trichostatin-a and Glioblastoma

Epigenetic mechanisms are increasingly recognized as a major factor contributing to pathogenesis of cancer including glioblastoma, the most common and most malignant primary brain tumour in adults. Enzymatic modifications of histone proteins regulating gene expression are being exploited for therapeutic drug targeting. Over the last decade, numerous studies have shown promising results with histone deacetylase (HDAC) inhibitors in various malignancies. This article provides a brief overview of mechanism of anti-cancer effect and pharmacology of HDAC inhibitors and summarizes results from pre-clinical and clinical studies in glioblastoma. It analyses experience with HDAC inhibitors as single agents as well as in combination with targeted agents, cytotoxic chemotherapy and radiotherapy. Hallmark features of glioblastoma, such as uncontrolled cellular proliferation, invasion, angiogenesis and resistance to apoptosis, have been shown to be targeted by HDAC inhibitors in experiments with glioblastoma cell lines. Vorinostat is the most advanced HDAC inhibitor that entered clinical trials in glioblastoma, showing activity in recurrent disease. Multiple phase II trials with vorinostat in combination with targeted agents, temozolomide and radiotherapy are currently recruiting. While the results from pre-clinical studies are encouraging, early clinical trials showed only modest benefit and the value of HDAC inhibitors for clinical practice will need to be confirmed in larger prospective trials. Further research in epigenetic mechanisms driving glioblastoma pathogenesis and identification of molecular subtypes of glioblastoma is needed. This will hopefully lead to better selection of patients who will benefit from treatment with HDAC inhibitors.

Topics: Animals; Brain Neoplasms; Clinical Trials as Topic; Depsipeptides; Epigenesis, Genetic; Glioblastoma; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Valproic Acid; Vorinostat

Glioblastoma is the most aggressive and fatal form of brain cancer. Despite new advancements in treatment, the desired outcomes have not been achieved. Temozolomide (TMZ) is the first-choice treatment for the last two decades and has improved survival rates. Emerging studies have shown that targeting epigenetics in glioblastoma can be beneficial when combined with clinically used treatments. Trichostatin A (TSA), a histone deacetylase inhibitor, has anti-cancer properties in various cancers. No data concerning the TMZ and TSA relationship was shown previously in glioblastoma therefore, we aimed to determine the likely therapeutic effect of the TMZ and TSA combination in glioblastoma. The T98G and U-373 MG, glioblastoma cell lines, were used in this study. TMZ and TSA cytotoxicity and combination index were performed by MTT assay. The expression of DNA repair genes (MGMT, MLH-1, PMS2, MSH2 and MSH6) was detected using RT-PCR. One-way analysis of variance (ANOVA) was used for statistical analysis. Combination index calculations revealed antagonistic effects of TMZ and TSA in terms of cytotoxicity. Antagonistic effects were more apparent in the T98G cell line, which is expressing MGMT relatively higher. MGMT and DNA Mismatch Repair (MMR) genes were upregulated in the T98G cell line, whereas downregulated in the U373-MG cell lines under TMZ and TSA combination treatment. It is concluded that MGMT might be playing a more active part than MMR genes in TMZ resistance to TMZ and TSA antagonism. This is the first study elucidating the TMZ and TSA relationship in cancer cell lines.

Topics: Antineoplastic Agents, Alkylating; Brain Neoplasms; Cell Line, Tumor; Dacarbazine; DNA Mismatch Repair; DNA Modification Methylases; DNA Repair Enzymes; Drug Resistance, Neoplasm; Glioblastoma; Humans; Temozolomide; Tumor Suppressor Proteins

Hurdled and marred by the notorious nature of glioblastomas (GBM) in terms of resistance to therapy and limited drug delivery into the brain, the anti-GBM drug pipeline is required to be loaded with mechanistically diverse agents. The consideration of HDAC inhibition as a prudent approach to circumvent the resistance issue in GBM spurred us to pragmatically design and synthesizes hydroxamic acids endowed with CNS penetrating ability. By virtue of the blood brain barrier permeability (BBB), memantine was envisioned as an appropriate CAP component for the construction of the HDAC inhibitors. Diverse linkers were stapled for the tetheration of the zinc binding motif with the CAP group to pinpoint an appropriate combination (CAP and linker) that could confer inhibitory preference to HDAC6 isoform (overexpressed in GBM). Resultantly, hydroxamic acid 16 was identified as a promising compound that elicited striking antiproliferative effects against Human U87MG GBM cells as well as TMZ-resistant GBM cells and P1S cells, a concurrent chemo radiotherapy (CCRT)-resistant/patient-derived glioma cell line mediated through preferential HDAC6 inhibition (IC

Topics: Animals; Antineoplastic Agents; Blood-Brain Barrier; Brain Neoplasms; Cell Proliferation; Dose-Response Relationship, Drug; Drug Design; Drug Screening Assays, Antitumor; Glioblastoma; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Humans; Male; Memantine; Mice; Mice, Inbred BALB C; Mice, Nude; Molecular Docking Simulation; Molecular Structure; Neoplasms, Experimental; Structure-Activity Relationship; Tumor Cells, Cultured

Glioblastoma multiforme (GBM) is a poor prognosis type of tumour due to its resistance to chemo and radiotherapy. SOCS1 and SOCS3 have been associated with tumour progression and response to treatments in different kinds of cancers, including GBM. In this study, cell lines of IDH-wildtype GBM from primary cultures were obtained, and the role of SOCS1 and SOCS3 in the radiotherapy response was analysed. Fifty-two brain aspirates from GBM patients were processed, and six new cell lines of IDH-wildtype GBM were established. These new cell lines were characterized according to the WHO classification of CNS tumours. SOCS1 and SOCS3 expression levels were determined, at mRNA level by Q-PCR, at protein level by immunocytochemistry, and Western blot analysis. The results showed that SOCS1 and SOCS3 are overexpressed in GBM, as compared to a non-tumoral brain RNA pool. SOCS1 and SOCS3 expression were reduced by siRNA, and it was found that SOCS3 inhibition increases radioresistance in GBM cell lines, suggesting a key role of SOCS3 in radioresistant acquisition. In addition, radioresistant clonal populations obtained by selective pressure on these cell cultures also showed a significant decrease in SOCS3 expression, while SOCS1 remained unchanged. Furthermore, the induction of SOCS3 expression, under a heterologous promoter, in a radiotherapy resistant GBM cell line increased its radiosensitivity, supporting an important implication of SOCS3 in radiotherapy resistance acquisition. Finally, the treatment with TSA in the most radioresistant established cell line produced an increase in the effect of radiotherapy, that correlated with an increase in the expression of SOCS3. These effects of TSA disappeared if the increase in the expression of SOCS3 prevented with an siRNA against SOCS3. Thus, SOCS3 signal transduction pathway (JAK/STAT) could be useful to unmask new putative targets to improve radiotherapy response in GBM.

Topics: Adult; Brain; Brain Neoplasms; Gene Expression Regulation, Neoplastic; Glioblastoma; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Janus Kinases; Primary Cell Culture; Radiation Tolerance; RNA, Small Interfering; Signal Transduction; STAT Transcription Factors; Suppressor of Cytokine Signaling 1 Protein; Suppressor of Cytokine Signaling 3 Protein; Tumor Cells, Cultured; Up-Regulation; Young Adult

Glioblastoma (GBM) ranks among the deadliest solid cancers worldwide and its prognosis has remained dismal, despite the use of aggressive chemo-irradiation treatment regimens. Limited drug delivery into the brain parenchyma and frequent resistance to currently available therapies are problems that call for a prompt development of novel therapeutic strategies. While only displaying modest efficacies as mono-therapy in pre-clinical settings, histone deacetylase inhibitors (HDACi) have shown promising sensitizing effects to a number of cytotoxic agents. Here, we sought to investigate the sensitizing effect of the HDACi trichostatin A (TSA) to the alkylating agent lomustine (CCNU), which is used in the clinic for the treatment of GBM.. Twelve primary GBM cell cultures grown as neurospheres were used in this study, as well as one established GBM-derived cell line (U87 MG). Histone deacetylase (HDAC) expression levels were determined using quantitative real-time PCR and Western blotting. The efficacy of either CCNU alone or its combination with TSA was assessed using various assays, i.e., cell viability assays (MTT), cell cycle assays (flow cytometry, FACS), double-strand DNA break (DSB) quantification assays (microscopy/immunofluorescence) and expression profiling assays of proteins involved in apoptosis and cell stress (Western blotting and protein array).. We found that the HDAC1, 3 and 6 expression levels were significantly increased in GBM samples compared to non-neoplastic brain control samples. Additionally, we found that pre-treatment of GBM cells with TSA resulted in an enhancement of their sensitivity to CCNU, possibly via the accumulation of DSBs, decreased cell proliferation and viability rates, and an increased apoptotic rate.. From our data we conclude that the combined administration of TSA and CCNU eradicates GBM cells with a higher efficacy than either drug alone, thereby opening a novel avenue for the treatment of GBM.

Topics: Antineoplastic Agents, Alkylating; Antineoplastic Combined Chemotherapy Protocols; Blotting, Western; Brain Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Survival; Flow Cytometry; Fluorescent Antibody Technique; Glioblastoma; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lomustine; Real-Time Polymerase Chain Reaction

There have been extensive efforts to improve the outcome of glioblastoma, but the prognosis of this disease has not been significantly altered to date. Histone deacetylase inhibitors (HDACIs) have been evaluated as promising anti-cancer drugs and regulate cell growth, cell cycle arrest and apoptosis in glioblastoma. Here, we demonstrated the therapeutic efficacy of a novel pan-HDACI, 7-ureido-N-hydroxyheptanamide derivative (CKD5), compared with traditional pan-HDACIs, such as suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA), in vitro and in vivo. Compared with SAHA and TSA, CKD5 had improved cytotoxic effects and induced apoptosis, anti-proliferative activity and cell cycle arrest at G2/M phase. Furthermore, CKD5 significantly reduced tumor volume and prolonged the survival in vivo compared with TSA, suggesting improved anti-cancer efficacy among HDACIs. Our results demonstrate that the novel HDACI CKD5 is a promising therapeutic candidate for glioblastoma.

Topics: Animals; Apoptosis; Brain Neoplasms; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Female; G2 Phase Cell Cycle Checkpoints; Glioblastoma; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Mice, Inbred BALB C; Mice, Nude; Time Factors; Tumor Burden; Urea; Vorinostat; Xenograft Model Antitumor Assays

Glioblastoma multiforme (GBM) is the most malignant brain tumor with limited effective treatment options. Cancer stem cells (CSCs), a subpopulation of cancer cells with stem cell properties found in GBMs, have been shown to be extremely resistant to radiation and chemotherapeutic agents and have the ability to readily reform tumors. Therefore, the development of therapeutic agents targeting CSCs is extremely important. In this study, we isolated glioblastoma-derived stem cells (GDSCs) from GBM tissue removed from patients during surgery and analyzed their gene expression using quantitative real-time PCR and immunocytochemistry. We examined the effects of histone deacetylase inhibitors trichostatin A (TSA) and valproic acid (VPA) on the proliferation and gene expression profiles of GDSCs. The GDSCs expressed significantly higher levels of both neural and embryonic stem cell markers compared to GBM cells expanded in conventional monolayer cultures. Treatment of GDSCs with histone deacetylase inhibitors, TSA and VPA, significantly reduced proliferation rates of the cells and expression of the stem cell markers, indicating differentiation of the cells. Since differentiation into GBM makes them susceptible to the conventional cancer treatments, we posit that use of histone deacetylase inhibitors may increase efficacy of the conventional cancer treatments for eliminating GDSCs.

Topics: Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Glioblastoma; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Neoplastic Stem Cells; Neural Stem Cells; Neurogenesis; Valproic Acid

Epigenetic alterations have been increasingly implicated in glioblastoma (GBM) pathogenesis, and epigenetic modulators including histone deacetylase inhibitors (HDACis) have been investigated as candidate therapies. GBMs are proposed to contain a subpopulation of glioblastoma stem cells (GSCs) that sustain tumor progression and therapeutic resistance and can form tumorspheres in culture. Here, we investigate the effects of the HDACi trichostatin A (TSA) in U87 GBM cultures and tumorsphere-derived cells. Using approaches that include a novel method to measure tumorsphere sizes and the area covered by spheres in GBM cultures, as well as a nuclear morphometric analysis, we show that TSA reduced proliferation and colony sizes, led to G2/M arrest, induced alterations in nuclear morphology consistent with cell senescence, and increased the protein content of GFAP, but did not affect migration, in cultured human U87 GBM cells. In cells expanded in tumorsphere assays, TSA reduced sphere formation and induced neuron-like morphological changes. The expression of stemness markers in these cells was detected by reverse transcriptase polymerase chain reaction. These findings indicate that HDACis can inhibit proliferation, survival, and tumorsphere formation, and promote differentiation of U87 GBM cells, providing further evidence for the development of HDACis as potential therapeutics against GBM.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Cellular Senescence; Glioblastoma; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Neoplastic Stem Cells; Spheroids, Cellular; Tumor Cells, Cultured

Histone deacetylase inhibitors (HDACi) have been described as multifunctional anticancer agents. The failure of conventional therapy for glioblastoma (GBM) renders this tumor an attractive target for immunotherapy. Innate immune cells, such as natural killer (NK) cells, play a crucial role in antitumor immune responses. Here, we describe how the HDACi trichostatin A (TSA) promotes apoptosis of tumor cells, as well as augments anti-GBM innate immune responses. In vitro treatment of GBM cells with TSA results in an up-regulation of the natural killer group-2 member-D (NKG2D) ligands major histocompatibility complex class I-related chain (MIC)-A and UL16 binding protein (ULBP)-2 at both mRNA and protein levels, rendering them susceptible to NK cell-mediated lysis. In vivo, TSA delays tumor growth of GBM xenografts. Both the in vitro and in vivo antitumor effect of TSA was significantly reduced by blocking NK cell activity. Our data suggest that HDACi, especially in combination with other clinical immunotherapeutical approaches, may be considered in a combined therapeutic approach for GBM.

Topics: Animals; Apoptosis; Blotting, Western; Brain Neoplasms; Cell Proliferation; Cytotoxicity, Immunologic; Female; Flow Cytometry; Glioblastoma; GPI-Linked Proteins; Histocompatibility Antigens Class I; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Intercellular Signaling Peptides and Proteins; Killer Cells, Natural; Mice; Mice, Nude; NK Cell Lectin-Like Receptor Subfamily K; Real-Time Polymerase Chain Reaction; Receptors, TNF-Related Apoptosis-Inducing Ligand; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand; Tumor Cells, Cultured

Nonsteroidal anti-inflammatory drug-activated gene, NAG-1, a transforming growth factor-β member, is involved in tumor progression and development. The association between NAG-1 expression and development and progression of glioma has not been well defined. Glioblastoma cell lines have lower basal expression of NAG-1 than other gliomas and normal astrocytes. Most primary human gliomas have very low levels of NAG-1 expression. NAG-1 basal expression appeared to inversely correlate with tumor grade in glioma. Aberrant promoter hypermethylation is a common mechanism for silencing of tumor suppressor genes in cancer cells. In glioblastoma cell lines, NAG-1 expression was increased by the demethylating agent, 5-aza-2'-deoxycytidine. To investigate whether the NAG-1 gene was silenced by hypermethylation in glioblastoma, we examined DNA methylation status using genomic bisulfite sequencing. The NAG-1 promoter was densely methylated in several glioblastoma cell lines as well as in primary oligodendroglioma tumor samples, which have low basal expression of NAG-1. DNA methylation at two specific sites (-53 and +55 CpG sites) in the NAG-1 promoter was strongly associated with low NAG-1 expression. The methylation of the NAG-1 promoter at the -53 site blocks Egr-1 binding and thereby suppresses Nag-1 induction. Treatment of cells with low basal NAG-1 expression with NAG-1 inducer also did not increase NAG-1. Incubation with a demethylation chemical increased Nag-1 basal expression and subsequent incubation with a NAG-1 inducer increased NAG-1 expression. We concluded from these data that methylation of specific promoter sequences causes transcriptional silencing of the NAG-1 locus in glioma and may ultimately contribute to tumor progression.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Azacitidine; Brain Neoplasms; Cell Growth Processes; Cell Line, Tumor; Decitabine; DNA Methylation; Early Growth Response Protein 1; Gene Expression Regulation, Neoplastic; Gene Silencing; Glioblastoma; Growth Differentiation Factor 15; Humans; Hydroxamic Acids; Promoter Regions, Genetic; Sulindac; Transfection

In this study we investigated epigenetic modifications such as DNA methylation, histone acetylation and histone methylation in the regulation of heparanase expression in glioblastoma. We found that heparanase promoters are differentially methylated among three glioblastoma cell lines; however, all these cells expressed baseline levels of heparanase. 5-Aza-2'-deoxycytidine (5-Aza-dC), a DNA methyltransferase inhibitor, revoked heparanase expression in all the examined cells. Trichostatin A (TSA), a histone deacetylase inhibitor, activated heparanase expression in promoter unmethylated LN229 and T98G cells but not in promoter methylated U251n cells. To identify the mechanisms of heparanase induction by 5-Aza-dC, heparanase expression-related transcription factors were examined. No detected transcription factors (EGR1, Ets1, GABPα and Sp1) were found to be induced either by 5-Aza-dC or TSA. Furthermore, we found that 5-Aza-dC increased acetylation of histone H3 and di-methylation of histone H3 lysine K4 (H3K4me2) in LN229 and T98G cells. The increased histone acetylation and H3K4me2 were also observed in heparanase-expressing tumor tissues by immunohistochemistry staining. Additionally, we found that nuclear factor κB (NFκB) p65 but not NFκB p50 was correlated with heparanase expression, which could be expressed both by neoplastic cells and angiogenesis-related neovessel cells. However, we did not observe any regulatory mechanism between heparanase and NFκB p65 via transient transfection of their cDNA in T98G and U251n cells. We concluded that heparanase expression is associated with histone modifications and promoter DNA methylation plays a role in the control of gene silencing. Overexpression of both heparanase and NFκB p65 may be the result of excessive histone modifications.

Topics: Azacitidine; Base Sequence; Cell Line, Tumor; CpG Islands; Decitabine; DNA Modification Methylases; Epigenesis, Genetic; Gene Expression; Gene Expression Regulation, Neoplastic; Glioblastoma; Glucuronidase; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Molecular Sequence Data; NF-kappa B p50 Subunit; Promoter Regions, Genetic; Protein Processing, Post-Translational; Transcription Factor RelA

Glioblastomas are known to be highly chemoresistant, but HDAC inhibitors (HDACi) have been shown to be of therapeutic relevance for this aggressive tumor type. We treated U87 glioblastoma cells with trichostatin A (TSA) to define potential epigenetic targets for HDACi-mediated antitumor effects. Using a cDNA array analysis covering 96 cell cycle genes, cyclin-dependent kinase inhibitor p21(WAF1) was identified as the major player in TSA-induced cell cycle arrest. TSA slightly inhibited proliferation and viability of U87 cells, cumulating in a G1/S cell cycle arrest. This effect was accompanied by a significant up-regulation of p53 and its transcriptional target p21(WAF1) and by down-regulation of key G1/S regulators, such as cdk4, cdk6, and cyclin D1. Nevertheless, TSA did not induce apoptosis in U87 cells. As expected, TSA promoted the accumulation of total acetylated histones H3 and H4 and a decrease in endogenous HDAC activity. Characterizing the chromatin modulation around the p21(WAF1) promoter after TSA treatment using chromatin immunoprecipitation, we found (1) a release of HDAC1, (2) an increase of acetylated H4 binding, and (3) enhanced recruitment of p53. p53-depleted U87 cells showed an abrogation of the G1/S arrest and re-entered the cell cycle. Immunofluorescence staining revealed that TSA induced the nuclear translocation of p21(WAF1) verifying a cell cycle arrest. On the other hand, a significant portion of p21(WAF1) was present in the cytoplasmic compartment causing apoptosis resistance. Furthermore, TSA-treated p53-mutant cell line U138 failed to show an induction in p21(WAF1), showed a deficient G2/M checkpoint, and underwent mitotic catastrophe. We suggest that HDAC inhibition in combination with other clinically used drugs may be considered an effective strategy to overcome chemoresistance in glioblastoma cells.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Chromatin Immunoprecipitation; Cyclin-Dependent Kinase Inhibitor p21; Flow Cytometry; Fluorescent Antibody Technique; Gene Expression Profiling; Glioblastoma; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunoblotting; Oligonucleotide Array Sequence Analysis; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Tumor Suppressor Protein p53

Recently, an important role of Notch activation for Ras-induced transformation of glial cells and for glioma growth and survival has been demonstrated. It was concluded that activation of Notch-signaling may represent a new target for glioblastoma multiforme (GBM) therapy. We now analyzed five GBM cell lines (Tx3095, Tx3868, U87, U118, U373) for key components of Notch-signaling pathways (Notch-1, Notch-2, Notch-3, Notch-4, Delta-like 1, Delta-like 3, Delta-like 4, Jagged-1, Jagged-2) using conventional RT-PCR. We found that some components (Notch-1, Notch-2, Notch-4, Jagged-1) were consistently expressed in all cell lines analyzed while, in contrast, other key components of Notch-signaling were differentially expressed. Notch-3 was expressed in three out of five cell lines (in U87, U118 and U373), but was missing in Tx3095 and Tx3868 cells. Jagged-2 was expressed in U87, U373 and Tx3868, but not in U118 or Tx3095 cells. Delta-like 1 and Delta-like 3 were not detected in Tx3905 cells, but in all other cell lines. RNA for Delta-like 4 was only found in U373 and Tx3868 GBM cell lines. Treating GBM cell lines with 1,25(OH)2D3 (10(-6), 10(-8), and 10(-10) M), the biologically active form of vitamin D, did not result in significant dose- or time-dependent antiproliferative effects, indicating that GBM cell lines are resistant against the antiproliferative activity of 1,25(OH)2D3. In vitro treatment of GBM cells with 1,25(OH)2D3 did not result in a modulation of the expression of key components of the Notch-signaling pathway. Treatment with HDAC-inhibitor TSA or DNA-methyltransferase inhibitor 5-aza exerted dose- and time-dependent antiproliferative effects on GBM cell lines. We asked the question whether the resistance against 1,25(OH)2D3 could be restored by co-treatment with TSA or 5-aza. However, combination therapy with 1,25(OH)2D3 and TSA or 5-aza did not result in enhanced antiproliferative effects as compared to treatment with TSA or 5-aza alone. In contrast, antiproliferative effects of TSA and 5-aza were partially antagonized by concomitant treatment with 1,25(OH)2D3, indicating a protective effect of 1,25(OH)2D3 against the antiproliferative effects of TSA and 5-aza in GBM cell lines. In conclusion, our findings point at a differential expression of key components of Notch-signaling in GBM cell lines that may be of importance for the growth characteristics of GBM. Our findings indicate that GBM cell lines are resistant against the antiproliferative e

Topics: Brain Neoplasms; Calcium-Binding Proteins; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Glioblastoma; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Intercellular Signaling Peptides and Proteins; Jagged-1 Protein; Membrane Proteins; Models, Biological; Receptors, Notch; Reverse Transcriptase Polymerase Chain Reaction; Serrate-Jagged Proteins; Signal Transduction; Steroid Hydroxylases; Vitamin D

Prognosis for patients with glioblastoma multiforme (GBM) is poor. Inhibitors of histone deacetylases (HDACi) like trichostatin A (TSA) are promising alternatives to conventional treatment. Deficient tumor suppressor functions, such as TP53 mutations and p14(ARF)/p16(INK4a) deletions, are characteristic for GBM and can cause resistance to DNA damaging agents such as cisplatin and to HDACi like TSA. The type II tumor suppressor Inhibitor of growth 1 (ING1) is involved in DNA damage response and histone modification. We have previously shown that ING1 is downregulated in GBM and involved in glioma-induced angiogenesis and in cisplatin-induced apoptosis in malignant glioma cells. Hence, the goal of our present study was to investigate whether TSA affects ING1 protein expression and also whether modulating ING1 levels affects TSA-induced apoptosis in malignant glioma cells that contain deficient p53 function and inactive pl4(ARF)/p16(INK4a) signaling. If so, we asked, which apoptotic pathway might be the major mediator beyond this interaction. To test whether ING1 proteins function in TSA-induced apoptosis in GBM, we analyzed TSA effects in LN229 GBM cells, which harbor TP53 mutations and INK4a deletion, following ING1 knockdown by siRNA. Expression of ING1, acetylated core histones H3 and H4, and the proapoptotic proteins caspase 3 and Fas-associated death domain (FADD) was determined by Western blotting. Percentages of apoptotic cells were obtained by flow cytometry. TSA induced the major ING1 isoform p33(ING1b) and increased levels of both histone acetylation and apoptosis in LN229 cells. ING1 knockdown cells revealed marked resistance to TSA-induced apoptosis, impairment of caspase 3 activation, and suppression of FADD. The data suggest that ING1 contributes to TSA-induced apoptosis in GBM cells with deficient p53 and p14(ARF)/p16(INK4a) functions, possibly by regulating FADD/caspase 3 signaling.

Topics: Acetylation; Apoptosis; Caspase 3; Cell Line, Tumor; Fas-Associated Death Domain Protein; Glioblastoma; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Inhibitor of Growth Protein 1; Intracellular Signaling Peptides and Proteins; Nuclear Proteins; Signal Transduction; Tumor Suppressor Protein p53; Tumor Suppressor Proteins

Nonsteroidal anti-inflammatory drug-activated gene (NAG-1) is a putative tumor suppressor whose expression can be increased by drug treatment. Glioblastoma is the most common central nervous system tumor, is associated with high morbidity and mortality, and responds poorly to surgical, chemical, and radiation therapy. The histone deacetylase inhibitors are under current consideration as therapeutic agents in treating glioblastoma. We investigated whether trichostatin A (TSA) would alter the expression of NAG-1 in glioblastoma cells. The DNA demethylating agent 5-aza-dC did not increase NAG-1 expression, but TSA up-regulated NAG-1 expression and acted synergistically with 5-aza-dC to induce NAG-1 expression. TSA indirectly increases NAG-1 promoter activity and increases NAG-1 mRNA and protein expression in the T98G human glioblastoma cell line. TSA also increases the expression of transcription factors Sp-1 and Egr-1. Small interfering RNA experiments link NAG-1 expression to apoptosis induced by TSA. Reporter gene assays, specific inhibition by small interfering RNA transfections, and chromatin immunoprecipitation assays indicate that Egr-1 and Sp-1 mediate TSA-induced NAG-1 expression. TSA also increases the stability of NAG-1 mRNA. TSA-induced NAG-1 expression involves multiple mechanisms at the transcriptional and post-transcriptional levels.

Topics: Azacitidine; Cell Line, Tumor; Decitabine; Early Growth Response Protein 1; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Glioblastoma; Growth Differentiation Factor 15; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Neoplasm Proteins; RNA, Messenger; RNA, Neoplasm; RNA, Small Interfering; Sp1 Transcription Factor; Up-Regulation

The anti-neoplastic effects of histone deacetylase inhibitors (HDACi), Trichostatin A (TSA) and 4-phenylbutyrate (4-PB) on the human glioblastoma cell lines GBM-29, U-343 MG and U-343 MGa Cl. 2:6 were investigated. TSA and 4-PB induced apoptosis in the three cell lines in a dose- and time-dependent manner. Whereas caspase-3 activation was detected in all three cell lines, U-343 MG cells were more sensitive to the apoptotic effect of HDACi compared with U-343 MGa Cl. 2:6. TSA and 4-PB induced differentiation in the three cell lines, each cell line developing unique phenotypic characteristics. During long-term treatment with a low dose of HDACi U-343 MGa Cl. 2:6 cells developed an astrocytic morphology with expression of glial fibrillary acidic protein (GFAP). GFAP-negative U-343 MG cells changed their morphology in response to HDACi and down-regulated their expression of vimentin. The nestin and vimentin positive GBM-29 cells also showed a morphological differentiation, while the expression of the two malignancy markers decreased. In summary, our results showed that these three glioblastoma cell lines display unique phenotypes and differentiation patterns in response to HDACi.

Topics: Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Survival; Enzyme Inhibitors; Glioblastoma; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Phenylbutyrates

High-grade gliomas are difficult to treat due to their location behind the blood-brain barrier and to inherent radioresistance and chemoresistance.. Because tumorigenesis is considered a multistep process of accumulating mutations affecting distinct signaling pathways, combinations of compounds, which inhibit nonoverlapping pathways, are being explored to improve treatment of gliomas. Histone deacetylase inhibitors (HDI) have proven antitumor activity by blocking cell proliferation, promoting differentiation, and inducing tumor cell apoptosis.. In this report, we show that the HDIs trichostatin A, sodium butyrate, and low nanomolar doses of LAQ824 combined with the glycolysis inhibitor 2-deoxy-d-glucose induce strong apoptosis in cancer cell lines of brain, breast, and cervix in a p53-independent manner. HDIs up-regulate p21, which is blocked by concomitant administration of 2-deoxy-d-glucose.. We propose simultaneous blockade of histone deacetylation and glycolysis as a novel therapeutic strategy for several major cancers.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Brain Neoplasms; Butyrates; Cell Line, Tumor; Deoxyglucose; Drug Synergism; Enzyme Inhibitors; Glioblastoma; Glycolysis; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids

Autophagy is a lysosome-dependent cellular catabolic mechanism mediating the turnover of intracellular organelles and long-lived proteins. Reduction of autophagy activity has been shown to lead to the accumulation of misfolded proteins in neurons and may be involved in chronic neurodegenerative diseases such as Huntington's disease and Alzheimer's disease. To explore the mechanism of autophagy and identify small molecules that can activate it, we developed a series of high-throughput image-based screens for small-molecule regulators of autophagy. This series of screens allowed us to distinguish compounds that can truly induce autophagic degradation from those that induce the accumulation of autophagosomes as a result of causing cellular damage or blocking downstream lysosomal functions. Our analyses led to the identification of eight compounds that can induce autophagy and promote long-lived protein degradation. Interestingly, seven of eight compounds are FDA-approved drugs for treatment of human diseases. Furthermore, we show that these compounds can reduce the levels of expanded polyglutamine repeats in cultured cells. Our studies suggest the possibility that some of these drugs may be useful for the treatment of Huntington's and other human diseases associated with the accumulation of misfolded proteins.

Topics: Autophagy; Calcium Channel Blockers; Cell Line, Tumor; Drug Evaluation, Preclinical; Fluspirilene; Glioblastoma; Green Fluorescent Proteins; Humans; Intracellular Membranes; Loperamide; Microtubule-Associated Proteins; Mycotoxins; Peptides; Phagosomes; Phosphatidylinositol Phosphates; Pimozide; Protein Kinases; Recombinant Fusion Proteins; Sirolimus; Small Molecule Libraries; TOR Serine-Threonine Kinases; Trifluoperazine; Zinc Fingers

Trichostatin A (TSA), histone deacetylase inhibitor, shows a promising therapeutic effect on cancer cells in combination with radiotherapy or chemotherapy. However, little has been reported on the combined treatment of TSA with hyperthermia. Here, we have assessed the effect of TSA/hyperthermia on human glioblastoma A172 cells and found that TSA increases the thermosensitivity of A172 cells, resulting in cellular apoptosis. The underlying mechanism of this effect consists of reduction in the level of phosphorylated STAT3 (Tyr705), a transcription factor required for survival of A172 cells, which leads to down-regulation of STAT3 target genes, cyclin D1 and Bcl-xL. Furthermore, the level of VEGF mRNA was also decreased by TSA/hyperthermia, suggesting the antiangiogenic effect of TSA/hyperthermia on human glioblastoma. Collectively, our results show the role of TSA as a chemical thermosensitizer, suggesting the possible therapeutic application of combined treatment of TSA/hyperthermia on STAT3-dependent tumors.

Topics: Blotting, Western; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Fever; Flow Cytometry; Glioblastoma; Humans; Hydroxamic Acids; Protein Synthesis Inhibitors; STAT3 Transcription Factor; Temperature; Thermosensing; Time Factors; Vascular Endothelial Growth Factor A

Resistance to radiation and chemotherapy remains an obstacle to the treatment of brain tumors. We have demonstrated that the replication-deficient adenovirus d1520, which lacks the E1A 13S protein, replicates efficiently and exhibits oncolytic potential in multidrug-resistant cells with nuclear localization of the human transcription factor YB-1. However, besides others, key factors regarding oncolytic virotherapy are limited tumor transduction rate and low replication efficiency. The objective of this study was to determine whether the chemotherapeutic agent irinotecan, by enhancing nuclear localization of YB-1, and the histone deacetylase inhibitor trichostatin A, by upregulating coxsackievirus-adenovirus receptor (CAR) expression, could augment replication of and cell lysis by adenovirus dl520 in glioblastomas in vitro. We found that trichostatin A upregulated CAR expression and that irinotecan caused increased nuclear localization of YB-1 in both glioblastoma cell lines. Irinotecan alone, and trichostatin A alone, enhanced replication of and cell lysis by dl520. Importantly, when combining both agents, the replication efficiency (maximum, 27-fold) and induction of cytopathic effect (maximum, 3.8-fold) of dl520 were further augmented significantly. These results support the hypothesis that the enhanced oncolytic effect of dl520, after incubation with chemotherapeutic agents, is mediated by an increased accumulation of YB-1 in the nucleus (due to irinotecan) and by upregulation of CAR (due to trichostatin A). Thus, therapy combining virotherapy, chemotherapy, and histone deacetylase inhibitor treatment is a novel approach to enhance the oncolytic efficacy of dl520.

Topics: Adenoviridae; Antineoplastic Agents, Phytogenic; Blotting, Southern; Brain Neoplasms; Camptothecin; Coxsackie and Adenovirus Receptor-Like Membrane Protein; Enzyme Inhibitors; Fibroblasts; Gene Deletion; Gene Expression; Gentian Violet; Glioblastoma; HeLa Cells; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunohistochemistry; Irinotecan; Oncolytic Viruses; Protein Synthesis Inhibitors; Receptors, Virus; Tumor Cells, Cultured

Malignant glioma is the most common central nervous system tumor of adults and is associated with a significant degree of morbidity and mortality. Gliomas are highly invasive and respond poorly to conventional treatments. Gliomas, like other tumor types, arise from a complex and poorly understood sequence of genetic and epigenetic alterations. Epigenetic alterations leading to gene silencing, in the form of aberrant CpG island promoter hypermethylation and histone deacetylation, have not been thoroughly investigated in brain tumors, and elucidating such changes is likely to enhance our understanding of their etiology and provide new treatment options. We used a combined approach of pharmacologic inhibition of DNA methylation and histone deacetylation, coupled with expression microarrays, to identify novel targets of epigenetic silencing in glioma cell lines. From this analysis, we identified >160 genes up-regulated by 5-aza-2'-deoxycytidine and trichostatin A treatment. Further characterization of 10 of these genes, including the putative metastasis suppressor CST6, the apoptosis-inducer BIK, and TSPYL5, whose function is unknown, revealed that they are frequent targets of epigenetic silencing in glioma cell lines and primary tumors and suppress glioma cell growth in culture. Furthermore, we show that other members of the TSPYL gene family are epigenetically silenced in gliomas and dissect the contribution of individual DNA methyltransferases to the aberrant promoter hypermethylation events. These studies, therefore, lay the foundation for a comprehensive understanding of the full extent of epigenetic changes in gliomas and how they may be exploited for therapeutic purposes.

Topics: Azacitidine; Brain Neoplasms; Cell Cycle Proteins; Cell Growth Processes; Cell Line, Tumor; Decitabine; DNA Methylation; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Silencing; Glioblastoma; Humans; Hydroxamic Acids; Up-Regulation

The carboxyl-terminal modulator protein (CTMP) has been identified as a negative regulator of protein kinase B/Akt. Aberrant Akt signaling is frequently observed in glioblastomas, the most common and most malignant glial brain tumors. Because loss of CTMP function and/or expression may remove the inhibitory effects on Akt and promote tumorigenesis, we studied 93 primary glioblastomas and nine glioblastoma cell lines for CTMP deletion, mutation, promoter hypermethylation, and mRNA expression. None of the tumors or cell lines had CTMP-homozygous deletions or coding sequence mutations. However, CTMP mRNA expression was lower by at least 50% relative to non-neoplastic brain tissue in 37 (40%) glioblastomas and six (67%) glioma cell lines. Reduced CTMP mRNA levels were closely associated with hypermethylation of the CTMP promoter. Furthermore, treatment of CTMP-hypermethylated A172 glioma cells with the demethylating agent 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor trichostatin A resulted in partial demethylation of the CTMP promoter and increased CTMP mRNA expression. Thus, epigenetic downregulation of CTMP transcription is a common aberration in glioblastomas.

Topics: Adaptor Proteins, Signal Transducing; Antibiotics, Antineoplastic; Antimetabolites, Antineoplastic; Azacitidine; Carrier Proteins; Cell Line, Tumor; Decitabine; DNA Methylation; Down-Regulation; Gene Deletion; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Hydroxamic Acids; Membrane Proteins; Mutation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thiolester Hydrolases; Transcription, Genetic

Histone deacetylase inhibitors (HDAC-Is) show in vitro and in vivo antitumor activity in various types of cancer cells and are being studied in clinical trials. However, studies addressing the combination of HDAC-I and radiation are lacking. The purpose of this study was to assess the effect of trichostatin A (TSA), an HDAC-I, on the radiosensitivity of U373MG and U87MG (human glioblastoma) cell lines.. Intrinsic TSA toxicity was determined by measuring survival in exponentially growing cells treated with 0-200 nM TSA for 0-24 h. To assay the radiosensitizing effect of TSA, cells were exposed to 0-200 nM TSA for 18 h before irradiation, and radiation survival curves were obtained. Radiation survival of TSA-treated cells was determined by clonogenic assay.. The human glioblastoma cells showed a dose-dependent reduction in survival and radiosensitization with TSA treatment in the range of 50-200 nM. Exposure to 200 nM TSA resulted in reduced survival of both cell lines, and survival was further reduced with time. Exposure of these cells to TSA before irradiation led to dose-dependent radiosensitization.. These results suggest that HDAC-Is may be a useful adjunct in the treatment of glioblastoma and merit further investigation. Given the limited efficacy of standard treatments for patients afflicted with glioblastoma, the results reported here provide support for clinical trials integrating HDAC-I with radiation therapy.

Topics: Apoptosis; Cell Cycle; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Glioblastoma; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Tumor Cells, Cultured

The expression of many cellular genes is modulated by DNA methylation and histone acetylation. These processes can influence malignant cell transformation and are also responsible for the silencing of DNA constructs introduced into mammalian cells for therapeutic or research purposes. As a better understanding of these biological processes may contribute to the development of novel cancer treatments and to study the complex mechanisms regulating gene silencing, we established a cellular system suitable to dissect the mechanisms regulating DNA methylation and histone acetylation. For this purpose, we stably transfected the neuroblastoma cell line U87 with a cytomegalovirus promoter-driven reporter gene construct whose expression was analyzed following treatment with the DNA methylation inhibitor 5'-aza-2'-deoxycytidine or histone deacetylation inhibitor trichostatin A. Both substances reactivated the silenced cytomegalovirus promoter, but with different reaction kinetics. Furthermore, whereas the kinetics of reactivation by trichostatin A did not substantially change over the time range considered (5 days), reactivation induced by 5'-aza-2'-deoxycytidine showed profound differences between day 1 and longer time points. We showed that this effect is related to the down-regulation of DNA replication by 5'-aza-2'-deoxycytidine. Finally, we have shown that the simultaneous administration of trichostatin A and 5'-aza-2'-deoxycytidine results in reactivation of the CMV promoter according to a cooperative, not synergistic or additive, mechanism. In conclusion, our cellular system should represent a powerful tool to investigate the complex mechanisms regulating gene silencing and to identify new anticancer drugs.

Topics: Animals; Azacitidine; Cytomegalovirus; DNA Methylation; DNA Replication; Flow Cytometry; Gene Expression Regulation, Neoplastic; Gene Silencing; Glioblastoma; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Microscopy, Fluorescence; Models, Theoretical; Plasmids; Polymerase Chain Reaction; Promoter Regions, Genetic; Protein Synthesis Inhibitors; Time Factors; Transfection; Tumor Cells, Cultured

We have shown previously that the tissue factor pathway inhibitor-2 (TFPI-2), a broad range proteinase inhibitor, is highly expressed in low-grade gliomas, but, minimally expressed or undetectable in glioblastomas, and that enforced expression of this gene reduces the invasive properties of brain tumor cells. Here, we examined the role of promoter methylation as a mechanism of TFPI-2 gene silencing. In SNB19 glioblastoma cells, which have no detectable TFPI-2 expression, 5-aza-2'-deoxycytidine (5aC), an inhibitor of DNA methyltransferase, induced TFPI-2 mRNA in a dose-dependent manner. Trichostatin A (TSA), the histone deacetylase (HDAC) inhibitor, by itself, was more efficient than 5aC in inducing TFPI-2 transcripts, and the 5aC+TSA combination resulted in highly synergistic reactivation of the gene, both at the transcript and protein levels. In Hs683 glioma cells, which express the TFPI-2 gene at high levels, transfection of the in vitro methylated TFPI-2 promoter constructs resulted in a drastic decrease of promoter activity compared to the unmethylated promoter. Further, the methylation-specific PCR in SNB19 and Hs683 cells showed that TFPI-2 gene repression was closely linked with methylation of the CpG islands in the promoter. Finally, the chromatin immunoprecipitation assays in SNB19 cells showed that the methylated and repressed TFPI-2 promoter was associated with the methyl-CpG binding protein 2 (MeCP2), and that gene reactivation resulted in the loss of MeCP2 from this site. These studies establish that TFPI-2 is transcriptionally silenced through promoter methylation in SNB19 cells.

Topics: Azacitidine; Brain Neoplasms; Chromatin; Chromosomal Proteins, Non-Histone; CpG Islands; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA-Binding Proteins; Enzyme Inhibitors; Gene Silencing; Glioblastoma; Glioma; Glycoproteins; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Methyl-CpG-Binding Protein 2; Polymerase Chain Reaction; Promoter Regions, Genetic; Repressor Proteins

Several anticancer drugs target DNA or enzymes acting on the DNA. Because chromatin DNA is tightly compacted, accessibility to the drug target may reduce the efficiency of these anticancer drugs. We thus treated four human cancer cell lines and two normal epithelial cell lines with either trichostatin A (TSA) or SAHA, two histone deacetylase inhibitors, before exposing the cells to VP-16, ellipticine, camptothecin, doxorubicin, cisplatin, 5-fluorouracil, or cyclophosmamide. Pretreatment with TSA or SAHA increased the killing efficiency of VP-16, ellipticine, doxorubicin, and cisplatin. The magnitude of sensitization is cell type specific and is >10-fold for VP-16 in D54, a brain tumor cell line intrinsically resistant to topoisomerase II inhibitors. Topoisomerase II levels and activity were not affected by this treatment, but p53, p21, and Gadd45 protein levels were markedly induced. Moreover, pretreatment with TSA also increased VP-16-induced apoptosis in a p53-dependent and -independent manner. Treating the cells in the reverse order (anticancer drug first, followed by TSA or SAHA) had no more cytotoxic effect than the drug alone. These data suggest that loosening-up the chromatin structure by histone acetylation can increase the efficiency of several anticancer drugs targeting DNA. This may be advantageous for treating tumors intrinsically resistant to these drugs.

Topics: Acetylation; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; DNA Topoisomerases, Type II; DNA, Neoplasm; Drug Synergism; Enzyme Inhibitors; Etoposide; Gene Expression; Glioblastoma; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Tumor Suppressor Protein p53; Vorinostat

We investigated the effects of histone deacetylase (HDAC) inhibitors such as sodium butyrate (SB) and trichostatin A (TSA) on the expression of vascular endothelial growth factor (VEGF) by human glioblastoma T98G, U251MG, and U87MG cells. The glioblastoma cells secreted three VEGF isoforms, VEGF (189), (165), and (121), although the expression levels of VEGF differed between the cell types. Treatment with either 5mM SB or 100 ng/ml TSA reduced VEGF secretion in conditioned media and reduced VEGF mRNA expression. We also studied the expression of VEGF-B, -C, and -D mRNA in human glioblastoma cells and their modulation by HDAC inhibitors. The PCR products of VEGF-B (357bp), VEGF-C (501bp), and VEGF-D (484bp) were amplified in all glioblastoma cells examined. Treatment with SB reduced the expression of VEGF-D mRNA in U251MG cells and the expression of VEGF-B mRNA in U87MG cells. TSA treatment reduced the expression of VEGF-D in U251MG cells. These results suggest that HDAC inhibitors reduce VEGF secretion and modulate the expression of the other VEGF family members, and therefore may inhibit angiogenesis in glioblastoma tissues.

Topics: Blotting, Western; Brain Neoplasms; Butyrates; Culture Media, Conditioned; Depression, Chemical; Endothelial Growth Factors; Enzyme Inhibitors; Glioblastoma; Glyceraldehyde-3-Phosphate Dehydrogenases; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Intercellular Signaling Peptides and Proteins; Lymphokines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The effects of sodium butyrate (SB) and trichostatin A (TSA) on cell proliferation andapoptosis against human glioma T98G, U251MG, and U877MG cells were investigated. Upon exposure to either SB or TSA, cell proliferation was reduced, and apoptosis detected by DNA fragmentation analysis and the cleavage of CPP32 was induced. Previously, we reported that SB increased the expression levels of p21 (WAF-1) and inhibited G1-S transition of the cell cycle. In this study, we showed that TSA also increased p21 expression, suggesting that histone deacetylase (HDAC) inhibitors may up-regulate p21 protein in common and thus arrest proliferation in the G1 phase of the cell cycle. To further determine the underlying molecular mechanisms of apoptosis with either SB or TSA treatment, we studied the expression levels of apoptosis-related proteins in human glioma cells. SB increased the expression of the Bad protein, although the expression of Bcl-2, Bcl-xL, Bax, and Fas was not changed by theaddition of SB. TSA treatment also up-regulated the expression of Bad protein. The results suggest that HDAC inhibitors such as SB and TSA induce apoptosis through an increase in Bad protein in human glioma cells in vitro.

Topics: Apoptosis; Brain Neoplasms; Butyrates; Cell Division; Cell Line, Tumor; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Glioblastoma; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Neoplasm Proteins; Nerve Tissue Proteins; Sodium