trichostatin-a and Carcinoma--Non-Small-Cell-Lung

Histone deacetylase (HDAC) inhibitors have a potential therapeutic role for non-small cell lung cancer (NSCLC). However, more preclinical studies of HDAC inhibitors in NSCLC and normal lung epithelial cells are required to evaluate their antitumor activities and mechanisms. The bicellular tight junction molecule claudin-2 (CLDN-2) is highly expressed in lung adenocarcinoma tissues and increase the proliferation of adenocarcinoma cells. Downregulation of the tricellular tight junction molecule angulin-1/LSR induces malignancy via EGF-dependent CLDN-2 and TGF-β-dependent cellular metabolism in human lung adenocarcinoma cells. In the present study, to investigate the detailed mechanisms of the antitumor activities of HDAC inhibitors in lung adenocarcinoma, human lung adenocarcinoma A549 cells and normal lung epithelial cells were treated with the HDAC inibitors Trichostatin A (TSA) and Quisinostat (JNJ-2648158) with or without TGF-β. Both HDAC inhibitors increased anguin-1/LSR, decrease CLDN-2, promoted G1 arrest and prevented the migration of A549 cells. Furthermore, TSA but not Quisinostat with or without TGF-β induced cellular metabolism indicated as the mitochondrial respiration measured using the oxygen consumption rate. In normal human lung epithelial cells, treatment with TSA and Quisinostat increased expression of LSR and CLDN-2 and decreased that of CLDN-1 with or without TGF-β in 2D culture. Quisinostat but not TSA with TGF-β increased CLDN-7 expression in 2D culture. Both HDAC inhibitors prevented disruption of the epithelial barrier measured as the permeability of FD-4 induced by TGF-β in 2.5D culture. TSA and Quisinostat have potential for use in therapy for lung adenocarcinoma via changes in the expression of angulin-1/LSR and CLDN-2.

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Movement; Cell Proliferation; Cells, Cultured; Drug Screening Assays, Antitumor; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lung Neoplasms; Tight Junction Proteins

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Catalytic Domain; Cell Line, Tumor; Cell Proliferation; Drug Screening Assays, Antitumor; Histone Deacetylase 6; Histone Deacetylase Inhibitors; HSP90 Heat-Shock Proteins; Humans; Hydroxamic Acids; Isoindoles; Lung Neoplasms; Male; Mice, Inbred BALB C; Molecular Docking Simulation; Protein Binding; Xenograft Model Antitumor Assays

We report structure-activity relationships of 1-arylsulfonyl indoline based benzamides. The benzamide (9) exhibits striking tubulin inhibition with an IC

Topics: A549 Cells; Animals; Antineoplastic Agents; Benzamides; Carcinoma, Non-Small-Cell Lung; Catalytic Domain; Cell Line, Tumor; Cell Proliferation; Heterografts; Histone Deacetylase Inhibitors; Humans; Indoles; Lymphoma, B-Cell; Mice; Models, Molecular; Protein Binding; Tubulin Modulators

Insulin-like growth factor (IGF) signaling plays an important role in tumorigenesis and metastasis. Here, we analyzed insulin-like growth factor (IGF) binding protein-2 (IGFBP2) expression in 81 lung cancer patients and 36 controls consisting of healthy and benign pulmonary lesion participants for comparison, then validated the IGFBP2 expression in additional 84 lung cancer patients, and evaluated the prognostic and chemoresistant significance of IGFBP2 in two cohorts respectively. Next we detected the reversal effect of trichostatin A (TSA) on chemoresistance in cell lines with high IGFBP2 expression. As a result, the mean expression of IGFBP2 in lung cancer patients was significantly higher than that in controls and increased with lung cancer progressed to advanced stage. In addition, high IGFBP2 expression was independently predictive for chemoresistance; over-expressed IGFBP2 enhances cell activity and TSA can reverse the chemoresistance induced by high IGFBP2 expression through enhancing autophagy. Furthermore, multivariate analysis showed that lung cancer patients whose blood IGFBP2 was higher had a poor survival outcome, with a hazard ratio of 8.22 (95%CI 1.78-37.92, P = 0.007) after adjustment for stage, histopathology, EGFR mutation, age, smoking and surgery.

Topics: Adenocarcinoma; Autophagy; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Case-Control Studies; Drug Resistance, Neoplasm; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Insulin-Like Growth Factor Binding Protein 2; Lung Neoplasms; Male; Middle Aged; Prognosis; Retrospective Studies; Survival Rate; Tumor Cells, Cultured

Recent studies indicated undisputed contribution of connective tissue growth factor (CTGF) in the development of many cancers, including non-small cell lung cancer (NSCLC). However, the functional role and regulation of CTGF expression during tumorigenesis remain elusive. Our goal was to determine CTGF transcript and protein levels in tumoral and matched control tissues from 98 NSCLC patients, to correlate the results with clinicopathological features and to investigate whether the CTGF expression can be epigenetically regulated in NSCLC.. We used quantitative PCR, Western blotting and immunohistochemistry to evaluate CTGF expression in lung cancerous and histopathologically unchanged tissues. We tested the impact of 5-Aza-2'-deoxycytidine (5-dAzaC) and trichostatin A (TSA) on CTGF transcript and protein levels in NSCLC cells (A549, Calu-1). DNA methylation status of the CTGF regulatory region was evaluated by bisulfite sequencing. The influence of 5-dAzaC and TSA on NSCLC cells viability and proliferation was monitored by the trypan blue assay.. We found significantly decreased levels of CTGF mRNA and protein (both p < 0.0000001) in cancerous tissues of NSCLC patients. Down-regulation of CTGF occurred regardless of gender in all histological subtypes of NSCLC. Moreover, we showed that 5-dAzaC and TSA were able to restore CTGF mRNA and protein contents in NSCLC cells. However, no methylation within CTGF regulatory region was detected. Both compounds significantly reduced NSCLC cells proliferation.. Decreased expression of CTGF is a common feature in NSCLC; however, it can be restored by the chromatin-modifying agents such as 5-dAzaC or TSA and consequently restrain cancer development.

Topics: Adult; Aged; Aged, 80 and over; Azacitidine; Biomarkers, Tumor; Carcinoma, Non-Small-Cell Lung; Connective Tissue Growth Factor; Decitabine; DNA Methylation; Down-Regulation; Epigenesis, Genetic; Female; Gene Expression Regulation, Neoplastic; HeLa Cells; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lung Neoplasms; Male; Middle Aged; Prognosis; Promoter Regions, Genetic; Tumor Cells, Cultured

Non-small-cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. NSCLC development and progression have recently been correlated with the heightened activation of histone deacetylases (HDACs) and PI3K/Akt signaling pathways. Targeted inhibition of these proteins is promising approach for the development of novel therapeutic strategies to treat patients with advanced NSCLC. For this reason, we combined a dual PI3K and mTOR inhibitor, BEZ235 with the HDAC inhibitor Trichostatin A (TSA), to determine their combined effects on human NSCLC. In this study, we initially discovered that co-treatment with BEZ235 and TSA showed a synergistic effect on inhibition of NSCLC cell proliferation and induction of apoptosis. The combination treatment also synergistically suppressed NSCLC migration, invasion and the NSCLC epithelial-mesenchymal transition (EMT) in vitro. The synergistic effect was also evidenced by declines in xenograft growth and metastasis rates and in ki-67 protein expression in vivo. Together, these results indicated that BEZ235 and TSA combination treatment significantly increased anti-tumor activities compared with BEZ235 and TSA alone, supporting a further evaluation of combination treatment for NSCLC.

Topics: A549 Cells; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Imidazoles; Lung Neoplasms; Mice, Inbred BALB C; Mice, Nude; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Quinolines; TOR Serine-Threonine Kinases; Treatment Outcome; Xenograft Model Antitumor Assays

To evaluate the potential therapeutic utility of histone deacetylase inhibitors (HDACi) in targeting VEGF receptors in non-small-cell lung cancer.. Non-small-cell lung cancer cells were screened for the VEGF receptors at the mRNA and protein levels, while cellular responses to various HDACi were examined.. Significant effects on the regulation of the VEGF receptors were observed in response to HDACi. These were associated with decreased secretion of VEGF, decreased cellular proliferation and increased apoptosis which could not be rescued by addition of exogenous recombinant VEGF. Direct remodeling of the VEGFR1 and VEGFR2 promoters was observed. In contrast, HDACi treatments resulted in significant downregulation of the Neuropilin receptors.. Epigenetic targeting of the Neuropilin receptors may offer an effective treatment for lung cancer patients in the clinical setting.

Topics: Acetylation; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chromatin Assembly and Disassembly; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Lung Neoplasms; Molecular Targeted Therapy; Receptors, Vascular Endothelial Growth Factor; Vascular Endothelial Growth Factor A; Vorinostat

DNA methylation plays a critical role during the development of acquired chemoresistance. The aim of this study was to identify candidate DNA methylation drivers of cisplatin (DDP) resistance in non-small cell lung cancer (NSCLC). The A549/DDP cell line was established by continuous exposure of A549 cells to increasing concentrations of DDP. Gene expression and methylation profiling were determined by high-throughput microarrays. Relationship of methylation status and DDP response was validated in primary tumor cell culture and the Cancer Genome Atlas (TCGA) samples. Cell proliferation, apoptosis, cell cycle, and response to DDP were determined in vitro and in vivo. A total of 372 genes showed hypermethylation and downregulation in A549/DDP cells, and these genes were involved in most fundamental biological processes. Ten candidate genes (S100P, GDA, WISP2, LOXL1, TIMP4, ICAM1, CLMP, HSP8, GAS1, BMP2) were selected, and exhibited varying degrees of association with DDP resistance. Low dose combination of 5-aza-2'-deoxycytidine (5-Aza-dC) and trichostatin A (TSA) reversed drug resistance of A549/DDP cells in vitro and in vivo, along with demethylation and restoration of expression of candidate genes (GAS1, TIMP4, ICAM1 and WISP2). Forced expression of GAS1 in A549/DDP cells by gene transfection contributed to increased sensitivity to DDP, proliferation inhibition, cell cycle arrest, apoptosis enhancement, and in vivo growth retardation. Together, our study demonstrated that a panel of candidate genes downregulated by DNA methylation induced DDP resistance in NSCLC, and showed that epigenetic therapy resensitized cells to DDP.

Topics: Antimetabolites, Antineoplastic; Antineoplastic Agents; Apoptosis; Azacitidine; Carcinoma, Non-Small-Cell Lung; Cell Cycle Checkpoints; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cisplatin; Decitabine; DNA Methylation; Drug Resistance, Neoplasm; Epigenesis, Genetic; Gene Expression Profiling; GPI-Linked Proteins; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lung Neoplasms; RNA, Messenger

Aggressive cancers in the epithelial-to-mesenchymal transition (EMT) phase are characterized by loss of cell adhesion, repression of E-cadherin, and increased cell mobility. Non-small cell lung cancer (NSCLC) differs in basal level of E-cadherin; predominantly exhibiting silenced expression due to epigenetic-related modifications. Accordingly, effective treatments are needed to modulate these epigenetic events that in turn can positively regulate E-cadherin levels. Herein, we investigated silibinin, a natural flavonolignan with anticancer efficacy against lung cancer, either alone or in combination with epigenetic therapies to modulate E-cadherin expression in a panel of NSCLC cell lines. Silibinin combined with HDAC inhibitor Trichostatin A [TSA; 7-[4-(dimethylamino)phenyl]-N-hydroxy-4,6-dimethyl-7-oxohepta-2,4-dienamide] or DNMT inhibitor 5'-Aza-deoxycytidine (Aza) significantly restored E-cadherin levels in NSCLC cells harboring epigenetically silenced E-cadherin expression. These combination treatments also strongly decreased the invasion/migration of these cells, which further emphasized the biologic significance of E-cadherin restoration. Treatment of NSCLC cells, with basal E-cadherin levels, by silibinin further increased the E-cadherin expression and inhibited their migratory and invasive potential. Additional studies showed that silibinin alone as well as in combination with TSA or Aza downmodulate the expression of Zeb1, which is a major transcriptional repressor of E-cadherin. Overall these findings demonstrate the potential of combinatorial treatments of silibinin with HDAC or DNMT inhibitor to modulate EMT events in NSCLC cell lines, leading to a significant inhibition in their migratory and invasive potentials. These results are highly significant, since loss of E-cadherin and metastatic spread of the disease via EMT is associated with poor prognosis and high mortalities in NSCLC.

Topics: Antioxidants; Azacitidine; Blotting, Western; Cadherins; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Decitabine; Drug Synergism; Enzyme Inhibitors; Epigenesis, Genetic; Gene Silencing; Histone Deacetylase Inhibitors; Homeodomain Proteins; Humans; Hydroxamic Acids; Neoplasm Invasiveness; Silybin; Silymarin; Transcription Factors; Up-Regulation; Zinc Finger E-box-Binding Homeobox 1

Cytoglobin (CYGB) is frequently downregulated in many types of human malignancies, and its exogenous overexpression reduces proliferation of cancer cells. Despite its implied tumour suppressor (TSG) functions, its exact role in carcinogenesis remains unclear as CYGB upregulation is also associated with tumour hypoxia and aggressiveness. In this study, we explore the TSG role of CYGB, its influence on the phenotype of cancerous cells under stress conditions and the clinical significance of CYGB expression and promoter methylation in non-small cell lung cancer (NSCLC). DNA methylation-dependent expression silencing of CYGB is demonstrated in both clinical samples and cell lines. CYGB promoter was more frequently methylated in lung adenocarcinomas (P = 1.4 × 10(-4)). Demethylation by 5'-azadeoxycytidine partially restored CYGB expression in cell lines. Interestingly, trichostatin A triggered upregulation of CYGB expression in cancer cell lines and downregulation in non-tumourigenic ones. CYGB mRNA expression in NSCLC surgical specimens correlated with that of HIF1α and VEGFa (P < 1 × 10(-4)). Overexpression of CYGB in cancer cell lines reduced cell migration, invasion and anchorage-independent growth. Moreover, CYGB impaired cell proliferation, but only in the lung adenocarcinoma cell line (H358). Upon hydrogen peroxide treatment, CYGB protected cell viability, migratory potential and anchorage independence by attenuating oxidative injury. In hypoxia, CYGB overexpression decreased cell viability, augmented migration and anchorage independence in a cell-type-specific manner. In conclusion, CYGB revealed TSG properties in normoxia but promoted tumourigenic potential of the cells exposed to stress, suggesting a bimodal function in lung tumourigenesis, depending on cell type and microenvironmental conditions.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Carcinoma, Non-Small-Cell Lung; Cell Hypoxia; Cell Line, Tumor; Cytoglobin; DNA Methylation; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, Tumor Suppressor; Globins; Humans; Hydroxamic Acids; Hypoxia-Inducible Factor 1, alpha Subunit; Lung Neoplasms; Oncogenes; Vascular Endothelial Growth Factor A

Histone deacetylases (HDACs) inhibitor is a promising new approach to the treatment of lung cancer therapy via inhibiting cell growth and inducing apoptosis. miR-15a and miR-16-1 are important tumor suppressors through modulating B cell lymphoma 2 (Bcl-2), Cyclin D1, D2, and others. However, whether HDACs inhibitor modulates the expression of miR-15a/16-1 in lung cancer is still unknown. The purpose of our study was to identify a new miRNA-mediated mechanism which plays an important role in the anti-cancer effects of HDACs inhibitor. We found HDACs inhibitors trichostatin A (TSA) and sodium butyrate upregulated the expression of miR-15a/16-1, residing in the host tumor suppressor Dleu2 gene, through increasing the histone acetylation in the region of Dleu2/miR-15a/16-1 promoter in lung cancer cells. Moreover, among class Ι HDACs subtypes, only knockdown of HDAC3 by specific siRNA increased the hyperacetylation of Dleu2/miR-15a/16-1 promoter region and finally resulted in the upregulation of miR-15a/16-1. Furthermore, overexpression of miR-15a/16-1, which were always deleted or downregulated in lung cancer cells, effectively suppressed cell growth and reduced colony formation. Finally, TSA reduced the expression of Bcl-2, an important survival protein in lung cancer cells, partly through upregulation of miR-15a/16-1. Therefore, this offers a therapeutic strategy that lung cancer patients who exhibit low level of miR-15a/16-1 or high activity of HDACs may benefit from HDACs inhibitor-based therapy.

Topics: Acetylation; Apoptosis; Butyric Acid; Carcinoma, Non-Small-Cell Lung; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Lung Neoplasms; MicroRNAs; Promoter Regions, Genetic; Proto-Oncogene Proteins c-bcl-2; RNA, Long Noncoding; Transferases; Tumor Stem Cell Assay; Tumor Suppressor Proteins; Up-Regulation

Non-small-cell lung cancer (NSCLC) represents approximately 80% of all types of lung cancer. Here, we report the chemotherapeutic effect of honokiol, a phytochemical from Magnolia grandiflora, on NSCLC cells and the molecular mechanisms underlying these effects using in vitro and in vivo models. Treatment of NSCLC cells (A549, H1299, H460 and H226) with honokiol (20, 40 and 60 µM) inhibited histone deacetylase (HDAC) activity, reduced the levels of class I HDAC proteins and enhanced histone acetyltransferase activity in a dose-dependent manner. These effects of honokiol were associated with a significant reduction in the viability of NSCLC cells. Concomitant treatment of cells with a proteasome inhibitor, MG132, prevented honokiol-induced degradation of class I HDACs, suggesting that honokiol reduced the levels of HDACs in NSCLC cells through proteasomal degradation. Valproic acid, an inhibitor of HDACs, exhibited a similar pattern of reduced viability and induction of death of NSCLC cells. Treatment of A549 and H1299 cells with honokiol resulted in an increase in G 1 phase arrest, and a decrease in the levels of cyclin D1, D2 and cyclin dependent kinases. Further, administration of honokiol by oral gavage significantly inhibited the growth of subcutaneous A549 and H1299 tumor xenografts in athymic nude mice, which was associated with the induction of apoptotic cell death and marked inhibition of class I HDACs proteins and HDAC activity in the tumor xenograft tissues. Together, our study provides new insights into the role of class I HDACs in the chemotherapeutic effects of honokiol on lung cancer cells.

Topics: Acetylation; Animals; Biphenyl Compounds; Bronchi; Carcinoma, Non-Small-Cell Lung; Cell Death; Cell Line, Tumor; Cell Proliferation; Cell Survival; Epithelial Cells; G1 Phase Cell Cycle Checkpoints; Histone Acetyltransferases; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Hydroxamic Acids; Lignans; Liver Neoplasms; Mice; Mice, Nude; Proteasome Endopeptidase Complex; Proteolysis; Valproic Acid; Xenograft Model Antitumor Assays

The major aim of this study was to investigate the role of DNA methylation (referred to as methylation) on miRNA silencing in non-small cell lung cancers (NSCLC).. We conducted microarray expression analyses of 856 miRNAs in NSCLC A549 cells before and after treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (Aza-dC) and with a combination of Aza-dC and the histone deacetylase inhibitor trichostatin A. miRNA methylation was determined in 11 NSCLC cell lines and in primary tumors and corresponding nonmalignant lung tissue samples of 101 patients with stage I-III NSCLC.. By comparing microarray data of untreated and drug-treated A549 cells, we identified 33 miRNAs whose expression was upregulated after drug treatment and which are associated with a CpG island. Thirty (91%) of these miRNAs were found to be methylated in at least 1 of 11 NSCLC cell lines analyzed. Moreover, miR-9-3 and miR-193a were found to be tumor specifically methylated in patients with NSCLC. We observed a shorter disease-free survival of patients with miR-9-3 methylated lung squamous cell carcinoma (LSCC) than patients with miR-9-3 unmethylated LSCC by multivariate analysis [HR = 3.8; 95% confidence interval (CI), 1.3-11.2, P = 0.017] and a shorter overall survival of patients with miR-9-3 methylated LSCC than patients with miR-9-3 unmethylated LSCC by univariate analysis (P = 0.013).. Overall, our results suggest that methylation is an important mechanism for inactivation of certain miRNAs in NSCLCs and that miR-9-3 methylation may serve as a prognostic parameter in patients with LSCC.

Topics: Antineoplastic Agents; Azacitidine; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Decitabine; DNA Methylation; Female; Gene Expression Profiling; Gene Silencing; Humans; Hydroxamic Acids; Lung; Lung Neoplasms; Male; MicroRNAs; Middle Aged; Molecular Targeted Therapy; Oligonucleotide Array Sequence Analysis

Our previous study has shown that genistein enhances apoptosis in A549 lung cancer cells induced by trichostatin A (TSA). The precise molecular mechanism underlying the effect of genistein, however, remains unclear. In the present study, we investigated whether genistein enhances the anti-cancer effect of TSA through up-regulation of TNF receptor-1 (TNFR-1) death receptor signaling. We incubated A549 cells with TSA (50 ng/mL) alone or in combination with genistein and then determined the mRNA and protein expression of TNFR-1 as well as the activation of downstream caspases. Genistein at 5 and 10 μM significantly enhanced the TSA-induced decrease in cell number and apoptosis in a dose-dependent manner. The combined treatment significantly increased mRNA and protein expression of TNFR-1 at 6 and 12h, respectively, compared with that of the control group; while TSA alone had no effect. TSA in combination with 10 μM of genistein increased TNFR-1 mRNA and protein expression by about 70% and 40%, respectively. The underlying mechanism for this effect of genistein may be partly associated with the estrogen receptor pathway. The combined treatment also increased the activation of caspase-3 and -10 as well as p53 protein expression in A549 cells. The enhancing effects of genistein on the TSA-induced decrease in cell number and on the expression of caspase-3 in A549 cells were suppressed by silencing TNFR-1 expression. These data demonstrated that the upregulation of TNFR-1 death receptor signaling plays an important role, at least in part, in the enhancing effect of genistein on TSA-induced apoptosis in A549 cells.

Topics: Apoptosis; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Caspases; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Synergism; Genistein; Humans; Hydroxamic Acids; Lung Neoplasms; Receptors, Tumor Necrosis Factor, Type I; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Up-Regulation

There is a renewed focus on targeted therapy against epigenetic events that are altered during the pathogenesis of lung cancer. However, the use of epigenomic modifiers as monotherapy lacks efficacy; thus, there is a need to develop safe and effective drug combinatorial regimens, which reverse epigenetic modifications and exhibit profound anticancer activity. Based on these perspectives, we evaluated, for the first time, the efficacy and associated mechanisms of a novel combinatorial regimen of histone deacetylase inhibitors (HDACi)-trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA)-with silibinin (a flavonolignan with established pre-clinical anti-lung cancer efficacy) against non-small cell lung cancer (NSCLC). Silibinin inhibited HDAC activity and decreased HDAC1-3 levels in NSCLC cells, leading to an overall increase in global histone acetylation states of histones H3 and H4. Combinations of HDCAi with silibinin synergistically augmented the cytotoxic effects of these single agents, which was associated with a dramatic increase in p21 (Cdkn1a). Subsequent ChIP assay indicated increased acetylated histone H3 and H4 levels on p21 promoter region, resulting in its increased transcription. The enhanced p21 levels promoted proteasomal degradation of cyclin B1, the limited supply of which halts the progression of cells into mitosis. Indeed, the resultant biological effect was a significant G 2/M arrest by the combination treatment, followed by apoptotic cell death. Similar epigenetic modulations were observed in vivo, together with a marked reduction in xenograft growth. These findings are both novel and highly significant in establishing that HDACi with silibinin would be safe and effective to suppress NSCLC growth.

Topics: Acetylation; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cyclin B1; Cyclin-Dependent Kinase Inhibitor p21; Drug Synergism; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Silybin; Silymarin; Vorinostat

Lung cancer is the leading cause of cancer-related mortality and requires more effective molecular markers of prognosis and therapeutic responsiveness. Special AT-rich binding protein 1 (SATB1) is a global genome organizer that recruits chromatin remodeling proteins to epigenetically regulate hundreds of genes in a tissue-specific manner. Initial studies suggest that SATB1 overexpression is a predictor of poor prognosis in breast cancer, but the prognostic significance of SATB1 expression has not been evaluated in lung cancer.. A cohort of 257 lung cancers was evaluated by immunohistochemistry. Epigenetic silencing of SATB1 was examined in cell lines by 5-Aza 2-deoxycytidine and trichostatin A treatment, and chromatin immunoprecipitation.. Significant loss of SATB1 expression was found in squamous preinvasive lesions (p < 0.04) and in non-small cell lung cancers (p < 0.001) compared with matched normal bronchial epithelium. Loss of SATB1 independently predicted poor cancer-specific survival in squamous cell carcinomas (SCCs; hazard ratio: 2.06, 95% confidence interval: 1.2-3.7, p = 0.016). Treatment of lung cancer cell lines with the histone deacetylase inhibitor trichostatin A resulted in up-regulation of SATB1. SATB1 was associated with a decrease in the active chromatin mark acetylated histone H3K9 and an increase in the repressive polycomb mark trimethylated H3K27 in a SCC cell line relative to a normal bronchial epithelial cell line.. This is the first study showing that SATB1 expression is lost in early preinvasive squamous lesions and that loss of SATB1 is associated with poor prognosis in lung SCC. We hypothesize that the SATB1 gene is epigenetically silenced through histone modifications.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Antimetabolites, Antineoplastic; Azacitidine; Blotting, Western; Carcinoma, Large Cell; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Line, Tumor; Chromatin Immunoprecipitation; Cohort Studies; Decitabine; DNA Methylation; Epigenesis, Genetic; Female; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunoenzyme Techniques; Lung Neoplasms; Male; Matrix Attachment Region Binding Proteins; Middle Aged; Neoplasm Staging; Prognosis; Tissue Array Analysis

Histone deacetylase inhibitors (HDACIs) induce growth arrest and apoptosis in cancer cells. In addition to their intrinsic anticancer properties, HDACIs modulate cellular responses to ionizing radiation (IR). We examined the molecular mechanism(s) associated with the radiosensitizing effects of HDACIs in human lung cancer cells.. Lung cancer cells were pretreated with the appropriate concentrations of suberoylanilide hydroxamic acid or trichostatin A. After 2 hours, cells were irradiated with various doses of γ-IR, and then we performed 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, fluorescence-activated cell sorting analysis, clonogenic assay, and Western blotting to detect cell viability or apoptosis and changes of specific proteins expression levels.. In this study, we showed that HDACIs (including suberoylanilide hydroxamic acid and trichostatin A) and IR synergistically trigger cell death in human non-small cell lung cancer cells. Cell viability and clonogenic survival were markedly decreased in cultures cotreated with HDACIs and IR. Interestingly, p53 acetylation at lysine 382 was significantly increased, and c-myc expression simultaneously down-regulated in cotreated cells. Radiosensitization by HDACIs was inhibited on transfection with small interfering RNA against p53 and c-myc overexpression, supporting the involvement of p53 and c-myc in this process. Furthermore, c-myc down-regulation and apoptotic cell death coinduced by IR and HDACI were suppressed in cells transfected with mutant K382R p53 and C135Y p53 displaying loss of acetylation at lysine 382 and DNA-binding activity, respectively.. Our results collectively demonstrate that the degree of radiosensitization by HDACIs is influenced by acetyl p53-mediated c-myc down-regulation.

Topics: Acetylation; Apoptosis; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Combined Modality Therapy; Down-Regulation; Flow Cytometry; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lung Neoplasms; Membrane Potential, Mitochondrial; Mutagenesis, Site-Directed; Mutation; Proto-Oncogene Proteins c-myc; Radiation-Sensitizing Agents; Radiation, Ionizing; Tumor Suppressor Protein p53; Vorinostat

The combination of anti-cancer drugs with nutritional factors is a potential strategy for improving the efficacy and decreasing the toxicity of chemotherapy. However, whether nutritional factors enhance the effect of trichostatin A (TSA), a novel anti-cancer drug, is unclear.. We investigated the individual enhancing effect and its possible mechanisms of genistein, daidzein, beta-carotene, retinoic acid, and alpha-tocopherol on the cell-growth-inhibitory effect of TSA in a human lung carcinoma cell line, A549.. A549 cells were incubated with TSA (50 ng/mL) alone or in combination with the various nutritional factors for various times, and cell growth was measured. IMR90 cells, human lung fibroblasts, were also incubated with TSA alone or in combination with genistein or beta-carotene to determine the selectivity of these treatments. In addition, we studied effects on the cell cycle, caspase-3 activity, and DNA damage (by comet assay) in A549 cells.. After treatment for 72 h, 10-microM genistein or beta-carotene significantly enhanced the growth-inhibitory effect of TSA in A549 cells. Daidzein, retinoic acid, and alpha-tocopherol at the same concentration had no significant effect. However, genistein and beta-carotene failed to enhance the cell-growth-arrest effect of TSA in IMR90 cells. Flow cytometric analysis showed that both genistein and beta-carotene significantly increased the TSA-induced apoptosis in A549 cells. Genistein significantly enhanced TSA-induced caspase-3 activity in A549 cells by 34% at 24 h, and the caspase-3 inhibitor partly inhibited the enhancing effect of genistein on TSA-induced apoptosis. beta-Carotene did not significantly affect TSA-induced caspase-3 activity. However, beta-carotene rather than genistein enhanced TSA-induced DNA damage.. Genistein and beta-carotene enhance the cell-growth-arrest effect of TSA on A549 cells. Genistein exerts its effect, at least partly, by increasing caspase-3 activity; whereas beta-carotene may enhance TSA-induced cell death mainly through a caspase-3-independent pathway.

Topics: Antineoplastic Agents; Apoptosis; beta Carotene; Carcinoma, Non-Small-Cell Lung; Caspase 3; Cell Cycle; Cell Division; Cell Line; Cell Line, Tumor; DNA Damage; Drug Interactions; Enzyme Activation; Fibroblasts; Genistein; Humans; Hydroxamic Acids; Lung

Cisplatin-based chemotherapy is the paradigm of non-small-cell lung cancer (NSCLC) treatment; however, it also induces de novo DNA-hypermethylation, a process that may be involved in the development of drug-resistant phenotypes by inactivating genes required for drug-cytotoxicity. By using an expression microarray analysis, we aimed to identify those genes reactivated in a set of two cisplatin (CDDP) resistant and sensitive NSCLC cell lines after epigenetic treatment. Gene expression, promoter methylation and CDDP-chemoresponse were further analyzed in three matched sets of sensitive/resistant cell lines, 23 human cancer cell lines and 36 NSCLC specimens. Results revealed specific silencing by promoter hypermethylation of IGFBP-3 in CDDP resistant cells, whereas IGFBP-3 siRNA interference, induced resistance to CDDP in sensitive cells (P<0.001). In addition, we found a strong correlation between methylation status and CDDP response in tumor specimens (P<0.001). Thus, stage I patients, whose tumors harbor an unmethylated promoter, had a trend towards increased disease-free survival (DFS). We report that a loss of IGFBP-3 expression, mediated by promoter-hypermethylation, results in a reduction of tumor cell sensitivity to cisplatin in NSCLC. Basal methylation status of IGFBP-3 before treatment may be a clinical biomarker and a predictor of the chemotherapy outcome, helping to identify patients who are most likely to benefit from CDDP therapy alone or in combination with epigenetic treatment.

Topics: Antineoplastic Agents; Azacitidine; Base Sequence; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Survival; Cisplatin; DNA Methylation; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; HeLa Cells; HT29 Cells; Humans; Hydroxamic Acids; Insulin-Like Growth Factor Binding Protein 3; Kaplan-Meier Estimate; Lung Neoplasms; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Tumor Cells, Cultured

Histone deacetylase (HDAC) inhibitors (HDIs) play an important role in the regulation of gene expression associated with cell cycle and apoptosis and have emerged as promising anticancer agents. In addition to their intrinsic anticancer properties, some studies have demonstrated that HDIs can modulate cellular responses to ionizing radiation (IR). Here we show evidence that co-treatment with the HDI trichostatin A (TSA) radiosensitizes human non-small cell lung cancer (NSCLC) A549 cells and H1650 cells. Cells were exposed to gamma-irradiation with or without TSA co-treatment. Clonogenic survival was significantly reduced in cells with TSA co-treatment. In A549 cells, TSA enhanced IR-induced accumulation of cells in G(2)/M phase, with upregulated expression of p21(waf1/cip1). In addition, TSA co-treatment caused pronounced apoptosis in irradiated cells, which was accompanied with p21(waf1/cip1) cleavage to a 15 kDa protein. The enhanced apoptotic effect was via mitochondrial pathway, as indicated by the increased dissipation of mitochondrial transmembrane potential (MMP) and release of cytochrome c from the mitochondria to the cytoplasm. Caspase-3 activation was also significantly increased, with accordingly more cleavage of PARP, associated with the repression of X-linked inhibitor of apoptosis protein (XIAP). Furthermore, TSA co-treatment impaired DNA repair capacity after IR by downregulation of Ku70, Ku80 and DNA-PKcs, reflected by enhanced and prolonged expression of gammaH2AX. Taken together, our results demonstrate that TSA acts as a powerful radiosensitizer in NSCLC cells by enhancing G(2)/M cell cycle arrest, promoting apoptosis through multiple pathways and interfering with DNA damage repair processes.

Topics: Apoptosis; Carcinoma, Non-Small-Cell Lung; Caspase 3; Cell Cycle; Cell Line, Tumor; Combined Modality Therapy; DNA Repair; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Down-Regulation; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Lung Neoplasms; Membrane Potential, Mitochondrial; Radiation Dosage; Radiation Tolerance; Radiation-Sensitizing Agents

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Non-Small-Cell Lung; Drug Screening Assays, Antitumor; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lung Neoplasms; Tumor Cells, Cultured; Vorinostat

Cyclooxygenase (COX)-2 is frequently overexpressed in non-small cell lung cancer (NSCLC) and results in increased levels of prostaglandin E2 (PGE(2)), an important signalling molecule implicated in tumourigenesis. PGE(2) exerts its effects through the E prostanoid (EP) receptors (EPs1-4).. The expression and epigenetic regulation of the EPs were evaluated in a series of resected fresh frozen NSCLC tumours and cell lines.. EP expression was dysregulated in NSCLC being up and downregulated compared to matched control samples. For EPs1, 3 and 4 no discernible pattern emerged. EP2 mRNA however was frequently downregulated, with low levels being observed in 13/20 samples as compared to upregulation in 5/20 samples examined. In NSCLC cell lines DNA CpG methylation was found to be important for the regulation of EP3 expression, the demethylating agent decitabine upregulating expression. Histone acetylation was also found to be a critical regulator of EP expression, with the histone deacteylase inhibitors trichostatin A, phenylbutyrate and suberoylanilide hydroxamic acid inducing increased expression of EPs2-4. Direct chromatin remodelling was demonstrated at the promoters for EPs2-4.. These results indicate that EP expression is variably altered from tumour to tumour in NSCLC. EP2 expression appears to be predominantly downregulated and may have an important role in the pathogenesis of the disease. Epigenetic regulation of the EPs may be central to the precise role COX-2 may play in the evolution of individual tumours.

Topics: Acetylation; Antineoplastic Agents; Azacitidine; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Chromatin Assembly and Disassembly; CpG Islands; Decitabine; DNA Methylation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Lung Neoplasms; Phenylbutyrates; Receptors, Prostaglandin E; RNA, Messenger; Tumor Cells, Cultured; Vorinostat

Loss of E-cadherin confers a poor prognosis in lung cancer patients and is associated with in vitro resistance to endothelial growth factor receptor inhibitors. Zinc finger E box-binding homeobox (ZEB)-1, the predominant transcriptional suppressor of E-cadherin in lung tumor lines, recruits histone deacetylases (HDACs) as co-repressors.. NSCLC cell lines were treated with HDAC inhibitors and analyzed for E-cadherin induction, growth inhibition and apoptosis. National Cancer Institute-H157 cells expressing ectopic E-cadherin were tested for tumorigenicity in murine xenografts.. We found that treatment with MS-275, compared to vorinostat (SAHA), valproic acid or trichostatin A, was most effective in E-cadherin up-regulation and persistence in non-small cell lung cancers. As with other tumor types and HDAC inhibitors, MS-275 inhibited growth and induced apoptosis. Importantly, blocking E-cadherin induction by short hairpin RNA resulted in less inhibition by MS-275, implicating the epithelial to mesenchymal phenotype process as a contributing factor. In contrast to H460 and H661, H157 cells were resistant to E-cadherin up-regulation by HDAC inhibitors. However, E-cadherin was restored, in a synergistic manner, by combined knockdown of ZEB-1 and ZEB-2. In addition, H157 cells stably transfected with E-cadherin were markedly attenuated in their tumor forming ability. Lastly, combining MS-275 with the microtubule stabilizing agent, paclitaxel, or 17-(allylamino)-17-demethoxygeldanamycin, a heat shock protein 90 inhibitor, resulted in synergistic growth inhibition. Since MS-275 has no reported activity against HDAC6, which regulates both microtubule and heat shock protein 90 functions, other mechanisms of synergy are anticipated.. These results support the role of ZEB proteins and HDAC inhibitors in the pathogenesis and treatment of lung cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; Benzamides; Benzoquinones; Blotting, Western; Cadherins; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Female; Flow Cytometry; Histone Deacetylase Inhibitors; Histone Deacetylases; Homeodomain Proteins; HSP90 Heat-Shock Proteins; Humans; Hydroxamic Acids; Lactams, Macrocyclic; Lung Neoplasms; Pyridines; Rats; Rats, Nude; Repressor Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Transcription Factors; Tumor Cells, Cultured; Up-Regulation; Vorinostat; Zinc Finger E-box Binding Homeobox 2; Zinc Finger E-box-Binding Homeobox 1

To ascertain the potential for histone deacetylase (HDAC) inhibitor-based treatment in non-small cell lung cancer (NSCLC), we analyzed the antitumor effects of trichostatin A (TSA) and suberoylanilide hydroxamic acid (vorinostat) in a panel of 16 NSCLC cell lines via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. TSA and vorinostat both displayed strong antitumor activities in 50% of NSCLC cell lines, suggesting the need for the use of predictive markers to select patients receiving this treatment. There was a strong correlation between the responsiveness to TSA and vorinostat (P < 0.0001). To identify a molecular model of sensitivity to HDAC inhibitor treatment in NSCLC, we conducted a gene expression profiling study using cDNA arrays on the same set of cell lines and related the cytotoxic activity of TSA to corresponding gene expression pattern using a modified National Cancer Institute program. In addition, pathway analysis was done with Pathway Architect software. We used nine genes, which were identified by gene-drug sensitivity correlation and pathway analysis, to build a support vector machine algorithm model by which sensitive cell lines were distinguished from resistant cell lines. The prediction performance of the support vector machine model was validated by an additional nine cell lines, resulting in a prediction value of 100% with respect to determining response to TSA and vorinostat. Our results suggested that (a) HDAC inhibitors may be promising anticancer drugs to NSCLC and (b) the nine-gene classifier is useful in predicting drug sensitivity to HDAC inhibitors and may contribute to achieving individualized therapy for NSCLC patients.

Topics: Algorithms; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Inhibitory Concentration 50; Lung Neoplasms; Models, Biological; Reproducibility of Results

Analysis of biopsies from a recent clinical trial suggested that Depsipeptide FK228 (DP) inhibits Aurora kinase expression in lung cancer cells. The present study was undertaken to confirm and extend these observations.. Aurora A and B mRNA levels in lung cancer cells were considerably higher than levels in normal pulmonary epithelia. DP, TSA and SAHA inhibited Aurora A, Aurora B and survivin expression with kinetics that were remarkably similar within individual cell lines, and appeared to coincide with p53 expression status. These effects were not observed following treatment with geldanamycins. Inhibition of Aurora B transcription coincided with decreased H3K9Ac and H3K4Me2 activation marks, and accumulation of H3K9Me3, as well as MBD1, MBD2 and MBD3 repression marks within the minimal Aurora B promoter. Knockdown of MBD1, -2 or -3 did not reproducibly abrogate inhibition of Aurora or survivin expression by DP or TSA. DP and TSA decreased expression and altered localization of Aurora kinases and survivin, resulting in mitotic catastrophe in lung cancer cells.. Aurora A, and Aurora B levels in lung cancer cells and normal respiratory epithelia were assessed using quantitative RT-PCR techniques. These methods, as well as as Western blots were used to examine expression of Auroras A/B, and several related genes/proteins in lung cancer cells exposed to DP, TSA, SAHA and geldanamycins. Transient transfection promoter-reporter assays, and chromatin immunoprecipitation (ChIP) techniques were used to examine DP-mediated changes in activity and chromatin structure of the Aurora B promoter. Confocal imaging techniques were used to examine the effects of DP and TSA on mitotic progression in lung cancer cells.. Novel transcriptional regulatory mechanisms involving Aurora kinase and survivin appear to contribute to cytotoxicity mediated by HDAC inhibitors in lung cancer cells.

Topics: Aurora Kinase B; Aurora Kinases; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Clinical Trials, Phase II as Topic; Depsipeptides; Drug Delivery Systems; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunohistochemistry; Inhibitor of Apoptosis Proteins; Kinetics; Lung Neoplasms; Microtubule-Associated Proteins; Neoplasm Proteins; Protein Serine-Threonine Kinases; RNA, Messenger; Survivin; Transcription, Genetic; Vorinostat

The insulin-receptor substrate family plays important roles in cellular growth, signaling, and survival. Two new members of this family have recently been isolated: IRS5/Dok4 and IRS6/Dok5. This study examines the expression of IRS5/DOK4 in a panel of lung cancer cell lines and tumor specimens. The results demonstrate that expression of IRS5/DOK4 is frequently altered with both elevated and decreased expression in non-small-cell lung cancer (NSCLC) tumor specimens. The altered expression of IRS5/DOK4 observed in tumor samples is not due to aberrant methylation. In vitro cell culture studies demonstrate that treatment of NSCLC cell lines with the histone deacetylase inhibitor trichostatin A (TSA) upregulates IRS5/DOK4. This finding indicates that expression is regulated epigenetically at the level of chromatin remodeling. Chromatin immunoprecipitation experiments confirm that the IRS5/DOK4 promoter has enhanced histone hyperacetylation following treatments with TSA. Finally, hypoxia was demonstrated to downregulate IRS5/DOK4 expression. This expression was restored by TSA. The clinical relevance of altered IRS5/DOK4 expression in NSCLC requires further evaluation.

Topics: Acetylation; Adaptor Proteins, Signal Transducing; Adenocarcinoma; Base Sequence; Bronchi; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Chromatin Assembly and Disassembly; Chromatin Immunoprecipitation; DNA Methylation; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Intracellular Signaling Peptides and Proteins; Lung; Lung Neoplasms; Molecular Sequence Data; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription, Genetic

Commonly used regimens in cancer therapy rely on the induction of apoptotic cell death, and drug resistance can be attributed, at least in part, to a disabled apoptotic program. Non-small cell lung carcinomas (NSCLC), exhibit an intrinsic resistance to chemotherapy. Here, we show that co-treatment with etoposide (VP16) and the pan-histone deacetylase (HDAC) inhibitor trichostatin A (TSA), but not valproic acid (VPA), induced apoptotic cell death in drug-resistant NSCLC cells. Co-treatment, but not single treatment, with VP16 and TSA induced apoptosis in a caspase-dependent manner accompanied by a crucial decrease in Bcl-xL expression allowing Bax activation and subsequent initiation of the apoptosis inducing factor (AIF)-dependent death pathway. Importantly, AIF proved to be required for the effects of TSA/VP16 as RNA knockdown of AIF resulted in a complete abolishment of TSA/VP16-induced apoptotic cell death in drug-resistant NSCLC cells. Our results thus provide evidence for the requirement of both caspase-dependent and caspase-independent apoptotic pathways in TSA/VP16-mediated death of drug-resistant NSCLC cells, and extend previous suggestions that HDAC inhibitors in combination with conventional chemotherapeutic drugs could be valuable in the treatment of NSCLC cancer and other malignancies in which Bcl-xL is overexpressed.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Inducing Factor; bcl-2-Associated X Protein; bcl-X Protein; Carcinoma, Non-Small-Cell Lung; Caspase Inhibitors; Caspases; Drug Evaluation, Preclinical; Etoposide; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lung Neoplasms; Models, Biological; RNA, Small Interfering; Signal Transduction; Tumor Cells, Cultured

New compounds derived from inhibitors of histone deacetylases (HDACs) have been synthesized and their antiproliferative activities towards non small lung cancer cell line H661 evaluated. Their design is based on hybrids between indanones to limit conformational mobility and other known HDAC inhibitors (SAHA, MS-275). The synthesis of these new derivatives was achieved by alkylation of appropriate indanones to introduce the side chain bearing a terminal ester group, the latter being a precursor of hydroxamic acid and aminobenzamide derivatives. These new analogues were found to be moderately active to inhibit H661 cell proliferation.

Topics: Antineoplastic Agents; Benzamides; Binding Sites; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Drug Screening Assays, Antitumor; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Indans; Molecular Structure; Pyridines; Structure-Activity Relationship; Vorinostat

Epigenetic changes involved in cancer development, unlike genetic changes, are reversible. DNA methyltransferase and histone deacetylase inhibitors show antiproliferative effects in vitro, through tumor suppressor reactivation and induction of apoptosis. Such inhibitors have shown activity in the treatment of hematologic disorders but there is little data concerning their effectiveness in treatment of solid tumors. FHIT, WWOX and other tumor suppressor genes are frequently epigenetically inactivated in lung cancers. Lung cancer cell clones carrying conditional FHIT or WWOX transgenes showed significant suppression of xenograft tumor growth after induction of expression of the FHIT or WWOX transgene, suggesting that treatments to restore endogenous Fhit and Wwox expression in lung cancers would result in decreased tumorigenicity. H1299 lung cancer cells, lacking Fhit, Wwox, p16(INK4a) and Rassf1a expression due to epigenetic modifications, were used to assess efficacy of epigenetically targeted protocols in suppressing growth of lung tumors, by injection of 5-aza-2-deoxycytidine (AZA) and trichostatin A (TSA) in nude mice with established H1299 tumors. High doses of intraperitoneal AZA/TSA suppressed growth of small tumors but did not affect large tumors (200 mm(3)); lower AZA doses, administered intraperitoneally or intratumorally, suppressed growth of small tumors without apparent toxicity. Responding tumors showed restoration of Fhit, Wwox, p16(INKa), Rassf1a expression, low mitotic activity, high apoptotic fraction and activation of caspase 3. These preclinical studies show the therapeutic potential of restoration of tumor suppressor expression through epigenetic modulation and the promise of re-expressed tumor suppressors as markers and effectors of the responses.

Topics: Acid Anhydride Hydrolases; Animals; Apoptosis; Azacitidine; Carcinoma, Non-Small-Cell Lung; Caspases; Cyclin-Dependent Kinase Inhibitor p16; Decitabine; DNA Methylation; DNA Modification Methylases; Enzyme Inhibitors; Epigenesis, Genetic; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Proteins; Oxidoreductases; Promoter Regions, Genetic; Transgenes; Transplantation, Heterologous; Tumor Cells, Cultured; Tumor Suppressor Proteins; WW Domain-Containing Oxidoreductase

Lung cancer is the leading cause of cancer-related deaths in the United States due, in large part, to the lack of early detection methods. Lung cancer arises from a complex series of genetic and epigenetic changes leading to uncontrolled cell growth and metastasis. Unlike genetic changes, epigenetic changes, such as DNA methylation and histone acetylation, are reversible with currently available pharmaceuticals and are early events in lung tumorigenesis detectable by non-invasive methods. In order to better understand how epigenetic changes contribute to lung cancer, and to identify new disease biomarkers, we combined pharmacologic inhibition of DNA methylation and histone deacetylation in non-small cell lung cancer (NSCLC) cell lines, with genome-wide expression profiling. Of the more than 200 genes upregulated by these treatments, three of these, neuronatin, metallothionein 3 and cystatin E/M, were frequently hypermethylated and transcriptionally downregulated in NSCLC cell lines and tumors. Interestingly, four other genes, cylindromatosis, CD9, activating transcription factor 3 and oxytocin receptor, were dominantly regulated by histone deacetylation and were also frequently downregulated in lung tumors. The majority of these genes also suppressed NSCLC growth in culture when ectopically expressed. This study therefore reveals new putative NSCLC growth regulatory genes and epigenetic disease biomarkers that may enhance early detection strategies and serve as therapeutic targets.

Topics: Acetylation; Adenocarcinoma; Azacitidine; Biomarkers, Tumor; Carcinoma, Large Cell; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; Chromatin Immunoprecipitation; Colony-Forming Units Assay; DNA Methylation; Enzyme Inhibitors; Epigenesis, Genetic; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lung Neoplasms; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured

Identification of a homozygous deletion in cancer cells provides strong evidence for the location of a tumor suppressor gene (TSG). We analyzed the 2p24 homozygous deletion of a non-small-cell lung cancer (NSCLC) cell line, NCI-H2882, and found that the deletion size was 3.7 Mbp. Since RhoB, which has been suggested to be a candidate TSG, was located in this region, we analyzed RhoB for alterations in NSCLC. Although we found no mutations in 48 cell lines including 20 NSCLCs, a loss of heterozygosity (LOH) analysis in 128 primary NSCLCs showed that 25 of 62 informative samples had LOH at the RhoB locus. Northern blot analysis of 28 cell lines (including 15 NSCLCs) indicated that RhoB expression was downregulated in 27. We analyzed RhoB expression in 112 primary NSCLCs with immunohistochemistry and found no or a weak RhoB expression in 33 (42%) of 78 adenocarcinomas, whereas we found it in 29 (94%) of 31 squamous cell carcinomas. No or a weak expression of RhoB was more frequently observed in poorly- or moderately-differentiated adenocarcinomas than in well-differentiated ones (p = 0.0014). Furthermore, no or a weak expression of RhoB indicated a tendency to poor patient prognosis. Although hypermethylation was not found at the promoter region, the RhoB expression in NSCLC cell lines was induced by histone deacetylase inhibition, suggesting that RhoB downregulation may be due to histone modification. The present study demonstrates that RhoB expression is frequently downregulated in NSCLCs by multiple mechanisms, suggesting that RhoB is a candidate TSG for NSCLC.

Topics: Aged; Blotting, Northern; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Chromosome Deletion; Chromosome Mapping; Chromosomes, Human, Pair 2; DNA Methylation; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunohistochemistry; Kaplan-Meier Estimate; Loss of Heterozygosity; Lung Neoplasms; Male; Microsatellite Repeats; Middle Aged; Prognosis; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; rhoB GTP-Binding Protein

A well-known histone deacetylase inhibitor, trichostatin A, was applied to non-small-cell lung cancer cells to determine whether inhibition of histone deacetylase leads to the production of proteins that either arrest tumor cell growth or lead to tumor cell death.. Trichostatin A (0.01 to 1.0 micromol/L) was applied to one normal lung fibroblast and four non-small-cell lung cancer lines, and its effect was determined by flow cytometry, annexin-V staining, immunoprecipitation, and Western blot analysis.. Trichostatin A demonstrated tenfold greater growth inhibition in all four non-small-cell lung cancer lines compared with normal controls, with a concentration producing 50% inhibition ranging from 0.01 to 0.04 micromol/L for the tumor cell lines and 0.7 micromol/L for the normal lung fibroblast line. Trichostatin A treatment reduced the percentage of cells in S phase (10% to 23%) and increased G1 populations (10% to 40%) as determined by flow cytometry. Both annexin-V binding assay and upregulation of the protein, gelsolin (threefold to tenfold), demonstrated that the tumor cells were apoptotic, whereas normal cells were predominantly in cell cycle arrest. Trichostatin A increased histone H4 acetylation and expression of p21 twofold to 15-fold without significant effect on p16, p27, CDK2, and cyclin D1.. Collectively, these data suggest that inhibition of histone deacetylation may provide a valuable approach for lung cancer treatment. We evaluated trichostatin A as a potential candidate for anticancer therapy in non-small-cell lung cancer.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Line; Cell Line, Tumor; Cell Survival; Cyclin D1; Enzyme Inhibitors; Gelsolin; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Lung; Lung Neoplasms

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Line; Cell Line, Tumor; Enzyme Inhibitors; Humans; Hydroxamic Acids; Lung; Lung Neoplasms; Protein Synthesis Inhibitors

Loss of tumor suppressor CCAAT/enhancer-binding protein-alpha (C/EBPalpha) expression is seen in several human malignancies, including acute myelogenous leukemia and lung cancer. We hypothesized that DNA methylation and histone acetylation of the C/EBPalpha promoter may modulate C/EBPalpha expression in lung cancer.. We analyzed C/EBPalpha expression in 15 human lung cancer cell lines and in 122 human lung primary tumors by northern blotting, immunoblotting, and immunohistochemistry. C/EBPalpha promoter methylation was assessed using bisulfite sequencing, combined bisulfite restriction analysis, methylation-specific polymerase chain reaction, and Southern blotting. We examined the acetylation status of histones H3 and H4 at the C/EBPalpha promoter by chromatin immunoprecipitation. Binding of methyl-CpG-binding proteins MeCP2 and MBD2 and upstream stimulatory factor (USF) to the C/EBPalpha promoter was assayed in cell lines that were untreated or treated with histone deacetylase inhibitor trichostatin A and demethylating agent 5-aza-2'-deoxycytidine (5-aza-dC) by chromatin immunoprecipitation and by electrophoretic mobility shift assays.. DNA methylation and histone acetylation in the upstream region (-1422 to -896) of the C/EBPalpha promoter were associated with low or absent C/EBPalpha expression in 12 of 15 lung cancer cell lines and in 81 of 120 primary lung tumors. MeCP2 and MBD binding to the upstream C/EBPalpha promoter was detected in C/EBPalpha-nonexpressing cell lines; USF binding was detected in C/EBPalpha-expressing cell lines; however, in C/EBPalpha-nonexpressing cell lines USF binding was detected only after trichostatin A and 5-aza-dC treatment.. DNA hypermethylation of the upstream C/EBPalpha promoter region, not the core promoter region as previously reported, is critical in the regulation of C/EBPalpha expression in human lung cancer.

Topics: Acetylation; Azacitidine; Blotting, Northern; Blotting, Southern; Carcinoma, Non-Small-Cell Lung; CCAAT-Enhancer-Binding Protein-alpha; Cell Line, Tumor; Chromatin Immunoprecipitation; CpG Islands; DNA Methylation; DNA-Binding Proteins; Electrophoretic Mobility Shift Assay; Enzyme Inhibitors; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunoblotting; Immunohistochemistry; Lung Neoplasms; Methyl-CpG-Binding Protein 2; Mutagenesis; Promoter Regions, Genetic; Sequence Analysis, DNA; Upstream Stimulatory Factors

Recently, the inhibition of histone deacetylase (HDAC) enzymes has attracted attention in the oncologic community as a new therapeutic opportunity for hematologic and solid tumors including non-small cell lung cancer (NSCLC). In hematologic malignancies, such as diffuse large B-cell lymphoma, the HDAC inhibitor (HDI), suberoylanilide hydroxamic acid (SAHA), has recently entered phase II and III clinical trials. To further advance our understanding of their action on tumor cells, we investigated the possible effect of HDI treatment on the functionality of the nuclear factor-kappaB (NF-kappaB) pathway in NSCLC. We found that in the NSCLC cell lines, A549 and NCI-H460, the NF-kappaB pathway was strongly inducible, for example, by stimulation with tumor necrosis factor-alpha (TNF-alpha). Incubation of several NSCLC cell lines with HDIs resulted in greatly reduced gene expression of TNF-alpha receptor-1. HDI-treated A549 and NCI-H460 cells down-regulated TNF-alpha receptor-1 mRNA and protein levels as well as surface exposure, and consequently responded to TNF-alpha treatment with reduced IKK phosphorylation and activation, delayed IkappaB-alpha phosphorylation, and attenuated NF-kappaB nuclear translocation and DNA binding. Accordingly, stimulation of NF-kappaB target gene expression by TNF-alpha was strongly decreased. In addition, we observed that SAHA displayed antitumor efficacy in vivo against A549 xenografts grown on nude mice. HDIs, therefore, might beneficially contribute to tumor treatment, possibly by reducing the responsiveness of tumor cells to the TNF-alpha-mediated activation of the NF-kappaB pathway. These findings also hint at a possible use of HDIs in inflammatory diseases, which are associated with the overproduction of TNF-alpha, such as rheumatoid arthritis or Crohn's disease.

Topics: Animals; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Growth Processes; Cell Line, Tumor; DNA, Neoplasm; Down-Regulation; Enzyme Inhibitors; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; I-kappa B Kinase; Lung Neoplasms; Mice; Mice, Nude; NF-kappa B; Phosphorylation; Receptors, Tumor Necrosis Factor, Type I; Tumor Necrosis Factor-alpha; Vorinostat; Xenograft Model Antitumor Assays

Although histone deacetylase (HDAC) inhibitors are emerging as a promising new treatment strategy in malignancy, how they exert their effect on human non-small cell lung cancer cells is as yet unclear. This study was undertaken to investigate the underlying mechanism of an HDAC inhibitor, Trichostatin A (TSA), -induced apoptosis in a human lung carcinoma cell line A549. The effects of this compound were also tested on cyclooxygenase (COX) activity. Treatment of A549 cells to TSA resulted in the inhibition of viability and the induction of apoptosis in a concentration-dependent manner, which could be proved by trypan blue counts, DAPI staining, agarose gel electrophoresis and flow cytometry analysis. Apoptosis of A549 cells by TSA was associated with a down-regulation of anti-apoptotic Bcl-2 protein and an up-regulation of pro-apoptotic Bax protein. TSA treatment induced the proteolytic activation of caspase-3 and caspase-9, and a concomitant degradation of poly(ADP-ribose)-polymerase protein. Furthermore, TSA decreased the levels of COX-2 mRNA and protein expression without significant changes in the levels of COX-1, which was correlated with an inhibition in prostaglandin E2 synthesis. Taken together, these findings provide important new insights into the possible molecular mechanisms of the anti-cancer activity of TSA.

Topics: Apoptosis; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Caspase 3; Caspase 9; Caspases; Cell Line, Tumor; Cell Survival; Dinoprostone; DNA Fragmentation; Dose-Response Relationship, Drug; Enzyme Inhibitors; Flow Cytometry; Gene Expression Regulation, Enzymologic; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Lung Neoplasms; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction

Topics: Azacitidine; beta Catenin; Carcinoma, Non-Small-Cell Lung; Cell Division; Cell Line, Tumor; Cytoskeletal Proteins; Decitabine; Desmoplakins; DNA Modification Methylases; gamma Catenin; Humans; Hydroxamic Acids; Lung Neoplasms; Neoplasm Proteins; Trans-Activators

Histone deacetylase (HDAC) inhibitors and ligands of the peroxisome proliferator-activated receptor gamma (PPARgamma) have been shown previously to induce growth arrest and differentiation in a variety of cancer cell lines. The purpose of this study was to determine whether HDAC inhibitors function similarly in non-small cell lung cancer (NSCLC) and whether combination treatment with HDAC inhibitors and PPARgamma ligands is more efficacious than either agent alone.. Nanomolar concentrations of trichostatin A induced growth arrest in five of seven NSCLC cell lines, whereas sodium phenylbutyrate (PB) was markedly less potent. In adenocarcinomas, trichostatin A up-regulated general differentiation markers (gelsolin, Mad, and p21/WAF1) and down-regulated markers of the type II pneumocyte progenitor cell lineage (MUC1 and SP-A), indicative of a more mature phenotype. PB had a similar effect. Simultaneous treatment with a PPARgamma ligand and PB enhanced the growth inhibition in adenocarcinomas but not in nonadenocarcinomas. Growth arrest was accompanied by markedly decreased cyclin D1 expression but not enhanced differentiation.. The present study demonstrates potent growth-inhibitory and differentiation-inducing activity of HDAC inhibitors in NSCLC and suggests that combination differentiation therapy should be explored further for the treatment of lung adenocarcinomas.

Topics: Adenocarcinoma; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Blotting, Western; Carcinoma, Non-Small-Cell Lung; Cell Differentiation; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA-Binding Proteins; Dose-Response Relationship, Drug; Drug Synergism; Gelsolin; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Ligands; Lung Neoplasms; Phenylbutyrates; Receptors, Cytoplasmic and Nuclear; Repressor Proteins; RNA; Thiazoles; Thiazolidinediones; Transcription Factors; Tumor Cells, Cultured