Apoptosis In U87 Glioblastoma Cells Research Articles

THE EFFECT OF 5-FU AND RUXOLITINIB ON MITOCHONDRIAL APOPTOSIS IN GLIOBLASTOMA U87 CELL LINE

Aims: The aim of this study is to carry out the effect of 5-Fluorouracil alone or combined with Ruxolitinib on both apoptosis and JAK/STAT pathway in U87 glioblastoma cells. Methods: We used U87 glioblastoma cell lines as the human brain cancer cells. We treated the cells with 5-Fluorouracil (3.125 μM-400 μM) alone and with a combination of Ruxolitinib (100 μM or 400 μM of Ruxolitinib with 3.125-25 μM 5-Fluorouracil), and performed the MTT test for calculating IC50 value. Molecular fluorescence staining was performed with Hoechst and acridine orange/ethidium bromide probes. The alteration in mitochondrial apoptosis and JAK/STAT pathways to drug treatment was analyzed by the qRT-PCR assay. Results: Decrease in cell viability was more prominent in U87 cells treated with a combination of 5-Fluorouracil and Ruxolitinib compared to those treated with 5-Fluoro- uracil alone. In gene expression analysis, apoptosis signals were observed in cells treated with 5-Fluorouracil alone and 5-Fluo- rouracil+Ruxolitinib treatment. Conclusion: Treatment with 5-Fluorouracil alone and 5-Fluorouracil+Ruxolitinib combination increased apoptosis in U87 glioblastoma cells. However, it is difficult to mention an evident difference between treatments. Therefore, further studies are needed.

Effects of Reduced Graphene Oxides on Apoptosis and Cell Cycle of Glioblastoma Multiforme.

Graphene (GN) and its derivatives (rGOs) show anticancer properties in glioblastoma multiforme (GBM) cells in vitro and in tumors in vivo. We compared the anti-tumor effects of rGOs with different oxygen contents with those of GN, and determined the characteristics of rGOs useful in anti-glioblastoma therapy using the U87 glioblastoma line. GN/ExF, rGO/Term, rGO/ATS, and rGO/TUD were structurally analysed via transmission electron microscopy, Raman spectroscopy, FTIR, and AFM. Zeta potential, oxygen content, and electrical resistance were determined. We analyzed the viability, metabolic activity, apoptosis, mitochondrial membrane potential, and cell cycle. Caspase- and mitochondrial-dependent apoptotic pathways were investigated by analyzing gene expression. rGO/TUD induced the greatest decrease in the metabolic activity of U87 cells. rGO/Term induced the highest level of apoptosis compared with that induced by GN/ExF. rGO/ATS induced a greater decrease in mitochondrial membrane potential than GN/ExF. No significant changes were observed in the cytometric study of the cell cycle. The effectiveness of these graphene derivatives was related to the presence of oxygen-containing functional groups and electron clouds. Their cytotoxicity mechanism may involve electron clouds, which are smaller in rGOs, decreasing their cytotoxic effect. Overall, cytotoxic activity involved depolarization of the mitochondrial membrane potential and the induction of apoptosis in U87 glioblastoma cells.

Brevilin A promotes oxidative stress and induces mitochondrial apoptosis in U87 glioblastoma cells.

BackgroundSesquiterpene lactones are plant-derived, natural, bioactive molecules often used against inflammatory diseases in traditional Chinese medicines. Recently, sesquiterpene lactones have been reported to exhibit potent anticancer activity. In the present study, we have investigated the anticancer activity of Brevilin A, a sesquiterpene lactone component of Centipeda minima, against U87 glioblastoma cells.Materials and methodsThe cell proliferation was determined by MTT assay. Cell morphological changes were observed by phase-contrast microscopy. Flow cytometry was used to measure apoptosis. Glutathione (GSH), ROS generation, and mitochondrial membrane potential were measured using commercially available kits. The expression of proteins was measured by Western blotting analysis.ResultsBrevilin A inhibited the proliferation of, and induced severe morphological changes and apoptotic cell death in, U87 glioblastoma cells in a dose-dependent manner. Further mechanistic study revealed that Brevilin A induces oxidative stress, as evident from ROS generation, GSH depletion, and increased phosphorylation of stress-activated proteins p38 and JNK. Furthermore, Brevilin A bcl-xl/bak ratio, decreased mitochondrial membrane potential and induced cytochrome c release from mitochondria into cytosol in a dose-dependent manner. Finally, Brevilin A decreased the expression of Xiap and increased the expression of cleaved forms of caspase-9 and -3 and PARP in a dose-dependent manner.ConclusionCollective findings demonstrated that Brevilin A is a potent, anticancer, bioactive molecule and it effectively induces apoptosis in U87 glioblastoma cells, which is associated with induction of oxidative stress and mitochondrial dysfunction.

Galectin-8 promotes migration and proliferation and prevents apoptosis in U87 glioblastoma cells.

BackgroundGlioblastoma is one of the most aggressive cancers of the brain. Malignant traits of glioblastoma cells include elevated migration, proliferation and survival capabilities. Galectins are unconventionally secreted glycan-binding proteins that modulate processes of cell adhesion, migration, proliferation and apoptosis by interacting with beta-galactosides of cell surface glycoproteins and the extracellular matrix. Galectin-8 is one of the galectins highly expressed in glioblastoma cells. It has a unique selectivity for terminally sialylated glycans recently found enhanced in these highly malignant cells. A previous study in glioblastoma cell lines reported that Gal-8 coating a plastic surface stimulates two-dimensional motility. Because in other cells Gal-8 arrests proliferation and induces apoptosis, here we extend its study by analyzing all of these processes in a U87 glioblastoma cell model.MethodsWe used immunoblot and RT-PCR for Gal-8 expression analysis, recombinant Gal-8 produced in a bacteria system for Gal-8 treatment of the cells, and shRNA in lentivirus transduction for Gal-8 silencing. Cell migration as assessed in transwell filters. Cell proliferation, cell cycle and apoptosis were analyzed by FACS.ResultsGal-8 as a soluble stimulus triggered chemotactic migration of U87 cells across the polycarbonate filter of transwell chambers, almost as intensively as fetal bovine serum. Unexpectedly, Gal-8 also enhanced U87 cell growth. Co-incubation of Gal-8 with lactose, which blocks galectin–glycan interactions, abrogated both effects. Immunoblot showed Gal-8 in conditioned media reflecting its secretion. U87 cells transduced with silencing shRNA in a lentiviral vector expressed and secreted 30–40 % of their normal Gal-8 levels. These cells maintained their migratory capabilities, but decreased their proliferation rate and underwent higher levels of apoptosis, as revealed by flow cytometry analysis of cell cycle, CFSE and activated caspase-3 staining. Proliferation seemed to be more sensitive than migration to Gal-8 expression levels.ConclusionsGal-8, either secreted or exogenously enriched in the media, and acting through extracellular glycan interactions, constitutes a strong stimulus of directional migration in glioblastoma U87 cells and for the first time emerges as a factor that promotes proliferation and prevents apoptosis in cancerous cells. These properties could potentially contribute to the exaggerated malignancy of glioblastoma cells.

Beclin 1, an autophagy-related gene, augments apoptosis in U87 glioblastoma cells

Beclin 1 acts as a tumor suppressor and is an essential mediator of autophagy. Beclin 1 also interacts with Bcl-2 and can induce apoptosis by activating the mitochondrion permeabilizing function of proapoptotic multidomain proteins from the Bcl-2 family. Moreover, these Bcl-2 family members can activate autophagy by liberating Beclin 1 from its inhibition by Bcl-2/Bcl-XL at the level of the endoplasmic reticulum. We found that overexpression of Beclin 1 in U87 glioblastoma cells enhanced the capacity for cellular autophagy and induced apoptosis. Silencing of Beclin 1 decreased autophagic capacity but had little effect on apoptosis and cell proliferation. Beclin 1-Bcl-2 and Beclin 1-Bcl-xL complexes were detected by immunoprecipitation in cells that overexpressed Beclin 1. Furthermore, the levels of cytochrome c in the cytosol and the activity of caspases-3/-9 in the cytosol increased after overexpression of Beclin 1. Our results suggest that Beclin 1 induces apoptosis via binding to Bcl-2 and Bcl-xL, followed by the release of cytochrome c into the cytosol and activation of caspases-3/-9.

Abstract 2952: The PKC-iota inhibitor ICA-1 combined with TRAIL for glioblastoma therapy.

Abstract Glioblastoma multiforme is a very aggressive brain tumor. Temozolimide is the standard clinical therapy following surgery which offers a limited survival advantage. The low survival rates of glioblastoma patients require further investigation into pre-clinical combination therapy options with apoptosis abilities. One such chemotherapeutic approach is the use of the novel PKC-iota inhibitor, ICA-1, combined with rhTRAIL. T98G or U87 glioblastoma cells were treated with ICA-1 for 48h or 72h, respectively, with or without rhTRAIL in a 24-well format. Based on flow cytometric analysis, the overall apoptosis rate for the T98G cells exposed to ICA-1 plus TRAIL was the most significant with no distinction between early and late apoptosis rates. In addition, the immunofluorescence analysis showed higher T98G cell death for the combination therapy. Further flow cytometric analysis showed that ICA-1 plus TRAIL generated significantly more late apoptosis for U87 cells than any other treatment. The mechanism of action for this combinatorial therapy is currently under investigation. However, our in vitro data demonstrated that ICA-1 plus TRAIL induced 60-70% apoptosis in U87 glioblastoma cells and T98G glioblastoma cells which may offer a better survival advantage in pre-clinical animal models. Citation Format: Andrea N. McCray, Shraddha Desai, Mildred Acevedo-Duncan. The PKC-iota inhibitor ICA-1 combined with TRAIL for glioblastoma therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2952. doi:10.1158/1538-7445.AM2013-2952

Alantolactone Induces Apoptosis in HepG2 Cells through GSH Depletion, Inhibition of STAT3 Activation, and Mitochondrial Dysfunction

Signal transducer and activator of transcription 3 (STAT3) constitutively expresses in human liver cancer cells and has been implicated in apoptosis resistance and tumorigenesis. Alantolactone, a sesquiterpene lactone, has been shown to possess anticancer activities in various cancer cell lines. In our previous report, we showed that alantolactone induced apoptosis in U87 glioblastoma cells via GSH depletion and ROS generation. However, the molecular mechanism of GSH depletion remained unexplored. The present study was conducted to envisage the molecular mechanism of alantolactone-induced apoptosis in HepG2 cells by focusing on the molecular mechanism of GSH depletion and its effect on STAT3 activation. We found that alantolactone induced apoptosis in HepG2 cells in a dose-dependent manner. This alantolactone-induced apoptosis was found to be associated with GSH depletion, inhibition of STAT3 activation, ROS generation, mitochondrial transmembrane potential dissipation, and increased Bax/Bcl-2 ratio and caspase-3 activation. This alantolactone-induced apoptosis and GSH depletion were effectively inhibited or abrogated by a thiol antioxidant, N-acetyl-L-cysteine (NAC). The data demonstrate clearly that intracellular GSH plays a central role in alantolactone-induced apoptosis in HepG2 cells. Thus, alantolactone may become a lead chemotherapeutic candidate for the treatment of liver cancer.

Phenylacetylglutaminate and Phenylacetate in Combination Upregulate VDUP1, Cause Cell Cycle Blockade and Apoptosis in U87 Glioblastoma Cells

Phenylacetylglutaminate (PG) and Phenylacetate (PN) are metabolites of Phenylbutyrate (PB) and are constituents of antineoplaston AS2-1. These are sodium salts of amino acid derivative and carboxylic acid that inhibit the growth of neoplastic cells without growth inhibitory effect in normal cells. The aim of this study was to identify molecular pathways involved in the anti-proliferative effect of antineoplastons. Using a total human genome microarray we have found that 1) Vitamin D3 upregulated protein (VDUP1) is significantly upregulated in response to PG and PN in the U87 glioblastoma cells; 2) Isobologram analysis shows that PG and PN act in an additive or synergistic manner to effectively suppress proliferation of U87 cells; 3) PG and PN cause cell cycle arrest, changes in expression of several cell cycle genes and suppress expression and activity of the G2/M checkpoint kinase, CHK1. The multiple cellular targets possibly make these compounds effective anti-proliferative agents. We propose that PG and PN in combination target important cellular pathways and upregulate VDUP1 leading to detachment-induced apoptosis in cancer cells.

Jaceosidin Induces Apoptosis in U87 Glioblastoma Cells through G2/M Phase Arrest.

Artemisia argyi is a widely used medicinal plant in China. The present study was designed to identify the bioactive constituents with antiglioma activity from leaves of Artemesia argyi. A bioactivity guided approach based on MTT assay for cells growth inhibition led to the isolation of a flavonoid, “jaceosidin” from ethanol extract of leaves of Artemesia argyi. The growth inhibitory effect of jaceosidin was explored using flow cytometry and Western blot studies. Our results showed that jaceosidin exerts growth inhibitory effect by arresting the cells at G2/M phase and induction of apoptosis. Furthermore, our study revealed that induction of apoptosis was associated with cell cycle arrest at G2/M phase, upregulation of p53 and Bax, decrease in mitochondrial membrane potential, release of cytochrome c, and activation of caspase 3. This mitochondrial-caspase-3-dependent apoptosis pathway was confirmed by pretreatment with caspase 3 inhibitor, Ac-DEVD-CHO. Our findings suggested that jaceosidin induces mitochondrial-caspase-3-dependent apoptosis in U87 cells by arresting the cell cycle at G2/M phase.

Oncolytic Vesicular Stomatitis Virus Induces Apoptosis in U87 Glioblastoma Cells by a Type II Death Receptor Mechanism and Induces Cell Death and Tumor Clearance In Vivo

Vesicular stomatitis virus (VSV) is a potential oncolytic virus for treating glioblastoma multiforme (GBM), an aggressive brain tumor. Matrix (M) protein mutants of VSV have shown greater selectivity for killing GBM cells versus normal brain cells than VSV with wild-type M protein. The goal of this research was to determine the contribution of death receptor and mitochondrial pathways to apoptosis induced by an M protein mutant (M51R) VSV in U87 human GBM tumor cells. Compared to controls, U87 cells expressing a dominant negative form of Fas (dnFas) or overexpressing Bcl-X(L) had reduced caspase-3 activation following infection with M51R VSV, indicating that both the death receptor pathway and mitochondrial pathways are important for M51R VSV-induced apoptosis. Death receptor signaling has been classified as type I or type II, depending on whether signaling is independent (type I) or dependent on the mitochondrial pathway (type II). Bcl-X(L) overexpression inhibited caspase activation in response to a Fas-inducing antibody, similar to the inhibition in response to M51R VSV infection, indicating that U87 cells behave as type II cells. Inhibition of apoptosis in vitro delayed, but did not prevent, virus-induced cell death. Murine xenografts of U87 cells that overexpress Bcl-X(L) regressed with a time course similar to that of control cells following treatment with M51R VSV, and tumors were not detectable at 21 days postinoculation. Immunohistochemical analysis demonstrated similar levels of viral antigen expression but reduced activation of caspase-3 following virus treatment of Bcl-X(L)-overexpressing tumors compared to controls. Further, the pathological changes in tumors following treatment with virus were quite different in the presence versus the absence of Bcl-X(L) overexpression. These results demonstrate that M51R VSV efficiently induces oncolysis in GBM tumor cells despite deregulation of apoptotic pathways, underscoring its potential use as a treatment for GBM.

Boron neutron capture therapy inducing apoptosis in U87 glioblastoma cells

Objective To explore the effect of boron neutron capture therapy(BNCT)on human brain glioma U87 cell line and its mechanisms.Method U87 cells in exponential phase were divided into 6 groups:untreated control,60Co γ 4 Gy,60Co γ 8 Gy,nuclear reactor exposure without boronophenylalanine (BPA)3.5 Gy,BNCT 4 Gy and BNCT 8 Gy.The anti-tumor effects were analyzed through cell morphology,the Annexin V/PI assay by flow cytometer(FCM),and methyl thiazolyl tetrazolium(MTT)assay.The expression of P53 protein was studied by immunocytochemistry and the expression of BCL-2 protein and BAX protein were measured by western blot.Results Typical morphological changes were observed after BNCT irradiation.The apoptotic rates were observed 48 h after irradiation with 65.1%and 85.9%for BNCT 4 Gy and 8 Gy.BNCT showed higher apoptotic rates than those of γ-ray control irradiation (P＜0.01).The expression level of P53 protein was negative in U87,while positive expression of P53 protein was observed in BNCT 4 Gy and 8 Gy groups.BNCT promoted BAX protein expression,at the same time it also inhibited BCL-2 expression.Conclusions BNCT can inhibit the growth of U87 significantly in a dose-dependent and time-dependent patterns. Key words: Boron neutron capture therapy; Glioma; Apoptosis

Induction of mitochondria-mediated apoptosis by methanol fraction of Ulmus davidiana Planch (Ulmaceae) in U87 glioblastoma cells

Mitochondria play a pivotal role in apoptosis of mammalian cells by releasing apoptogenic proteins such as cytochrome c into the cytoplasm. Mitochondrial membrane permeability during apoptosis is regulated directly by the Bcl-2 family of proteins. In glioma cells, there are no specific features of apoptosis compared with apoptosis in other cell types. Ulmus davidiana Planch (Ulmaceae) is a deciduous tree, which is widely distributed in Korea. The barks of the stem and the root of the plant have been used in oriental traditional medicine for the treatment of oedema, mastitis, gastric cancer, and inflammation. Our results demonstrate that the methanol fraction of the stem bark extracts of Ulmus davidiana Planch induce mitochondria-mediated apoptosis in U87 glioblastoma cells. The methanol fraction exhibited a comparatively higher cytotoxic activity (IC 50 = 7.5 ± 0.7 μg/ml) in a dose-dependent manner than chloroform, and hexane fractions. The results show the typical ladder profile of oligonucleosomal fragments characteristics of apoptosis and the secreted cytosolic cytochrome c level was increased by treatment of methanol fraction of UD. Moreover, the expressional changes of the Bcl-2 family protein occurred within 30 min after treatment with methanol fraction. All results indicate that the methanol fraction of U. davidiana Planch (the barks of the stem) is capable of inducing apoptosis in U87 glioblastoma cells.