcumin can inhibit the growth and induce apoptosis of the C6 glioma cells.

Compound K attenuates stromal cell-derived growth factor 1 (SDF-1)-induced migration of C6 glioma cells

BACKGROUND/OBJECTIVES: Stromal cell-derived growth factor 1 (SDF-1), also known as chemokine ligand 12, and chemokine receptor type 4 are involved in cancer cell migration. Compound K (CK), a metabolite of protopanaxadiol-type ginsenoside by gut microbiota, is reported to have therapeutic potential in cancer therapy. However, the inhibitory effect of CK on SDF-1 pathway-induced migration of glioma has not yet been established. MATERIALS/METHODS: Cytotoxicity of CK in C6 glioma cells was determined using an EZ-Cytox cell viability assay kit. Cell migration was tested using the wound healing and Boyden chamber assay. Phosphorylation levels of protein kinase C (PKC)α and extracellular signal-regulated kinase (ERK) were measured by western blot assay, and matrix metallopeptidases (MMP) were measured by gelatin-zymography analysis. RESULTS: CK significantly reduced the phosphorylation of PKCα and ERK1/2, expression of MMP9 and MMP2, and inhibited the migration of C6 glioma cells under SDF-1-stimulated conditions. CONCLUSIONS: CK is a cell migration inhibitor that inhibits C6 glioma cell migration by regulating its downstream signaling molecules including PKCα, ERK1/2, and MMPs.

Compound K attenuates stromal cell-derived growth factor 1 (SDF-1)-induced migration of C6 glioma cells

BACKGROUND/OBJECTIVES: Stromal cell-derived growth factor 1 (SDF-1), also known as chemokine ligand 12, and chemokine receptor type 4 are involved in cancer cell migration. Compound K (CK), a metabolite of protopanaxadiol-type ginsenoside by gut microbiota, is reported to have therapeutic potential in cancer therapy. However, the inhibitory effect of CK on SDF-1 pathway-induced migration of glioma has not yet been established. MATERIALS/METHODS: Cytotoxicity of CK in C6 glioma cells was determined using an EZ-Cytox cell viability assay kit. Cell migration was tested using the wound healing and Boyden chamber assay. Phosphorylation levels of protein kinase C (PKC)α and extracellular signal-regulated kinase (ERK) were measured by western blot assay, and matrix metallopeptidases (MMP) were measured by gelatin-zymography analysis. RESULTS: CK significantly reduced the phosphorylation of PKCα and ERK1/2, expression of MMP9 and MMP2, and inhibited the migration of C6 glioma cells under SDF-1-stimulated conditions. CONCLUSIONS: CK is a cell migration inhibitor that inhibits C6 glioma cell migration by regulating its downstream signaling molecules including PKCα, ERK1/2, and MMPs.

Effect of Isoliquiritigenin on C6 glioma cell proliferation and differentiation / 中国药理学通报

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.

Expression of IL-12 family subunit genes in mice C6 glioma cells / 中国免疫学杂志

Objective:To observe the expression of IL-12 family subunit genes by real-time quantitative PCR in mice C6 glioma cells,construct the basis of the brain glioma research on IL-12 family in the future.Methods:Mice C6 glioma RNA was abstracted and reversed transcription cDNA.The mice C6 glioma cells mRNA expression influence of IL-12 family subunit genes was compared and analyzed by real-time quantitative PCR.Results: In mice C6 glioma cells, high expression abundances in IL-23a, IL-12a, midlde expression abundances in EBI3, IL-27, low expression abundance in IL-12b.Conclusion: IL-12 families are closely related to the occurrence and development of glioma,IL-12,IL-23 are regarded as the most potential anti-glioma cytokines among them,research de-velopments will bring a new way of brain glioma immune therapy.

The effect of yacon (Samallanthus sonchifolius) ethanol extract on cell proliferation and migration of C6 glioma cells stimulated with fetal bovine serum

BACKGROUND/OBJECTIVES: Yacon (Samallanthus sonchifolius), a common edible plant grown throughout the world, is well known for its antidiabetic properties. It is also known to have several other pharmacological properties including anti-inflammatory, anti-oxidant, anti-allergic, and anti-cancer effects. To date, the effect of yacon on gliomas has not been studied. In this study, we investigated the effects of yacon on the migration and proliferation of C6 glioma cells stimulated by fetal bovine serum (FBS). MATERIALS/METHODS: Cell growth and proliferation were determined by evaluating cell viability using an EZ-Cytox Cell Viability Assay Kit. FBS-induced migration of C6 glioma cells was evaluated by performing the scratch wound healing assay and the Boyden chamber assay. We also used western blot analysis to determine the expression levels of extracellular signal-regulated kinase 1/2 (ERK1/2), a major regulator of migration and proliferation of glioma cells. Matrix metallopeptidase (MMP) 9 and TIMP-1 levels were measured by performing reverse transcription PCR. RESULTS: Yacon (300 microg/mL) reduced both the FBS-induced proliferation of C6 glioma cells and the dose-dependent migration of the FBS-stimulated C6 cells. FBS-stimulated C6 glioma cells treated with yacon (200 and 300 microg/mL) showed reduced phosphorylation of ERK1/2 and inhibition of MMP 9 expression compared to those shown by the untreated FBS-stimulated C6 cells. In contrast, yacon (200 and 300 microg/mL) induced TIMP-1 expression. CONCLUSIONS: On the basis of these results, we suggest that yacon may exert an anti-cancer effect on FBS-stimulated C6 glioma cells by inhibiting their proliferation and migration. The most likely mechanism for this is down-regulation of ERK1/2 and MMP9 and up-regulation of TIMP-1 expression levels.

Extracellular space diffusion during progression of rat C6 glioma quantificated by magnetic resonance imaging / 中国比较医学杂志

Objective To compare the extracellular space diffusion at different stages of rat C6-gliomas determined by MRI tracer method and analyze the influencing effect of extracellular matrix ( ECM) on the diffusion process.Methods Introducing adolinium-diethylene triaminepentaacetic acid ( Gd-DTPA) into extracellular space ( ECS) as a tracer.The diffusion parameters and half-life time were quantified according to mathematical model of diffusion.The main ECM components ( e.g. chordroitin sulfate proteoglycans ( CSPGs ) , collagen IV tenascin C ) were detected by immunohistochemical and immunoblot analysis.Results Gd-DTPA introduced into 20-day glioma in the rats diffused more slowly [(6.67 ±1.78) ×10 -5 mm2/s vs.(1.26 ±0.27) ×10-4 mm2/s; t =4.265; P<0.01)], deriving a larger tortuosity [(3.99 ±0.57) vs.(2.83 ±0.29);t=4.11;P<0.01)], localized within the tumor with a smaller clearance rate [(7.67 ±2.29) ×10 -5mm2/s)vs.(1.46 ±0.36) ×10 -4mm2/s);t=3.87;P<0.05), and a longer half-life time ((0.86 ±0.23 h)vs.(1.64 ±0.12 h);(t=5.91;p<0.01)] compared with 10-day gliomas in the rats.The increased levels of extracellular matrix of glioma were associated with different diffusion and clearance parameters of 20-day gliomas in the rats in comparision with those in the 10-day rat gliomas, in which the chordroitin sulfate proteoglycans[(0.48 ±0.07) vs.(0.32 ±0.09);t=4.663;P<0.01)], tenascin C [(0.29 ±0.04) vs.(0.58 ±0.11);t =6.50;P<0.01] and collagen IV [(0.24 ±0.07)vs.(0.33 ±0.06);t=3.81;P<0.05] were tested.Conclusions The ECS parameters are changed with the C6 glioma progression due to the increased ECM content.The results of our study may help us to better understanding the glioma micro-environment and provide beneficial references for the brain interstitial drug delivery to treat gliomas.

The inhibitory effect of photodynamic therapy and of an anti-VCAM-1 monoclonal antibody on the in vivo growth of C6 glioma xenografts

We investigated the effect of photodynamic therapy (PDT) and of an anti-vascular cell adhesion molecule-1 (VCAM-1) monoclonal antibody on the in vivo growth of C6 glioma. Seven days after inoculation with C6 cells, adult male Wistar rats weighing 280-300 g with MRI-confirmed glioma were randomly assigned to 4 groups (N = 15 per group): PDT + VCAM-1 antibody group; PDT group; VCAM-1 antibody group; control group. Eight days after inoculation, hematoporphyrin monomethyl ether (HMME) was administered as a photosensitizer and PDT was performed at 630 nm (illumination intensity: 360 J/cm²) for 10 min. VCAM-1 antibody (50 µg/mL) was then administered (0.5 mL) through the tail vein every other day from day 8 to day 16. At day 21, 5 rats in each group were sacrificed and cancers were harvested for immunohistochemistry and Western blot assay for the detection of VCAM-1, and TUNEL assay was used to detect apoptosis. Survival and tumor volume were recorded in the remaining 10 rats in each group. In the PDT group, tumor growth was significantly suppressed (67.2 percent) and survival prolonged (89.3 percent), accompanied by an increase in apoptosis (369.5 percent), when compared to control. Furthermore, these changes were more pronounced in the PDT + VCAM-1 antibody group. After PDT, VCAM-1 expression was markedly increased (121.8 percent) and after VCAM-1 monoclonal antibody treatment, VCAM-1 expression was significantly reduced (58.2 percent). PDT in combination with VCAM-1 antibody can significantly inhibit the growth of C6 glioma and prolong survival. This approach may represent a promising strategy in the treatment of glioma.

Thrombin-induced Migration and Matrix Metalloproteinase-9 Expression Are Regulated by MAPK and PI3K Pathways in C6 Glioma Cells

Glioblastoma multiforme is one of the most common and aggressive tumors in central nervous system. It often possesses characteristic necrotic lesions with hemorrhages, which increase the chances of exposure to thrombin. Thrombin has been known as a regulator of MMP-9 expression and cancer cell migration. However, the effects of thrombin on glioma cells have not been clearly understood. In the present study, influences of thrombin on glioma cell migration were examined using Boyden chamber migration assay and thrombin-induced changes in MMP-9 expression were measured using zymography, semi-quantitative RT-PCR, and Western blotting. Furthermore, underlying signaling pathways by which thrombin induces MMP-9 expression were examined. Thrombin-induced migration and MMP-9 expression were significantly potentiated in the presence of wortmannin, a PI3K inhibitor, whereas MAPK inhibitors suppressed thrombin-induced migration and MMP-9 expression in C6 glioma cells. The present data strongly demonstrate that MAPK and PI3K pathways evidently regulate thrombin-induced migration and MMP-9 expression of C6 glioma cells. Therefore, the control of these pathways might be a beneficial therapeutic strategy for treatment of invasive glioblastoma multiforme.

Induction of Inducible Nitric Oxide Synthase Expression by Manganese in C6 Glioma Cells / 대한산업의학회지

OBJECTIVE: It is well established that manganese neurotoxicity is associated with clinical symptoms similar to those of idiopathic Parkinson's disease. Recent research has shown that the exposure to manganese (MnCl2) leads to induction of iNOS in BV2 microglial cells via iNOS transcriptional up-regulation and activation of both MAPKs and PI3K/Akt signaling pathways. Here, we further investigated the effect and the action mechanism of MnCl2 on iNOS expression in C6 glioma cells. METHODS: Western blot analyses demonstrated that treatment with MnCl2 at 250 micronmeter was sufficient to induce iNOS at both the protein and mRNA levels in C6 cells. RESULTS: These studies demonstrated that the induction of iNOS protein and mRNA was visible after 4h- and 2 h-treatment with MnCl2, respectively. MnCl2 treatment led to strong phosphorylation of JNKs and ERKs, members of MAP kinases (MAPKs), and Akt, a PI3-kinase (PI3K) downstream effector, in C6 cells. MnCl2 treatment had no effect on I kappa B-alpha in C6 cells. Notably, pretreatment with LY294002 (a PI3K inhibitor), which inhibited phosphorylation of Akt by MnCl2, caused strong suppression of MnCl2- induced iNOS protein and mRNA expression in C6 cells. Moreover, pretreatment with SP600125 (an inhibitor of JNKs) and PD98050 (an inhibitor of ERKs), which respectively interfered with MnCl2-mediated phosphorylation of JNKs and ERKs, led to the partial suppression of MnCl2-induced iNOS protein. Interestingly, pretreatment with LY294002 inhibited phosphorylation of not only Akt, but also ERKs and JNKs, in response to MnCl2. Moreover, there was an effective suppression of MnCl2-mediated phosphorylation of AKT by SP600125. CONCLUSION: These results collectively suggest that MnCl2 induces iNOS expression in C6 glioma cells via activation of PI3K/Akt and JNK-ERK MAPK signaling proteins, whose activations seem to be mutually interconnected in response to MnCl2.

Inhibition of Pingliukang Medicated Serum on Proliferation of Rat C6 Glioma Cells in Vitro / 中国康复理论与实践

@#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.

Induction of apoptosis and inhibition of proliferation of C6 glioma cells in vitro by tamoxifen / 药物分析学报

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.

Effects of Recombinant Disintegrin rAdinbitor on FAK-Ras/MAPK Pathway in C6 Glioma Cells / 生物化学与生物物理进展

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.

Group Ⅱ and Ⅲ metabotropic glutamate receptors agonists reverse 1-methyl-4-phenylpyridinium-induced glutamate uptake inhibition in C6 glioma cells / 中国临床药理学与治疗学

AIM: To study the effect of group Ⅱ and Ⅲ metabotropic glutamate receptors (mGluRs) agonists on 1-methyl-4-phenylpyridinium (MPP~+)-induced glutamate uptake inhibition in C6 glioma cells. METHODS: The glutamate uptake into astrocytes was measured by using radio-ligand binding assay method. RESULTS: It was shown that Group Ⅱ mGluRs agonist (2' S, 2' R, 3 ' R) -2- (2', 3 ' -dicarboxycyclopropyl) glycine (DCG-Ⅳ) (100 ?mol?L~(-1)) and Group Ⅲ mGluRs agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4) (100 ?mol?L~(-1)) significantly reversed MPP~+-induced glutamate uptake inhibition. Furthermore, the enhancement effects of DCG-Ⅳ and L-AP4 were blocked by their respective antagonists, (RS)-1 -Amino-5-phosphonoinan-1-carboxylic acid (APICA) and (RS)-?-methylserine-O-phosphate (MSOP). CONCLUSION: Group Ⅱ and Ⅲ mGluRs agonists produce neuroprotective effects by enhancing the activity of glutamate transporters.