Apoptosis In U251 Glioma Cells Research Articles

Interference with the HSF1/HSP70/BAG3 Pathway Primes Glioma Cells to Matrix Detachment and BH3 Mimetic-Induced Apoptosis.

Malignant gliomas exhibit a high intrinsic resistance against stimuli triggering apoptotic cell death. HSF1 acts as transcription factor upstream of HSP70 and the HSP70 co-chaperone BAG3 that is overexpressed in glioblastoma. To specifically target this resistance mechanism, we applied the selective HSF1 inhibitor KRIBB11 and the HSP70/BAG3 interaction inhibitor YM-1 in combination with the pan-Bcl-2 inhibitor AT-101. Here, we demonstrate that lentiviral BAG3 silencing significantly enhances AT-101-induced cell death and reactivates effector caspase-mediated apoptosis in U251 glioma cells with high BAG3 expression, whereas these sensitizing effects were less pronounced in U343 cells expressing lower BAG3 levels. KRIBB11 decreased protein levels of HSP70, BAG3, and the antiapoptotic Bcl-2 protein Mcl-1, and both KRIBB11 and YM-1 elicited significantly increased mitochondrial dysfunction, effector caspase activity, and apoptotic cell death after combined treatment with AT-101 and ABT-737. Depletion of BAG3 also led to a pronounced loss of cell-matrix adhesion, FAK phosphorylation, and in vivo tumor growth in an orthotopic mouse glioma model. Furthermore, it reduced the plating efficiency of U251 cells in three-dimensional clonogenic assays and limited clonogenic survival after short-term treatment with AT-101. Collectively, our data suggest that the HSF1/HSP70/BAG3 pathway plays a pivotal role for overexpression of prosurvival Bcl-2 proteins and cell death resistance of glioma. They also support the hypothesis that interference with BAG3 function is an effective novel approach to prime glioma cells to anoikis. Mol Cancer Ther; 16(1); 156-68. ©2016 AACR.

Synergistic anti-glioma effect of a coloaded nano-drug delivery system.

The anti-glioma effect of temozolomide (Tem) is sometimes undermined by the emerging resistance. Recently, resveratrol (Res), herbal medicine extracted from grape seeds, has been demonstrated for its potential use in chemosensitization. In the current study, both these drugs were loaded simultaneously into nanoparticles with methoxy poly(ethylene glycol)-poly epsilon caprolactone (mPEG-PCL) as drug carriers in order to achieve better antitumor efficiency. Tem/Res-coloaded mPEG-PCL nanoparticles were constructed, characterized, and tested for antitumor effect on glioma cells by using in vitro and xenograft model system. The nanoparticle constructs were satisfactory with drug loading content (Res =~12.4%; Tem =~9.3%) and encapsulation capacity of >85% for both the drugs. In addition, the coencapsulation led to better in vitro stability of the nanoparticles than Tem-loaded nanoparticles. An in vitro uptake study demonstrated a high uptake efficiency of the nanoparticles by glioma cells. The synergistic antitumor effect against glioma cells was observed in the combinational treatment of Res and Tem. Tem/Res-coloaded nanoparticles induced higher apoptosis in U87 glioma cells as compared to cells treated by the combination of free drugs. Tem/Res-coloaded particles caused more effective inhibition of phosphor-Akt, leading to upregulation of the downstream apoptotic proteins. In addition, the in vivo study showed the superior tumor delaying effect of coloaded nanoparticles than that of free drug combination. These results suggest that Tem/Res-coloaded nanoparticles could be a potential useful chemotherapeutic formulation for glioma therapy.

RLIP76 Depletion Enhances Autophagic Flux in U251 Cells.

Our previous study showed that RalA-binding protein 1 (RLIP76) is overexpressed in gliomas and is associated with higher tumour grade and decreased patient survival. Furthermore, RLIP76 downregulation increases chemosensitivity of glioma cells to temozolomide by inducing apoptosis. However, other mechanisms underlying RLIP76-associated chemoresistance are unknown. In this study, we investigated the effect of RLIP76 depletion on autophagy. RLIP76 was knocked down in U251 glioma cells using shRNA and autophagy-related proteins, and PI3K/Akt signalling components were evaluated. RLIP76 depletion significantly increased cell autophagy as demonstrated by a significant increase in LC3 II, autophagy protein 5 (ATG-5), and Beclin1, and a decrease in p62 expression levels. Furthermore, RLIP76 knockdown increased autophagic flux in U251 cells as autolysosome numbers increased relative to autophagosome numbers. Autophagy induced by RLIP76 knockdown resulted in increased apoptosis that was independent of temozolomide treatment. Moreover, RLIP76 knockdown decreased PI3K and Akt activation. RLIP76 depletion also resulted in decreased levels of the anti-apoptotic protein Bcl2. LY294002, a PI3K/Akt pathway inhibitor, led to increased autophagy and apoptosis in U251 RLIP76-depleted cells. Therefore, RLIP76 knockdown increased autophagic flux and apoptosis in U251 glioma cells, possibly through inhibition of the PI3K/Akt pathway. Thus, this study provides a novel mechanism for the role of RLIP76 in glioma pathogenesis and chemoresistance.

MiR-17 enhances proliferation and migration and inhibits apoptosis in glioma cells by regulating SASH1 expression

Background: To investigate the mechanisms underlying the regulation of human glioma cell growth, proliferation, and apoptosis by microRNA-17 (miR-17) to provide targets for novel human glioma therapies. Methods: The expression of miR-17 in human glioma tissues and cell lines was detected using real-time quantitative PCR (qRT-PCR). A miR-17 inhibitor was transfected into the U251 human glioma cell line using liposomes, and changes in miR-17 expression were detected using qRT-PCR. The effects of miR-17 on numerous biological characteristics of U251 cells, including viability, proliferation, apoptosis, and migration, were analyzed using MTT, flow cytometry, and Transwell migration chamber assays. A luciferase reporter gene system was used to validate SAM- and SH3-domain containing 1 ( SASH1 ) as a true target of miR-17. The effects of miR-17 on SASH1 protein expression were determined using western blotting. Results: qRT-PCR results showed that compared with adjacent tissues, miR-17 expression was significantly increased in human glioma tissues and cells lines (P Conclusions: miR-17 promotes viability, proliferation, and migration and inhibits apoptosis in U251 glioma cells, and these mechanisms could be mediated by the regulation of SASH1 gene expression.

DT(270-326) , a Truncated Diphtheria Toxin, Increases Blood-Tumor Barrier Permeability by Upregulating the Expression of Caveolin-1.

The nontoxic mutant of diphtheria toxin (DT) has been demonstrated to act as a receptor-specific carrier protein to delivery drug into brain. Recent research showed that the truncated "receptorless" DT was still capable of being internalized into cells. This study investigated the effects and potential mechanisms of DT(270-326) , a truncated "receptorless" DT, on the permeability of the blood-tumor barrier (BTB). BTB and GECs were subjected to DT(270-326) treatment. HRP flux assays, immunofluorescent, co-immunoprecipitation, Western blot, CCK-8, and Flow cytometry analysis were used to evaluate the effects of DT(270-326) administration. Our results revealed that 5 μM of DT(270-326) significantly increased the permeability of BTBin vitro, which reached its peak at 6 h. The permeability was reduced by pretreatment with filipinIII. DT(270-326) co-localized and interacted with caveolin-1 via its caveolin-binding motif. The mRNA and protein expression levels of caveolin-1 were identical with the changes of BTB permeability. The upregulated expression of caveolin-1 was associated with Src kinase-dependent tyrosine phosphorylation of caveolin-1, which subsequently induced phosphorylation and inactivation of the transcription factor Egr-1. The combination of DT(270-326) with doxorubicin significantly enhanced the loss of cell viability and apoptosis of U87 glioma cells in contrast to doxorubicin alone. DT(270-326) might provide a novel strategy to increase the delivery of macromolecular therapeutic agents across the BTB.

Role of Autophagy in Capsaicin-Induced Apoptosis in U251 Glioma Cells.

In recent years, the role of capsaicin in cancer prevention and treatment has gained people's attention. However, the mechanism of anti-glioma cells by capsaicin has not been elucidated. Here, we discuss the mechanism of capsaicin in U251 cells. Cell viability was detected by MTT and extracellular LDH measurements, while immunofluorescence was performed to measure changes of LC3 in U251 cells. The expressions of LC3II, Puma-α, Beclin1, P62, Procaspase-3, and P53 were observed by immunoblotting. The cell viability decreased and the punctate patterns of LC3 in U251 cells were observed after Capsaicin treatment. Meanwhile, the expressions of Beclin1, P62, and Puma-α increased. After using 3-MA, the expressions of Beclin1 and Procaspase-3 were reduced while those of P53 and Puma-α increased. The expression of LC3II was increased after Pifithrin-α treatment. Therefore, we believed that capsaicin could induce apoptosis in U251 cells, and the inhibition of autophagy could contribute to apoptosis.

Effect of 3-methyladenine on cisplatin induced apoptosis in human glioma U251 cells

Objective 3-methyl adenine (3-methyladenine, 3-MA) was used to test the effect on cisplatin induced U251 cell apoptosis. Methods MTT assay was used to test the effect of 3-MA on cisplatin cytotoxicity in U251 cells. Hoechst33342 staining was used to test the effect of 3-MA on cisplatin induced apoptosis. Western bolt was used to detect the expression of ER stress associated proteins, ubiquitinated protein, protein disulfide isomerase (PDI), glucose-regulated protein78 (Grp78), C/EBP homologous protein (CHOP), and activation of caspase-4 and caspase-3. The expression of microtubule-associated protein 1 light chain 3 (LC3) and PDI were observed by immunofluorescence staining and confocal microscopy. Results MTT assay showed that 3-MA could enhance cisplatin induced growth inhibition rate in human glioma U251 cells (P=0.00324 cisplatin troup vs. control group; P=0.02249 3-MA+ cisplatin group vs. cisplatin group). Hoechst33342 staining showed that 3-MA could increase cisplatin induced apoptotic effect in U251 cells. Western blot showed that 3-MA increases the expressions of ubiquitinated proteins, ERS marker proteins PDI, Grp78(P=0.00294, 0.0012, 0.00179 cisplatin group vs. control group; P=0.01896, 0.01515, 0.00734 3-MA+ cisplatin group vs cisplatin group), CHOP, and active Caspase-4 and active Caspase-3 (P=0.00081, 0.00017, 0.00018 cisplatin group vs. control group; P=0.00671, 0.00934, 0.01124 3-MA+ cisplatin group vs. cisplatin group). 3-MA could inhibit the expression of LC3II induced by cisplatin in U251 cell (P=0.00447 cisplatin group vs. control group; P=0.01588 3-MA+ cisplatin group vs. cisplatin group). Indirect immunofluorence staining showed that 3-MA inhibited the aggregation of LC3 and increased the expression of PDI. Conclusion 3-MA combined with cisplatin enhances cisplatin induced apoptosis by increasing ER stress. Key words: Cisplatin; Autophagy; ER stress; Apoptosis; Glioma

Mangiferin regulates proliferation and apoptosis in glioma cells by induction of microRNA-15b and inhibition of MMP-9 expression.

Mangiferin, a flavonoid extracted from the leaves of the Anacardiaceae plant, the mango tree, has physiological activity and pharmacological effects in many aspects. The present study aimed to clarify the effect of mangiferin on proliferation and apoptosis of glioma cells and the mechanism of these curative effects of mangiferin. In this experiment, we detected the proliferation using 3-(4,5-dimethylthylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. Then, cell apoptosis of U87 glioma cells was measured with the Annexin V-FITC/propidium iodide (PI) apoptosis detection kit, DAPI staining assay and the caspase-3 and caspase-9 activity assay kit. Next, quantitative real-time PCR and gelatin zymography were used to analyze the expression of microRNA-15b (miR-15b) and matrix metalloproteinase-9 (MMP-9), respectively. MMP-9 agonist, miR-15b mimics and anti-miR-15b mimics were added to the U87 glioma cells for elucidating the mechanisms involved in the curative effects of mangiferin. In the present study, mangiferin notably restrained the proliferation and increased the apoptosis of the U87 glioma cells. Meanwhile, mangiferin specifically promoted the expression of miR-15b and suppressed the level of MMP-9 in the U87 glioma cells. miR-15b regulated the expression of MMP-9 in the U87 glioma cells. MMP-9 agonist and anti-miR‑15b reduced the curative effects of mangiferin in the U87 glioma cells. In summary, mangiferin regulates proliferation and apoptosis in glioma cells by induction of miR-15b and inhibition of MMP-9 expression.

Effect of microRNA-874 on proliferation and apoptosis in U87 glioma cells

Objective To observe the effects of microRNA(miR)- 874 on proliferation and apoptosis in U87 glioma cells. Methods The expression of miR- 874 was detected in 12 cases of gliomas and 6 nontumor control brain tissues by using real- time fluorescent quantitative polymerase chain reaction(FQPCR). The expression of miRNA- 874 was tested by FQ- PCR after synthesized miR- 874 mimics was transfected into U87 glioma cells and cell proliferation was detect by methyl thiazol tetrazolium(MTT).Flow cytometry was applied to detect the cell cycle and apoptosis. Results MiR- 874 was down- regulated in glioblastoma samples compared with non- tumor control brain tissues. The expression of miR- 874 increased significantly after transfection of miR- 874 mimics in U87 cells compared to control group(638.00±80.63 vs. 0.17±0.04,P< 0.05), meanwhile suppressed cells proliferation(48 h:0.357±0.026 vs.0.589±0.021, P<0.05;72 h:0.522±0.034 vs.0.834±0.035, P< 0.05).Up- regulation of miR- 874 raised the percentage of G0/G1 phase cells[(56.28±1.62)% vs.(48.57±0.88)%, P<0.05]and induced cells apoptosis [(12.50±0.66)% vs.(4.91±0.15)%, P< 0.05]. Conclusion The expression of miR- 874 was low in glioblastoma. Loss of miR- 874 is correlated with Tumorigenesis and development. The miR- 874 might be a valid target for the diagnosis and treatment of glioblastoma. Key words: MicroRNA-874; Glioblastoma; Proliferation; Apoptosis

MiR-133b contributes to arsenic-induced apoptosis in U251 glioma cells by targeting the hERG channel.

Substantial evidence indicates that the human ether-a-go-go-related gene potassium channel (hERG, Kv11.1, KCNH2) is overexpressed in human glioblastoma multiforme (GBM) specimens and plays an essential role in the malignant proliferation of glioma cells. However, its upstream regulator in glioma cells is not fully elucidated. The present study was designed to determine whether the expression of hERG gene is regulated by miR-133b or miR-34a, thereby contributing to the anti-proliferation effect of arsenic trioxide (ATO) in U251 human glioma cells. Real-time polymerase chain reactions (qRT-PCR) and Western blot results demonstrated that hERG mRNA and protein levels were dramatically upregulated in clinical GBM specimens. Conversely, both miR-133b and miR-34a were markedly downregulated in clinical GBM specimens by qRT-PCR. The hERG gene was a direct target of miR-133b and miR-34a by bioinformatics analyses and luciferase reporter assays. Moreover, ATO, which is an emerging chemotherapy drug for glioma disease, remarkably elevated the level of miR-133b, but not miR-34a in U251 glioma cells. The level of miR-133b upstream transactivator serum response factor (SRF) was also suppressed by ATO. The transfection of anti-miR-133b oligonucleotide (AMO-133b) remarkably prevented the decrease of hERG protein by 5μM ATO treatment for 24h in U251 cells, whereas anti-miR-34a oligonucleotide (AMO-34a) did not exhibit recuperated effect. Finally, the transient overexpression by miR-133b mimics and treatment with the hERG channel-specific blocker E4031 markedly facilitated the ATO inhibition of proliferation of and induced apoptosis in U251 cells, whereas AMO-miR-133b attenuated these changes. Our study provided the evidence for the pathological role of miR-133b and miR-34a in the development of GBM and thus expanded our understanding of the hERG gene expression and ATO chemotherapeutic roles of miRNAs. Targeting miR-133b/hERG pathway may be a new strategy for chemotherapy of malignant gliomas.

Polyphyllin D induces apoptosis in U87 human glioma cells through the c-Jun NH2-terminal kinase pathway.

Polyphyllin D (PD), an active component from a traditional medicinal herb Paris polyphylla, which has long been used for the treatment of cancer in Asian countries, has been found to hold significant antitumor activity in vivo or in vitro. However, there were few reports on the effects and underlying mechanism of PD on apoptosis in U87 human glioma cells. The present study was conducted to evaluate apoptotic induction of PD in U87 human glioma cells, and explore its underlying pathway. U87 glioma cells were cultured and treated with varied concentrations of PD (from 10(-8) to 10(-4) M). The inhibition of U87 glioma cell proliferation by PD was assessed by MTT assay. The apoptosis of U87 glioma cells was detected by flow cytometry, and western blot analysis was used to examine human B-cell leukemia/lymphoma 2 (Bcl-2), human Bcl-2 associated X protein (Bax), caspase-3, total-c-jun NH2-terminal kinase (t-JNK), and phosphorylation-JNK (p-JNK) protein expression in U87 human glioma cells. The treatment with PD for 24 h significantly inhibited the proliferation of U87 human glioma cells in a concentration-dependent manner. PD increased apoptosis and significantly upregulated the expression of Bax, caspase-3, and p-JNK associated with apoptosis, but downregulated antiapoptotic Bcl-2 expression in U87 human glioma cells. Our data provided evidences that PD induces apoptosis in U87 human glioma cells. This effect might be associated with the JNK pathway.

Staurosporine induced apoptosis rapidly downregulates TDP- 43 in glioma cells.

TDP-43 is a ubiquitously expressed DNA/RNA binding protein that has recently attracted attention for its involvement in neurodegenerative diseases. While TDP-43 has been found to participate in various important cellular activities including stress and apoptosis, little is known about its role in cancer cells. Here we report that staurosporine (STS) induced apoptosis in U87 glioma cells is associated with rapid downregulation of TDP-43 at both mRNA and protein levels. The latter is dependent on activation of caspase 3. More importantly, we have shown that knockdown of TDP-43 by specific siRNA dramatically enhanced cytotoxicity of STS. These results suggest that normal level of TDP-43 may be protective for cancer cells under apoptotic insult.

Transient axonal glycoprotein-1 induces apoptosis-related gene expression without triggering apoptosis in U251 glioma cells.

Previous studies show that transient axonal glycoprotein-1, a ligand of amyloid precursor protein, increases the secretion of amyloid precursor protein intracellular domain and is involved in apoptosis in Alzheimer's disease. In this study, we examined the effects of transient axonal glycoprotein-1 on U251 glioma cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that transient axonal glycoprotein-1 did not inhibit the proliferation of U251 cells, but promoted cell viability. The terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that transient axonal glycoprotein-1 did not induce U251 cell apoptosis. Real-time PCR revealed that transient axonal glycoprotein-1 substantially upregulated levels of amyloid precursor protein intracellular C-terminal domain, and p53 and epidermal growth factor receptor mRNA expression. Thus, transient axonal glycoprotein-1 increased apoptosis-related gene expression in U251 cells without inducing apoptosis. Instead, transient axonal glycoprotein-1 promoted the proliferation of these glioma cells.

Epigallocatechin-3-gallate induces apoptosis, inhibits proliferation and decreases invasion of glioma cell

Epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, has been considered a potential therapeutic and chemopreventive agent for cancer. Glioma is a malignant tumor with high mortality but effective therapy has not yet been developed. In this study, we found that EGCG induced apoptosis in U251 glioma cells via the laminin receptor (molecular weight 67 kDa) in a time- and dose-dependent manner, decreased their invasiveness and inhibited their proliferation. The mitogen-activated protein kinase pathway was shown to be involved in glioma cell apoptosis and proliferation. Furthermore, the mRNA levels of matrix metalloproteinase (MMP)-2 and MMP-9 were reduced after EGCG treatment. These results suggest that EGCG has important therapeutic effects with low toxicity and side-effects, and could be used in cancer chemoprevention.

Effect of RhoE on apoptosis of human glioma U251 cells and the mechanism

Objective To investigate the effects of Ras homolog gene family,member E (RhoE)overexpression on the apoptosis of human glioma U251 cells and the mechanism.Methods The Myc-N1 and Myc-RhoE plasmids were transfected into human glioma U251 cells by Lipofectamine technology.The changes of RhoE and Cyclin D1 in transfected glioma cells were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting.The cell proliferaion and apopotosis were analyzed by methyl thiazol tetrazolium (MTF) assay and flow cytometry.Results The RhoE mRNA and protein levels in RhoE-U251 group was significantly up-regulated compared to N1-U251 group and control group (P ＜0.05),while the Cyclin D1 mRNA and protein levels in RhoE-U251 group were significantly suppressed.After overexpression of RhoE was up-regulated,the growth of these cells was inhibited and their apoptosis was obviously enhanced.Conclusion RhoE may be a tumor suppressor gene.Up-regulation of RhoE expression can down-regulate the Cyclin D1 expression,and then promote the apoptosis and inhibit the growth of human glioma U251 cells. Key words: Glioma; Apoptosis ; RhoE ; Cyclin D1

Affect of miR-195 on proliferation and apoptosis of glioma U251 cells

Objective To explore the function and mechanism of miR-195 over-expression in glioblastoma U251 cell proliferation and apoptosis.Methods The experiment is divided into Blank,NC and miR-195.Blank is treated with equal volume PBS.Negative control or miR-195 mimics were transfected into U251 ceils by Lipofectamine RNAiMAX.miR-195 expression levels were detected by Real -time PCR.Cell growth inhibition rates and proliferation were analyzed by CCK-8 assay and colony formation.Flow cytometry was used to monitor changes in cell apoptosis and cycle.Apoptotic cells and necrotic cells were also qualified by Hoechst 33342 and propidium iodide staining.The target of miR-195 was forecasted by bioinformatics.The expression level of Bcl-2 protein was detected through Western blotting.Results After the over-expression of miR-195,Real-time PCR showed that miR-195 expression level was up-regulated about six times.Cell growth inhibition rate and colony formation revealed that the growth and proliferation ability of the miR-195 group were significantly suppressed(compared with NC,P ＜0.001).Over-expression of miR-195 could promote cell apoptosis and death(compared with Blank and NC,P ＜ 0.001).Predicting outcomes of TargetScan indicated that Bcl-2 was a target of miR 195.Furthermore,the over-expression of miR-195 could block G0/G1 transition and dowm-regulate the Bcl-2 expression (compared with Blank and NC,P ＜ 0.05).Conclusion Over-expression of miR-195 could inhibit proliferation,induce apoptosis and death,and block G0/S1 transition.miR-195 may serve as an attractive target of gene therapy for glioblastoma. Key words: Glioma; MiR-195 ; Proliferation; Apoptosis; Bcl-2

A preliminary study about the apoptostic mechanism of RNA targeting basic fibroblast growth factor in glioma U251 cells

To preliminarily investigate the mechanism of small interfering RNA (siRNA) induced apoptosis in glioma U251 cells by silencing basic fibroblast growth factor (bFGF). U251 cells were divided into the normal control group, the mock group and experiment group, the mock and experiment group were transfected with mock vector (Ad-null) and the recombinant adenovirus carrying bFGF-siRNA (Ad-bFGF-siRNA) respectively at a multiplicity of infection (MOI) of 100. After 72 hours, the expression of related proteins was revealed by the method of Western blot. Mitochondrial transmembrane potential (ΔΨm) was measured with flow cytometry and confocal microscopy, Groups were compared using single factor analysis of variance (One-way ANOVA). After U251 cells were transfected with bFGF-siRNA, the results of Western blot showed that after 72 hours of transfection the bFGF protein in the experiment group decreased obviously, meanwhile Cytochrome C, Caspase-3 and Bax showed increased expression while in the Bcl-xl and Bcl-2 proteins decreased expression. The proportion of high mitochondrial membrane potential of cells by flow cytometry, the experimental group was 74.4% ± 4.7% decreased significantly compared with the control group 92.1% ± 2.5%, the mock group 90.9% ± 1.8% (F = 28.805, P < 0.05); laser scanning confocal microscopy results showed that the red fluorescence and green fluorescence ratio of the experimental group was 0.83 ± 0.12 decreased significantly compared with 1.36 ± 0.40 of the control group and 1.32 ± 0.35 of the mock group(F = 7.920, P < 0.05). siRNA targeting bFGF induced U251 cell apoptosis may be achieved through the mitochondrial pathway.

AMP-activated protein kinase-dependent and -independent mechanisms underlying in vitro antiglioma action of compound C

We investigated the effect of compound C, a well-known inhibitor of the intracellular energy sensor AMP-activated protein kinase (AMPK), on proliferation and viability of human U251 and rat C6 glioma cell lines. Compound C caused G 2/M cell cycle block, accompanied by apoptotic glioma cell death characterized by caspase activation, phosphatidylserine exposure and DNA fragmentation. The mechanisms underlying the pro-apoptotic action of compound C involved induction of oxidative stress and downregulation of antiapoptotic molecule Bcl-2, while no alteration of pro-apoptotic Bax was observed. Compound C diminished AMPK phosphorylation and enzymatic activity, resulting in reduced phosphorylation of its target acetyl CoA carboxylase. AMPK activators metformin and AICAR partly prevented the cell cycle block, oxidative stress and apoptosis induced by compound C. The small interfering RNA (siRNA) targeting of human AMPK mimicked compound C-induced G 2/M cell cycle arrest, but failed to induce oxidative stress and apoptosis in U251 glioma cells. In conclusion, our data indicate that AMPK inhibition is required, but not sufficient for compound C-mediated apoptotic death of glioma cells.

Adenovirus-mediated transfer of p53 augments hyperthermia-induced apoptosis in U251 glioma cells

Purpose: Hyperthermia kills glioma cells by inducing apoptosis and is thereby an effective therapeutic modality for the treatment of malignant gliomas. However, cells harboring mutated p53 are refractory to hyperthermia-induced apoptosis. In this study, we assessed whether or not adenovirus (Adv)-mediated transduction of p53 overrides this resistant mechanism. Methods and Materials: We transduced the p53 wild-type tumor suppressor gene into U251 glioma cells harboring mutated p53 using Adv vectors in combination with hyperthermia (43, 44.5°C), and evaluated the degree of cell death and apoptosis. Results: The percentage of cells that had died, as measured by trypan blue staining, among U251 cells infected with the Adv for p53 (Adv-p53) and treated with hyperthermia, was significantly higher than the percentage of cells that had died among U251 cells infected with Adv-p53 and not treated with hyperthermia, or those infected with the control Adv for dE (Adv-dE) and treated with hyperthermia. The degree of apoptosis, measured at 24 h after treatment, in hyperthermia-treated U251 cells infected with Adv-p53 (43°C, 73%; 44.5°C, 92%) was much higher than that infected with Adv-p53 (41%), or that infected with control Adv-dE and treated with hyperthermia (43°C, 1.3%; 44.5°C, 19%). Treatment with combined hyperthermia and Adv-p53 infection induced cleavage of caspase-3 in U251 cells. Conclusion: These results indicate that Adv-mediated transduction of p53 would render glioma cells highly sensitive to hyperthermia.