Apoptosis In Human Glioblastoma Cells Research Articles

Overexpression of WW domain-containing oxidoreductase WOX1 preferentially induces apoptosis in human glioblastoma cells harboring mutant p53

PurposeHuman WWOX gene encoding WW domain-containing oxidoreductase, named WWOX, FOR, or WOX1, has been studied in various types of cancer cells and shown to be a tumor suppressor with pro-apoptotic properties. Mutation or gain-of-function of p53 in glioma cells is associated with resistance to radiation therapy and poor prognosis. In this study, we overexpressed WOX1 to examine the pro-apoptotic activity against human glioblastoma cells harboring mutant p53. MethodsOverexpression of WOX1 in glioblastoma cell lines and apoptosis-related assays were performed. ResultsOur results showed that overexpressed WOX1 induced apoptosis of glioblastoma U373MG harboring mutant p53 by causing hypoploidy and DNA fragmentation. However, ectopic WOX1 had no effect with U87MG possessing wild type p53. Unlike temozolomide, WOX1 induced apoptosis of U373MG cells via a mitochondria-independent and caspase-3-independent pathway. ConclusionsOverexpression of WOX1 preferentially inhibited viability and induced apoptosis in human glioblastoma cells expressing mutant p53 via a mechanism independent of the intrinsic apoptotic pathway. Conceivably, the survival of human glioblastoma cells depends upon interactions between the gain-of-function of p53 and WOX1. This suggests that modulation of WOX1 expression may be a novel strategy for treating human glioblastoma cells with mutant p53.

Effect of Girdin on apoptosis of human glioblastoma cells

Objective To investigate the effects of Girdin on apoptosis of LN229 glioblastoma cells by Girdin small RNA interference (RNAi) technology and the molecular mechanisms. Methods Girdin expression was knocked down in LN229 glioblastoma cells by Girdin small interfering RNA (siRNA). The expression levels of Girdin, cytochrome C (Cyt-C) and Bad proteins in glioblastoma LN229 cells were detected by Western blotting. Stable clones were used to measure cells survival ratio by proliferation assay and methyl thiazol tetrazolium ( MTT) assay. Flow Cytometry was used to examine the content of Cyt-C in siGirdin/LN229 and scr/LN229. 20 male athymic Nu/Nu mice were subcutaneously injected with siGirdin/LN229 or scr/LN229 cells respectively to study the growth of tumor in each group. Results Western blotting showed that the expression of Girdin was decreased significantly. The relative intensity of Cyt-C and Bad in siGirdin/LN229 cells was 3. 57 ±0.43 and 4. 78 ± 1. 03 respectively, and the relative intensity of Cyt-C and Bad in scr/LN229 was 1. 13 ±0. 11,1. 78 ±0.20 respectively (P＜0. 05). The mean fluorescence density of Cyt-C in siGirdin/LN229 and scr/LN229 by flow cytometry was 12 531.00 ±1412. 98 and 183. 67 ±41. 55 respectively. Meanwhile, the number of siGirdin/LN229 cells was obviously decreased as compared with control group in proliferation assay. The number of siGirdin/LN229 cells on the 6th day was (15. 08 ± 1.17) x 104, and that of scr/LN229 cells was (53. 93 ± 6. 26) x 104. MTT assay revealed the survival rate of siGirdin/LN229 on the 5th day was (323. 15 ± 57. 01) % , and that of scr/LN229 was (640.67 ±59.66)%, P＜0.05. Additionally, the volume of xenograft tumors in siGirdin/LN229 group was decreased significantly as compared with control group. The volume of xenograft tumors in siGirdin/LN229 group was (36.42 ±2. 00) mm3, and that in scr/LN229 group was (262. 42 ±24. 12) mm3 at the 7.5th week. Conclusion Girdin plays a critical role in apoptosis of glioblastoma cells. Inhibition of Girdin by siRNA could promote the apoptosis of glioblastoma LN229 cells through the release of Cyt-C from mitochondria and up-regulation of Bad. Key words: Glioma; Girdin; Apoptosis

Thymoquinone Induces Telomere Shortening, DNA Damage and Apoptosis in Human Glioblastoma Cells

BackgroundA major concern of cancer chemotherapy is the side effects caused by the non-specific targeting of both normal and cancerous cells by therapeutic drugs. Much emphasis has been placed on discovering new compounds that target tumour cells more efficiently and selectively with minimal toxic effects on normal cells.Methodology/Principal FindingsThe cytotoxic effect of thymoquinone, a component derived from the plant Nigella sativa, was tested on human glioblastoma and normal cells. Our findings demonstrated that glioblastoma cells were more sensitive to thymoquinone-induced antiproliferative effects. Thymoquinone induced DNA damage, cell cycle arrest and apoptosis in the glioblastoma cells. It was also observed that thymoquinone facilitated telomere attrition by inhibiting the activity of telomerase. In addition to these, we investigated the role of DNA-PKcs on thymoquinone mediated changes in telomere length. Telomeres in glioblastoma cells with DNA-PKcs were more sensitive to thymoquinone mediated effects as compared to those cells deficient in DNA-PKcs.Conclusions/SignificanceOur results indicate that thymoquinone induces DNA damage, telomere attrition by inhibiting telomerase and cell death in glioblastoma cells. Telomere shortening was found to be dependent on the status of DNA-PKcs. Collectively, these data suggest that thymoquinone could be useful as a potential chemotherapeutic agent in the management for brain tumours.

α5β1 integrin antagonists reduce chemotherapy‐induced premature senescence and facilitate apoptosis in human glioblastoma cells

The alpha5beta1 integrin represent a new therapeutic target for glioblastoma, which are malignant brain tumors difficult to cure with conventional therapies. Glioblastoma are known to be highly resistant to chemotherapy. We, therefore, investigated whether blocking alpha5beta1 integrin with specific nonpeptidic antagonists concomitantly with chemotherapy (ellipticine and temozolomide) may impact the response to chemotherapy of human glioblastoma. Here we show that inhibiting alpha5beta1 integrin with 2 selective ligands (SJ749 and K34c) decreases chemotherapy-induced premature senescence and facilitates cell apoptosis in a functional p53 background (U87MG cells). When p53 is mutated and inactive (U373 cells), chemotherapy induces p53-independent cell apoptosis instead of senescence that is not improved by integrin antagonists. Silencing p53 in U87MG cells with siRNA as well as evaluating HCT116 p53+/+ and p53-/- colon carcinoma cell behavior support the hypothesis of an as yet unknown effect of alpha5beta1 integrin antagonists on the control of chemotherapy-induced premature senescence and apoptosis. alpha5beta1 integrin antagonists modulate the p53 signaling induced by chemotherapy. Our results highlight a new role of the alpha5beta1 integrin in the control of glioblastoma aggressiveness and responsiveness to chemotherapy, which may have a crucial impact in the clinical management of patients suffering from brain tumors.

585 Absence or low expression of Fas-associated protein with death domain in acute leukemia and lymphoma cell lines

The study aimed at exploring the effects involved in Fas-Associated protein with Death Domain (FADD) expression and cysteine-aspartic acid specific protease-8 (Caspase-8) in relation to the proliferation and apoptosis of human glioblastoma (GBM) cells.93 GBM tissues and 64 normal brain tissues were the central mediums used for the investigation of the study. Cultured human GBM SC189 cells were divided into separate groups including the blank negative control (NC), FADD and Caspase-8 groups. The mRNA and protein expressions of FADD and Caspase-8 in tissues and human glioblastoma (GBM) cells were detected using qRT-PCR and Western blotting techniques. Cell proliferation was tested by CCK-8. Flow cytometry was used for the measure of cell cycle and apoptosis rates.The mRNA and protein expressions of FADD and Caspase-8 in GBM tissues were less than the levels of expression displayed in normal brain tissues. Correlations between the expressions of FADD and Caspase-8 in GBM tissues were analyzed as being linked with the clinical grades of GBM patients. Patients in stage III + IV displayed lower expressions of FADD and Caspase-8 than patients in stage I + II. In comparison with the blank group, the FADD and Caspase-8 groups showed decreased proliferation rates of SHG44 cells and lower ratios of cells in the S phase and Bcl-2 expression. Greater ratios of cells in the G0/G1 stage as well as increased cell apoptosis and expressions of Caspase-8 and Bax were exhibited. The expression of FADD in the FADD group was higher than the blank group, however no significant differences in FADD expression was observed between the blank and Caspase-8 groups.The data obtained during the study demonstrated that overexpression of FADD and Caspase-8 suppresses proliferation whilst promoting the apoptosis of human GBM cells.

583 Antiapoptotic effect of aminoguanidine on doxorubicin induced apoptosis

The study aimed at exploring the effects involved in Fas-Associated protein with Death Domain (FADD) expression and cysteine-aspartic acid specific protease-8 (Caspase-8) in relation to the proliferation and apoptosis of human glioblastoma (GBM) cells.93 GBM tissues and 64 normal brain tissues were the central mediums used for the investigation of the study. Cultured human GBM SC189 cells were divided into separate groups including the blank negative control (NC), FADD and Caspase-8 groups. The mRNA and protein expressions of FADD and Caspase-8 in tissues and human glioblastoma (GBM) cells were detected using qRT-PCR and Western blotting techniques. Cell proliferation was tested by CCK-8. Flow cytometry was used for the measure of cell cycle and apoptosis rates.The mRNA and protein expressions of FADD and Caspase-8 in GBM tissues were less than the levels of expression displayed in normal brain tissues. Correlations between the expressions of FADD and Caspase-8 in GBM tissues were analyzed as being linked with the clinical grades of GBM patients. Patients in stage III + IV displayed lower expressions of FADD and Caspase-8 than patients in stage I + II. In comparison with the blank group, the FADD and Caspase-8 groups showed decreased proliferation rates of SHG44 cells and lower ratios of cells in the S phase and Bcl-2 expression. Greater ratios of cells in the G0/G1 stage as well as increased cell apoptosis and expressions of Caspase-8 and Bax were exhibited. The expression of FADD in the FADD group was higher than the blank group, however no significant differences in FADD expression was observed between the blank and Caspase-8 groups.The data obtained during the study demonstrated that overexpression of FADD and Caspase-8 suppresses proliferation whilst promoting the apoptosis of human GBM cells.

Reduction of intersectin1-s induced apoptosis of human glioblastoma cells

Malignant gliomas have a high proliferation ability and high tendency to invade diffusely into surrounding healthy brain tissues, thereby precluding their successful surgical removal. Intersectin1 (also called ITSN1) as a molecular linker in the central nervous system is well known as an important regulator of endocytosis and exocytosis. ITSN1 has two isoforms: ITSN1-l and ITSN1-s. In this study, we show that siRNA-mediated down regulation of ITSN1-s induced glioma cells apoptosis. In addition, we demonstrate the possible mechanisms by which ITSN1-s functions in glioma cells apoptosis. Our data demonstrate that several key proteins, including FAK, Akt, Bcl-2, BAD which are critical for cells apoptosis were probably involved in ITSN1-s signaling pathways. Our results indicate that ITSN1-s is an effecter in regulation of gliomas cells apoptosis, and identify that ITSN1-s may be a new potentially anti-apoptosis target for therapeutic of gliomas.

Sorafenib Induces Growth Arrest and Apoptosis of Human Glioblastoma Cells through the Dephosphorylation of Signal Transducers and Activators of Transcription 3

Glioblastoma is the most common type of primary brain tumor and is rapidly progressive with few treatment options. Here, we report that sorafenib (< or =10 micromol/L) inhibited cell proliferation and induced apoptosis in two established cell lines (U87 and U251) and two primary cultures (PBT015 and PBT022) from human glioblastomas. The effects of sorafenib on these tumor cells were associated with inhibiting phosphorylated signal transducers and activators of transcription 3 (STAT3; Tyr705). Expression of a constitutively activated STAT3 mutant partially blocked the effects of sorafenib, consistent with a role for STAT3 inhibition in the response to sorafenib. Phosphorylated Janus-activated kinase (JAK)1 was inhibited in U87 and U251 cells, whereas phosphorylated JAK2 was inhibited in primary cultures. Sodium vanadate, a general inhibitor of protein tyrosine phosphatases, blocked the inhibition of phosphorylation of STAT3 (Tyr705) induced by sorafenib. These data indicate that the inhibition of STAT3 activity by sorafenib involves both the inhibition of upstream kinases (JAK1 and JAK2) of STAT3 and increased phosphatase activity. Phosphorylation of AKT was also reduced by sorafenib. In contrast, mitogen-activated protein kinases were not consistently inhibited by sorafenib in these cells. Two key cyclins (D and E) and the antiapoptotic protein Mcl-1 were downregulated by sorafenib in both cell lines and primary cultures. Our data suggest that inhibition of STAT3 signaling by sorafenib contributes to growth arrest and induction of apoptosis in glioblastoma cells. These findings provide a rationale for potential treatment of malignant gliomas with sorafenib. Mol Cancer Ther; 9(4); 953-62. (c)2010 AACR.

Differential role of cathepsins B and L in autophagy-associated cell death induced by arsenic trioxide in U87 human glioblastoma cells

Arsenic trioxide (arsenite) was the first chemotherapeutic drug to be described and is now being rediscovered in cancer treatment, including glioblastoma multiforme. Arsenite toxicity triggers autophagy in cancer cells, although final stages of the process involve executive caspases, suggesting an interplay between autophagic and apoptotic pathways that awaits to be explained at a molecular level. We evaluated the contribution of the lysosomal cathepsins (Cat) L and B, which are upregulated in glioblastomas, in the mechanism of arsenite toxicity in human glioblastoma cells. Arsenite treatment induced autophagosome formation and permeabilization of mitochondria, followed by caspase 3/7-mediated apoptosis. The autophagy inhibitor 3-methyladenine protected from arsenite toxicity, whereas bafilomycin A1 did not. Furthermore, arsenite significantly decreased CatB levels and selectively inhibited its cellular and recombinant protein activity, while not affecting CatL. However, downregulation of CatL greatly enhanced apoptosis by arsenite. Our results show that arsenite toxicity involves a complex interplay between autophagy and apoptosis in human glioblastoma cells and is associated with inhibition of CatB, and that this toxicity is highly exacerbated by simultaneous CatL inhibition. The latter points to a synergy that could be used in clinical treatment to lower the therapeutic dose, thus avoiding the toxic side effects of arsenite in glioblastoma management.

Flavonoids activated caspases for apoptosis in human glioblastoma T98G and U87MG cells but not in human normal astrocytes

Human glioblastoma is a deadly brain cancer that continues to defy all current therapeutic strategies. The authors induced apoptosis in human glioblastoma T98G and U87MG cells after treatment with apigenin, (-)-epigallocatechin, (-)-epigallocatechin-3-gallate (EGCG), and genistein, which did not induce apoptosis in human normal astrocytes. Induction of apoptosis was examined using Wright staining and ApopTag assay. Production of reactive oxygen species (ROS) and increase in intracellular free Ca(2+) were measured by fluorescent probes. Analysis of mRNA and Western blotting indicated increases in expression and activities of the stress kinases and cysteine proteases for apoptosis. JC-1 showed changes in mitochondrial membrane potential (DeltaPsi(m)), and use of specific inhibitors confirmed activation of kinases and proteases in apoptosis. Treatment of glioblastoma cells with apigenin, (-)-epigallocatechin, EGCG, or genistein triggered ROS production that induced apoptosis with phosphorylation of p38 mitogen-activated protein kinase (MAPK) and activation of the redox-sensitive c-Jun N-terminal kinase 1 pathway. Pretreatment of cells with ascorbic acid attenuated ROS production and p38 MAPK phosphorylation. Increases in intracellular free Ca2+ and activation of caspase-4 indicated involvement of endoplasmic reticulum stress in apoptosis. Other events in apoptosis included overexpression of Bax, loss of DeltaPsi(m), mitochondrial release of cytochrome c and Smac into the cytosol, down-regulation of baculoviral inhibitor-of-apoptosis repeat-containing proteins, and activation of calpain, caspase-9, and caspase-3. (-)-Epigallocatechin and EGCG also induced caspase-8 activity. Apigenin, (-)-epigallocatechin, EGCG, and genistein did not induce apoptosis in human normal astrocytes. Results strongly suggest that flavonoids are potential therapeutic agents for induction of apoptosis in human glioblastoma cells.

Combination of taxol and Bcl‐2 siRNA induces apoptosis in human glioblastoma cells and inhibits invasion, angiogenesis and tumour growth

Taxol is a powerful chemotherapeutic agent that binds to microtubules to prevent tumour cell division. However, a traditional high dose of taxol may also induce apoptosis in normal cells. The anti-apoptotic molecule Bcl-2 is up-regulated in tumour cells to prevent apoptosis. We designed this study to determine whether use of a low dose of taxol and anti-apoptotic Bcl-2 gene silencing would effectively induce apoptosis in human glioblastoma U251MG cells and also inhibit invasion, angiogenesis and intracranial as well as subcutaneous tumour growth. We treated the cells with either 100 nM taxol or transfected with a plasmid vector expressing Bcl-2 siRNA or both agents together for 72 h. Knockdown of Bcl-2 potentiated efficacy of taxol for cell death. Fluorescence-activated cell sorting analysis, double immunofluorescent staining and TUNEL assay demonstrated apoptosis in about 70% of the cells after treatment with the combination of taxol and Bcl-2 siRNA. In vitro Matrigel invasion assay demonstrated dramatic decrease in glioblastoma cell invasion and in vivo angiogenesis assay showed complete inhibition of neovascularization in athymic nude mice after treatment with the combination. Further, treatment with the combination of taxol and Bcl-2 siRNA caused suppression of intracranial tumour growth and subcutaneous solid tumour development. In conclusion, our results indicate that the combination of taxol and Bcl-2 siRNA effectively induces apoptosis and inhibits glioblastoma cell invasion, angiogenesis and intracranial as well as subcutaneous tumour growth. Therefore, the combination of a low dose of taxol and Bcl-2 siRNA is a promising therapeutic strategy for controlling the aggressive growth of human glioblastoma.

N-(4-Hydroxyphenyl) retinamide induced both differentiation and apoptosis in human glioblastoma T98G and U87MG cells

N-(4-Hydroxyphenyl) retinamide (4-HPR) is a synthetic retinoid that has shown biological activity against several malignant tumors and minimal side effects in humans. To explore the mechanisms underlying the chemotherapeutic effects of 4-HPR in glioblastoma, we used two human glioblastoma T98G and U87MG cell lines. In situ methylene blue staining showed the morphological features of astrocytic differentiation in glioblastoma cells following exposure to 1 μM and 2 μM 4-HPR for a short duration (24 h). Astrocytic differentiation was associated with an increase in expression of glial fibrillary acidic protein (GFAP) and downregulation of telomerase. Wright staining and ApopTag assay indicated appearance of apoptotic features in glioblastoma cells following exposure to 1 μM and 2 μM 4-HPR for a long duration (72 h). We found that 4-HPR caused apoptosis with activation of caspase-8 and cleavage of Bid to truncated Bid (tBid). Besides, apoptosis was associated with alterations in expression of pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins resulting in an increase in Bax:Bcl-2 ratio, mitochondrial release of cytochrome c and Smac , downregulation of selective baculoviral inhibitor-of-apoptosis repeat containing (BIRC) molecules, an increase in intracellular free [Ca 2+], and activation of calpain and caspase-3. Taken together, these results strongly suggested that 4-HPR could be used at low doses for induction of both differentiation and apoptosis in human glioblastoma cells.

Molecular Mechanisms of the Combination of Retinoid and Interferon-gamma for Inducing Differentiation and Increasing Apoptosis in Human Glioblastoma T98G and U87MG Cells

Glioblastoma is the deadliest brain tumor that remains incurable. We examined efficacy of combination of retinoid and interferon-gamma (IFN-gamma) in human glioblastoma T98G and U87MG cells. We conjectured that retinoid could induce differentiation with down regulation of telomerase activity to increase sensitivity to IFN-gamma for apoptosis in glioblastoma cells. Indeed, treatment of cells with 1 muM all-trans retinoic acid (ATRA) or 1 muM 13-cis retinoic acid (13-CRA) for 7 days induced astrocytic differentiation with upregulation of glial fibrillary acidic protein (GFAP) and down regulation of telomerase activity. Wright staining and ApopTag assay showed, respectively, morphological and biochemical features of apoptosis in glioblastoma cells following exposure to 200 units/ml IFN-gamma for 48 h. Induction of differentiation was associated with decreases in levels of nuclear factor kappa B (NFkappaB), inducible nitric oxide synthase (iNOS), and production of nitric oxide (NO) so as to increase sensitivity to IFN-gamma for apoptosis. Notably, IFN-gamma induced signal transducer and activator of transcription-1 (STAT-1) to bind to gamma-activated sequence (GAS) of the target gene. Also, IFN-gamma activated caspase-8 and cleaved Bid to truncated Bid (tBid) for translocation to mitochondria. Fura-2 assay showed increases in intracellular free [Ca2+] and activation of calpain in apoptotic cells. Besides, increases in Bax:Bcl-2 ratio and mitochondrial release of cytochrome c and Smac into the cytosol activated caspase-9 and caspase-3 for apoptosis. Taken together, our results showed that retinoid induced astrocytic differentiation with down regulation of telomerase activity and enhanced sensitivity to IFN-gamma for increasing apoptosis in human glioblastoma cells.

N-(4-Hydroxyphenyl)retinamide induced differentiation with repression of telomerase and cell cycle to increase interferon-γ sensitivity for apoptosis in human glioblastoma cells

Glioblastoma is the most malignant and prevalent brain tumor in humans. It is composed of heterogenic abnormal astroglial cells that avoid differentiation, maintain proliferation, and hardly commit apoptosis. N-(4-Hydroxyphenyl)retinamide (4-HPR) induced astrocytic differentiation and increased sensitivity to interferon-γ (IFN-γ) for apoptosis in human glioblastoma A172, LN18, and SNB19 cells. Combination of 4-HPR and IFN-γ significantly inhibited human telomerase reverse transcriptase (hTERT), cyclin dependent kinase 2 (CDK2), and survivin to up-regulate caspase-8, caspase-9, and caspase-3 for increasing apoptosis in all glioblastoma cell lines. Hence, combination of 4-HPR and IFN-γ should be considered for controlling growth of different human glioblastoma cells.

Viral protein synthesis is required for Enterovirus 71 to induce apoptosis in human glioblastoma cells.

Human glioblastoma cells (SF268) develop apoptosis, as characterized by DNA fragmentation and caspase activation, upon infection with Enterovirus 71 (EV71). To determine the step in virus replication that triggers apoptosis, the authors used ultraviolet (UV)-inactivated virus, inhibitors of protein and viral RNA synthesis, and chloroquine to block virus uncoating. Activation of caspase-3 was detected 24 h after infection with EV71 but not with UV-inactivated EV71. Apoptosis was inhibited when EV71-infected cells were treated with chloroquine, guanidine HCl, or cycloheximide. In summary, the authors studied the event(s) required to induce apoptosis in EV71-infected human glioblastoma cells, a subject much less studied than the possible role of viral proapoptotic genes, concluding that EV71 induces apoptosis in the infected SF268 cell in the presence of viral protein synthesis and virus replication, whereas virus adsorption, internalization, entry, uncoating, and viral RNA replication are all not required to trigger the apoptosis.

Ligands of the mitochondrial 18 kDa translocator protein attenuate apoptosis of human glioblastoma cells exposed to erucylphosphohomocholine.

Background: We have previously shown that the anti-neoplastic agent erucylphosphohomocholine (ErPC3) requires the mitochondrial 18 kDa Translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor (PBR), to induce cell death via the mitochondrial apoptosis pathway. Methods: With the aid of the dye JC-1 and cyclosporin A, applied to glioblastoma cells, we now investigated the significance of opening of the mitochondrial permeability transition pore (MPTP) for ErPC3-induced apoptosis in interaction with the TSPO ligands, PK 11195 and Ro5 4864. Furthermore, we measured cytochrome c release, and caspase-9 and -3 activation in this paradigm. Results: The human glioblastoma cell lines, U87MG, A172 and U118MG express the MPTP-associated TSPO, voltage-dependent anion channel and adenine nucleotide transporter. Indeed, ErPC3-induced apoptosis was inhibited by the MPTP blocker cyclosporin A and by PK 11195 and Ro5 4864 in a concentration-dependent manner. Furthermore, PK 11195 and Ro5 4864 inhibited collapse of the mitochondrial membrane potential, cytochrome c release, and caspase-9 and -3 activation caused by ErPC3 treatment. Conclusions: This study shows that PK 11195 and Ro5 4864 inhibit the pro-apoptotic function of ErPC3 by blocking its capacity to cause a collapse of the mitochondrial membrane potential. Thus, the TSPO may serve to open the MPTP in response to anti-cancer drugs such as ErPC3.

Combination of sublethal concentrations of epidermal growth factor receptor inhibitor and microtubule stabilizer induces apoptosis of glioblastoma cells

The oncogenic epidermal growth factor receptor (EGFR) pathway triggers downstream phosphatidylinositol 3-kinase (PI3K)/RAS-mediated signaling cascades. In transgenic mice, glioblastoma cannot develop on single but only on simultaneous activation of the EGFR signaling mediators RAS and AKT. However, complete blockade of EGFR activation does not result in apoptosis in human glioblastoma cells, suggesting additional cross-talk between downstream pathways. Based on these observations, we investigated combination therapies using protein kinase inhibitors against EGFR, platelet-derived growth factor receptor, and mammalian target of rapamycin, assessing glioblastoma cell survival. Clinically relevant doses of AEE788, Gleevec (imatinib), and RAD001 (everolimus), alone or in combinations, did not induce glioblastoma cell apoptosis. In contrast, simultaneous inactivation of the EGFR downstream targets mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase and PI3K by U0126 and wortmannin triggered rapid tumor cell death. Blocking EGFR with AEE788 in combination with sublethal concentrations of the microtubule stabilizer patupilone also induced apoptosis and reduced cell proliferation in glioblastoma cells, accompanied by reduced AKT and ERK activity. These data underline the critical role of the PI3K/AKT and the RAS/RAF/mitogen-activated protein/ERK kinase/ERK signaling cascades in the cell-intrinsic survival program of sensitive glioblastoma cell lines. We conclude that drug combinations, which down-regulate both ERK and protein kinase B/AKT activity, may prove effective in overcoming cell resistance in a subgroup of glioblastoma.

Dietary Isothiocyanate Iberin Inhibits Growth and Induces Apoptosis in Human Glioblastoma Cells

In this study, we evaluated the antiproliferative and proapoptotic effects of the isothiocyanate iberin, a bioactive agent in Brassicaceae species, in human glioblastoma cells. The human glioblastoma cell cultures were treated with different concentrations of iberin and tested for growth inhibition, cytotoxicity, induction of apoptosis, and activation of caspases. Iberin inhibited growth of tumor cells in cell proliferation assays, enhanced cytotoxicity, and induced apoptosis by activation of caspase-3 and caspase-9. Findings from this study could provide a basis for potential usefulness of the diet-derived isothiocyanate iberin as a promising therapeutic micronutrient in the prevention/intervention of brain tumors.

Apoptosis in human glioblastoma cells produced using embryonic stem cell–derived astrocytes expressing tumor necrosis factor–related apoptosis-inducing ligand

Embryonic stem (ES) cell-derived astrocytes have several theoretical and practical advantages as gene therapy vectors in the treatment of malignant gliomas. The aim of this study was to test the proapoptotic effects of ES cell-derived astrocytes expressing transgenic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in human malignant glioma cells. Mouse ES cells containing a doxycycline-inducible transgene were engineered with human TRAIL (hTRAIL) and then directed to differentiate into astrocytes. The ES cell-derived-TRAIL-expressing astrocytes were cocultured with human malignant glioma cells. Reverse transcriptase polymerase chain reaction, immunocytochemistry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling, and flow cytometry were used to quantify results. In vitro coculture of ES cell-derived astrocytes expressing hTRAIL with A172 human malignant glioma cells after doxycycline induction caused a significant decrease in cell viability from 85 +/- 2% at baseline to 8 +/- 2% posttreatment (p < 0.001). Labeling with apoptotic markers showed that cell death occurred by means of apoptosis. A significant increase in apoptotic rate (88 +/- 3%) from baseline (4 +/- 2%) was found in A172 cells after doxycycline induction (p < 0.005). This effect was superior to the apoptotic rate seen after treatment with recombinant TRAIL (57 +/- 2%). A decrease in cell viability and an increase in the apoptotic rate were not found in TRAIL-expressing-ES cell-derived astrocytes after induction with doxycycline or in A172 cells exposed to doxycycline alone. Engineering of transgenic hTRAIL by using ES cell-derived astrocytes induced apoptosis in human malignant glioma cells while sparing nontumor astrocytes. The apoptotic effects of transgenic hTRAIL are greater than those of recombinant hTRAIL. Analysis of these results suggests that hTRAIL-expressing-ES cell-derived astrocytes should be considered in the development of new in vivo strategies to treat malignant human gliomas.

Involvement of mitochondrial aggregation in arsenic trioxide (As2O3)‐induced apoptosis in human glioblastoma cells

Arsenic trioxide (As(2)O(3)) is effective against acute promyelocytic leukemia and has potential as a novel treatment against malignant solid tumors. As(2)O(3) induces differentiation and inhibits growth. It also causes mitochondrial damage mediated by the production of reactive oxygen species (ROS) and the dissipation of mitochondrial transmembrane potential (DeltaPsi(m)), leading to apoptosis. Mitochondria might be the key target of antitumor activity by As(2)O(3); however, its mechanisms have not been completely elucidated. Using two human glioblastoma cell lines, A172 and T98G, we found that As(2)O(3) induced apoptosis in A172 cells but not in T98G cells. As(2)O(3)-induced ROS production was observed in both cell lines; however, the dissipation of DeltaPsi(m), Bax oligomerization and caspase activation occurred only in As(2)O(3)-sensitive A172 cells. To determine the mechanisms of As(2)O(3)-induced apoptosis after ROS generation, we examined the change of mitochondrial morphology. As we reported previously, mitochondrial aggregation occurs before cytochrome c release during apoptosis, thus playing a role in cell death progression. We observed mitochondrial aggregation in As(2)O(3)-sensitive A172 cells but not in T98G cells treated with As(2)O(3). Using laser scanning cytometry, we quantitatively confirmed the results, which indicate that mitochondrial aggregation plays an important role in regulating sensitivity to As(2)O(3)-induced apoptosis. We propose a sequential process involving ROS generation, mitochondrial aggregation, Bax oligomerization and DeltaPsi(m) dissipation, and caspase activation during As(2)O(3)-induced apoptosis.