benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Glioma

Sirtuin-1 (Sirt-1), an NAD-dependent deacetylase, promotes tumorigenesis in glioma; however, whether the Sirt-1 specific inhibitor, EX527 exerts antitumor effects and the underlying mechanism in glioma requires further investigation. In the present study, the proliferative and colony formation abilities of two glioma cell lines (U87MG and LN-299) were inhibited by EX527. Treatment with EX527 increased the number of apoptotic cells (Annexin V-fluorescein isothiocyanate/propidium iodide); pretreatment with the caspase inhibitor Z-VAD-FMK suppressed EX527-induced apoptosis, suggesting that EX527 induced caspase-dependent apoptosis. In addition, western blotting revealed that EX527 treatment increased the expression of cleaved-caspase-3, poly (ADP-ribose) polymerase-1, B-cell lymphoma 2 (Bcl-2)-associated-X-protein and Bcl-2-like 11 but decreased that of Bcl-2. p53 is deacetylated by Sirt-1, attenuating its function. Furthermore, EX527 upregulated the expression of p53, acetylated p53 and the p53 target gene p21. This result suggests that EX527 induced cell apoptosis by activating p53 in glioma. Of note, EX527 exhibited antitumor effects on patient-derived glioma cells under three-dimensional culture conditions. Collectively, the results of the present study indicated that EX527 may be used as an effective compound in the treatment of glioma.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Carbazoles; Caspase 3; Caspase Inhibitors; Cell Growth Processes; Cell Line, Tumor; Cell Proliferation; Glioma; Humans; Neoplastic Stem Cells; Poly (ADP-Ribose) Polymerase-1; Signal Transduction; Sirtuin 1; Tumor Suppressor Protein p53; Up-Regulation

Pediatric high grade glioma is refractory to conventional multimodal treatment, highlighting a need to develop novel efficacious therapies. We investigated tumor metabolism as a potential therapeutic target in a panel of diverse pediatric glioma cell lines (SF188, KNS42, UW479 and RES186) using metformin and 2-deoxyglucose. As a single agent, metformin had little effect on cell viability overall. SF188 cells were highly sensitive to 2-deoxyglucose however, combination of metformin with 2-deoxyglucose significantly reduced cell proliferation compared to either drug alone in all cell lines tested. In addition, the combination of the two agents was associated with a rapid decrease in cellular ATP and subsequent AMPK activation. However, increased cell death was only observed in select cell lines after prolonged exposure to the drug combination and was caspase independent. Anti-apoptotic BCL-2 family proteins have been indicated as mediators of resistance against metabolic stress. Therefore we sought to determine whether pharmacological inhibition of BCL-2/BCL-xL with ABT-263 could potentiate apoptosis in response to these agents. We found that ABT-263 increased sensitivity to 2-deoxyglucose and promoted rapid and extensive cell death in response to the combination of 2-deoxyglucose and metformin. Furthermore, cell death was inhibited by the pan-caspase inhibitor, z-VAD-FMK suggesting that ABT-263 potentiated caspase-dependent cell death in response to 2-deoxyglucose or its combination with metformin. Overall, these data provide support for the concept that targeting metabolic and anti-apoptotic pathways may be an effective therapeutic strategy in pediatric glioma.

Topics: Adenosine Triphosphate; Amino Acid Chloromethyl Ketones; Aniline Compounds; Apoptosis; Blotting, Western; Caspases; Cell Line, Tumor; Cell Proliferation; Child; Deoxyglucose; Drug Synergism; Flow Cytometry; Glioma; Humans; Immunohistochemistry; Metformin; Statistics, Nonparametric; Sulfonamides

Malignant gliomas are the leading cause of morbidity and mortality in brain and central nervous system tumors. Recently, casticin has drawn wide attention to its critical role in tumor progression. However, the effect of casticin on glioma remains undefined.. Following treatment with casticin, cell viability, apoptosis, and cell cycle arrest were examined in U251 glioma cells. Additionally, the involved molecular mechanism was assessed by western blotting and flow cytometry.. Casticin triggered an obvious dose-dependent decrease in U251, U87 and U373 glioma cell viability, and the growth inhibitory effect of casticin was correlated with cell cycle arrest and cell apoptosis. Further mechanistic analysis indicated that casticin induced G2/M phase arrest by attenuating the polymerization of tubulin. Furthermore, striking apoptosis was also confirmed, accompanied by the up-regulation of caspase-3, p53 and proapoptotic protein Bax. These effects were absent when the caspase inhibitor z-VAD-fmk or p53 inhibitor PFTα were applied, suggesting that casticin could trigger cell apoptosis in a caspase-3 and p53-dependent manner.. These findings provide a prominent insight into how casticin abrogates the pathogenesis of glioma, and support its potential clinical prospect for further development of anti-brain cancer therapy.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; bcl-2-Associated X Protein; Benzothiazoles; Caspase 3; Cell Cycle Checkpoints; Cell Line, Tumor; Flavonoids; G2 Phase Cell Cycle Checkpoints; Glioma; Humans; M Phase Cell Cycle Checkpoints; Toluene; Tubulin; Tumor Suppressor Protein p53; Up-Regulation

Isoliquiritigenin (ISL), a member of the flavonoids, has been demonstrated to possess antitumor activity in various cancer cell lines in vitro and in vivo. In this study, we investigated the antitumor effects of ISL on U87 glioma cells in vitro. As determined by MTT assay, ISL inhibited the proliferation of U87 cells in a time-dependent and dose-dependent manner. The results of fluorescence-activated cell sorting (FACS) analysis suggested that ISL induced the apoptosis of the U87 cells and blocked cell cycle progression at the S and G2/M phases. Moreover, it was identified that ISL induced the apoptosis of the U87 cells in a caspase-dependent manner. Although treatment with the pan-caspase inhibitor Z-VAD-FMK efficiently blocked the ISL-induced caspase activation, it did not eliminate the ISL-induced cell death. Further examination using western blot analysis revealed that ISL upregulated p21/WAF1 and p27. These results indicate that cell cycle arrest and the caspase-mediated apoptosis pathway may participate in the antiproliferative activity of ISL in U87 cells by regulating the expression of specific molecules.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Caspases; Cell Cycle Checkpoints; Cell Line, Tumor; Chalcones; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Enzyme Activation; Glioma; Humans; Up-Regulation

Orexin A and orexin B (also known as hypocretins) are closely related peptides synthesized by hypothalamic neurons. They orchestrate diverse central and peripheral processes by stimulation of two G-protein coupled receptors, OX(1)R and OX(2)R. Recent studies have demonstrated the ability of orexins to promote a robust apoptosis in different cancer cells in culture and a potent growth reduction of human colon tumors in mice xenografts. Here we report effects of orexins on survival of rat C6 glioma cells, an experimental model for studies on glioblastoma multiforme (GBM). Quantitative real-time PCR demonstrated the expression of both types of orexin receptors in C6 cells. Orexin A and orexin B did not affect rat C6 glioma cell proliferation as assessed by [(3)H]thymidine incorporation assay. Incubation of the cells with orexin A (0.001-1 μM) resulted in a marked decrease of cell viability. The observed effect was caspase-dependent, as it was blocked by Z-VAD-fmk, a pan caspase inhibitor. In addition to that, a parallel increase in caspase-3 activity was observed. It is suggested that stimulation of orexin receptors induces death of rat C6 glioma cells through activation of caspase pathway.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase Inhibitors; Caspases; Cell Division; Cell Line, Tumor; Glioma; Intracellular Signaling Peptides and Proteins; Neoplasm Proteins; Neuropeptides; Orexin Receptors; Orexins; Rats; Receptors, G-Protein-Coupled; Receptors, Neuropeptide; RNA, Messenger; RNA, Neoplasm

Autophagy can promote cell survival or cell death, but the molecular basis underlying its dual role in cancer remains obscure. Here we demonstrate that delta(9)-tetrahydrocannabinol (THC), the main active component of marijuana, induces human glioma cell death through stimulation of autophagy. Our data indicate that THC induced ceramide accumulation and eukaryotic translation initiation factor 2alpha (eIF2alpha) phosphorylation and thereby activated an ER stress response that promoted autophagy via tribbles homolog 3-dependent (TRB3-dependent) inhibition of the Akt/mammalian target of rapamycin complex 1 (mTORC1) axis. We also showed that autophagy is upstream of apoptosis in cannabinoid-induced human and mouse cancer cell death and that activation of this pathway was necessary for the antitumor action of cannabinoids in vivo. These findings describe a mechanism by which THC can promote the autophagic death of human and mouse cancer cells and provide evidence that cannabinoid administration may be an effective therapeutic strategy for targeting human cancers.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Autophagy; Basic Helix-Loop-Helix Transcription Factors; Cannabinoids; Caspase 3; Cell Cycle Proteins; Cell Death; Cell Line, Transformed; Cell Line, Tumor; Dronabinol; Endoplasmic Reticulum; Enzyme Inhibitors; Eukaryotic Initiation Factor-2; Glioma; Humans; Mechanistic Target of Rapamycin Complex 1; Mice; Microtubule-Associated Proteins; Models, Biological; Multiprotein Complexes; Neoplasm Proteins; Phosphorylation; Protein Serine-Threonine Kinases; Proteins; Proto-Oncogene Proteins c-akt; Repressor Proteins; Ribosomal Protein S6 Kinases; TOR Serine-Threonine Kinases; Transcription Factors; Xenograft Model Antitumor Assays

Marine sponges have been prominently featured in the area of cancer research. Here, we examined the anti-proliferative effects of crude extracts (aqueous and organic) of the Brazilian marine sponge Polymastia janeirensis in the U138MG human glioma cell line. Moreover, we examined the effects of extracts on selective cytotoxicity in the glioma cells in comparison with a normal cell culture. Exposure of glioma cells to treatments (24 h) resulted in cell number decrease at all doses tested, with both aqueous and organic extracts (IC(50) <20 and <30 microg/ml, respectively). Parallel to this result, sponge extracts reduced glioma cell viability (IC(50) <15 microg/ml for both extracts). However, higher doses (50 and 100 microg/ml) induced a stronger cytotoxic effect when compared to the lower dose tested (10 microg/ml), inhibiting more than 80% of cellular growth and viability. Propidium iodide uptake and flow cytometry analysis further showed that sponge extracts caused necrosis in the glioma cell line at higher doses, while a high percentage of apoptotic glioma cells were observed at 10 microg/ml. Moreover, apoptosis was prevented by the pan-caspase inhibitor Z-VAD, suggesting that marine sponge extracts, at lower doses, induce caspase-dependent apoptosis in U138MG glioma cells. Surprisingly the extracts herein tested were more effective than temozolomide, a potent inductor of apoptosis used for the treatment of malignant gliomas. Furthermore, our results suggested a selectivity cytotoxic effect on glioma cell line in comparison with a normal cell culture, since the effect on viability found in glioma cells was not observed in astrocyte cultures with the lower dose (10 microg/ml). Thus, this marine sponge may be considered a good candidate for development of new cancer medicines with antitumor activity against gliomas.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Agents; Apoptosis; Astrocytes; Brazil; Cell Line, Transformed; Cell Line, Tumor; Complex Mixtures; Dacarbazine; Glioma; Humans; Porifera; Temozolomide

The novel protein kinase C-beta inhibitor enzastaurin (ENZA) induced apoptosis in LNT-229 and T98G cells whereas A172 cells were resistant. Further, ENZA reduced proliferation in glioblastoma-initiating cells T 269 and T 323 but did not induce apoptosis. ENZA-induced apoptosis involved cleavage of caspases 3, 8, and 9 and led to mitochondrial cytochrome c release and was strongly suppressed by the broad spectrum caspase inhibitor zVAD-fmk but only slightly by the expression of the viral caspase 1/8 inhibitor cytokine response modifier-A. ENZA did not reduce the phosphorylation of protein kinase B (Akt), but of p70 S6 kinase and of its substrate S6 protein in T98G cells. Inhibition of the phosphatidylinositol 3 kinase signaling pathway did not restore sensitivity of A172 cells towards ENZA, and constitutively active Akt did not protect LNT-229 and T98G cells from ENZA-induced apoptosis. Dephosphorylation of glycogen synthase kinase 3beta, a biomarker of ENZA action, and cell death induction by ENZA were separately regulated. Inhibition or activation of Akt only weakly modulated ENZA-induced dephosphorylation of glycogen synthase kinase 3beta. In ENZA-resistant A172 cells, apoptosis ligand 2 (Apo2L.0)-induced cleavage of caspases 3, 8, and 9 was increased by ENZA, resulting in synergistic activity of ENZA and Apo2L.0.

Topics: Adult; Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; bcl-X Protein; Brain Neoplasms; Caspases; Cell Line, Tumor; Cytochromes c; Drug Resistance, Neoplasm; Enzyme Inhibitors; Glioma; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Indoles; Mitochondria; Protein Kinase C; Proto-Oncogene Proteins c-akt; Receptors, TNF-Related Apoptosis-Inducing Ligand

20S-Protopanaxadiol (1) is an aglycon metabolic derivative of the protopanaxadiol-type ginseng saponins. In the present study, 1 was used to induce cytotoxicity for two human glioma cell lines, SF188 and U87MG. For the SF188 cells, 1 activated caspases-3, -8, -7, and -9 within 3 h and induced rapid apoptosis, which could be partially inhibited by a general caspase blocker and completely abolished when the caspase blocker was used in combination with an antioxidant. Compound 1 also induced cell death in U87MG cells but did not activate any caspases in these cells. Monodansylcadaverine staining showed that 1 induced dramatic autophagy in both cell lines. Elevated levels of superoxide anion in both cells and reduced levels of phosphorylated Akt in U87MG cells were also demonstrated. These results showed that 20S-protopanaxadiol (1) induces different forms of programmed cell death, including both typical apoptosis and autophagy through both caspase-dependent and -independent mechanisms.

Topics: Caspases; Cell Death; Glioma; Humans; Molecular Structure; Sapogenins; Triterpenes

The expression of inhibitor of apoptosis (IAP) family members contributes to the resistance of human cancers to apoptosis induced by radiotherapy and chemotherapy. We report that the infection of malignant glioma cells and several other tumor cell lines with adenoviruses encoding antisense RNA to X-linked IAP (XIAP) depletes endogenous XIAP levels and promotes global caspase activation and apoptosis. In contrast, non-neoplastic SV-FHAS human astrocytes and other non-neoplastic cells express XIAP at very low levels and resist these effects of adenovirus-expressing XIAP antisense RNA (Ad-XIAP-as). Caspase inhibitors such as z-Val-Ala-DL-Asp(OMe)-fluoromethylketone (zVAD-fmk) delay caspase processing and XIAP depletion, suggesting that XIAP depletion results both from antisense-mediated interference with protein synthesis and proteolytic cleavage by activated caspases. However, zVAD-fmk neither prevents nor delays cell death, indicating a caspase-independent pathway to cell death triggered by IAP depletion. Similarly, B-cell lymphoma-X(L) (BCL-X(L)) inhibits caspase activity, but fails to rescue from apoptosis. Loss of p65/nuclear factor-kappaB (NF-kappaB) protein and NF-kappaB activity is an early event triggered by Ad-XIAP-as and probably involved in Ad-XIAP-as-induced apoptosis. Finally, Ad-XIAP-as gene therapy induces cell death in intracranial glioma xenografts, prolongs survival in nude mice and may reduce tumorigenicity in synergy with Apo2L/TNF-related apoptosis-inducing ligand (TRAIL) in vivo. Altogether, these data define a powerful survival function for XIAP and reinforce its possible role as a therapeutic target in human glioma cells.

Topics: Adenoviridae; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase Inhibitors; Caspases; Enzyme Activation; Female; Gene Expression; Genetic Therapy; Genetic Vectors; Glioma; Humans; Immunohistochemistry; Mice; Mice, Nude; NF-kappa B; Proto-Oncogene Proteins c-bcl-2; RNA, Antisense; X-Linked Inhibitor of Apoptosis Protein; Xenograft Model Antitumor Assays

The present study reports for the first time a dual antiglioma effect of the well-known antidiabetic drug metformin. In low-density cultures of the C6 rat glioma cell line, metformin blocked the cell cycle progression in G(0)/G(1) phase without inducing significant cell death. In confluent C6 cultures, on the other hand, metformin caused massive induction of caspase-dependent apoptosis associated with c-Jun N-terminal kinase (JNK) activation, mitochondrial depolarization and oxidative stress. Metformin-triggered apoptosis was completely prevented by agents that block mitochondrial permeability transition (cyclosporin A) and oxygen radical production (N-acetylcisteine), while the inhibitors of JNK activation (SP600125) or glycolysis (sodium fluoride, iodoacetate) provided partial protection. The antiglioma effect of metformin was reduced by compound C, an inhibitor of AMP-activated protein kinase (AMPK), and was mimicked by the AMPK agonist AICAR. Similar effects were observed in the human glioma cell line U251, while rat primary astrocytes were completely resistant to the antiproliferative and proapoptotic action of metformin.

Topics: Amino Acid Chloromethyl Ketones; AMP-Activated Protein Kinases; Animals; Antineoplastic Agents; Apoptosis; Caspase Inhibitors; Caspases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Drug Synergism; Flow Cytometry; Glioma; Glycolysis; Humans; Hydrogen Peroxide; Hypoglycemic Agents; JNK Mitogen-Activated Protein Kinases; Membrane Potential, Mitochondrial; Metformin; Mitochondria; Multienzyme Complexes; Protein Serine-Threonine Kinases; Rats; Time Factors

Resveratrol (trans-3,4',5-trihydroxystilbene) is a naturally occurring polyphenolic compound highly enriched in grapes, peanuts, red wine, and a variety of food sources. Resveratrol has antiinflammatory and antioxidant properties, and also has potent anticancer properties. Human glioma U251 cells were used to understand the molecular mechanisms by which resveratrol acts as an anticancer agent, since glioma is a particularly difficult cancer to treat and eradicate. Our data show that resveratrol induces dose- and time-dependent death of U251 cells, as measured by lactate dehydrogenase release and internucleosomal DNA fragmentation assays. Resveratrol induces activation of caspase-3 and increases the cleavage of the downstream caspase substrate, poly(ADP-ribose) polymerase. Resveratrol-induced DNA fragmentation can be completely blocked by either a general caspase inhibitor (Z-VAD-FMK) or a selective caspase-3 inhibitor (Z-DEVD-FMK), but not by a selective caspase-1 inhibitor. Resveratrol induces cytochrome c release from mitochondria to the cytoplasm and activation of caspase-9. Resveratrol also increases expression of proapoptotic Bax and its translocation to the mitochondria. Resveratrol inhibits U251 proliferation, as measured by MTS assay [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt], and induces G0/G1 growth arrest, as determined by flow cytometry. The cyclin-dependent kinase inhibitor, olomoucine, prevents cell cycle progression and resveratrol-induced apoptosis. These results suggest that multiple signaling pathways may underlie the apoptotic death of U251 glioma induced by resveratrol, which warrants further exploration as an anticancer agent in human glioma.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspase 3; Caspase 9; Caspase Inhibitors; Caspases; Cell Cycle; Cell Line, Tumor; Cytochromes c; Cytoplasm; DNA Fragmentation; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Glioma; Humans; Kinetin; L-Lactate Dehydrogenase; Phenols; Poly(ADP-ribose) Polymerases; Polyphenols; Proto-Oncogene Proteins c-bcl-2; Purines; Resveratrol; Signal Transduction; Stilbenes; Subcellular Fractions; Time Factors; Up-Regulation

CD40, a TNF-R-related cell surface receptor, is shown here to be expressed by glioma cells in vitro and in vivo. Glioma cell lines expressing low levels of CD40 at the cell surface resist cytotoxic effects of CD40L. CD40 gene transfer sensitizes glioma cells to CD40L. Inhibition of protein synthesis potentiates cell death which involves CD40 clustering and caspases 8 and 3 processing. CD40-transfected LN-18 cells acquire resistance to CD95L. In contrast, subtoxic concentrations of CD40L strongly sensitize these cells for TNF-alpha-induced apoptosis. Bispecific CD40xCD95 antibodies specifically kill glioma cells, disclosing the property of endogenous CD40 to facilitate death signalling.

Topics: Amino Acid Chloromethyl Ketones; Antibodies; Antineoplastic Agents; Apoptosis; Blotting, Northern; Blotting, Western; Caspases; CD40 Antigens; CD40 Ligand; Cell Line, Tumor; Cell Survival; Collagen Type XI; Dose-Response Relationship, Drug; Drug Interactions; Fas Ligand Protein; fas Receptor; Gene Expression Regulation; Glioma; Humans; Immunohistochemistry; Immunoprecipitation; Leupeptins; Membrane Glycoproteins; Neuroprotective Agents; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Messenger; Time Factors; Transfection; Tumor Necrosis Factor-alpha

Taurolidine, a derivative of the amino acid taurin, was recently found to display a potent antineoplastic effect both in vitro and in vivo. The authors therefore initiated studies to assess the potential antineoplastic activity of taurolidine in human glioma cell lines and in ex vivo malignant cell cultures. They also studied the mechanisms that induce cell death and the impact of taurolidine on tumor-derived vascular endothelial growth factor (VEGF) production.. Cytotoxicity and clonogenic assays were performed using crystal violet staining. In the cytotoxicity assay 100% of glioma cell lines (eight of eight) and 74% of ex vivo glioma cultures (14 of 19) demonstrated sensitivity to taurolidine, with a mean median effective concentration (EC50) of 51 +/- 28 microg/ml and 56 +/- 23 microg/ml, respectively. Colony formation was inhibited by taurolidine, with a mean EC50 of 7 +/- 3 microg/ml for the cell lines and a mean EC50 of 3.5 +/- 1.7 microg/ml for the ex vivo glioma cultures. On observing this high activity of taurolidine in both assays, the authors decided to evaluate its cell death mechanisms. Fragmentation of DNA, externalization of phosphatidylserine, activation of poly(adenosine diphosphate-ribose) polymerase, loss of the mitochondrial membrane potential followed by a release of apoptosis-inducing factor, and typical apoptotic features were found after taurolidine treatment. Cell death was preceded by the generation of reactive O2 intermediates, which was abrogated by N-acetylcysteine but not by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Moreover, taurolidine also induced suppression of VEGF production on the protein and messenger RNA level, as shown by an enzyme-linked immunosorbent assay and by reverse transcription-polymerase chain reaction.. Given all these findings, taurolidine may be a promising new agent in the treatment of malignant gliomas; it displays a combination of antineoplastic and antiangiogenic activities, inducing tumor cell apoptosis and inhibiting tumor-derived VEGF production.

Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Brain Neoplasms; Caspase Inhibitors; Cell Line, Tumor; Collagen Type XI; Cysteine Proteinase Inhibitors; Fas Ligand Protein; Gene Expression; Glioma; Humans; Membrane Glycoproteins; Mitochondria; Phosphatidylserines; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species; Taurine; Thiadiazines; Vascular Endothelial Growth Factor A

Adenoviral replacement of the p53 gene has already been proved effective for the treatment of various tumours, including malignant gliomas. However, it is difficult to treat malignant glioma with p53 gene therapy alone because of problems with resistance or a less-than-satisfactory response to the treatment. This study investigated whether heat shock at 43 degrees C (mild hyperthermia) augments the cytotoxic effect of p53 gene transfer on malignant glioma cells expressing wild-type p53 (D54) or mutant p53 (U373-MG and U251-MG). The combination of mild hyperthermia and adenoviral p53 over-expression had an additive inhibitory effect on cellular proliferation in all three cell lines studied. Further, both cell cycle analysis and a DNA fragmentation assay showed that apoptosis was induced by p53 over-expression alone but not by heat shock at 43 degrees C alone. However, p53 over-expression followed by mild hyperthermia additively increased the proportion of cells in which apoptosis was induced, regardless of the endogenous p53 status of the tumour cells. Interestingly, a caspase-independent mechanism was observed to be involved in the p53-induced apoptosis in U251-MG and D54 cells. Taken together, the findings showed that combining adenoviral p53 transfer with mild hyperthermia inhibits the proliferation of malignant glioma cells in an additive manner, irrespective of their endogenous p53 status, suggesting a novel treatment strategy for this malignancy.

Topics: Adenoviridae; Amino Acid Chloromethyl Ketones; Apoptosis; Caspase Inhibitors; Cell Survival; Combined Modality Therapy; Cyclin-Dependent Kinase Inhibitor p21; Genes, p53; Genetic Therapy; Glioma; Humans; Hyperthermia, Induced; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The nervous system is frequently the site of symptomatic toxicity of antineoplastic agents. However, there is limited information about the differential vulnerability of neurons, astrocytes and glioma cells. We have analyzed the effects of four chemotherapeutic drugs (lomustine, cisplatin, topotecan and vincristine) on primary cerebellar granule neurons and astrocytes derived from rats. All drugs led to cell death in cerebellar granule neurons in a concentration-dependent manner. Comparison of the EC50 values for cerebellar neurons and astrocytes with the median EC50 values of 12 malignant glioma cell lines demonstrated a large therapeutic range for lomustin and cisplatin. Further, this comparison revealed a 100-fold higher sensitivity of cerebellar neurons towards vincristine and 10-fold higher sensitivity towards topotecan compared with glioma cells. Astrocytes were generally resistant to vincristine. In cerebellar granule neurons, vincristine and to a lesser extent topotecan induced caspase 3 and caspase 9 cleavage, and enhanced caspase activity and Akt-dependent expression of phosphorylated BAD. zVAD-fmk, a caspase inhibitor and brain-derived neurotrophic factor (BDNF), but not MK-801, a non-competitive NMDA receptor antagonist, significantly reduced vincristine- or topotecan-induced cell death.

Topics: Amino Acid Chloromethyl Ketones; Analysis of Variance; Animals; Animals, Newborn; Antineoplastic Agents; Astrocytes; ATP Binding Cassette Transporter, Subfamily B, Member 1; bcl-Associated Death Protein; bcl-X Protein; Blotting, Western; Brain-Derived Neurotrophic Factor; Carrier Proteins; Caspase 3; Caspase 9; Caspases; Cell Death; Cell Size; Cell Survival; Cells, Cultured; Cerebellum; Chemokines, CC; Dose-Response Relationship, Drug; Drug Interactions; Gene Expression Regulation; Glioma; Necrosis; Neurons; Oligodeoxyribonucleotides, Antisense; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transfection

Recent clinical data shows that arsenic trioxide (As(2)O(3)) causes remission in patients with acute promyelocytic leukemia and multiple myeloma without severe side effects. Laboratory data suggest that As(2)O(3) induces apoptosis or cell differentiation of hematopoietic or solid tumor cells. To date, there has been no study on the effects of As(2)O(3) on glioma cells. In this study, we investigated the in vitro effect of As(2)O(3) on cell growth inhibition and cell death mechanisms in human glioma cells. As(2)O(3) significantly inhibited the proliferation of all six of the glioma cell lines (U373, U87, U251, GB1, A-172, and T98G) tested in this study in a dose-dependent manner. The IC(50) of As(2)O(3) for all of the tumor cell lines was <2 micro M. Previous studies have shown that this is a clinically safe concentration. Treatment with 2 micro M As(2)O(3) induced G(2)/M arrest in all of the glioma cell lines. Autophagy (programmed cell death type II), but not apoptosis (programmed cell death type I), was detected by electron microscopy in U-373-MG cells treated with 2 micro M As(2)O(3). Caspase inhibitors did not halt As(2)O(3)-induced cell death. Furthermore, combination of As(2)O(3) with bafilomycin A1 autophagy inhibitor enhanced the antitumor effect of As(2)O(3) through induction of apoptosis. These findings suggest that As(2)O(3) at a clinically safe concentration may be an effective chemotherapeutic agent for malignant gliomas.

Topics: Amino Acid Chloromethyl Ketones; Anti-Bacterial Agents; Antineoplastic Agents; Apoptosis; Arsenic Trioxide; Arsenicals; Autophagy; Caspase Inhibitors; Cell Division; Cytoplasm; Drug Interactions; Enzyme Inhibitors; G2 Phase; Glioma; Humans; Macrolides; Mitosis; Oxides; Tumor Cells, Cultured

Erucylphosphocholine (ErPC) is a promising anti-neoplastic drug for the treatment of malignant brain tumours. It exerts strong anti-cancer activity in vivo and in vitro and induces apoptosis even in chemoresistant glioma cell lines. The purpose of this study was to expand on our previous observations on the potential mechanisms of ErPC-mediated apoptosis with a focus on death receptor activation and the caspase network. A172 and T98G glioma cells were treated with ErPC for up to 48 h. ErPC effects on the expression of the tumour necrosis factor (TNF) and TNF-related apoptosis-inducing ligand (TRAIL) receptor system, and on caspase activation were determined. ErPC had no effect on the expression of TNFalpha or TRAIL. Inhibition of the TNF or TRAIL signalling pathway with antagonistic antibodies or fusion proteins did not affect apoptosis induced by ErPC, and a dominant-negative FADD construct did not abolish ErPC-induced effects. Western blot analysis indicated that ErPC-triggered apoptosis resulted in a time-dependent processing of caspases-3, -7, -8 and -9 into their respective active subunits. Co-treatment of A172 cells with different caspase inhibitors prevented apoptosis but did not abrogate cell death. These data suggest that A172 cells might have an additional caspase-independent pathway that insures cell death and guarantees killing of those tumour cells whose caspase pathway is incomplete.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspases; Cell Death; Enzyme Activation; Enzyme Inhibitors; Glioma; HeLa Cells; Humans; Ligands; Phosphorylcholine; Receptors, Tumor Necrosis Factor; Signal Transduction; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

A growing body of evidence now suggests that programmed cell death (PCD) occurs via non-apoptotic mechanisms as well as by apoptosis. In contrast to apoptosis, however, the molecular mechanisms involved in the regulation of non-apoptotic PCD remain only poorly understood. Here we show that ceramide induces a non-apoptotic PCD with a necrotic-like morphology in human glioma cells. Characteristically, the cell death was not accompanied by loss of the mitochondrial transmembrane potential, cytosolic release of cytochrome c from mitochondria, or the activation of the caspase cascade. Consistent with these characteristics, this ceramide-induced cell death was inhibited neither by the overexpression of Bcl-xL nor by the pan-caspase inhibitor zVAD-fmk. However, strikingly, the ceramide-induced non-apoptotic cell death was inhibited by the activation of the Akt/protein kinase B pathway through the expression of a constitutively active version of Akt. The results for the first time indicate that the Akt kinase, known to play an essential role in survival factor-mediated inhibition of apoptotic cell death, is also involved in the regulation of non-apoptotic PCD.

Topics: Adenoviridae; Amino Acid Chloromethyl Ketones; Apoptosis; bcl-X Protein; Caspases; Cell Death; Cell Membrane; Cell Survival; Ceramides; Cysteine Proteinase Inhibitors; Cytochrome c Group; Cytosol; Enzyme Inhibitors; Glioma; Humans; Immunoblotting; Membrane Potentials; Microscopy, Electron; Mitochondria; Protein Binding; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tumor Cells, Cultured

Chemotherapeutic drug-induced apoptosis of human malignant glioma cells involves the death receptor-independent activation of caspases other than caspases 3 or 8 (Glaser et al., Oncogene 18, 5044-5053, 1999). Here, we report that caspases 1, 2, 3, 7, 8, and 9 are constitutively expressed in most human malignant glioma cell lines. Cytotoxic drug-induced apoptosisinvolves delayed activation of caspases 2, 7, and 9, but not 8 and 3, and is blocked by a broad spectrum caspase inhibitor, zVAD-fmk. Cytochrome c release from mitochondria precedes caspase activation during drug-induced apoptosis and is unaffected by zVAD-fmk or ectopic expression of the viral caspase inhibitor, crm-A. In contrast, ectopic expression of BCL-X(L) prevents drug-induced cytochrome c release, caspase activation and cell death. Thus, cancer chemotherapy targets the mitochondrial, caspase-dependent death pathway in human malignant glioma cells.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Agents; Apoptosis; bcl-X Protein; Caspase 1; Caspase 2; Caspase 3; Caspase 7; Caspase 8; Caspase 9; Caspases; Cytochrome c Group; DNA, Recombinant; Glioma; Humans; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Serpins; Teniposide; Transfection; Tumor Cells, Cultured; Vincristine; Viral Proteins

We investigated the role of radiation-induced mitogen activated protein kinase (MAPK) pathway activity in the regulation of proliferation, cell survival and vascular endothelial growth factor (VEGF) production in primary astrocytes and in T9 and RT2 glioblastoma cells derived from Fisher 344 rats. In these cells, ionizing radiation (2 Gy) caused activation of the MAPK pathway which was blocked by specific inhibitor drugs. Blunting of radiation-induced MAPK activity weakly enhanced radiation-induced apoptosis 24 h after exposure in RT2 cells. Furthermore, blunting of MAPK activation weakly enhanced the ability of radiation to reduce RT2 cell growth in clonogenic growth assays. These findings argue that inhibition of MAPK signaling reduces proliferation and enhances cell killing by ionizing radiation in transformed astrocytes. Proliferation and survival of cancer cells has been linked in vivo to enhanced expression of angiogenic growth factors. Recently we demonstrated that the gene product of a novel rodent radiation-responsive gene, progression elevated gene 3 (PEG-3), could enhance vascular endothelial growth factor (VEGF) promoter activity in rodent fibroblasts, leading to increased VEGF protein levels and tumorigenic behavior in vivo. Thus PEG-3 and VEGF expression could be expected to directly correlate with the oncogenic potential of transformed cells. RT2 cells expressed more PEG-3 and VEGF protein than T9 cells, and were more tumorigenic in vivo than T9 cells. Radiation activated the PEG-3 promoter via MAPK signaling and ectopic over-expression of PEG-3 enhanced both basal MAPK activity and basal VEGF promoter activity. Basal MAPK activity partially correlated with basal VEGF promoter activity and VEGF protein levels in primary astrocytes, T9 and RT2 cells. Radiation increased the activity of the VEGF promoter and VEGF protein levels in primary astrocytes, T9 and RT2 cells which were dependent upon MAPK function. Furthermore, inhibition of AP-1 transcription factor signaling by dominant negative c-Jun (TAM67) also significantly reduced basal, and to a lesser extent radiation-induced, VEGF promoter function in RT2 cells. Collectively, our data demonstrate that radiation-induced MAPK signaling can both protect cells from radiation-induced cell death as well as enhance protein levels of pro-angiogenic factors such as VEGF. Enhanced VEGF expression in RT2 cells may be mediated via MAPK and JNK pathway signaling which converges upon the AP-1 transcripti

Topics: Amino Acid Chloromethyl Ketones; Animals; Antigens, Differentiation; Apoptosis; Astrocytes; Cell Survival; Clone Cells; Endothelial Growth Factors; Flavonoids; Glioblastoma; Glioma; Lymphokines; Mitogen-Activated Protein Kinases; Neoplasm Proteins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-jun; Radiation, Ionizing; Rats; Rats, Inbred F344; Signal Transduction; Transcription Factor AP-1; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors