Effects of polyphyllin Ⅵ on the proliferation and apoptosis of glioma cells and potential mechanism / 中国药房

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.

Effect of shRNA silencing of Sp1 on radiosensitivity of glioma cells / 中华放射肿瘤学杂志

Objective:To evaluate the effect of down-regulation of Sp1 expression on the radiosensitivity of glioma cells.Methods:The oligonucleotide sequence encoding shRNA was designed and synthesized, and cloned into LV3 (H1/GFP & Puro) vector to construct the recombinant. U251 and U87 cells were infected with recombinant lentivirus, then the stably-transfected cell lines were obtained by puromycin screening. The expression levels of Sp1 mRNA and protein were detected by RT-PCR and Western blot. Cell survival was detected by clonal survival assay, cell cycle was determined by flow cytometry, and DNA damage was measured by immunofluorescence assay, respectively.Results:At 72 h after infection, high expression of Sp1 lentiviral vector was observed in two cell lines under fluorescence microscope. RT-PCR and Western blot confirmed that the expression levels of Sp1 mRNA and protein were significantly down-regulated in both transfected cells (both P<0.01) and the silencing rates of Sp1 were above 90%. The sensitization enhancement ratio (SER) of shRNA-U251 and shRNA-U87 cells at 10% cell survival level were 1.39 and 1.18, respectively. After irradiation, the G 2/M phase ratio and the number of γ-H2AX foci in two Sp1 knockout groups were significantly increased. Conclusion:shRNA silencing of Sp1 increases the G 2/M phase arrest induced by X-ray, aggravates the degree of DNA double-strand breaks, and improves the radiosensitivity of glioma cells.

miR-205-5p/E2F1 signal axis is involved in the regulation of radiosensitivity of glioma cells through suppressing the classical Wnt/β-catenin signaling pathway / 中华放射肿瘤学杂志

Objective:To explore the mechanism of miR-205-5p/E2F1 signal axis in regulating the glioma U251, U87 radiotherapy resistance.Methods:X-ray gradual ascending and intermittent induction method was used to irradiate the glioma U251 cells to establish U251/TR, U87/TR radiation-resistant cell lines. Then, the morphology, migration, invasion and proliferation abilities of cells (U251/TR, U87/TR radiation-resistant cells and U251, U87 radiation-sensitive cells) were analyzed. Luciferase gene detection system and point mutation technique were employed to analyze the mechanism of miR-205-5p and E2F1 gene activity on U251 and U87 radiation-resistant cell lines.Results:Compared with the radiation-sensitive U251 cells, the radiation-resistant cells U251/TR, U87/TR showed increased proliferation activity, enhanced migration and invasion abilities and decreased apoptosis under X-ray irradiation. miR-205-5p mimics transfection could down-regulate the expression of E2F1 factor in U251/TR cells, inhibit cell proliferation, invasion and migration and increase the radiosensitivity of U251/TR cells. miR-205-5p mimics transfection combined with with E2F1 down-regulation exerted anti-tumor effect and decreased cell tolerance by suppressing the Wnt/β-catenin signaling pathway activity.Conclusions:The glioma radiation-resistant cell line U251/TR, U87/TR can be established by X-ray gradual ascending and intermittent induction method. The miR-205-5p/E2F1 signal axis exerts tumor-suppressing effect through the classical Wnt/β-catenin signaling pathway, which can be used as an therapeutic target to increase the radiosensitivity of glioma.

Silencing lncRNA UCA1 affects radiosensitivity of glioma cells by up-regulating miR-873-5p expression / 中华放射肿瘤学杂志

Objective:To investigate the effect of lncRNA UCA1 on the radiosensitivity of in vitro cultured glioma cell lines SHG-44, U87 and U251 by regulating the miR-873-5p expression. Methods:The survival of glioma cells SHG-44, U87 and U251 treated with different radiation intensities (0, 2, 4, 6 and 8 Gy) was detected by colony formation assay. The expression levels of UCA1 in glioma cells SHG-44, U87 and U251 were measured by qRT-PCR. The radiation-resistant glioma cells U87 and U251 were selected for subsequent study. After silencing UCA1 expression and/or over-expressing miR-873-5p, the cell survival rate was detected by colony formation assay, and the cell apoptosis rate was determined by flow cytometry. The dual luciferase reporter gene assay and qRT-PCR were employed to verify the targeting relationship between UCA1 and miR-873-5p.Results:UCA1 was up-regulated in the radiation-resistant U87 and U251 cells. Silencing UCA1 or over-expressing miR-873-5p inhibited the survival of U87 and U251 cells, and promoted the cell apoptosis induced by radiation exposure. miR-873-5p was a target gene of UCA1, and UCA1 negatively regulated the expression of miR-873-5p. The inhibition of miR-873-5p could reverse the effect of silencing UCA1 on the radiosensitivity of glioma cells. Silencing UCA1 increased the inhibitory effect of radiation on the glioma cell U251 xenografts.Conclusion:Silencing UCA1 inhibits the survival of glioma cells and promotes the cell apoptosis by up-regulating the expression of miR-873-5p, thereby increasing the radiosensitivity of glioma cells.

Mechanisms for propofol in inhibiting the proliferation and invasion of glioma U87 cells and its effect on miR-134 expression / 中南大学学报(医学版)

OBJECTIVES@#To investigate the effects of propofol on the proliferation and invasion of glioma U87 cells and to explore the possible anti-tumor mechanisms.@*METHODS@#The glioma U87 cells was divided into a blank group, a positive control group, and the propofol groups (1.00, 2.00 or 5.00 mmol/L). Cell counting kit-8 (CCK-8) was used to detect cell proliferation; Transwell method was used to detect the effect of propofol on invasion and migration of U87 cells; real-time PCR was used to detect the expression of microRNA-134 (miR-134); Western blotting was used to detect the expression levels of reproduction-related protein Ki-67, invasion-related protein metalloproteinase-2 (MMP-2), metalloproteinase-9 (MMP-9) and phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) signaling pathway-related protein.@*RESULTS@#Compared with the blank group, the proliferation, invasion and migration capacity of U87 cells were reduced in the positive control group and the propofol groups after 48 hours (all @*CONCLUSIONS@#Propofol can decrease the proliferation rate, and the invasion and migration abilities of U87 cells, which may be achieved by up-regulation of miR-134 and suppression of PI3K/Akt signaling pathway.

Two new mixed-ligand coordination polymers based on multi-N chelating ligand inhibit YAP expression and induce caspase-mediated spinal tumor cell apoptosis

Two new coordination polymers [Zn (bdc)(bpybzimH2)](DMF)0.5 (1, H2bdc=1,4-dicarboxybenzene, bpybzimH2=6,6′-bis-(1H-benzoimidazol-2-yl)-2,2′-bipyridine, DMF=N,N-dimethylformamide) and [Co (bpybzimH2)(sbc)]H2O (2, H2sbc=4-mercaptobenzoic acid) have been successfully prepared under solvothermal conditions using the multi-N chelating organic ligand bpybzimH2 as the foundational building block. In addition, the Cell Counting Kit-8 assay was conducted to evaluate the anti-proliferation activity of compounds 1 and 2 against human spinal tumor cells OPM-2. The cell viability curves showed that the two compounds have anti-proliferation activity on spinal tumor cells, and the activity of compound 1 is higher than compound 2. The annexin V-FITC/PI assay and western blot were used to detect the apoptotic percentage of OPM-2 cells incubated with compounds 1 and 2. The YAP protein expression and its role in cell apoptosis were further studied with qRT-PCR, immunoblotting, and flow cytometer.

The expression characteristics and co-localization of HIV capsid protein p24 and TRIM22 in U-251 glioma cells / 西安交通大学学报(医学版)

Objective: To observe the expression characteristics and co-localization of exogenous TRIM22 and HIV capsid protein p24 in glioma cells. Methods: The vectors of pEGFP-N3-TRIM22 or pDsRed1-p24 were transfected into U-251 glioma cells respectively to examine the expression of TRIM22-EGFP or p24-DsRed1 by confocal microscopy. Moreover, we used a confocal z-stacking program to achieve series of optical sections and to rebuild 3-D images by ImageJ 1.50i software to detect the expression characteristics of p24-DsRed1 in U-251 cells. In the end, the vectors of pEGFP-N3-TRIM22 and pDsRed1-p24 were co-transfected into U-251 cells to detect the co-localization between TRIM22 and p24 by confocal microscopy. Results: Confocal microscopy results showed that TRIM22-EGFP or p24-Dsred1 was localized to the cell body as well as to protuberance in U-251 cells, and 3D structural reconstruction showed that p24-Dsred1 could be transferred to foot processes of U-251 cells. Simultaneously, confocal microscopy results also showed that TRIM22 and p24 could be co-localized and their combination could be released by budding in U-251 cells co-transfected with pEGFP-N3-TRIM22 and pDsRed1-p24. Conclusion: TRIM22 co-localized to HIV capsid protein p24 and their combination can be released by budding in glioma cells.

Impact of calcineurin inhibitors on rat glioma cells viability / 영남의대학술지

BACKGROUND: Although kidney transplantation outcomes have improved dramatically after using calcineurin inhibitors (CNIs), CNI toxicity continues to be reported and the mechanism remains uncertain. Here, we investigated the neurotoxicity of CNIs by focusing on the viability of glioma cells.METHODS: Glioma cells were treated with several concentrations of CNIs for 24 hours at 37℃ and their cell viability was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.RESULTS: Exposure to 0, 0.25, 0.5, 2.5, 5.0, and 10.0 mM concentrations respectively showed 100%, 64.3%, 61.3%, 68.1%, 62.4%, and 68.6% cell viability for cyclosporine and 100%, 38.6%, 40.8%, 43.7%, 37.8%, and 43.0% for tacrolimus. The direct toxic effect of tacrolimus on glioma cell viability was stronger than that of cyclosporine at the same concentration.CONCLUSION: CNIs can cause neurological side effects by directly exerting cytotoxic effects on brain cells. Therefore, we should carefully monitor the neurologic symptoms and level of CNIs in kidney transplant patients.

Effects of novel smo inhibitor si-4650 on proliferation, apoptosis and autophagy of human glioma u87mg cells / 中国药理学通报

Aim To investigate the effects of a novel spermine oxidase (SMO) inhibitor SI-4650 on proliferation, apoptosis and autophagy of human glioma U87MG cells as well as its possible mechanism. Methods MIT assay was used to detect cell proliferation. The chemiluminescence assay was used to determine enzyme activities of SMO and N'-acetylpolyamine oxidase (APAO). High performance liquid chromatography (HPI.C) was performed to detect cellular polyamine content. Transwell assay was used to evaluate the migration ability of U87MG cells. PI single-staining/flow cytometry( FCM ) were used to analyze cell cycle. PI/ FITC-Annexin V double-staining/ FCM and Western blot were used to analyze apoptosis. Confocal laser scanning microscopy ( LSCM) and Western blot were used to analyze autophagy. Results SI-4650 could significantly inhibit the activities of SMO and APAO, interfere with polyamine metabolism and reduce the content of total polyamine in U87MG cells. Treatment of U87MG cells with SI-4650 inhibited the proliferation and migration, caused G0/G, cell cycle arrest and induced apoptotic and autophagic cell death. Conclusions SI-4650 has the pharmacological activity of killing glioma U87MG cells, and its mechanism may be related to the interference of polyamine metabolism and induction of cell apoptosis and autophagy.

Stem cell-like properties of glioma cells undergoing epithelial to mesenchymal transition and inhibitory effect of resveratrol on their stem cell-like properties / 吉林大学学报(医学版)

Objective: To investigate the stem cell-like properties of glioma U87 cells undergoing epithelial to mesenchymal transition (EMT), and to clarify the inhibitory effect of resveratrol (Res) on their stem cell-like properties. Methods; The glioma U87 cells were treated with transforming growth factor-β1 (TGF-β1) to induce EMT, and the morphology of U87 cells was observed under inverted microscope. Western blotting method was used to detect the expression levels of mesenchymal markers (N-cadherin, Vimentin) and EMT-inducing transcription factors (Snail, Slug, Zebl, Twistl). After the EMT of glioma cells was confirmed, the experiment was divided into control group (U87 cells didn't receive any treatment), EMT group (the glioma cells presented EMT induced by TGF-β1) and Res treatment group (the glioma cells with EMT were treated with Res). The number of secondgeneration gliospheres and the expression levels of stem cell markers (Bmil and Sox2) in glioma U87 cells in various groups were detected. Results: The glioma U87 cells treated with TGF-β1 for 48 h showed dispersed, elongated, and similar to the appearance of fibroblasts. The expression levels of N-cadherin, Vimentin, Snail, Slug, Zebl and Twistl in glioma cells in different concentrations of TGF-β1 groups were significantly increased, suggesting the presence of EMT in glioma U87 cells. Compared with control group, the number of second-generation gliospheres in the U87 cells in EMT group was increased (P<0. 01), and the expression levels of stem cell markers (Bmil and Sox2) in the glioma U87 cells in EMT group were markedly increased (P<0. 05 or P<0. 01). Compared with EMT group, the number of second-generation gliospheres in the glioma U87 cells in Res treatment group was significantly decreased (P<0. 01), and the expression levels of Bmil and Sox2 in the glioma U87 cells in Res treatment group were decreased (P < 0. 01). Conclusion: The glioma U87 cells undergoing EMT exhibit the stem cell-like properties, and Res can inhibit their stem-like properties.

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>

Effect of Elemene Emulsion Injection Combined with Ganciclovir on Invasive Ability and TIMP-1 mRNA Expression in C6 Brain Tumor Stem Cells / 中国药房

OBJECTIVE:To study the effect of Elemene emulsion combined with ganciclovir on invasive ability and TIMP-1 mRNA expression in C6 brain tumor stem cells. METHODS:Cells with 3 generations were isolated and cultured from rats'C6 ma-lignant glioma cell lines,and immunocytochemistry was adopted to detect the protein expressions of stem cell markers CD133,Nes-tin. 20 rats were randomly divided into blank control group(normal saline),ganciclovir group(intragastrically administrated,216 mg/kg),Elemene emulsion injection group (intravenously injected in tail,36 mg/kg) and combination group (intragastrically ad-ministrated 216 mg/kg of ganciclovir+intravenously injected 36 mg/kg of Elemene emulsion injection in tail),5 in each group, once every day,for 10 d. After 2 h of last administration,the blood of rats in each group was collected,serum was isolated and co-cultured 24 h with C6 BTSCs in in vitro culture medium. Boyden invasion test was used to detect the invasive ability of C6 BTSCs,and real-time quantitative polymerase chain reaction(RT-PCR)method was used to determine the TIMP-1 mRNA expres-sion of C6 BTSCs. RESULTS:Cells with 3 generations had obvious protein expressions in CD133 and Nestin,indicating they were BTSCs. Compared with blank control group,the cell invasion rates of C6 BTSCs in ganciclovir group,Elemene emulsion in-jection group and combination group were obviously decreased,TIMP-1 mRNA expression was obviously enhanced,with statisti-cal significances(P<0.05). Compared with ganciclovir group and Elemene emulsion injection group,the cell invasion rate of C6 BTSCs in combination group was obviously decreased,TIMP-1 mRNA expression was obviously enhanced,with statistical signifi-cances(P<0.05). CONCLUSIONS:Elemene emulsion injection combined with ganciclovir can obviously inhibit the cell invasion of C6 BTSCs,the mechanism may be associated with promoting the TIMP-1 generation,and combination use shows better effect than single use.

Influence of Bufalin on the Growth and Proliferation of Human Glioma Cell Line U251 / 中国药师

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.

Establishment of human brain glioma cell SHG-44 subcutaneous model in nude mice and observation of growth characteristics / 中华行为医学与脑科学杂志

Objective To establish nude mouse model with human brain glioma SHG-44 and understand its growing characteristics in vivo.Methods The 4-week-old male mice were randomly divided into high density cell suspension inoculation group(n=10),low density cell suspension group(n=10),the tumor tissue mass vaccination group(n=10)and the blank control group with normal saline injection(n=10).The SHG-44 human brain glioma cell suspension was injected into the subcutaneous of the nude mice' s armpit.The tumor tissue was cut into 1 mm3 after tumor tissue growth and formation,and re-inoculated into the subcutaneous of the new nude mice' s armpits.Apart from daily observation,the long and short diameters of tumor were recorded every 5 days after graft.All the mice were sacrificed at 60 days and the tumor tissues were harvested for pathological examination.Results With a longer incubation period and slower growth rate,the tumor formation rate in high density cell suspension inoculation group and low density cell suspension group was lower compared with that in the tumor tissue mass vaccination group.Around day 20,grafted tumor appeared remarkably big((41.51 ±6.42)mm3) with good morphology.On day 50,the tumor derived from group the tumor tissue mass vaccination group((565.69± 123.36)mm3) showed a bigger size in comparison with that from high density cell suspension inoculation group((203.85±104.63) mm3) and low density cell suspension group ((153.02± 31.76) mm3,all P＜0.05).The tumors in three groups were well defined with a rich vascularity and no apparent invasion was observed.The positive expression of GFAP and S-100 in a large body of tumor cells was observed under optical microscope.Conclusion With a shorter incubation period and faster growth,the mouse tumor models established with tissue pieces from the tumor-bearing mice are much better compared to those with cell suspension.

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.

Autophagic Cell Death in Glioma Cell Induced by Ceramide through JNK/c-Jun Pathway / 中国康复理论与实践

@#Objective To observe the autophagy of 87-MG and U251 glioma cells induced by ceramide and explore the possible mechanism. Methods The viability and apoptosis of 87-MG and U251 cells were detected by MTT assay and flow cytometry, respectively. Autophagic- related protein expressions of LC3B /LC3A and Beclin-1 were determined by Western blotting. The activation of JNK/c-Jun signaling pathway induced by ceramide with or without the treatment of JNK specific inhibitor SP600125 was also measured. Results 24 hours after treatment of ceramide, the growth of 87-MG and U251 cells was significantly inhibited time-dependently (P<0.05); and the number of autophagic cells increased dose-dependently (P<0.05). The levels of LC3B/LC3A and Beclin-1 significantly increased after ceramide treatment (P<0.05). JNK signaling pathway was activated in the 87-MG and U251 cells and the phosphorylation of c-Jun also increased after ceramide treatment. This activation of autophagy could be reversed by the pre-treatment of SP600125. Conclusion Ceramide may induce autophagy in 87-MG and U251 glioma cells and the mechanism may be related to the activation of JNK/c-Jun signaling pathway.

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.

Autophagic Cell Death in Glioma Cell Induced by Ceramide through JNK/c-Jun Pathway / 中国康复理论与实践

Objective To observe the autophagy of 87-MG and U251 glioma cells induced by ceramide and explore the possible mecha-nism. Methods The viability and apoptosis of 87-MG and U251 cells were detected by MTT assay and flow cytometry, respectively. Autoph-agic-related protein expressions of LC3B/LC3A and Beclin-1 were determined by Western blotting. The activation of JNK/c-Jun signaling pathway induced by ceramide with or without the treatment of JNK specific inhibitor SP600125 was also measured. Results 24 hours after treatment of ceramide, the growth of 87-MG and U251 cells was significantly inhibited time-dependently (P<0.05);and the number of au-tophagic cells increased dose-dependently (P<0.05). The levels of LC3B/LC3A and Beclin-1 significantly increased after ceramide treat-ment (P<0.05). JNK signaling pathway was activated in the 87-MG and U251 cells and the phosphorylation of c-Jun also increased after ce-ramide treatment. This activation of autophagy could be reversed by the pre-treatment of SP600125. Conclusion Ceramide may induce au-tophagy in 87-MG and U251 glioma cells and the mechanism may be related to the activation of JNK/c-Jun signaling pathway.

The effect of etomidate on the gap junctional intercellular communication between human malignant glioma cells / 中国癌症杂志

Background and purpose:Gap junctions (GJ) could enhance cytotoxicity induced by chemo-therapeutic agents. Previous studies have showed that some of anesthetics exerted effect on GJ and thereby affected the cytotoxicity of X-ray. However, it is still unclear whether etomidate, a commonly used anesthesia adative agent, could alter GJ intercellular communication in tumor cells. This study explored the effect of etomidate on GJ composed of connexin 43 in U87 malignant glioma cells to provide mechanism clues for studies on the effect of anesthetics on the toxicity induced by chemotherapeutic agents.Methods:Sulforhodamine B was used to examine the toxicity of etomidate on U87 malignant gli-oma cells. The effect of etomidate on GJ function was determined by parachute dye-coupling assay.Results:Pretreatment of etomidate at the concentration of 0.1, 0.5, 1 or 5 μmol/L for 4 h did not induce cytotoxicity in U87 cells. So etomidate at these concentrations would not reduce the amount of GJ on U87 cell membranes. Parachute dye-coupling assay had showed that treatment with 0.5 and 1 μmol/L etomidate for 4 h significantly decreased the dye spread through GJ in U87 cells, while 0.1 μmol/L etomidate had no effect on dye spread of U87 cells through GJ.Conclusion:Etomidate inhibits GJ function in glioma cells.

DAPT suppresses the proliferation of human glioma cell line SHG-44

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.