trichostatin-a and Prostatic-Neoplasms

Both tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and histone deacetylase inhibitors (HDIs) show promise for the treatment of cancer. However, in a number of reports they have been proven ineffective to induce cell death when applied as single agents. In this study, we show that A549 lung carcinoma cells and PC-3 prostate carcinoma cells underwent substantial apoptosis when coexposed to TRAIL and either suberoylanilide hydroxamic acid, sodium butyrate or trichostatin A. HDIs and TRAIL synergized in activation of capase-3, induction of internucleosomal DNA fragmentation and promoting mitochondrial damage. Significantly, cotreatment with minimally toxic doses of HDIs and TRAIL resulted in a marked apoptotic response in both cell lines. These data provide a rationale for a more in-depth exploration into the potential of combining TRAIL and HDIs as a valuable anticancer strategy.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Butyric Acid; Caspase 3; Caspases; Cell Line, Tumor; Drug Synergism; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Lung Neoplasms; Male; Membrane Glycoproteins; Prostatic Neoplasms; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha; Vorinostat

Overcoming drug resistance is critical for increasing the survival rate of prostate cancer (PCa). Docetaxel is the first cytotoxic chemotherapeutical approved for treatment of PCa. However, 99% of PCa patients will develop resistance to docetaxel within 3 years. Understanding how resistance arises is important to increasing PCa survival.. In this study, we modeled docetaxel resistance using two PCa cell lines: DU145 and PC3. Using the Passing Attributes between Networks for Data Assimilation (PANDA) method to model transcription factor (TF) activity networks in both sensitive and resistant variants of the two cell lines. We identified edges and nodes shared by both PCa cell lines that composed a shared TF network that modeled changes which occur during acquisition of docetaxel resistance in PCa. We subjected the shared TF network to connectivity map analysis (CMAP) to identify potential drugs that could disrupt the resistant networks. We validated the candidate drug in combination with docetaxel to treat docetaxel-resistant PCa in both in vitro and in vivo models.. In the final shared TF network, 10 TF nodes were identified as the main nodes for the development of docetaxel resistance. CMAP analysis of the shared TF network identified trichostatin A (TSA) as a candidate adjuvant to reverse docetaxel resistance. In cell lines, the addition of TSA to docetaxel enhanced cytotoxicity of docetaxel resistant PCa cells with an associated reduction of the IC50 of docetaxel on the resistant cells. In the PCa mouse model, combination of TSA and docetaxel reduced tumor growth and final weight greater than either drug alone or vehicle.. We identified a shared TF activity network that drives docetaxel resistance in PCa. We also demonstrated a novel combination therapy to overcome this resistance. This study highlights the usage of novel application of single cell RNA-sequencing and subsequent network analyses that can reveal novel insights which have the potential to improve clinical outcomes.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Docetaxel; Drug Resistance, Neoplasm; Humans; Hydroxamic Acids; Male; Mice; Prostatic Neoplasms; Protein Interaction Maps; RNA-Seq; Single-Cell Analysis; Transcription Factors

Maspin repression is frequently observed in prostate cancer; however, the molecular mechanism(s) causing the loss is not completely understood. Here, we demonstrate that inhibition of class I histone deacetylases (HDACs) mediates re-expression of maspin which plays an essential role in suppressing proliferation and migration capability in prostate cancer cells. Human prostate cancer LNCaP and DU145 cells treated with HDAC inhibitors, sodium butyrate, and trichostatin A, resulted in maspin re-expression. Interestingly, an exploration into the molecular mechanisms demonstrates that maspin repression in prostate tumor and human prostate cancer cell lines occurs via epigenetic silencing through an increase in HDAC activity/expression, independent of promoter DNA hypermethylation. Furthermore, transcriptional activation of maspin was accompanied with the suppression of HDAC1 and HDAC8 with significant p53 enrichment at the maspin promoter associated with an increase in histone H3/H4 acetylation. Our results provide evidence of maspin induction as a critical epigenetic event altered by class I HDACs in the restoration of balance to delay proliferation and migration ability of prostate cancer cells.

Topics: Apoptosis; Biomarkers, Tumor; Cell Proliferation; DNA Methylation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Male; Prognosis; Promoter Regions, Genetic; Prostatic Neoplasms; Serpins; Tumor Cells, Cultured

Some drugs can be used to treat multiple diseases, suggesting potential patterns in drug treatment. Determination of drug treatment patterns can improve our understanding of the mechanisms of drug action, enabling drug repurposing. A drug can be associated with a multilayer tissue-specific protein-protein interaction (TSPPI) network for the diseases it is used to treat. Proteins usually interact with other proteins to achieve functions that cause diseases. Hence, studying drug treatment patterns is similar to studying common module structures in multilayer TSPPI networks. Therefore, we propose a network-based model to study the treatment patterns of drugs. The method was designated SDTP (studying drug treatment pattern) and was based on drug effects and a multilayer network model. To demonstrate the application of the SDTP method, we focused on analysis of trichostatin A (TSA) in leukemia, breast cancer, and prostate cancer. We constructed a TSPPI multilayer network and obtained candidate drug-target modules from the network. Gene ontology analysis provided insights into the significance of the drug-target modules and co-expression networks. Finally, two modules were obtained as potential treatment patterns for TSA. Through analysis of the significance, composition, and functions of the selected drug-target modules, we validated the feasibility and rationality of our proposed SDTP method for identifying drug treatment patterns. In summary, our novel approach used a multilayer network model to overcome the shortcomings of single-layer networks and combined the network with information on drug activity. Based on the discovered drug treatment patterns, we can predict the potential diseases that the drug can treat. That is, if a disease-related protein module has a similar structure, then the drug is likely to be a potential drug for the treatment of the disease.

Topics: Breast Neoplasms; Drug Repositioning; Female; Gene Ontology; Gene Regulatory Networks; Humans; Hydroxamic Acids; Leukemia; Male; Models, Biological; Prostatic Neoplasms; Protein Interaction Maps; Protein Synthesis Inhibitors; Transcriptome

Therapeutic agents are urgently needed for treating metastatic castration-refractory prostate cancer (mCRPC) that is unresponsive to androgen deprivation and chemotherapy. Our screening assays demonstrated that chemotherapy-resistant prostate cancer (PCa) cells are more sensitive to HDAC inhibitors than paired sensitive PCa cells, as demonstrated by cell proliferation and apoptosis in vitro and in vivo. Kinetic study revealed that TSA-induced apoptosis was significantly dependent on enhanced transcription and protein synthesis in an early stage, which subsequently caused ER stress and apoptosis. ChIP analysis indicated that TSA increased H4K16 acetylation, promoting ER stress gene transcription. The changes in Ac-H4K16, ATF3 and ATF4 were also validated in TSA-treated animals. Further study revealed the higher enzyme activity of HDACs and an increase in acetylated proteins in resistant cells. The higher nucleocytoplasmic acetyl-CoA in resistant cells was responsible for elevated acetylation status of protein and a more vigorous growth state. These results strongly support the pre-clinical application of HDAC inhibitors for treating chemotherapy-resistant mCRPC.

Topics: Acetyl Coenzyme A; Adaptor Proteins, Signal Transducing; Allografts; Animals; Antineoplastic Agents; Apoptosis; Carrier Proteins; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cisplatin; Docetaxel; Doxorubicin; Drug Resistance, Neoplasm; Endoplasmic Reticulum Stress; Eukaryotic Initiation Factors; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Male; Mice; Mice, Inbred C57BL; Phosphoproteins; Prostatic Neoplasms; Protein Isoforms; Proto-Oncogene Proteins c-akt; Signal Transduction; TOR Serine-Threonine Kinases; Tumor Burden

Estrogens act as key factors in prostate biology, cellular proliferation and differentiation as well as cancer development and progression. The expression of estrogen receptor (ER)-β appears to be lost during prostate cancer progression through hypermethylation mechanism. Epigenetic drugs such as 5-aza-2'-deoxycytidine (5-AZAC) and Trichostatin A (TSA) showed efficacy in restoring ERβ expression in prostate cancer cells. This study was designed to explore the potential anti-carcinogenic effects resulting from re-expressing ERβ1 using 5-AZAC and/or TSA, followed by its stimulation with Diarylpropionitrile (DPN), a selective ERβ1 agonist, in prostate cancer cell line PC-3.. Cells were treated with 5-AZAC, TSA, DPN and their combination. Subsequently, they were subjected to proliferation assays, determinations of ERβ1 expression, protein levels of active caspase-3, cyclin D1, β-catenin and VEGF.. Treatment with these drugs exhibited an increase in ERβ1 expression to different extents as well as active caspase-3 levels. Meanwhile, a significant reduction in cyclin D1, VEGF and β-catenin levels was achieved as compared to the vehicle control group (p < 0.05). Interestingly, the triple combination regimen led to the most prominent anti-tumor responses in terms of increased apoptosis, reduced proliferation as well as angiogenesis.. The results support the notion that ERβ1 acts as a tumor suppressor protein and suggest that sequential ERβ1 expression and activation can offer significant anti-tumor responses. The study highlights that the strategy of merging epigenetic and hormonal therapies may be beneficial in treating advanced prostate cancer.

Topics: Anticarcinogenic Agents; Azacitidine; beta Catenin; Caspase 3; Cell Proliferation; Cyclin D1; Decitabine; Disease Progression; Epigenesis, Genetic; Estrogen Receptor beta; Hormones; Humans; Hydroxamic Acids; Male; Methylation; Nitriles; Propionates; Prostate; Prostatic Neoplasms; Vascular Endothelial Growth Factor A

A series of 3-aroylindole hydroxamic acids (10-17) were developed based on the concept of a structural combination of tubulin and histone deacetylase (HDAC) inhibitors. This was accomplished by introducing hydroxamic acid-containing moieties at the N1 position of the tubulin assembly inhibitor, compound 9 (SCB01A, BPR0L075, phase II trial). Most of synthetic compounds produced in this way displayed comparable HDAC inhibitory activity, and four (10, 12-14) of them also inhibit tubulin assembly. Notably, compound 12 possesses not only tubulin and HDAC inhibitory activity but also shows HDAC6 selectivity over other HDAC isoforms. In addition, it exhibits remarkable inhibitory activity against the growth cancer cells in vitro and in vivo (PC3 and RPMI-8226 cells). Notably, it suppresses the growth of multiple myeloma xenografts without leading to the death of teated animals like reference compound. In sum, this study provided potential compounds with safer profiles for cancer treatment.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Drug Design; HeLa Cells; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Indoles; Male; Mice, Nude; Prostate; Prostatic Neoplasms; Tubulin; Tubulin Modulators

While enzalutamide and abiraterone are approved for treatment of metastatic castration-resistant prostate cancer (mCRPC), approximately 20-40% of patients have no response to these agents. It has been stipulated that the lack of response and the development of secondary resistance to these drugs may be due to the presence of AR splice variants. HDAC6 has a role in regulating the androgen receptor (AR) by modulating heat shock protein 90 (Hsp90) acetylation, which controls the nuclear localization and activation of the AR in androgen-dependent and independent scenarios. With dual-acting AR-HDAC6 inhibitors it should be possible to target patients who don't respond to enzalutamide. Herein, we describe the design, synthesis and biological evaluation of dual-acting compounds which target AR and are also specific towards HDAC6. Our efforts led to compound 10 which was found to have potent dual activity (HDAC6 IC

Topics: Androgen Antagonists; Animals; Cell Line, Tumor; Crystallography, X-Ray; Histone Deacetylase 6; Histone Deacetylase Inhibitors; Histone Deacetylases; HSP90 Heat-Shock Proteins; Humans; Male; Mice; Models, Molecular; Prostatic Neoplasms

Sex steroid hormones have been reported to induce inflammation causing dysregulation of cytokines in prostate cancer cells. However, the underlying epigenetic mechanism has not well been studied. The objective of this study was to evaluate the effect of sex steroid hormones on epigenetic DNA methylation changes in prostate cancer cells using a signature PCR methylation array panel that correspond to 96 genes with biological function in the human inflammatory and autoimmune signals in prostate cancer. Of the 96-gene panel, 32 genes showed at least 10% differentially methylation level in response to hormonal treatment when compared to untreated cells. Genes that were hypomethylated included CXCL12, CXCL5, CCL25, IL1F8, IL13RAI, STAT5A, CXCR4 and TLR5; and genes that were hypermethylated included ELA2, TOLLIP, LAG3, CD276 and MALT1. Quantitative RT-PCR analysis of select genes represented in a cytokine expression array panel showed inverse association between DNA methylation and gene expression for TOLLIP, CXCL5, CCL18 and IL5 genes and treatment of prostate cancer cells with 5'-aza-2'-deoxycytidine with or without trichostatin A induced up-regulation of TOLLIP expression. Further analysis of relative gene expression of matched prostate cancer tissues when compared to benign tissues from individual patients with prostate cancer showed increased and significant expression for CCL18 (2.6-fold; p<0.001), a modest yet significant increase in IL5 expression (1.17-fold; p=0.015), and a modest increase in CXCL5 expression (1.4-fold; p=0.25). In conclusion, our studies demonstrate that sex steroid hormones can induce aberrant gene expression via differential methylation changes in prostate carcinogenesis.

Topics: Azacitidine; Biomarkers, Tumor; Cell Line, Tumor; Chemokine CXCL5; Chemokines, CC; Cytokines; Decitabine; DNA Methylation; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Gonadal Steroid Hormones; Humans; Hydroxamic Acids; Inflammation; Interleukin-5; Male; Polymerase Chain Reaction; Promoter Regions, Genetic; Prostatic Neoplasms; Tissue Array Analysis; Tumor Cells, Cultured

This study determined the anti-neoplastic activity and nephrotoxicity of epigenetic inhibitors in vitro. The therapeutic efficacy of epigenetic inhibitors was determined in human prostate cancer cells (PC-3 and LNCaP) using the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-Aza) and the histone deacetylase inhibitor trichostatin A (TSA). Cells were also treated with carbamazepine (CBZ), an anti-convulsant with histone deacetylase inhibitor-like properties. 5-Aza, TSA or CBZ alone did not decrease MTT staining in PC-3 or LNCaP cells after 48 h. In contrast, docetaxel, a frontline chemotherapeutic induced concentration-dependent decreases in MTT staining. Pretreatment with 5-Aza or TSA increased docetaxel-induced cytotoxicity in LNCaP cells, but not PC-3 cells. TSA pretreatment also increased cisplatin-induced toxicity in LNCaP cells. Carfilzomib (CFZ), a protease inhibitor approved for the treatment of multiple myeloma had minimal effect on LNCaP cell viability, but reduced MTT staining 50% in PC-3 cells compared to control, and pretreatment with 5-Aza further enhanced toxicity. Treatment of normal rat kidney (NRK) and human embryonic kidney 293 (HEK293) cells with the same concentrations of epigenetic inhibitors used in prostate cancer cells significantly decreased MTT staining in all cell lines after 48 h. Interestingly, we found that the toxicity of epigenetic inhibitors to kidney cells was dependent on both the compound and the stage of cell growth. The effect of 5-Aza and TSA on DNA methyltransferase and histone deacetylase activity, respectively, was confirmed by assessing the methylation and acetylation of the CDK inhibitor p21. Collectively, these data show that combinatorial treatment with epigenetic inhibitors alters the efficacy of chemotherapeutics in cancer cells in a compound- and cell-specific manner; however, this treatment also has the potential to induce nephrotoxic cell injury.

Topics: Acetylation; Animals; Antineoplastic Agents; Azacitidine; Cell Death; Cell Line, Tumor; Cell Proliferation; Cisplatin; Cyclin-Dependent Kinase Inhibitor p21; Docetaxel; Epigenesis, Genetic; HEK293 Cells; Histones; Humans; Hydroxamic Acids; Kidney; Male; Methylation; Neoplasms; Oligopeptides; Prostatic Neoplasms; Rats; Taxoids

Paclitaxel (PTX) is a microtubule-targeting drug widely used for the treatment of a variety of cancers. However, drug resistance can emerge after a series of treatments, and this can seriously affect the patient's prognosis. Here, we analyzed the mechanism of PTX resistance using a human prostate cancer cell line, PC3, and its PTX-resistant subline, PC3-PR. Compared with PC3, PC3-PR exhibited some unique phenotypes that might be associated with PTX resistance, including decreased expression of acetylated α-tubulin and the cell cycle regulator p21, and increased expression of βIII tubulin, histone deacetylase 6 (HDAC6), and the anti-apoptotic protein Bcl2. The drug exporters MDR1 and MRP1 were not involved in PTX resistance. Although cabazitaxel (CTX), a novel taxoid, has been reported to overcome PTX resistance, its mechanism of action is unknown. We found that treatment of PC3-PR cells with CTX induced expression of acetylated α-tubulin and p21, but not the related regulators p27, p15, and p16 or the Bcl2 family proteins. The pan-HDAC inhibitors trichostatin A and suberanilohydroxamic acid and the HDAC6-specific inhibitor tubacin inhibited PC3-PR proliferation and increased expression of p21 and acetylated α-tubulin in a manner similar to CTX. Our data shed light on the cellular response to PTX and CTX.

Topics: Acetylation; Anilides; Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; Drug Resistance, Neoplasm; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Paclitaxel; Prostatic Neoplasms; Protein Stability; Taxoids; Tubulin; Tubulin Modulators; Vorinostat

Trichostatin A (TSA) is a kind of classical histone deacetylase (HDAC) inhibitor. In this study, we reported the reversal effects of TSA on EMT and investigated the possible involved molecular mechanisms in SW480 and PC3 cells. Firstly, we observed that TSA induced the reversal process of epithelial-mesenchymal transition (EMT) in SW480 and PC3 cells, resulting in attenuated cell invasion and migration abilities. TSA-induced EMT reversal was characterized by up-regulation of E-cadherin and down-regulation of Vimentin. Then, treatment with TSA also decreased the expression of transcription factor Slug. Furthermore, over-expression of Slug significantly caused down-regulation of E-cadherin and up-regulation of Vimentin. Meanwhile, TSA treatment in Slug-expressing cells could prevent these changes. These findings suggested that Slug played a crucial role in TSA-induced EMT reversal. Additionally, the study showed that TSA could induce the increase of HDAC1 and HDAC2 on the Slug gene promoter, which might be responsible for the suppression of Slug. Overall, TSA could reverse EMT in SW480 and PC3 cells and TSA-mediated down-regulation of Slug was involved in the reversal process.

Topics: Antigens, CD; Cadherins; Cell Line, Tumor; Cell Movement; Colorectal Neoplasms; Dose-Response Relationship, Drug; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Histone Deacetylase 1; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Microscopy, Confocal; Neoplasm Invasiveness; Promoter Regions, Genetic; Prostatic Neoplasms; Snail Family Transcription Factors; Transcription Factors; Vimentin

Prostate cancer is the second most common malignancy in men worldwide. Abnormal epigenetic alterations such as DNA methylation and histone modification play an important role in tumor initiation, progression and regulation of cancer-related genes such as integrin α4 and E-cadherin. Expression of these genes was determined by semi-quantitative reverse transcriptase-PCR in prostate cancer cell lines, DU145 and PC3, before and after treatment with 5-aza-2-deoxycytidine and trichostatin A. Laser capture microdissection microscopy was used to obtain exclusively affected epithelial cells from prostate gland biopsies of 30 patients with prostate cancer and 40 with benign prostate hyperplasia. DNA bisulfite modifications followed by methylation-specific PCR were used to evaluate the promoter methylation status of E-cadherin and α4 integrin genes in extracted DNA from patients and aforementioned cell lines. The integrin α4 promoter in DU145 was fully methylated, whereas in PC3 cells, partial methylation was detected. E-cadherin was expressed in both cell lines; trichostatin A and 5-aza-2-deoxycytidine treatment had no effect on E-cadherin expression, however the combined treatment of both drugs or 5-aza-2-deoxycytidine alone increased integrin α4 expression. Integrin α4 and E-cadherin were hypermethylated in 66.6 % and 6.6 % of prostate cancer cases, respectively; no hypermethylation was observed in patients with benign prostate hyperplasia. These results together suggest that aberrant DNA methylation is one of the mechanisms involved in integrin α4 expression and may play an important role in human prostate carcinogenesis. In addition, the higher rate of integrin α4 gene methylation in prostate cancer patients elects it as a potential molecular tumor marker.

Topics: Antimetabolites, Antineoplastic; Azacitidine; Biomarkers, Tumor; Cadherins; CpG Islands; Decitabine; DNA Methylation; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Integrin alpha4; Male; Promoter Regions, Genetic; Prostatic Hyperplasia; Prostatic Neoplasms; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured

Increasing evidence suggests that aberrant DNA methylation changes may contribute to prostate cancer (PCa) ethnic disparity. To comprehensively identify DNA methylation alterations in PCa disparity, we used the Illumina 450K methylation platform to interrogate the methylation status of 485,577 CpG sites focusing on gene-associated regions of the human genome. Genomic DNA from African-American (AA; 7 normal and 3 cancers) and Caucasian (Cau; 8 normal and 3 cancers) was used in the analysis. Hierarchical clustering analysis identified probe-sets unique to AA and Cau samples, as well as common to both. We selected 25 promoter-associated novel CpG sites most differentially methylated by race (fold change > 1.5-fold; adjusted P < 0.05) and compared the β-value of these sites provided by the Illumina, Inc. array with quantitative methylation obtained by pyrosequencing in 7 prostate cell lines. We found very good concordance of the methylation levels between β-value and pyrosequencing. Gene expression analysis using qRT-PCR in a subset of 8 genes after treatment with 5-aza-2'-deoxycytidine and/or trichostatin showed up-regulation of gene expression in PCa cells. Quantitative analysis of 4 genes, SNRPN, SHANK2, MST1R, and ABCG5, in matched normal and PCa tissues derived from AA and Cau PCa patients demonstrated differential promoter methylation and concomitant differences in mRNA expression in prostate tissues from AA vs. Cau. Regression analysis in normal and PCa tissues as a function of race showed significantly higher methylation prevalence for SNRPN (P = 0.012), MST1R (P = 0.038), and ABCG5 (P < 0.0002) for AA vs. Cau samples. We selected the ABCG5 and SNRPN genes and verified their biological functions by Western blot analysis and siRNA gene knockout effects on cell proliferation and invasion in 4 PCa cell lines (2 AA and 2 Cau patients-derived lines). Knockdown of either ABCG5 or SNRPN resulted in a significant decrease in both invasion and proliferation in Cau PCa cell lines but we did not observe these remarkable loss-of-function effects in AA PCa cell lines. Our study demonstrates how differential genome-wide DNA methylation levels influence gene expression and biological functions in AA and Cau PCa.

Topics: Antimetabolites, Antineoplastic; Azacitidine; Black or African American; Cell Line, Tumor; Decitabine; DNA Methylation; Gene Expression Regulation, Neoplastic; Genome, Human; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Organ Specificity; Promoter Regions, Genetic; Prostatic Neoplasms; White People

Commonly occurred in aged males, the incidence of prostate carcinoma is increasing by years. Histone deacetylase (HDACs) as one key enzyme in regulating gene transcription has been found to be related with cancer occurrence. Trichostatin A (TSA) is one HDAC inhibitor for suppressing tumor growth. This study thus treated prostate carcinoma cell line PC3 with TSA, to analyze the effect of HDAC on the occurrence and progression of HDAC. PC3 cells were treated with gradient concentrations of TSA. MTT assay was employed to detect the proliferation of PC3 cells, while flow cytometry was used to detect the cell apoptosis and cell cycle. Apoptotic proteins including caspase-3, caspase-9 and bcl-2 were further quantified by Western blotting. MTT assays showed a dose- and time-dependent manner of TSA in inhibiting PC3 cell proliferation. Most of PC3 cells were arrested at G1 phase after treating with TSA. The apoptotic ratio of cells was also elevated by higher concentrations of drugs. Apoptotic proteins including caspase-3, caspase-9 and bcl-2 were all up-regulated by TSA. HDAC inhibitor can effectively suppress the proliferation of prostate carcinoma cells, which can be arrested at G1 phase. The elevated apoptotic ratio was caused by up-regulation of apoptosis-related proteins caspase-3, caspase-9 and bcl-2, in both dose- and time-dependent manners.

Topics: Adenocarcinoma; Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Flow Cytometry; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Male; Prostatic Neoplasms

Prostate cancer is one of the most common types of urological cancers. Despite the implementation of effective radiotherapy and chemotherapy methods, prostate cancer cells can still show resistance to treatment. In recent years, a combination of proteasome and histone deacetylase inhibitors has been used to treat various malignancies. In this study, we examined the cytotoxic and apoptotic effects of the proteasome inhibitor bortezomib (Velcade/PS-341) and histone deacetylase inhibitor trichostatin A (TSA), used either alone or in combination, on the human prostate LNCaP and PC3 cell lines. We investigated the cytotoxic activity of these inhibitors using a WST-1 assay, IkBα and caspase-3 mRNA levels by real-time polymerase chain reaction, and caspase-3 activity and activation of phosphorylated (p-IkBα) protein by Western blotting. Low-dose bortezomib and TSA synergistically induced apoptosis in both prostate cancer cell lines. Combination treatment with TSA with bortezomib effectively inactivated NFkB signaling, upregulated the predominant endogenous apoptotic factor caspase-3, and disrupted the NFkB pathway in the androgen-independent PC3 cell line. In contrast, androgen-dependent LNCaP cells showed upregulation of caspase-3 through a pathway other than NFkB. This study examined the possible clinical use of bortezomib and TSA, together with reduced doses of chemotherapeutic agents with high cytotoxicity, to determine their apoptotic effects on the NFkB pathway in prostate cancer cell lines. Therefore, combination bortezomib and TSA treatment may represent a novel therapeutic strategy for prostate cancer.

Topics: Antineoplastic Agents; Apoptosis; Boronic Acids; Bortezomib; Caspase 3; Cell Line, Tumor; Cell Survival; Drug Resistance, Neoplasm; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; NF-kappa B; Prostatic Neoplasms; Pyrazines

Ultraconserved regions (UCR) are genomic segments of more than 200 base pairs that are evolutionarily conserved among mammalian species. They are thought to have functions as transcriptional enhancers and regulators of alternative splicing. Recently, it was shown that numerous RNAs are transcribed from these regions. These UCR-encoded transcripts (ucRNAs) were found to be expressed in a tissue- and disease-specific manner and may interfere with the function of other RNAs through RNA: RNA interactions. We hypothesized that ucRNAs have unidentified roles in the pathogenesis of human prostate cancer. In a pilot study, we examined ucRNA expression profiles in human prostate tumors.. Using a custom microarray with 962 probesets representing sense and antisense sequences for the 481 human UCRs, we examined ucRNA expression in resected, fresh-frozen human prostate tissues (57 tumors, 7 non-cancerous prostate tissues) and in cultured prostate cancer cells treated with either epigenetic drugs (the hypomethylating agent, 5-Aza 2'deoxycytidine, and the histone deacetylase inhibitor, trichostatin A) or a synthetic androgen, R1881. Expression of selected ucRNAs was also assessed by qRT-PCR and NanoString®-based assays. Because ucRNAs may function as RNAs that target protein-coding genes through direct and inhibitory RNA: RNA interactions, computational analyses were applied to identify candidate ucRNA:mRNA binding pairs.. We observed altered ucRNA expression in prostate cancer (e.g., uc.106+, uc.477+, uc.363 + A, uc.454 + A) and found that these ucRNAs were associated with cancer development, Gleason score, and extraprostatic extension after controlling for false discovery (false discovery rate < 5% for many of the transcripts). We also identified several ucRNAs that were responsive to treatment with either epigenetic drugs or androgen (R1881). For example, experiments with LNCaP human prostate cancer cells showed that uc.287+ is induced by R1881 (P < 0.05) whereas uc.283 + A was up-regulated following treatment with combined 5-Aza 2'deoxycytidine and trichostatin A (P < 0.05). Additional computational analyses predicted RNA loop-loop interactions of 302 different sense and antisense ucRNAs with 1058 different mRNAs, inferring possible functions of ucRNAs via direct interactions with mRNAs.. This first study of ucRNA expression in human prostate cancer indicates an altered transcript expression in the disease.

Topics: Adenocarcinoma; Aged; Azacitidine; Case-Control Studies; Cell Line, Tumor; Conserved Sequence; Decitabine; Epigenesis, Genetic; Gene Expression; Gene Expression Regulation, Neoplastic; Genome, Human; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Metribolone; Middle Aged; Oligonucleotide Array Sequence Analysis; Prostate; Prostatic Neoplasms; RNA, Messenger; RNA, Neoplasm; RNA, Untranslated; Testosterone Congeners; Transcriptome

Sialyl Lewis antigens are selectin ligands involved in leukocyte trafficking and cancer metastasis. Biosynthesis of these selectin ligands occurs by the sequential actions of several glycosyltransferases in the Golgi apparatus following synthesis of the protein backbone in the endoplasmic reticulum. In this study, we examine how the synthesis of sialyl Lewis a (sLe(a)) is regulated in prostatic cells and identify a mucin that carries this glycotope. We treat human prostatic cells including one normal and three cancerous cells with histone deacetylase inhibitors, valproic acid, tricostatin A (TSA), and suberoylanilide hydroxamic acid (SAHA), and then monitor the expression of sLe(a). We have found that SAHA enhances the production of sLe(a) in normal prostatic RWPE-1 cells but not prostatic cancer cells. Employing siRNA technology and co-immunoprecipitation, we show that the sLe(a) is associated with MUC1, which is confirmed by confocal immunofluorescence microscopy and proximity ligation assay. The SAHA-induced production of sLe(a) in RWPE-1 cells is resulted from upregulation of B3GALT1 gene via enhancement of acetylated histone-3 and histone-4. Interestingly, PC3 and LNCaP C-81 cells do not produce detectable amounts of sLe(a) despite expressing high levels of B3GALT1. However, the MUC1-associated sLe(a) is generated in these cells after introduction of MUC1 cDNA. We conclude that the synthesis of sLe(a) is controlled by not only peptide backbone of the glycoprotein but also glycoprotein-specific glycosyltransferases involved in the synthesis of sLe(a). Further, the SAHA induction of this selectin ligand in normal prostatic cells may pose a potentially serious side effect of this drug recently approved by the US Food and Drug Administration.

Topics: CA-19-9 Antigen; Cell Line; Glycoproteins; Glycosyltransferases; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Male; Mucin-1; Oligosaccharides; Prostate; Prostatic Neoplasms; Tumor Cells, Cultured; Valproic Acid; Vorinostat

Clinically relevant prostate cancer (PCa) is more frequent in Westernised societies and increasingly men have co-morbidities associated with a Western lifestyle, primarily diabetes, characterised by hyperinsulinaemia and hyperglycaemia. IGFs and their binding proteins (IGFBPs) are important mediators of the effects of nutrition on growth and play a key role in the development of PCa. We used DU145, PC3 and LNCaP PCa cell lines to examine how hyperglycaemia altered their response to docetaxel. Trypan Blue dye-exclusion assay was used to determine the percentage of cell death. Protein abundance was determined using western immunoblotting. Levels of IGFBP2 were measured using an ELISA. IGFBP2 gene silencing was achieved using siRNA technology. DNA methylation was assessed using combined bisulphide restriction analysis. Acetylation status of histones H3 and H4 associated with IGFBP2 gene was assessed using chromatin immunoprecipitation assay. Hyperglycaemia reduced docetaxel-induced apoptosis by 40% for DU145 cells and by 88% for LNCaP cells. This reduced cell death was mediated by a glucose-induced up-regulation of IGFBP2, as silencing IGFBP2 negated the survival effect of high glucose. Glucose increased IGFBP2 via increasing the acetylation of histones associated with the IGFBP2 gene promoter. This finding could have important implications in relation to therapeutic strategies as epigenetic modulation could be reversible.

Topics: Acetylation; Antineoplastic Agents; Benzamides; Cell Line, Tumor; Docetaxel; Drug Resistance, Neoplasm; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Hyperglycemia; Insulin-Like Growth Factor Binding Protein 2; Male; Naphthols; Promoter Regions, Genetic; Prostatic Neoplasms; RNA, Small Interfering; Sirtuin 1; Sirtuin 2; Taxoids

Radiotherapy can be an effective treatment for prostate cancer, but radiorecurrent tumours do develop. Considering prostate cancer heterogeneity, we hypothesised that primitive stem-like cells may constitute the radiation-resistant fraction.. Primary cultures were derived from patients undergoing resection for prostate cancer or benign prostatic hyperplasia. After short-term culture, three populations of cells were sorted, reflecting the prostate epithelial hierarchy, namely stem-like cells (SCs, α2β1integrin(hi)/CD133(+)), transit-amplifying (TA, α2β1integrin(hi)/CD133(-)) and committed basal (CB, α2β1integrin(lo)) cells. Radiosensitivity was measured by colony-forming efficiency (CFE) and DNA damage by comet assay and DNA damage foci quantification. Immunofluorescence and flow cytometry were used to measure heterochromatin. The HDAC (histone deacetylase) inhibitor Trichostatin A was used as a radiosensitiser.. Stem-like cells had increased CFE post irradiation compared with the more differentiated cells (TA and CB). The SC population sustained fewer lethal double-strand breaks than either TA or CB cells, which correlated with SCs being less proliferative and having increased levels of heterochromatin. Finally, treatment with an HDAC inhibitor sensitised the SCs to radiation.. Prostate SCs are more radioresistant than more differentiated cell populations. We suggest that the primitive cells survive radiation therapy and that pre-treatment with HDAC inhibitors may sensitise this resistant fraction.

Topics: Aged; Aged, 80 and over; Cell Differentiation; Comet Assay; DNA Damage; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Middle Aged; Neoplastic Stem Cells; Prostatic Hyperplasia; Prostatic Neoplasms; Radiation-Sensitizing Agents; Tumor Cells, Cultured

GRP78, a major endoplasmic reticulum chaperone and signaling regulator, is commonly overexpressed in cancer. Moreover, induction of GRP78 by a variety of anti-cancer drugs, including histone deacetylase inhibitors, confers chemoresistance to cancer, thereby contributing to tumorigenesis. Thus, therapies aimed at decreasing GRP78 levels, which results in the inhibition of tumor cell proliferation and resensitization of tumor cells to chemotherapeutic drugs may hold promise for cancer treatment. Despite advances in our understanding of GRP78 actions, little is known about endogenous inhibitors controlling its expression. As endogenous regulators, microRNAs (miRNAs) play important roles in modulating gene expression; therefore, we sought to identify miRNA(s) that target GRP78, under the hypothesis that these miRNAs may serve as therapeutic agents. Here, we report that three miRNAs (miR-30d, miR-181a, miR-199a-5p) predicted to target GRP78 are down-regulated in prostate, colon and bladder tumors, and human cancer cell lines. We show that in C42B prostate cancer cells, these miRNAs down-regulate GRP78 and induce apoptosis by directly targeting its 3' untranslated region. Importantly, we demonstrate that the three miRNAs act cooperatively to decrease GRP78 levels, suggesting that multiple miRNAs may be required to efficiently control the expression of some genes. In addition, delivery of multiple miRNAs by either transient transfection or lentivirus transduction increased the sensitivity of cancer cells to the histone deacetylase inhibitor, trichostatin A, in C42B, HCT116 and HL-60 cells. Together, our results indicate that the delivery of co-transcribed miRNAs can efficiently suppress GRP78 levels and GRP78-mediated chemoresistance, and suggest that this strategy holds therapeutic potential.

Topics: 3' Untranslated Regions; Adenocarcinoma; Animals; Apoptosis; Cell Line, Tumor; Colonic Neoplasms; Drug Resistance, Neoplasm; Endoplasmic Reticulum Chaperone BiP; Gene Expression Regulation, Neoplastic; Genes, Reporter; Genetic Vectors; Heat-Shock Proteins; Histone Deacetylase Inhibitors; HL-60 Cells; Humans; Hydroxamic Acids; Lentivirus; Male; Mice; Mice, Nude; MicroRNAs; Neovascularization, Pathologic; Prostatic Neoplasms; RNA; RNA, Messenger; Thapsigargin; Transcription, Genetic; Transfection; Tumor Stem Cell Assay; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

Green tea polyphenols (GTPs) reactivate epigenetically silenced genes in cancer cells and trigger cell cycle arrest and apoptosis; however, the mechanisms whereby these effects occur are not well understood. We investigated the molecular mechanisms underlying the antiproliferative effects of GTP, which may be similar to those of histone deacetylase (HDAC) inhibitors. Exposure of human prostate cancer LNCaP cells (harboring wild-type p53) and PC-3 cells (lacking p53) with 10-80 μg/ml of GTP for 24 h resulted in dose-dependent inhibition of class I HDAC enzyme activity and its protein expression. GTP treatment causes an accumulation of acetylated histone H3 in total cellular chromatin, resulting in increased accessibility to bind with the promoter sequences of p21/waf1 and Bax, consistent with the effects elicited by an HDAC inhibitor, trichostatin A. GTP treatment also resulted in increased expression of p21/waf1 and Bax at the protein and message levels in these cells. Furthermore, treatment of cells with proteasome inhibitor, MG132 together with GTP prevented degradation of class I HDACs, compared with cells treated with GTP alone, indicating increased proteasomal degradation of class I HDACs by GTP. These alterations were consistent with G(0)-G(1) phase cell cycle arrest and induction of apoptosis in both cell lines. Our findings provide new insight into the mechanisms of GTP action in human prostate cancer cells irrespective of their p53 status and suggest a novel approach to prevention and/or therapy of prostate cancer achieved via HDAC inhibition.

Topics: Acetylation; Apoptosis; bcl-2-Associated X Protein; Cell Cycle Checkpoints; Cell Proliferation; Chromatin; Cyclin-Dependent Kinase Inhibitor p21; Down-Regulation; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Leupeptins; Male; Polyphenols; Promoter Regions, Genetic; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Protein Binding; Proteolysis; Tea; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Histone deacetylase (HDAC) inhibitors are emerging as a novel class of anti-tumor agents and have manifested the ability to induce apoptosis of cancer cells, and a significant number of genes have been identified as potential effectors responsible for HDAC inhibitor-induced apoptosis. However, the mechanistic actions of these HDAC inhibitors in this process remain largely undefined. We here report that the treatment of LNCap prostate cancer cells with HDAC inhibitor trichostatin A (TSA) resulted in downregulation of the Jumonji domain-containing protein 2B (JMJD2B). We also found that the TSA-mediated decrease in survivin expression in LNCap cells was partly attributable to downregulation of JMJD2B expression. This effect was attributable to the promoted degradation of survivin protein through inhibition of Cyclin B1/Cdc2 complex-mediated survivin Thr34 phosphorylation. Consequently, knockdown of JMJD2B enhanced TSA-induced apoptosis by regulating the Cyclin B1-dependent survivin degradation to potentiate the apoptosis pathways.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Cyclin B1; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Inhibitor of Apoptosis Proteins; Jumonji Domain-Containing Histone Demethylases; Male; Phosphorylation; Prostatic Neoplasms; RNA Interference; RNA, Small Interfering; Survivin

Deleted in liver cancer (DLC1), a tumor suppressor gene in multiple cancers, is recurrently down regulated or inactivated by epigenetic mechanisms in primary prostate carcinomas (PCAs). In this study the methylation and acetylation profile of the DLC1 promoter region was examined in three PCA cell lines with low or undetectable DLC1 expression: LNCaP, its derivative C4-2B-2, and 22Rv1. Two histone deacetylase inhibitors (HDAC), suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA) induced histone acetylation of the DLC1 promoter in all three lines. DLC1 promoter methylation and deacetylation were detected in LNCaP and C4-2B-2 cells while in 22Rv1 cells DLC1 is silenced by deacetylation. Treatment with SAHA or TSA efficiently increased DLC1 expression in all lines, particularly in 22Rv1 cells, and activated the DLC1 promoter through the same Sp1 sites. The 22Rv1 cell line was selected to evaluate the efficacy of combined DLC1 transduction and SAHA treatment on tumor growth in athymic mice. Individually, DLC1 transduction and SAHA exposure reduced the tumor size by 75-80% compared to controls and in combination almost completely inhibited tumor growth. The antitumor effect was associated with the induction of apoptosis and inhibition of RhoA activity. SAHA alone significantly reduced RhoA activity, showing that this RhoGTPase is a target for SAHA. These results, obtained with a reliable preclinical in vivo test, predict that combined therapeutic agents targeting the pathways governing DLC1 function and HDAC inhibitors may be beneficial in management of prostate cancer.

Topics: Animals; Antineoplastic Agents; BALB 3T3 Cells; Cell Line, Tumor; Combined Modality Therapy; DNA Methylation; Drug Evaluation, Preclinical; GTPase-Activating Proteins; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Mice; Prostatic Neoplasms; Transduction, Genetic; Tumor Suppressor Proteins; Vorinostat

DNA methylation is an important epigenetic mechanism in prostate cancer (PCa) progression. Given the role of even-skipped homeobox 1 (EVX1) in the regulation of multiple genes during embryogenesis, we postulated that EVX1 methylation is altered in PCa progression.. Bisulphite sequencing and quantitative MethyLight were used to assess methylation in human prostate epithelial cells, four PCa cell lines, liver, lung, spleen, kidney, 35 paired tumour and tumour-associated benign tissues, and 11 normal prostate tissues. Prostate cancer cell lines were treated with 5-azacytidine (AzaC) or trichostatin A (TSA), and expression of EVX1 transcript and variants was assessed by qPCR. Hypermethylation was compared with clinicopathological features in a validation set of 58 patients using microarray.. Even-skipped homeobox 1 hypermethylation was observed in all four PCa cell lines and 57% of tumours. High-grade tumours exhibited increased methylation compared with intermediate-grade tumours. Even-skipped homeobox 1 expression was induced in PCa cell lines after treatment with AzaC or TSA. In the validation set, 83% of tumours were hypermethylated and hypermethylation was associated with worse recurrence-free survival.. In this first evaluation of EVX1 methylation in human cancer, EVX1 is one of the most commonly hypermethylated genes observed in PCa and predicted treatment failure in moderate risk patients.

Topics: Azacitidine; Cell Line, Tumor; Disease-Free Survival; DNA Methylation; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Homeodomain Proteins; Humans; Hydroxamic Acids; Male; Prognosis; Prostate; Prostate-Specific Antigen; Prostatic Neoplasms; Recurrence

Complex mechanisms of prostate cancer progression prompt to novel therapeutic strategies concerning a combination of drugs or of single molecules able to interact with more crucial targets. Histone deacetylase inhibitors and sigma ligands with mixed σ(1) antagonist and σ(2) agonist properties were proposed as new potential tools for treatment of prostate cancer. (±)-MRJF4 was synthesized as phenylbutyrate ester of haloperidol metabolite II, which is a molecule consisting of a histone deacetilase inhibitor (4-phenylbutyric acid) and a sigma ligand (haloperidol metabolite II). Antiproliferatives activities of 4-phenylbutyric acid, haloperidol metabolite II, equimolar mixture of both compounds and (±)-MRJF4 were evaluated in vitro on LNCaP and PC3 prostate cancer cells. Preliminary binding studies of (±)-MRJF4 for σ(1), σ(2), D(2) and D(3) receptors and inhibition HDAC activity were reported. MTT cell viability assays highlighted a notable increase of antiproliferative activity of (±)-MRJF4 (IC(50) = 11 and 13 μM for LNCaP and PC3, respectively) compared to 4-phenylbutyric acid, haloperidol metabolite II and the respective equimolar pharmacological association. (±)-MRJF4 was also used in combination with σ(1) agonist (+)-pentazocine and σ(2) antagonist AC927 in order to evaluate the role of σ receptor subtypes in prostate cancer cell death.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Guinea Pigs; Haloperidol; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Male; Phenylbutyrates; Prostatic Neoplasms; Receptors, Dopamine D2; Receptors, Dopamine D3; Receptors, sigma

PCDH10 (protocadherin-10), a novel tumour suppressor gene, is down-regulated in several human cancers due to hypermethylation of promoter CGIs (CpG islands). Here, we investigated the expression of PCDH10 in different normal adult tissues and in a panel of prostate cancer cell lines. PCDH10 was widely expressed in normal tissues with higher levels in the prostate. The expression of PCDH10 was markedly reduced or silenced in prostate cancer cell lines compared with normal adult prostate tissue. Decreased PCDH10 expression was correlated with the methylation status of the PCDH10 promoter. Furthermore, the DNA demethylating agent 5'-azacytidin restored PCDH10 expression by suppressing PCDH10 promoter methylation in prostate cancer cell lines. Treatment with Trichostatin A alone had no significant effect on the expression of PCDH10 but enhanced the effect of 5'-azacytidin. In conclusion, we found that the decreased PCDH10 expression in prostate cancer cells was associated with the aberrant methylation of PCDH10 promoter CGI. Our results may contribute to the understanding of the role of PCDH10 inactivation in the progression of prostate cancers.

Topics: Adult; Blotting, Western; Cadherins; Cell Line, Tumor; CpG Islands; Cytidine Monophosphate; DNA Methylation; Drug Synergism; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Male; Polymerase Chain Reaction; Promoter Regions, Genetic; Prostatic Neoplasms; Protocadherins

An ETS family member, ETS Related Gene (ERG) is involved in the Ewing family of tumors as well as leukemias. Rearrangement of the ERG gene with the TMPRSS2 gene has been identified in the majority of prostate cancer patients. Additionally, overexpression of ERG is associated with unfavorable prognosis in prostate cancer patients similar to leukemia patients. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) regulate transcription as well as epigenetic status of genes through acetylation of both histones and transcription factors. Deregulation of HATs and HDACs is frequently seen in various cancers, including prostate cancer. Many cellular oncogenes as well as tumor viral proteins are known to target either or both HATs and HDACs. Several studies have demonstrated that there are alterations of HDAC activity in prostate cancer cells. Recently, we found that ERG binds and inhibits HATs, which suggests that ERG is involved in deregulation of protein acetylation. Additionally, it has been shown that ERG is associated with a higher expression of HDACs. In this study, we tested the effect of the HDAC inhibitors valproic acid (VPA) and trichostatin-A (TSA) on ERG-positive prostate cancer cells (VCaP). We found that VPA and TSA induce apoptosis, upregulate p21/Waf1/CIP1, repress TMPRSS2-ERG expression and affect acetylation status of p53 in VCaP cells. These results suggest that HDAC inhibitors might restore HAT activity through two different ways: by inhibiting HDAC activity and by repressing HAT targeting oncoproteins such as ERG.

Topics: Acetylation; Animals; Apoptosis; Caspase 3; Caspase 7; Cell Line, Tumor; Cell Survival; Chlorocebus aethiops; COS Cells; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Male; Models, Biological; p21-Activated Kinases; Prostatic Neoplasms; Proto-Oncogene Proteins c-ets; Tumor Suppressor Protein p53; Valproic Acid

Aberrant up-regulation of P-Rex1 expression plays important roles in cancer progression and metastasis. The present study investigated the regulatory mechanism underlying P-Rex1 gene expression in prostate cancer cells. We showed that P-Rex1 expression was much higher in metastatic prostate cancer cells than in prostate epithelial cells and non-metastatic prostate cancer cells. Histone deacetylase (HDAC) inhibitors or silence of endogenous HDAC1 and HDAC2 markedly elevated P-Rex1 transcription in non-metastatic prostate cancer cells, whereas overexpression of recombinant HDAC1 in metastatic prostate cancer cells suppressed P-Rex1 expression. HDAC inhibitor trichostatin A (TSA) also significantly increased P-Rex1 promoter activity and caused acetylated histones to accumulate and associate with the P-Rex1 promoter. One Sp1 site, essential for basal promoter activity, was identified as critical for the TSA effect. TSA treatment did not alter the DNA-binding activity of Sp1 toward the P-Rex1 promoter; however, it facilitated the dissociation of the repressive HDAC1 and HDAC2 from the Sp1 binding region. Interestingly, HDAC1 association with Sp1 and with the P-Rex1 promoter were much weaker in metastatic prostate cancer PC-3 cells than in non-metastatic prostate cancer cells, and HDAC inhibitors only had very modest stimulatory effects on P-Rex1 promoter activity and P-Rex1 expression in PC-3 cells. Altogether, our studies demonstrate that HDACs could regulate P-Rex1 gene transcription by interaction with Sp1 and by region-specific changes in histone acetylation within the P-Rex1 promoter. Disassociation of HDACs from Sp1 on the P-Rex1 promoter may contribute to aberrant up-regulation of P-Rex1 in cancer.

Topics: Animals; Base Sequence; Binding Sites; Cell Line, Tumor; Epigenesis, Genetic; Epithelial Cells; Gene Expression Regulation, Neoplastic; Guanine Nucleotide Exchange Factors; Histone Deacetylase 1; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Neoplasm Metastasis; Promoter Regions, Genetic; Prostatic Neoplasms; Sp1 Transcription Factor; Substrate Specificity; Transcriptional Activation; Up-Regulation

The three main types of urological cancers are mostly curable by surgical resection, if early detected. We aimed to identify novel DNA methylation biomarkers common to these three urological cancers, potentially suitable for non-invasive testing. From a candidate list of markers created after gene expression assessment of pharmacologically treated cell lines and tissue samples, two genes were selected for further validation. Methylation levels of these genes were quantified in a total of 12 cancer cell lines and 318 clinical samples. PCDH17 and TCF21 methylation levels provided a sensitivity rate of 92% for bladder cancer, 67% for renal cell tumors and 96% for prostate cancer. Methylation levels were significantly different from those of cancer free individuals (n = 37) for all tumor types (p < 0.001), providing 83% sensitivity and 100% specificity for cancer detection. Although in urine samples the sensitivity was 60%, 32% and 26% for bladder, renal, and prostate tumors, respectively (39% overall), absolute specificity was retained. We identified novel and highly specific methylation markers common to the three main urological cancers. However, additional efforts are required to increase the assay's sensitivity, enabling the simultaneous non-invasive screening of urological tumors in a single voided urine analysis.

Topics: Adult; Aged; Aged, 80 and over; Azacitidine; Basic Helix-Loop-Helix Transcription Factors; Biomarkers; Cadherins; Carcinoma, Renal Cell; Case-Control Studies; Cell Line, Tumor; Decitabine; DNA Methylation; Epigenesis, Genetic; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Hydroxamic Acids; Male; Middle Aged; Promoter Regions, Genetic; Prostatic Neoplasms; ROC Curve; Sensitivity and Specificity; Urinary Bladder Neoplasms

Histone acetylation is a fundamental mechanism in the regulation of local chromatin conformation and gene expression. Research has focused on the impact of altered epigenetic environments on the expression of specific genes and their pathways. However, changes in histone acetylation also have a global impact on the cell. In this study we used digital texture analysis to assess global chromatin patterns following treatment with trichostatin A (TSA) and have observed significant alterations in the condensation and distribution of higher-order chromatin, which were associated with altered gene expression profiles in both immortalised normal PNT1A prostate cell line and androgen-dependent prostate cancer cell line LNCaP. Furthermore, the extent of TSA-induced disruption was both cell cycle and cell line dependent. This was illustrated by the identification of sub-populations of prostate cancer cells expressing high levels of H3K9 acetylation in the G(2)/M phase of the cell cycle that were absent in normal cell populations. In addition, the analysis of enriched populations of G(1) cells showed a global decondensation of chromatin exclusively in normal cells.

Topics: Acetylation; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Chromatin; Dose-Response Relationship, Drug; Flow Cytometry; G1 Phase; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Histones; Humans; Hydroxamic Acids; Lysine; Male; Prostatic Neoplasms

The minichromosome maintenance (MCM) gene family is essential for DNA replication and is frequently upregulated in various cancers. Here, we examined the role of MCM2 in prostate cancer and the effect of microRNA-1296 (miR-1296), genistein, and trichostatin A (TSA) on the MCM complex. Profiling results showed that expression of MCM genes was higher in tumor samples. Genistein and TSA significantly downregulated the expression of all MCM genes. Genistein, TSA, and small interfering RNA duplexes caused a significant decrease in the S phase of the cell cycle. There was also downregulation of CDT1, CDC7, and CDK2 genes, which govern loading of the MCM complex on chromatin. We also found that miR-1296 was significantly downregulated in prostate cancer samples. In PC3 cells, inhibition of miR-1296 upregulated both MCM2 mRNA and protein, whereas overexpression caused a significant decrease in MCM2 mRNA, protein, and the S phase of the cell cycle. MCM genes are excellent anticancer drug targets because they are essential DNA replication factors that are highly expressed in cancer cells. This is the first report showing anti-MCM effect by miR-1296, genistein, and TSA. TSA is undergoing clinical trials as a prostate cancer treatment but has high toxicity. Genistein, a natural, nontoxic dietary isoflavone, may be an advantageous therapeutic agent for treating prostate cancer. The use of RNA interference is currently being implemented as a gene-specific approach for molecular medicine. The specific downregulation of oncogenes by miR may contribute to novel therapeutic approaches in the treatment of prostate cancer.

Topics: Apoptosis; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Chromatin; DNA Replication; DNA-Binding Proteins; Down-Regulation; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Genistein; Humans; Hydroxamic Acids; Male; MicroRNAs; Minichromosome Maintenance Complex Component 2; Minichromosome Maintenance Complex Component 7; Nuclear Proteins; Prostatic Neoplasms; RNA Interference; RNA, Small Interfering; Transfection

Polymer-mediated gene delivery is an attractive alternative to viral vectors, but is limited by low efficacies of transgene expression. We report that polymers possess differential efficacies for transfecting closely related human prostate cancer cells, which correlates with dramatically different intracellular fate of nanoscale cargo in these cells. Sequestration of nanoscale cargo (27 nm quantum dots and 150-250 nm polyplexes) at a single location near the microtubule organizing compartment (MTOC) in PC3-PSMA human prostate cancer cells correlated with lower polymer-mediated transgene expression compared to PC3 cells, which showed distributed localization throughout the cytoplasm. We show, for the first time, that treatment with the histone deacetylase 6 (HDAC6) inhibitor tubacin, which acetylates tubulin of microtubules in the cytoplasm, abolished quantum dot and polyplex sequestration at the perinuclear recycling compartment/microtubule organizing center (PNRC/MTOC) and increased polymer-mediated transgene expression by up to forty-fold compared to cells not treated with the HDAC6 inhibitor drug. Treatment with the class I and II HDAC inhibitor trichostatin A (TSA) demonstrated similar levels of transgene expression enhancement. These results indicate that mediators of intracellular trafficking can be employed to modulate nanoparticle fate and enhance the efficacy of nanoscale therapeutics in cells. Simultaneous use of high-efficacy polymers along with mediators of intracellular trafficking is an attractive synergistic strategy for enhancing polymer-mediated transgene expression.

Topics: Anilides; Biological Transport; Cell Line, Tumor; Cytoplasm; DNA; Gene Expression; Gene Transfer Techniques; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Plasmids; Polymers; Prostatic Neoplasms; Quantum Dots; Transgenes

Signaling through the androgen receptor (AR) plays a critical role in prostate cancer progression. The AR is a classical nuclear receptor (NR) providing a link between signaling molecule and transcription response. Histone deacetylase inhibitors- (HDACI) have antiproliferative and proapoptotic effects on prostate cancer cells and their implication in silence AR signaling may have potential therapeutic use. We aimed to study the inhibitory effects of the corepressor SMRT (Silencing Mediator for Retinoid and Thyroid -hormone receptors) which forms a complex together with nuclear receptor corepressor (N-CoR) and with histone deacetylase 3 (HDAC3) on AR activity.The androgen-sensitive prostate cancer cell line LNCaP and androgen-insensitive prostate cancer cell line C4-2 both AR-positive, and androgen-insensitive DU145 and PC3 prostate cancer cell lines were treated with two HDACIs, sodium butyrate (NaB) and/or trichostatin A (TSA). We amplified immunoprecipitated DNA by conventional PCR and in the -following step we used the chromatin immunoprecipitation (ChIP) analysis coupled with quantitative PCR for monitoring NaB induced formation of AR-SMRT/N-CoR complex binding on the PSA promoter. The co-immunoprecipitation assay revealed increase in AR-SMRT formation in NaB treated cells. Simultaneously, the Western blot analysis showed a significant decrease in AR protein expression. In conclusion, the inhibitory effect of NaB on AR gene expression seems to be specific and unique for prostate cancer AR-positive cell lines and corresponds with its ability to stimulate AR-SMRT complex formation. We suggest that AR and SMRT/N-CoR corepressors may form a stable complex in vitro and NaB may facilitate the interaction between AR nuclear steroid receptor and SMRT corepressor prote.

Topics: Butyrates; Cell Line, Tumor; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Male; Multidrug Resistance-Associated Proteins; Nuclear Receptor Co-Repressor 1; Promoter Regions, Genetic; Prostate-Specific Antigen; Prostatic Neoplasms; Receptors, Androgen

Calcitriol, a regulator of calcium homeostasis with antitumor properties, is degraded by the product of the CYP24A1 gene, which is downregulated in human prostate cancer by unknown mechanisms. We found that CYP24A1 expression is inversely correlated with promoter DNA methylation in prostate cancer cell lines. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (DAC) activates CYP24A1 expression in prostate cancer cells. In vitro methylation of the CYP24A1 promoter represses its promoter activity. Furthermore, inhibition of histone deacetylases by trichostatin A (TSA) enhances the expression of CYP24A1 in prostate cancer cells. Quantitative chromatin immunoprecipitation-PCR (ChIP-qPCR) reveals that specific histone modifications are associated with the CYP24A1 promoter region. Treatment with TSA increases H3K9ac and H3K4me2 and simultaneously decreases H3K9me2 at the CYP24A1 promoter. ChIP-qPCR assay reveals that treatment with DAC and TSA increases the recruitment of vitamin D receptor to the CYP24A1 promoter. Reverse transcriptase-PCR analysis of paired human prostate samples revealed that CYP24A1 expression is downregulated in prostate malignant lesions compared with adjacent histologically benign lesions. Bisulfite pyrosequencing shows that CYP24A1 gene is hypermethylated in malignant lesions compared with matched benign lesions. Our findings indicate that repression of CYP24A1 gene expression in human prostate cancer cells is mediated in part by promoter DNA methylation and repressive histone modifications.

Topics: Azacitidine; Calcitriol; Cell Line, Tumor; Decitabine; DNA Methylation; Down-Regulation; Epigenesis, Genetic; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Promoter Regions, Genetic; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Steroid Hydroxylases; Vitamin D3 24-Hydroxylase

MicroRNAs (miRNAs) are small noncoding RNAs that have important roles in numerous cellular processes. Recent studies have shown aberrant expression of miRNAs in prostate cancer tissues and cell lines. On the basis of miRNA microarray data, we found that miR-145 is significantly downregulated in prostate cancer.. We investigated the expression and functional significance of miR-145 in prostate cancer. The expression of miR-145 was low in all the prostate cell lines tested (PC3, LNCaP and DU145) compared with the normal cell line, PWR-1E, and in cancerous regions of human prostate tissue when compared with the matched adjacent normal. Overexpression of miR-145 in PC3-transfected cells resulted in increased apoptosis and an increase in cells in the G2/M phase, as detected by flow cytometry. Investigation of the mechanisms of inactivation of miR-145 through epigenetic pathways revealed significant DNA methylation of the miR-145 promoter region in prostate cancer cell lines. Microarray analyses of miR-145-overexpressing PC3 cells showed upregulation of the pro-apoptotic gene TNFSF10, which was confirmed by real-time PCR and western analysis.. One of the genes significantly upregulated by miR-145 overexpression is the proapoptotic gene TNFSF10. Therefore, modulation of miR-145 may be an important therapeutic approach for the management of prostate cancer.

Topics: Apoptosis; Azacitidine; Cell Cycle; Cell Line, Tumor; Decitabine; DNA Methylation; Down-Regulation; Gene Silencing; Genistein; Humans; Hydroxamic Acids; Male; Microarray Analysis; MicroRNAs; Prostatic Neoplasms; TNF-Related Apoptosis-Inducing Ligand; Up-Regulation

Prostate cancer is the second leading cause of cancer-associated deaths among men in the western countries. Here, we report that human RecQL4 helicase, which is implicated in the pathogenesis of a subset of cancer-prone Rothmund-Thomson syndrome, is highly elevated in metastatic prostate cancer cell lines. Increased RecQL4 expression was also detected in human prostate tumor tissues as a function of tumor grade with the highest expression level in metastatic tumor samples, suggesting that RecQL4 may be a potential prognostic factor for advanced stage of prostate cancer. Transient and stable suppression of RecQL4 by small interfering RNA and short hairpin RNA vectors drastically reduced the growth and survival of metastatic prostate cancer cells, indicating that RecQL4 is a prosurvival factor for prostate cancer cells. RecQL4 suppression led to increased poly(ADP-ribose) polymerase (PARP) synthesis and RecQL4-suppressed prostate cancer cells underwent an extensive apoptotic death in a PARP-1-dependent manner. Most notably, RecQL4 knockdown in metastatic prostate cancer cells drastically reduced their cell invasiveness in vitro and tumorigenicity in vivo, showing that RecQL4 is essential for prostate cancer promotion. Observation of a direct interaction of retinoblastoma (Rb) and E2F1 proteins with RecQL4 promoter suggests that Rb-E2F1 pathway may regulate RecQL4 expression. Collectively, our study shows that RecQL4 is an essential factor for prostate carcinogenesis.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Chromatin Immunoprecipitation; DNA Damage; E2F1 Transcription Factor; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; HeLa Cells; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunohistochemistry; In Situ Hybridization, Fluorescence; Male; Promoter Regions, Genetic; Prostatic Neoplasms; Protein Binding; RecQ Helicases; Retinoblastoma Protein; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Up-Regulation

Signaling through the androgen receptor (AR) plays a critical role in prostate cancer progression. The AR is a classical nuclear receptor (NR) providing a link between signaling molecule and transcription response. Histone deacetylase inhibitors (HDACI) have antiproliferative and proapoptotic effects on prostate cancer cells and their implication in silence AR signaling may have potential therapeutic use. We aimed to study the inhibitory effects of the corepressor SMRT (Silencing Mediator for Retinoid and Thyroid hormone receptors) which forms a complex together with nuclear receptor corepressor (N-CoR) and with histone deacetylase 3 (HDAC3) on AR activity. The androgen-sensitive prostate cancer cell line LNCaP and androgen-insensitive prostate cancer cell line C4-2 both AR-positive, and androgen-insensitive DU145 and PC3 prostate cancer cell lines were treated with two HDACIs, sodium butyrate (NaB) and/or trichostatin A (TSA). We amplified immunoprecipitated DNA by conventional PCR and in the following step we used the chromatin immunoprecipitation (ChIP) analysis coupled with quantitative PCR for monitoring NaB induced formation of AR-SMRT/N-CoR complex binding on the PSA promoter. The co-immunoprecipitation assay revealed increase in AR-SMRT formation in NaB treated cells. Simultaneously, the Western blot analysis showed a significant decrease in AR protein expression. Furthermore, we estimated the reduced presence of HDAC2 and HDAC3 proteins by NaB and TSA treatment in AR-negative DU145 cell line. In conclusion, the inhibitory effect of NaB on AR gene expression seems to be specific and unique for prostate cancer AR-positive cell lines and corresponds with its ability to stimulate AR-SMRT complex formation. We suggest that AR and SMRT/N-CoR corepressors may form a stable complex in vitro and NaB may facilitate the interaction between AR nuclear steroid receptor and SMRT corepressor protein.

Topics: Butyrates; Cell Line, Tumor; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Immunoprecipitation; Male; Nuclear Receptor Co-Repressor 2; Prostatic Neoplasms; Protein Binding; Receptors, Androgen; Time Factors

Glutathione-S-transferase (Gst) genes are downregulated in human prostate cancer, and GSTP1 silencing is mediated by promoter DNA hypermethylation in this malignancy. We examined Gst gene expression and Gst promoter DNA methylation in normal murine prostates and Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) tumors.. Primary and metastatic tumors were obtained from TRAMP mice, and normal prostates were obtained from strain-matched WT mice (n = 15/group). Quantitative real-time RT-PCR was used to measure GstA4, GstK1, GstM1, GstO1, and GstP1 mRNA expression, and Western blotting and immunohistochemical staining was used to measure GstM1 and GstP1 protein expression. MassARRAY Quantitative Methylation Analysis was used to measure DNA methylation of the 5' CpG islands of GstA4, GstK1, GstM1, GstO1, and GstP1. TRAMP-C2 cells were treated with the epigenetic remodeling drugs decitabine and trichostatin A (TSA) alone and in combination, and Gst gene expression was measured.. Of the genes analyzed, GstM1 and GstP1 were expressed at highest levels in normal prostate. All five Gst genes showed greatly reduced expression in primary tumors compared to normal prostate, but not in tumor metastases. Gst promoter methylation was unchanged in TRAMP tumors compared to normal prostate. Combined decitabine + TSA treatment significantly enhanced the expression of 4/5 Gst genes in TRAMP-C2 cells.. Gst genes are extensively downregulated in primary but not metastatic TRAMP tumors. Promoter DNA hypermethylation does not appear to drive Gst gene repression in TRAMP primary tumors; however, pharmacological studies using TRAMP cells suggest the involvement of epigenetic mechanisms in Gst gene repression.

Topics: Adenocarcinoma; Animals; Antimetabolites, Antineoplastic; Azacitidine; Cell Line, Tumor; CpG Islands; Decitabine; DNA Methylation; Drug Combinations; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Gene Silencing; Glutathione S-Transferase pi; Glutathione Transferase; Hydroxamic Acids; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasm Metastasis; Prostate; Prostatic Neoplasms; RNA, Messenger

Cystatin E/M (CST6) is a natural inhibitor of lysosomal cysteine proteases. Recent studies have shown that experimental manipulation of CST6 expression alters the metastatic behavior of human breast cancer cells. However, the association of CST6 with prostate cancer invasion and progression remains unclear. Here, we show that CST6 is robustly expressed in normal human prostate epithelium, whereas its expression is downregulated in metastatic prostate cell lines and prostate tumor tissues. Treatment of metastatic prostate cell lines with the histone deacetylase inhibitor trichostatin A resulted in significant induction of CST6 mRNA levels and increased CST6 protein expression, indicating that epigenetic silencing may play a role in the loss of CST6 expression observed in prostate cancer. CST6 overexpression in human prostate cancer cells significantly reduced in vitro cell proliferation and matrigel invasion. Furthermore, the results from a bioluminescence tumor/metastasis model showed that the overexpression of CST6 significantly inhibits tumor growth and the incidence of lung metastasis. These results suggest that the downregulation of the CST6 gene is associated with promoter histone modifications and that this association plays an important role in prostate cancer progression during the invasive and metastatic stages of the disease.

Topics: Acetylation; Animals; Azacitidine; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Chromatin; Cystatin M; Decitabine; Disease Progression; DNA Methylation; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Immunohistochemistry; Male; Mice; Mice, Nude; Neoplasms, Experimental; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Transplantation, Heterologous

The development of castration-resistant prostate cancer (PCa) requires that under castration conditions, the androgen receptor (AR) remains active and thus nuclear. Heat shock protein 90 (Hsp90) plays a key role in androgen-induced and -independent nuclear localization and activation of AR. Histone deacetylase 6 (HDAC6) is implicated, but has not been proven, in regulating AR activity via modulating Hsp90 acetylation. Here, we report that knockdown of HDAC6 in C4-2 cells using short hairpin RNA impaired ligand-independent nuclear localization of endogenous AR and inhibited PSA expression and cell growth in the absence or presence of dihydrotestosterone (DHT). The dose-response curve of DHT-stimulated C4-2 colony formation was shifted by shHDAC6 such that approximately 10-fold higher concentration of DHT is required, indicating a requirement for HDAC6 in AR hypersensitivity. HDAC6 knockdown also inhibited C4-2 xenograft tumor establishment in castrated, but not in testes-intact, nude mice. Studies using HDAC6-deficient mouse embryonic fibroblasts cells showed that inhibition of AR nuclear localization by HDAC6 knockdown can be largely alleviated by expressing a deacetylation mimic Hsp90 mutant. Taken together, our studies suggest that HDAC6 regulates AR hypersensitivity and nuclear localization, mainly via modulating HSP90 acetylation. Targeting HDAC6 alone or in combination with other therapeutic approaches is a promising new strategy for prevention and/or treatment of castration-resistant PCa.

Topics: Acetylation; Animals; Blotting, Western; Castration; Cell Line, Tumor; Cell Proliferation; Dihydrotestosterone; Gene Expression; Histone Deacetylase 6; Histone Deacetylases; HSP90 Heat-Shock Proteins; Humans; Hydroxamic Acids; Immunoprecipitation; Male; Mice; Prostate-Specific Antigen; Prostatic Neoplasms; Receptors, Androgen; Reverse Transcriptase Polymerase Chain Reaction; Xenograft Model Antitumor Assays

Somatostatin (SMS), binds to its specific receptors (SSTRs) and transduces growth inhibitory, anti-secretory and apoptotic signals. Several human cancers express SSTRs, including prostate cancer, and therefore SMS is of interest for anti-cancer therapy. DNA methylation and histone modifications are involved in normal cell development, gene imprinting and human carcinogenesis. Reversing DNA methylation is an attractive therapeutic possibility, since epigenetic modifications change gene expression without changing the gene function. DNA methylation inhibitors such as 5-aza-2'-deoxycytidine (5'-aza, decitabine) have been used to treat several types of haematological malignancies. Histone deacetylase inhibitors such as trichostatin (TSA), are a new class of 'targeted anti-cancer agents'. TSA and decitabine can induce growth arrest, apoptosis or terminal differentiation in a variety of solid and haematological cancers in advanced disease patients. In the present study, the LNCaP cell line (prostate cancer) was incubated with SMS or Somadex (an SMS polymer conjugate) for three days, 1 nM per day, and the untreated cells were the negative control. For DNA demethylation, cells were grown in the presence of 2.5 microM 5-aza for 120 h, and re-fed with 5-aza-containing fresh medium at day 3. The total incubation time with 5-aza was 120 h. TSA at 1.0 microM was added into the cultured cells for 24 h. The combined treatment of 5-aza and TSA was performed by incubating the cells with 5-aza for 120 h followed by a 24-h exposure to TSA. Using cDNA obtained from these cell lines, the difference in the expression level of SSTR mRNA transcripts before and after 5-aza and TSA treatments was analyzed by RT-PCR. An increased induction of mRNA expression of the five SSTR subtypes was observed in the LNCaP cells when incubated with SMS/Somadex (dose-dependent). The inhibition of DNA methylation and histone acetylation resulted in the up-regulation of SSTR5 mRNA expression. The results demonstrate a positive feedback loop between SMS and its receptors. This regulation pathway may enhance the anti-tumor activity of somatostatin. To benefit from this effect in a clinical setting, the dose, dose frequency and pan affinity of the SMS derivative are important factors. The epigenetic manipulation with DNA methylation or histone deacetylase inhibitors, combined with SMS, may offer a novel alternative for the treatment of advanced prostate cancer.

Topics: Azacitidine; Cell Line, Tumor; Decitabine; DNA Methylation; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Prostatic Neoplasms; Receptors, Somatostatin; RNA, Messenger; Somatostatin; Up-Regulation

Previous studies comparing the effects of two histone deacetylase (HDAC) inhibitors, trichostatin A (TSA) and CG-1521, have shown that these compounds selectively inhibit HDAC and induce differentially acetylated p53 isoforms and assembly of mutually exclusive transcriptional complexes on the p21 promoter. To determine whether the differential transcriptional regulation seen in p21 gene is unique or whether it is representative of the genome-wide effects of these two HDAC inhibitors, we have used microarray and Ingenuity pathway analysis to compare the effects of TSA and CG-1521 on gene expression on LNCaP cells. Gene array analysis confirmed by quantitative real-time PCR shows that CG-1521 modulates the expression of a highly circumscribed group of genes involved in cell cycle progression and cell death. In contrast, TSA appears to induce widespread transrepression of many genes and does not modulate the expression of the same cohort as CG-1521. These data show that the selective effects of CG-1521 and TSA on the assembly of transcription complexes are not unique to the p21 gene and suggest that selective inhibition of HDAC can lead to significant changes in gene expression through the acetylation of transcription factors including but not limited to p53.

Topics: Cell Cycle; Cell Death; Cell Line, Tumor; G2 Phase; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Kinetochores; Male; Mitosis; Oligonucleotide Array Sequence Analysis; Prostatic Neoplasms; RNA, Messenger; Spindle Apparatus; Tumor Suppressor Protein p53

Gene fusions between prostate-specific, androgen responsive TMPRSS2 gene and oncogenic ETS factors, such as ERG, occur in up to 50% of all prostate cancers. We recently defined a gene signature that was characteristic to prostate cancers with ERG activation. This suggested epigenetic reprogramming, such as upregulation of histone deactylase 1 (HDAC1) gene and downregulation of its target genes. We then hypothesized that patients with ERG-positive prostate cancers may benefit from epigenetic therapy such as HDAC inhibition (HDACi), especially in combination with antiandrogens. Here, we exposed ERG-positive prostate cancer cell lines to HDAC inhibitors Trichostatin A (TSA), MS-275 and suberoylanilide hydroxamic acid (SAHA) with or without androgen deprivation. We explored the effects on cell phenotype, gene expression as well as ERG and androgen receptor (AR) signaling. When compared with 5 other prostate cell lines, ERG-positive VCaP and DuCap cells were extremely sensitive to HDACi, in particular TSA, showing synergy with concomitant androgen deprivation increasing apoptosis. Both of the HDAC inhibitors studied caused repression of the ERG-fusion gene, whereas the pan-HDAC inhibitor TSA prominently repressed the ERG-associated gene signature. Additionally, HDACi and flutamide caused retention of AR in the cytoplasm, indicating blockage of androgen signaling. Our results support the hypothesis that HDACi, especially in combination with androgen deprivation, is effective against TMPRSS2-ERG-fusion positive prostate cancer in vitro. Together with our previous in vivo observations of an "epigenetic reprogramming gene signature" in clinical ERG-positive prostate cancers, these studies provide mechanistic insights to ERG-associated tumorigenesis and suggest therapeutic paradigms to be tested in vivo.

Topics: Androgen Antagonists; Anilides; Antineoplastic Agents, Hormonal; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Benzamides; Biomarkers, Tumor; Blotting, Western; Cell Line, Tumor; Drug Synergism; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Gene Silencing; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Nitriles; Oncogene Proteins, Fusion; Polymerase Chain Reaction; Prostatic Neoplasms; Protein Synthesis Inhibitors; Pyridines; Receptors, Androgen; Tosyl Compounds; Up-Regulation

Epigenetic silencing mechanisms are increasingly thought to play a major role in the development of human cancers, including prostate cancer. Promoter CpG island hypermethylation and histone hypoacetylation, catalyzed by DNA methyltransferase (DNMT) and histone deacetylase (HDAC), respectively, are associated with transcriptional repression in a number of cancers. Evidence is accumulating the two mechanisms are dynamically linked, yet few studies have examined a potential interaction in prostate cancer.. LNCaP, DU-145, and PC-3 prostate cancer cells were co-treated with a DNMT inhibitor, 5'-aza-2'-deoxycytidine (5-AZAC), and an HDAC inhibitor, trichostatin A (TSA). Following treatment cells were processed for cell proliferation/apoptosis assays, or harvested for real-time RT-PCR. Assessed target genes were estrogen receptor beta (ERbeta), estrogen receptor alpha (ERalpha), androgen receptor (AR), progesterone receptor (PGR), and prostate specific antigen (PSA).. In all cell-lines, co-treatment was associated with reduced cell proliferation compared with control groups (P<0.05). A reciprocal rise in caspase activation was identified, indicating apoptosis was the major mechanism of cell death. Most marked effects were seen in the androgen-dependent, AR-positive LNCaP cell-line. In all cell-lines, an additive re-expression of ERbeta was identified in the co-treatment group, a finding not seen for either AR or PSA.. At concentrations associated with gene re-expression, the DNA demethylating agent 5-AZAC and the HDAC inhibitor TSA co-operate to induce apoptosis in prostate cancer cell-lines. Increased apoptosis in the co-treatment group was associated with marked re-expression of ERbeta, raising the possibility of further targeting of prostate cancer cells with ERbeta-selective agents.

Topics: Apoptosis; Azacitidine; Cell Line, Tumor; Cell Proliferation; Decitabine; DNA Methylation; DNA Modification Methylases; Enzyme Inhibitors; Estrogen Receptor alpha; Estrogen Receptor beta; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Male; Prostate-Specific Antigen; Prostatic Neoplasms; Receptors, Androgen; Receptors, Progesterone

Genistein is a phytoestrogen that has been reported to suppress the AKT signaling pathway in several malignancies. However, the molecular mechanism of genistein action is not known. We tested the hypothesis that genistein activates expression of several aberrantly silenced tumor suppressor genes (TSGs) that have unmethylated promoters such as PTEN, CYLD, p53 and FOXO3a. We report here that genistein activates TSGs through remodeling of the heterochromatic domains at promoters in prostate cancer cells by modulating histone H3-Lysine 9 (H3-K9) methylation and deacetylation. Genistein activation involved demethylation and acetylation of H3-K9 at the PTEN and the CYLD promoter, while acetylation of H3-K9 at the p53 and the FOXO3a promoter occurred through reduction of endogenous SIRT1 activity. There was a decrease of SIRT1 expression and accumulation of SIRT1 in the cytoplasm from the nucleus. Increased expression of these TSGs was also reciprocally related to attenuation of phosphorylated-AKT and NF-kappaB binding activity in prostate cancer cells. This is the first report describing a novel epigenetic pathway that activates TSGs by modulating either histone H3-Lysine 9 (H3-K9) methylation or deacetylation at gene promoters leading to inhibition of the AKT signaling pathway. These findings strengthen the understanding of how genistein may be chemoprotective in prostate cancer.

Topics: Acetylation; Anticarcinogenic Agents; Antimetabolites, Antineoplastic; Azacitidine; Blotting, Western; Cell Line, Tumor; Chromatin Immunoprecipitation; Chromones; CpG Islands; Decitabine; Deubiquitinating Enzyme CYLD; Down-Regulation; Electrophoretic Mobility Shift Assay; Enzyme Inhibitors; Forkhead Box Protein O3; Forkhead Transcription Factors; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Genes, p53; Genes, Tumor Suppressor; Genistein; Histones; Humans; Hydroxamic Acids; Male; Methylation; Morpholines; NF-kappa B; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phytoestrogens; Prostatic Neoplasms; Protein Kinase Inhibitors; PTEN Phosphohydrolase; Reverse Transcriptase Polymerase Chain Reaction; Sirtuin 1; Sirtuins; Tumor Suppressor Proteins; Up-Regulation

Prostate carcinoma is characterized by the silencing of pi-class glutathione S-transferase gene (GSTP1), which encodes a detoxifying enzyme. The silencing of GSTP1, due to aberrant methylation at the CpG island in the promoter/5'-UTR, occurs in the vast majority of prostate tumors and precancerous lesions. It is a pathologic marker and probably an underlying cause of oxidative damage and inflammation at tumor initiation. Inhibition of the aberrant promoter methylation could therefore be an effective mean to prevent carcinogenesis. Several isothiocyanates, including phenethyl isothiocyanate (PEITC), found naturally in cruciferous vegetables, induced growth arrest and apoptosis in prostate cancer cells in culture and xenografts. The effects of PEITC to reactivate GSTP1 were investigated. Exposure of prostate cancer LNCaP cells to PEITC inhibited the activity and level of histone deacetylases (HDACs), and induced selective histone acetylation and methylation for chromatin unfolding. Concurrently PEITC demethylated the promoter and restored the unmethylated GSTP1 in both androgen-dependent and -independent LNCaP cancer cells to the level found in normal prostatic cells, as quantified by methylation-specific PCR and pyrosequencing. The dual action of PEITC on both the DNA and chromatin was more effective than 5'-Aza-2'-deoxycytidine, sodium butyrate, or trichostatin A (TSA), and may de-repress the methyl-binding domain (MBD) on gene transcription. The PEITC-mediated cross-talk between the DNA and chromatin in demethylating and reactivating GSTP1 genes, which is critically inactivated in prostate carcinogenesis, underlines a primary mechanism of cancer chemoprevention. Consequently, new approaches could be developed, with isothiocyanates to prevent and inhibit malignancies.

Topics: Acetylation; Anticarcinogenic Agents; Butyrates; Chromatin; CpG Islands; Deoxycytidine; DNA Methylation; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Gene Silencing; Glutathione S-Transferase pi; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Isothiocyanates; Male; Neoplasms, Hormone-Dependent; Promoter Regions, Genetic; Prostatic Neoplasms; Transcription, Genetic; Tumor Cells, Cultured

Histone deacetylase (HDAC) inhibitors are being investigated as possible adjuvant therapies for a number of diseases, including cancer. In addition to stabilization of acetylated histones, HDAC inhibitors stabilize the acetylation of a number of transcription factors, including p53. This study investigates the action of two HDAC inhibitors, CG-1521 and trichostatin A, which stabilize Ac-Lys-373 p53 and Ac-Lys-382 p53, respectively, in LNCaP prostate cancer cells. Real-time PCR demonstrates that CG-1521 induces p21 transcription whereas trichostatin A does not alter the steady state level of p21 mRNA. Co-immunoprecipitation demonstrates that the selective acetylation of p53 directs the recruitment of mutually exclusive coactivator complexes on the p53 response elements in the p21 promoter. Furthermore, the co-activator complexes initiate the recruitment of the components of the basal transcription apparatus to the basal promoter with markedly different outcomes because only Ac-Lys-373 p53 promotes the assembly of the basal transcriptional apparatus on the p21 promoter. These data highlight the profound effects of post-translational modification, including acetylation, on the function of p53. The data also suggest a novel and critically important role for protein acetylation/deacetylation in the assembly of active transcription processes that may be as important as classical phosphorylation/dephosphorylation.

Topics: Acetylation; Cell Line, Tumor; Chemotherapy, Adjuvant; Cyclin-Dependent Kinase Inhibitor p21; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Male; Promoter Regions, Genetic; Prostatic Neoplasms; Protein Processing, Post-Translational; RNA, Messenger; RNA, Neoplasm; Transcription, Genetic; Tumor Suppressor Protein p53

Increased expression of IFI16 protein (encoded by the IFI16 gene) in normal human prostate epithelial cells is associated with cellular senescence-associated cell growth arrest. Consistent with a role for IFI16 protein in cellular senescence, the expression of IFI16 protein is either very low or not detectable in human prostate cancer cell lines. We now report that treatment of DU-145 and LNCaP prostate cancer cell lines with histone deacetylase inhibitor trichostatin A (TSA) or CGK1026 resulted in transcriptional activation of the IFI16 gene. The induction of IFI16 protein in LNCaP cells was dependent on the duration of TSA treatment. Furthermore, TSA treatment of LNCaP cells up-regulated the expression of Janus-activated kinase 1 protein kinase and modulated the transcription of certain IFN-activatable genes. However, overexpression of exogenous Janus-activated kinase 1 protein in LNCaP cells and treatment of cells with IFNs (alpha and gamma) did not increase the expression of IFI16. Instead, the transcriptional activation of IFI16 gene by TSA treatment of LNCaP cells was dependent on transcriptional activation by c-Jun/activator protein-1 transcription factor. Importantly, increased expression of IFI16 in LNCaP cells was associated with decreases in the expression of androgen receptor and apoptosis of cells. Conversely, knockdown of IFI16 expression in TSA-treated LNCaP cells increased androgen receptor protein levels with concomitant decreases in apoptosis. Together, our observations provide support for the idea that histone deacetylase-dependent transcriptional silencing of the IFI16 gene in prostate epithelial cells contributes to the development of prostate cancer.

Topics: Cell Line, Tumor; Down-Regulation; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Gene Silencing; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Janus Kinase 1; Male; Nuclear Proteins; Phosphoproteins; Prostatic Neoplasms; RNA, Messenger; Transcriptional Activation; Up-Regulation

HLA class I down-regulation in cancer cells confers immunological escape from cytotoxic T lymphocytes. We assessed the frequency of down-regulation of HLA class I antigens in a large series of prostate cancer tissues and determined the mechanism of up-regulation by investigating prostate cancer cell lines.. Immunohistochemical staining for HLA class I was done in specimens of 419 prostate cancers. We also investigated clinicopathological parameters, and the relationships between HLA class I down-regulation and the parameters. Furthermore, we examined whether HLA down-regulation was caused by epigenetic changes in vitro.. HLA class I was down-regulated in 311 prostate cancers (74.2%) and it significantly correlated with beta2-microglobulin down-regulation and a higher clinical stage. Flow cytometric analysis revealed a low level of HLA class I in LNCaP cells, which was up-regulated by the histone deacetylase inhibitor trichostatin A (Sigma). Trichostatin A up-regulated LNCaP beta2-microglobulin at the protein level. Furthermore, chromatin immunoprecipitation assay using an anti-acetylated histone H3 antibody provided direct evidence that trichostatin A up-regulated beta2-microglobulin by modulating the acetylation status of the promoter region in LNCaP cells.. The current study shows that the prevalence of HLA class I down-regulation is high in prostate cancer but histone deacetylase inhibitors can up-regulate HLA class I in LNCaP cells by up-regulating beta2-microglobulin. We suggest that the combination of an immunotherapeutic approach and histone deacetylase inhibition would accentuate the effects of current immunotherapies for prostate cancer.

Topics: Adenocarcinoma; beta 2-Microglobulin; Cell Line, Tumor; Down-Regulation; Enzyme Inhibitors; Flow Cytometry; Histocompatibility Antigens Class I; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Prostate; Prostatic Neoplasms; Up-Regulation

Transcription factor haploinsufficiency plays a role in the pathogenesis of many diseases, including cancer. In a mouse model of prostate tumor initiation, loss of a single allele of the tumor suppressor Nkx3.1 stochastically inactivates the expression of a class of dosage-sensitive target genes. Here we show that dosage sensitivity is associated with the differential histone H3/H4 acetylation states of Nkx3.1 target genes. When histone acetylation is induced in Nkx3.1+/- mouse prostates with the histone deacetylase inhibitor Trichostatin A, Nkx3.1 can bind to and reactivate the expression of dosage-sensitive target genes. We incorporated our findings into a mathematical model that entails the association of Nkx3.1 with histone acetyltransferase activity. Subsequent experiments indicate that Nkx3.1 associates with and recruits the histone acetyltransferase p300/CREB-binding protein-associated factor to chromatin. Finally, we demonstrate a role for the dosage-sensitive target gene intelectin/omentin in suppressing prostate tumorigenicity. Our results reveal how the interplay between transcription factor dosage and chromatin affects target gene expression in tumor initiation.

Topics: Acetylation; Animals; Cell Transformation, Neoplastic; Chromatin; Chromatin Assembly and Disassembly; Disease Models, Animal; Gene Dosage; Gene Expression Regulation, Neoplastic; Histone Acetyltransferases; Histones; Homeodomain Proteins; Hydroxamic Acids; Lectins; Loss of Heterozygosity; Male; Mice; Mice, Mutant Strains; Models, Biological; p300-CBP Transcription Factors; Prostatic Neoplasms; Protein Processing, Post-Translational; Protein Synthesis Inhibitors; Transcription Factors; Tumor Suppressor Proteins

In quantitative RT-PCR (qRT-PCR), analysis of gene expression is dependent on normalization using housekeeping genes such as 18S rRNA, GAPDH and beta actin. However, variability in their expression has been reported to be caused by factors like drug treatment, pathological states and cell-cycle phase. An emerging area of cancer research focuses on identifying the role of epigenetic alterations such as histone modifications and DNA methylation in the initiation and progression of cancer. Histone acetylation is the best studied modification so far and has been probed through the use of histone deacetylase inhibitors (HDACi). Further, modulation of histone acetylation is currently being explored as a therapeutic strategy in the treatment of cancer and HDACis have shown promise in inhibiting tumorigenesis and metastasis. Trichostatin-A (TSA) is the most widely used HDACi. Therefore, we were driven to identify a suitable internal control for RT-PCR following TSA treatment. We performed quantitative RT-PCR analysis using mouse prostate tissue explants, human prostate cancer (LNCaP) cells and human breast cancer (T-47D and ZR-75-1) cells following TSA treatment. Expression of housekeeping genes including 18S rRNA, beta actin, GAPDH and ribosomal highly-basic 23-kDa protein (rb 23-kDa, RPL13A) were compared in vehicle versus TSA treated samples. Our results showed marked variations in 18S rRNA, beta actin mRNA and GAPDH mRNA levels in mouse prostate explants and a human prostate cancer (LNCaP) cell line following TSA treatment. Furthermore, in two human breast cancer cell lines (T-47D and ZR-75-1) 18S rRNA, beta actin mRNA and GAPDH mRNA levels varied significantly. However, RPL13A mRNA levels remained constant in all the conditions tested. Therefore, we recommend use of RPL13A as a standard for normalization during TSA treatment.

Topics: Actins; Animals; Breast Neoplasms; Cell Line, Tumor; Female; Gene Expression; Glyceraldehyde-3-Phosphate Dehydrogenases; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Mice; Prostate; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Ribosomal Proteins; RNA, Ribosomal, 18S

Histone deacetylase inhibitors (HDACI) are potential therapeutic agents that inhibit tumor cell growth and survival. Although there are several publications regarding the effects of HDACIs on prostate cancer cell growth, their mechanism(s) of action remains undefined. We treated several human prostate cancer cell lines with the HDACI trichostatin A and found that trichostatin A induced cell death in androgen receptor (AR)-positive cell lines to higher extent compared with AR-negative cell lines. We then discovered that trichostatin A and other HDACIs suppressed AR gene expression in prostate cancer cell lines as well as in AR-positive breast carcinoma cells and in mouse prostate. Trichostatin A also induced caspase activation, but trichostatin A-induced AR suppression and cell death were caspase independent. In addition, we found that doxorubicin inhibited AR expression, and p21 protein completely disappeared after simultaneous treatment with trichostatin A and doxorubicin. This effect may be attributed to the induction of protease activity under simultaneous treatment with these two agents. Further, simultaneous treatment with trichostatin A and doxorubicin increased cell death in AR-positive cells even after culturing in steroid-free conditions. The protease/proteasome inhibitor MG132 protected AR and p21 from the effects of trichostatin A and doxorubicin and inhibited trichostatin A-induced cell death in AR-positive prostate cells. Taken together, our data suggest that the main mechanism of trichostatin A-induced cell death in AR-positive prostate cancer is inhibition of AR gene expression. The synergistic effect of simultaneous treatment with trichostatin A and doxorubicin is mediated via inhibition of AR expression, induction of protease activity, increased expression of p53, and proteolysis of p21.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Blotting, Western; Breast Neoplasms; Caspases; Cyclin-Dependent Kinase Inhibitor p21; Cysteine Proteinase Inhibitors; Doxorubicin; Drug Synergism; Drug Therapy, Combination; Enzyme Activation; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leupeptins; Luciferases; Male; Mice; Promoter Regions, Genetic; Prostate; Prostatic Neoplasms; Receptors, Androgen; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The connexin 43 gene (cx43, GJA1) mediates gap junctional intercellular communication (GJIC), which regulates tissue homeostasis. cx43 is frequently downregulated in prostate cancer. We investigated the role of a histone deacetylase (HDAC)-dependent mechanism in the transcriptional repression of cx43 in a panel of prostate cancer cells.. The impact of Trichostatin A (TSA), an inhibitor of HDAC, on exogenous and endogenous cx43 gene transcription was examined by the luciferase assay, Northern blot, nuclear run-on, Western blot, and chromatin immunoprecipitation assays.. Trichostatin A induces transcription of cx43 gene and GJIC. The co-activator p300/CBP synergizes with TSA for cx43 promoter activation. We identified a promoter region where cooperation between Ap1 and Sp1 elements was essential for TSA-induced cx43 transcription. TSA increased the level of hyperacetylated histones bound to cx43 promoter.. Our results highlight the potential utility of inhibitors of HDAC to restore cx43 gene expression in prostate cancer.

Topics: Acetylation; Animals; Blotting, Northern; Blotting, Western; Cell Line, Tumor; Connexin 43; Down-Regulation; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Immunosorbent Techniques; Male; Mice; NIH 3T3 Cells; Promoter Regions, Genetic; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic; Transfection

Fibroblast growth factor 8 (FGF8) is over-expressed in prostate cancer (CaP) correlating with high-grade disease and reduced survival. The role of acetylation in transcriptional regulation of FGF8 was investigated using the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA).. FGF8 transcriptional response to TSA was investigated by gene reporter assays, RT-PCR, and Western blotting. Chromatin immunoprecipitation (ChIP) assays were also performed.. FGF8 is upregulated in response to TSA treatment along with NF-kappaB transcriptional activity. Over-expression of p65 activated FGF8 transcription. ChIP assays revealed p65 recruitment to the fgf8 promoter, containing putative NF-kappaB binding sites, post TSA stimulation. PI-3K activity is required for TSA mediated FGF8 upregulation.. Using TSA treatment in prostate cancer cells, a requirement of PI-3K activity in mediating TSA function is demonstrated and a novel role for NF-kappaB in the regulation of FGF8 expression is uncovered.

Topics: Acetylation; Base Sequence; Binding Sites; Blotting, Western; Cell Line, Tumor; Chromatin; DNA; Enzyme Inhibitors; Fibroblast Growth Factor 8; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunosorbent Techniques; Male; Molecular Sequence Data; NF-kappa B; Phosphatidylinositol 3-Kinases; Polymerase Chain Reaction; Promoter Regions, Genetic; Prostatic Neoplasms; Recombinant Fusion Proteins; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factor RelA; Transcription, Genetic; Transfection; Up-Regulation

Chemoresistance has been one of the major problems in anticancer therapy. In our effort to find a potential molecular target for overcoming the chemoresistance in prostate cancer, a promising anticancer drug trichostatin A (TSA) induced cell death was found to be compromised by enhanced NF-kappaB activation in 267B1/K-ras human prostate epithelial cancer cells. However, both the NF-kappaB activation and chemoresistance were reduced by pretreatment with proteasome inhibitor-I (ProI), accompanied by accumulations of both IkappaBalpha and p65/RelA (but not p50/NF-kappaB1) in the cytoplasm. Clonogenic cell survival and soft agar assays further confirmed the increased TSA chemosensitivity of 267B1/K-ras cells by ProI treatment. Moreover, dominant negative mutant of IKKbeta, IkappaBalpha and p65 enhanced the chemosensitization, too. Unexpectedly, using LY294002 and PD98059, phosphatidylinositol-3-kinase and mitogen-activated protein kinase were also implied in TSA chemoresistance through NF-kappaB activation, while these compounds had showed no effect on radiosensitization in the cells. On the other hand, together with TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay, activations of caspase-8 and caspase-3 by TSA and ProI were noticed, suggesting the involvement of apoptotic process in chemosensitization of 267B1/K-ras cells. Altogether, these results suggest that blocking the NF-kappaB activation pathway could be an efficient target for improving the TSA chemosensitization and applying to the development of anticancer therapeutics in Ki-Ras-overexpressing tumorigenic cells, including prostate cancer.

Topics: Animals; Apoptosis; Cell Line, Transformed; Cell Survival; Cell Transformation, Neoplastic; Humans; Hydroxamic Acids; Male; Mice; Mice, Nude; NF-kappa B; Prostatic Neoplasms; Protein Synthesis Inhibitors; ras Proteins; Transfection

Transcriptional silencing associated with aberrant promoter methylation has been established as an alternate pathway for the development of cancer by inactivating tumor suppressor genes. TMS1 (Target of Methylation induced Silencing), also known as ASC (Apoptosis Speck like protein containing a CARD) is a tumor suppressor gene which encodes for a CARD (caspase recruitment domain) containing regulatory protein and has been shown to promote apoptosis directly and by activation of downstream caspases. This study describes the methylation induced silencing of TMS1/ASC gene in prostate cancer cell lines. We also examined the prevalence of TMS1/ASC gene methylation in prostate cancer tissue samples in an effort to correlate race and clinico-pathological features with TMS1/ASC gene methylation.. Loss of TMS1/ASC gene expression associated with complete methylation of the promoter region was observed in LNCaP cells. Gene expression was restored by a demethylating agent, 5-aza-2'deoxycytidine, but not by a histone deacetylase inhibitor, Trichostatin A. Chromatin Immunoprecipitation (ChIP) assay showed enrichment of MBD3 (methyl binding domain protein 3) to a higher degree than commonly associated MBDs and MeCP2. We evaluated the methylation pattern in 66 prostate cancer and 34 benign prostatic hyperplasia tissue samples. TMS1/ASC gene methylation was more prevalent in prostate cancer cases than controls in White patients (OR 7.6, p 0.002) while no difference between the cases and controls was seen in Black patients (OR 1.1, p 0.91).. Our study demonstrates that methylation-mediated silencing of TMS1/ASC is a frequent event in prostate cancer, thus identifying a new potential diagnostic and prognostic marker for the treatment of the disease. Racial differences in TMS1/ASC methylation patterns implicate the probable role of molecular markers in determining in susceptibility to prostate cancer in different ethnic groups.

Topics: Aged; Aged, 80 and over; Azacitidine; CARD Signaling Adaptor Proteins; Cell Line, Tumor; Chromatin Immunoprecipitation; CpG Islands; Cytoskeletal Proteins; Decitabine; DNA Methylation; DNA Modification Methylases; DNA, Neoplasm; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Gene Silencing; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Middle Aged; Promoter Regions, Genetic; Prostatic Hyperplasia; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction

The histone deacetylase inhibitor Trichostatin A (TSA) has previously been found to induce caspase activity in the human prostate cancer cell lines DU145 and LNCaP. TSA treatment resulted in the release of cytochrome c and Smac/DIABLO from mitochondria in DU145, and activation of caspase-9 in both cell lines. We concluded that TSA mediated its effect via the mitochondrial pathway. The aim of the current study was to determine how TSA initiated the caspase cascade. The results revealed that caspase-2 plays an important role in TSA-induced apoptosis. Inhibition of caspase-2 by siRNA or expression of caspase-2dn substantially decreased caspase activity after TSA treatment in both cell lines, siRNA caspase-2 also inhibited TSA-induced cell death. Caspase-2 acts upstream of caspase-8 and -9 and mediates mitochondrial cytochrome c release. Coimmunoprecipitation experiments show that caspase-2 formed protein complexes with RADD/RAIDD and PIDD. Together, these data indicate that caspase-2 initiates caspase cascade after TSA treatment and involves the formation of the PIDDosome.

Topics: Carrier Proteins; Caspase 2; Caspase 8; Caspase 9; Cell Death; Cell Line, Tumor; CRADD Signaling Adaptor Protein; Cytochromes c; Death Domain Receptor Signaling Adaptor Proteins; Enzyme Activation; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Mitochondria; Prostatic Neoplasms; RNA, Small Interfering; Transfection

Histone deacetylase (HDAC) inhibitors have shown significant anti-proliferative and apoptotic properties on various cancer cells, including prostate cancer, and are therefore being evaluated as treatment modalities. However, the specific effect of HDAC inhibitors on androgen-sensitive and androgen-independent cell lines have not been thoroughly studied which we hypothesized could be different. We therefore assessed whether three structurally unrelated HDAC inhibitors, trichostatin A (TSA), depsipeptide (FR901228), and sodium butyrate, affect cell death in the prostate cancer cell lines LNCaP, DU-145, and PC-3.. To investigate the extent and the nature of cell death, we used Trypan blue exclusion assay, phase-contrast light microscopy, fluorescence microscopy, and Western blot analyses.. At concentrations where they potentiate transcriptional activation, all three HDAC inhibitors induced cell death in LNCaP and DU-145 cells, but not in PC-3 cells, within the timeline of the experiments. HDAC inhibitor-induced cell death in LNCaP and DU-145 cells showed several characteristic apoptotic features, such as cell shrinkage, nuclear condensation, and poly(ADP) ribose polymerase cleavage. However, there were differences in the way LNCaP and DU-145 cells responded to treatment with various HDAC inhibitors. For example, whereas TSA and FR901228 were more effective in inducing apoptosis in LNCaP cells compared with DU-145 cells, the reverse was true for sodium butyrate. Moreover, within the same cell line, TSA, FR901228, and sodium butyrate exhibited different potencies for induction of apoptosis.. Collectively, these results suggest that the response of prostate cancer cells to HDAC inhibitors is not uniform, but cell line and inhibitor specific. Given that prostate cancer is generally a multiclonal disease representing different cell lineages, it is important to develop HDAC inhibitors that will be effective against all of these cell types.

Topics: Apoptosis; Blotting, Western; Butyrates; Cell Line, Tumor; Depsipeptides; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Indoles; Male; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Poly Adenosine Diphosphate Ribose; Prostatic Neoplasms; Trypan Blue

The human prostatic carcinoma cell line DU145 has previously been found to be resistant to treatment with TNF-family ligands. However, TRAIL, TNF-alpha and anti-Fas antibodies (Ab) treatment in combination with the histone deacetylase inhibitor Trichostatin A (TSA) converted the phenotype of DU145 from resistant to sensitive. TSA induced 15% cell death but simultaneous treatment with TRAIL, TNF-alpha and anti-Fas Ab resulted in 55%, 70% and 40% cell death, respectively. Simultaneous treatment did not increase the level of TSA-induced histone acetylation, but induced the release of acetylated histones from chromatin into the cytosol. This release was caspase dependent since it was abrogated by Z-VAD-fmk. In addition, treatment with TSA induced caspase-9 activation and resulted in the release of cytochrome c and Smac/DIABLO from mitochondria. To further investigate the role of caspase-9 in TSA-mediated apoptosis we used two different approaches: (1) cells were pretreated with the caspase-9 inhibitor Z-LEHD-fmk, and (2) cells were transfected with a dominant-negative form of caspase-9. Both approaches gave similar results: cells became resistant to treatment with TSA. These data indicate that TSA mediates its effect via the mitochondrial pathway. This was confirmed by examining DU145 overexpressing Bcl-2. These transfectants were resistant to TSA treatment. Taken together, our data shows that only simultaneous treatment with TNF-family ligands and TSA in DU145 resulted in caspase activity sufficient to induce apoptosis. The combination of TSA and TNF-family ligands could potentially be the basis for the treatment of prostate cancer.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Cell Line, Tumor; Cell Survival; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Ligands; Male; Membrane Glycoproteins; Models, Biological; Prostatic Neoplasms; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-X Protein; Caspases; Cell Proliferation; Collagen Type XI; Cytochromes c; Down-Regulation; Drug Resistance, Neoplasm; Enzyme Activation; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Ligands; Male; Membrane Glycoproteins; Prostatic Neoplasms; RNA, Small Interfering; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

Quantitative structure-activity relationships (QSAR) for a series of new trichostatin A (TSA)-like hydroxamic acids for the inhibition of cell proliferation of the PC-3 cell line have been developed using molecular descriptors from Qikprop and electronic structure calculations. The best regression model shows that the PM3 atomic charge on the carbonyl carbon in the CONHOH moiety(Qco), globularity (Glob), and the hydrophilic component of the solvent-accessible surface area (FISA) describe the IC(50) of 19 inhibitors of the PC-3 cell line with activities ranging over five orders of magnitude with an R(2)=0.92 and F=59.2. This information will be helpful in the further design of novel anticancer drugs for treatment of prostate cancer and other diseases affected by HDAC inhibition.

Topics: Antineoplastic Agents; Cell Division; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Male; Molecular Structure; Prostatic Neoplasms; Quantitative Structure-Activity Relationship; Tumor Cells, Cultured; Vorinostat

Pathogenesis of prostate cancer is paralleled by aberrant transcriptional regulation which involves gene silencing by histone deacetylases. In cancer cells, inhibitors of histone deacetylases such as valproic acid can act as differentiation agents which relieve pro-apoptotic factors from transcriptional repression. We investigated the potential of the well-tolerated anticonvulsant valproic acid in prostate cancer cell line LNCaP and analyzed the activation of pro-apoptotic factors and resulting apoptosis. We used real time RT-PCR to quantify the mRNA expression of prostate-specific antigen, prostate-derived Ets transcription factor, tissue inhibitor of matrix metalloproteinase-3 and insulin-like growth factor binding protein-3. An automated sandwich-ELISA was used to measure secretion of prostate-specific antigen in conditioned cell culture media of LNCaP prostate cancer cells. Apoptotic cells were detected cytochemically and by applying immunocytochemistry. Activity of histone deacetylases in nuclear extracts was measured with a colorimetric assay kit. Valproic acid treatment caused a marked inhibition of histone deacetylases activity. Expression of prostate-derived Ets transcription factor and consequently prostate-specific antigen were down-regulated to basal levels in LNCaP cells. Pro-apoptotic factor caspase-3, tissue inhibitor of matrix metalloproteinase-3 and insulin-like growth factor binding protein-3 were up-regulated resulting in apoptosis of tumor cells. Valproic acid mediates marked effects on the expression of genes relevant in proliferation and apoptosis. Our study provides strong evidence that prostate cancer may benefit particularly from anti-proliferative stimuli from this well established drug.

Topics: Apoptosis; Caspase 3; Caspases; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Neoplastic; HeLa Cells; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Insulin-Like Growth Factor Binding Protein 3; Male; Prostate-Specific Antigen; Prostatic Neoplasms; Proto-Oncogene Proteins c-ets; Receptors, Androgen; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tissue Inhibitor of Metalloproteinase-3; Transcription Factors; Valproic Acid

The androgen receptor (AR) regulates ligand-dependent gene transcription upon binding specific DNA sequences. The AR conveys both trans-activation and trans-repression functions, which together contribute to prostate cellular growth, differentiation, and apoptosis. Like histone H3, the AR is post-translationally modified by both acetylation and phosphorylation. The histone acetyltransferase p300 transactivates the AR and directly acetylates the AR in vitro at a conserved motif. Point mutations of the AR acetylation motif that abrogate acetylation reduce trans-activation by p300 without affecting the trans-repression function of the AR. The current studies assessed the functional relationship between acetylation and phosphorylation of the AR. Herein trans-activation of the AR acetylation site mutants were enhanced by the p42/p44 MAPK pathway but were defective in regulation by protein kinase A (PKA) signaling. PKA inhibition augmented ARwt activity but not AR acetylation mutant gene reporter activity and association at an androgen response element in chromatin immunoprecipitation assays. Mutations of the lysine residues at the AR acetylation site reduced trichostatin A (TSA) responsiveness and ligand-induced phosphorylation of the AR. The AR acetylation site mutant formed ligand-induced phosphorylation-dependent isoforms with distinguishable characteristics from wild type AR as determined with two-dimensional electrophoresis. Conversely, point mutation of a subset of AR phosphorylation sites reduced trichostatin A responsiveness and trans-activation by histone acetyltransferases. Together these studies suggest that acetylation and phosphorylation of the AR are linked events and that the conserved AR lysine motif contributes to a select subset of pathways governing AR activity.

Topics: Acetylation; Animals; Cell Line, Tumor; Cyclic AMP; Enzyme Inhibitors; Gene Expression Regulation; Genes, Reporter; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; JNK Mitogen-Activated Protein Kinases; Ligands; Lysine; Male; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Phosphates; Point Mutation; Prostatic Neoplasms; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Receptors, Androgen; Transcription, Genetic

Down-regulation of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) tumor suppressor gene expression is common in several malignancies including prostate, colon, and breast cancer. The mechanism that mediates this down-regulation is not known. Here, we report that down-regulation of CEACAM1 expression in prostate cancer cells occurs primarily at the transcriptional level and is mediated by Sp2, a member of the Sp family of transcription factors. Sp2 binds to the CEACAM1 promoter in vitro and in vivo, and transient overexpression of Sp2 down-regulates endogenous CEACAM1 expression in normal prostate epithelial cells. Sp2 appears to repress CEACAM1 gene expression by recruiting histone deacetylase activity to the CEACAM1 promoter. In human prostate cancer specimens, Sp2 expression is high in prostate cancer cells but low in normal prostate epithelial cells and is inversely correlated with CEACAM1 expression. Our studies show that transcriptional repression by Sp2 represents one mechanism by which CEACAM1 tumor suppressor gene is down-regulated in prostate cancer.

Topics: Acetyltransferases; Animals; Antigens, CD; Antigens, Differentiation; Carcinoembryonic Antigen; Cell Adhesion Molecules; Cell Line, Tumor; DNA-Binding Proteins; Down-Regulation; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Histone Acetyltransferases; Humans; Hydroxamic Acids; Male; Mice; Mice, Inbred BALB C; Promoter Regions, Genetic; Prostatic Neoplasms; Sp2 Transcription Factor; Transcription Factors; Transcription, Genetic

Androgen receptor (AR) overexpression is one of the characteristics of prostate cancer (PC) that progresses to hormone independence. An androgen-independent (AI) derivative, with much higher AR-mRNA and protein levels than the parental LNCaP cell line, whose proliferation was androgen dependent (AD), was used to explore the mechanism of AR overexpression. We found that a suppressor element (ARS), previously identified in mouse AR and located in the 5'-untranslated region of human AR gene, malfunctions in AI cells. Transfection of constructs that included ARS element into AD cells reduced the transactivating activities of both AR promoter and a heterologous SV40 promoter. The deletion of ARS resulted in an eightfold increase in AR-promoter activity in AD cells, but had no effect in AI cells. Moreover, the nuclear extracts of AD cells contained proteins that produced a specific, ARS-binding complex, while this complex appeared to have been lost from AI cells. Most importantly, treatment of AI cells with a demethylating agent or histone deacetylase inhibitors restored the lost ARS-binding complex. The restoration of the complex coincided with a reduced expression of AR-mRNA and protein and a reduced rate of AR-gene transcription, determined by nuclear run-on experiment. Thus, epigenetic transcriptional silencing of the suppressor protein(s) may be responsible for AR overexpression in AI cells, and its reversal in hormone-independent PC may normalize AR levels and restore their hormone dependence.

Topics: 5' Untranslated Regions; Carcinoma; Cell Division; Cell Line, Tumor; Cell Nucleus; Enzyme Inhibitors; Gene Deletion; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, Reporter; Genes, Tumor Suppressor; Humans; Hydroxamic Acids; Luciferases; Male; Promoter Regions, Genetic; Prostatic Neoplasms; Proteins; Receptors, Androgen; RNA, Messenger; Transcription, Genetic; Transcriptional Activation; Transfection

Androgens are critical in the development and maintenance of the male reproductive system and important in the progression of prostate cancer. The effects of androgens are mediated by the androgen receptor (AR), which is a ligand-modulated transcription factor that belongs to the nuclear receptor superfamily. We and others have previously shown that CREB-binding protein (CBP) can function as a coactivator for AR. Similar to some other nuclear receptor coactivators and/or the proteins that they interact with, CBP has histone acetyl transferase (HAT) activity that is thought to contribute to transcriptional activation by nuclear receptors. We have therefore assessed whether an increase in the histone acetylation status in the cell can influence AR transcriptional activity, by using the histone deacetylase (HDAC) inhibitors (HDACIs) trichostatin A (TSA), sodium butyrate (Na-But) and depsipeptide (FR901228). We found that inhibition of HDAC activity significantly increased the ability of endogenous AR in LNCaP cells, or ectopically expressed AR in HeLa cells, to activate transcription from AR-dependent reporter constructs. In addition, HDACIs increased the androgen-dependent activation of the prostate-specific antigen (PSA) gene in LNCaP cells, an increase that was not due to an increase in nuclear AR protein levels. Moreover, the viral oncoprotein E1A that inhibits CBP HAT activity fully repressed the ability of HDACIs to stimulate AR-mediated transcription, indicating that CBP is involved in this process. Deletional mutagenesis of AR indicated that whereas the AF-2 domain in the C-terminus is dispensable, the AF-1 domain in the N-terminus is required for augmentation of AR action by HDACIs, an observation which is in concordance with the reduced ability of CBP to activate AR N-terminal deletion mutants. Furthermore, HDACI treatment rescued the deficiency in the transactivation potential of AF-2 mutants. Taken together, our findings suggest that a change in the level of histone acetylation of target genes is an important determinant of AR action, possibly mediated by CBP.

Topics: Acetylation; Animals; Blotting, Northern; Blotting, Western; Butyrates; Depression, Chemical; Depsipeptides; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Male; Mutation; Peptides, Cyclic; Prostatic Neoplasms; Receptors, Androgen; Transcription, Genetic

In human prostate cancer cells, the availability of the steroid hormone 1,25-dihydroxyvitamin D(3) for antimitotic action is determined through the activity of the two enzymes CYP24 and CYP27B1, viz. 25-hydroxyvitamin D-24-hydroxylase and 25-hydroxyvitamin D-1alpha-hydroxylase. High performance liquid chromatography (HPLC) analysis of [(3)H]25(OH)D(3) metabolism in human prostate cancer DU-145 cells revealed that genistein and other isoflavonoids, such as dihydrogenistein and daidzein, as well as the antiestrogenic compound ICI 182,780, inhibited Vitamin D-metabolizing enzyme activities. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that only in case of genistein this was due to transcriptional inhibition of CYP24 and CYP27B1 gene expressions. In case of CYP27B1, reduction of gene activity involves histone deacetylation because genistein was inactive in the presence of the histone deactylase inhibitor trichostatin A. In contrast, under the same condition, CYP24 gene activity was largely suppressed. In summary, our results suggest that a combined effect of genistein and trichostatin A could increase the responsiveness of human prostate cancer cells to the antiproliferative action of 1,25-dihydroxyvitamin D(3).

Topics: 25-Hydroxyvitamin D3 1-alpha-Hydroxylase; Cell Division; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme Inhibitors; Enzyme Inhibitors; Estradiol; Estrogen Antagonists; Estrogens, Non-Steroidal; Fulvestrant; Genistein; Histone Deacetylases; Humans; Hydroxamic Acids; Isoflavones; Male; Phytoestrogens; Plant Preparations; Prostatic Neoplasms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Steroid Hydroxylases; Time Factors; Transcription, Genetic; Tumor Cells, Cultured; Vitamin D; Vitamin D3 24-Hydroxylase

Transcriptional silencing of cancer-related genes by DNA methylation is observed in various cancers. To identify genes controlled by methylation in prostate cancer, we used cDNA microarray analysis to investigate gene expression in prostate cancer cell lines LNCaP and DU145 treated with a methyltransferase inhibitor alone or together with a histone deacetylase inhibitor. We detected significant changes (3.4-5.7%) in gene expression in prostate cancer cell lines with the drug treatments. Among the affected genes, that for the vascular endothelial growth factor receptor 1 (VEGFR-1) was re-expressed in LNCaP and DU145 after the drug treatments. Bisulfite sequencing revealed the promoter and exon 1 of the VEGFR-1 to be hypermethylated in the cell lines. These results support the idea that methylation is associated with loss of VEGFR-1 mRNA expression in prostate cancer cell lines. Combined bisulfite restriction analysis (COBRA) showed the gene to be methylated in 24 (38.1%) of 63 primary local prostate cancer samples, while in all 13 benign prostate samples it was not. These findings indicate that methylation of VEGFR-1 is related with prostatic carcinogenesis.

Topics: Antimetabolites, Antineoplastic; Azacitidine; Decitabine; DNA Methylation; Enzyme Inhibitors; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Oligonucleotide Array Sequence Analysis; Polymerase Chain Reaction; Promoter Regions, Genetic; Prostatic Hyperplasia; Prostatic Neoplasms; Sulfites; Tumor Cells, Cultured; Vascular Endothelial Growth Factor Receptor-1

Breast and prostate cancer are leading causes of cancer death in the Western world. Hormone ablation is the primary therapy for invasive disease, but the tumour often recurs in an androgen or oestrogen receptor negative form for which novel therapies are sought urgently. The vitamin D receptor (VDR) may provide an important alternative therapeutic target. However, cancer cell line models from these tissues display a range of sensitivities to the antiproliferative effects of 1alpha,25dihydroxyvitamin D3 (1alpha,25(OH)2D3). The reason for apparent 1alpha,25(OH)2D3 insensitivity is currently unknown and we have investigated epigenetic mechanisms that may suppress the transcriptional activity of the VDR. Nuclear co-repressors have associated histone deacetylase (HDAC) activity, which keeps chromatin in a closed, transcriptionally silent state. We have found that the aggressive cancer cell lines with relative insensitivity to 1alpha,25(OH)2D3 have elevated nuclear co-repressor levels. For example, PC-3 prostate cancer cells have a significant 1.8-fold elevation in the co-repressor SMRT compared to normal epithelial cells (P < 0.05). We believe that a combination of elevated co-repressor level with reduced VDR content can cause 1alpha,25(OH)2D3 resistance. Consistent with this, we have shown that combining a low dose of HDAC inhibitor Trichostatin A (15 nM TSA) with 1alpha,25(OH)2D3 (100 nM) synergistically inhibits the proliferation of PC-3 prostate and MDA-MB-231 breast cancer cell lines. The inhibition of proliferation was potentiated further by treating cells with 19-nor-hexafluoride vitamin D3 analogues instead of 1alpha,25(OH)2D3, plus TSA. For example, the combination of 1alpha,25(OH)2D3 and TSA-inhibited MDA-MB-231 cell proliferation by 38% (+/-5%), whereas Ro26-2198 (1alpha,25-(OH)2-16,23Z-diene-26,27-F6-19-nor-D3) and TSA inhibited growth by 62% (+/-2%). Therapeutically the hypercalcaemic side effects associated with 1alpha,25(OH)2D3 could be minimized by combining low doses of potent 1a,25(OH)2D3 analogues with HDAC inhibitors as a novel anticancer regime for hormone-insensitive prostate and breast cancer.

Topics: Androgens; Antineoplastic Agents; Breast Neoplasms; Cell Division; Cholecalciferol; DNA-Binding Proteins; Drug Synergism; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Ligands; Male; Neoplasms, Hormone-Dependent; Nuclear Receptor Co-Repressor 2; Prostatic Neoplasms; Repressor Proteins; Tumor Cells, Cultured; Vitamin D

Although prostate-specific antigen (PSA) is considered a uniquely important tumor marker and is broadly used for early detection of prostate cancer, the molecular mechanisms underlying its elevated expression in tumors have been unknown. By using cDNA microarray gene expression profiling, we found a fourfold increase in the PSA mRNA level in prostatic carcinoma cell line LNCaP, in which the p53 pathway was suppressed by a dominant negative p53 mutant. Consistently, p53 suppression caused a 4-8-fold increase in secretion of PSA protein in culture medium, suggesting that PSA gene expression is under negative control of p53. While wild type p53 strongly repressed, dominant negative p53 mutants stimulated PSA promoter-driven transcription and secretion of PSA in transient transfection experiments. The inhibitory effect of wild type p53 was undetectable in the presence of trichostatin A, suggesting the involvement of histone deacetylation in negative regulation of PSA promoter activity. Thus, PSA is likely to be a tissue specific indicator of transformation-associated p53 suppression in prostate cells. This finding provides a plausible explanation for a frequent increase of PSA levels in advanced prostate cancer.

Topics: Acetylation; Adenocarcinoma; Chloramphenicol O-Acetyltransferase; Culture Media, Conditioned; DNA, Complementary; Gene Expression Regulation, Neoplastic; Genes, Dominant; Genes, p53; Genes, Reporter; Humans; Hydroxamic Acids; Male; Neoplasm Proteins; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; Prostate-Specific Antigen; Prostatic Neoplasms; Protein Processing, Post-Translational; Recombinant Fusion Proteins; RNA, Messenger; RNA, Neoplasm; Transcription, Genetic; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Telomerase activity is involved in cellular immortality. We have recently demonstrated that telomerase activity is closely associated with cell proliferation in prostate cancers. Telomerase is composed primarily of the catalytic subunit (hTERT) and the RNA template (hTERC), and hTERT expression is regulated by several factors such as c-MYC and p21(Waf1). Histone deacetylase (HDAC) inhibitors are known to modulate transcription and exhibit antiproliferative effects on cancer cells. The present study was designed to evaluate the effects of HDAC inhibitors on hTERT mRNA expression in prostate cancer cells. LNCaP and PC-3 cells were treated with HDAC inhibitors, trichostatin A (TSA) and sodium butyrate (NaB); mRNA expression and telomerase activity were evaluated by RT-PCR and the TRAP assay, respectively. In LNCaP cells, hTERT mRNA expression was suppressed at 1 and 3 hr after treatment with 1 microM TSA and 4 mM NaB, respectively, followed by inhibition of telomerase activity. The inhibition of hTERT mRNA expression preceded suppression of cell proliferation. In PC-3 cells, TSA and NaB also inhibited cell proliferation, hTERT mRNA expression and telomerase activity. In both cell lines, TSA and NaB had no effect on hTERC expression, or on expression of c-myc and p21(Waf1) mRNA. These effects of TSA and NaB were unlikely to be consequences of cell cycle arrest, apoptosis, or cell differentiation. Thus, HDAC inhibitors down-regulated telomerase activity via suppression of hTERT mRNA expression. Our study identified a novel mechanism for the antiproliferative effects of HDAC inhibitors on prostate cancer cells.

Topics: Butyrates; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA-Binding Proteins; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Neoplasms, Experimental; Prostate-Specific Antigen; Prostatic Neoplasms; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-myc; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Telomerase; Tumor Cells, Cultured

Prostate cancer is a major cause of male cancer death. In vitro and in vivo data support a role for 1 alpha,25 Dihydroxyvitamin D(3) (1 alpha,25(OH)(2)D(3)) in regulating the growth and differentiation of the normal prostate gland yet prostate cancer cells appear significantly less sensitive to this action. Vitamin D(3) receptor (VDR) content or mutational status do not correlate clearly with the antiproliferative effects of 1 alpha,25(OH)(2)D(3) and therefore it is unclear why prostate cancer cell lines are significantly less sensitive to this action. We hypothesized that the antiproliferative responses of prostate cancer cells to 1 alpha,25(OH)(2)D(3) are suppressed by a process involving histone deacetylation. Sodium butyrate (NaB) and trichostatin A (TSA) are inhibitors of histone deacetylase (HDAC) activity. Low doses of NaB or TSA (300 microM and 15 nM respectively), which alone were relatively inactive, synergized with 1 alpha,25(OH)(2)D(3) in liquid and semi-solid agar to inhibit the growth of LNCaP, PC-3 and DU-145 prostate cancer cells. Still greater synergy was observed between vitamin D(3) hexafluoride analogs and either NaB or TSA. The mechanism appeared to involve neither the cyclin-dependent kinase inhibitor, p21((waf1/cip1)) nor cell cycle arrest, but rather induction of apoptosis. These data suggest that cells dysregulate the normal pro-apoptotic signals of 1 alpha,25(OH)(2)D(3) during prostate cancer development by a mechanism involving histone deacetylation. Combination therapy with potent vitamin D(3) analogs and clinically approved HDAC inhibitors may overcome this lesion and improve the treatment of both androgen-dependent and independent prostate cancer.

Topics: Antineoplastic Agents; Apoptosis; Butyric Acid; Calcitriol; Cell Cycle; Cholecalciferol; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cytochrome P-450 Enzyme System; Drug Synergism; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Prostatic Neoplasms; Steroid Hydroxylases; Transcriptional Activation; Tumor Cells, Cultured; Vitamin D3 24-Hydroxylase

The neuroendocrine (NE) cell is a minor cell population in normal human prostate glands. The number of NE cells is increased in advanced hormone-refractory prostate carcinomas (PCA). The mechanism of increased NE cell population in these advanced tumors is poorly understood. We examined molecular mechanisms which may be involved in the regulation of the transdifferentiation process of human PCA cells leading to a NE phenotype. We compared PCA cell lines LNCaP and PC-3 in the following medium conditions: steroid-reduced (SR), interleukin-6 (IL-6)-supplemented, or dibutyrate cAMP (db-cAMP)-supplemented. We found that androgen-responsive C-33 LNCaP cells responded to all treatments, having a neuronal-like morphology. In contrast, C-81 LNCaP cells, having a decreased androgen responsiveness, had a less pronounced effect although followed a similar trend. Androgen-unresponsive PC-3 cells showed little change in their morphology. Grown in the SR condition, the level of neuron-specific enolase (NSE), a marker of neuronal cells, was upregulated in C-33 LNCaP cells, while to a lesser degree in the presence of IL-6. In the presence of db-cAMP, the NSE level in C-33 cells was decreased, lower than that in control cells. An opposite effect was observed for C-81 LNCaP cells. Nevertheless, the NSE level was only elevated in db-cAMP-treated PC-3 cells, but no change was found in PC-3 cells grown in the SR- or IL-6-supplemented medium. Thus, a similar gross phenotypic change may correlate with differential molecular expressions. We also analyzed the expression of protein tyrosine phosphatase alpha (RPTPalpha) since it plays a critical role in normal neuronal differentiation and signaling. Our results showed that the expression of RPTPalpha correlates with the NE phenotypic change of LNCaP cells in the SR condition. In summary, our data clearly show that the molecular process by which cultured human prostate cancer cells undergo a transdifferentiation process to a NE cell-like phenotype is accompanied by differential expressions of different markers, and a gross NE cell-like phenotype can occur by exposing PCA cells to different pharmacological agents.

Topics: Androgens; Bucladesine; Carcinoma; Cell Differentiation; Culture Media; Humans; Hydroxamic Acids; Interleukin-6; Male; Neurosecretory Systems; Phenotype; Phosphopyruvate Hydratase; Prostate-Specific Antigen; Prostatic Neoplasms; Protein Tyrosine Phosphatases; Receptor-Like Protein Tyrosine Phosphatases, Class 4; Receptors, Androgen; Receptors, Cell Surface; Staining and Labeling; Tumor Cells, Cultured

Methylation of the glutathione S-transferase P1 (GSTP1) gene has been described as a highly specific and sensitive biomarker for prostate cancer. However, at present, it is not known whether methylation represses GSTP1 gene expression in human prostate cancer. We found the GSTP1 gene promoter to be completely methylated in the LNCaP prostate cancer cell line, where this gene is transcriptionally inactive. In contrast, Du145 and PC3 prostate cancer cells express the GSTP1 gene and exhibit methylated and unmethylated GSTP1 alleles. In a transient transfection assay using LNCaP cells, methylation of the GSTP1 promoter-driven luciferase reporter vector (GSTP1-pGL3) resulted in a >20-fold inhibition of transcription, and this repression was not relieved by the presence of a histone deacetylase inhibitor, trichostatin A (TSA). Treatment of LNCaP cells with a DNA methyltransferase inhibitor, 5-Aza-2'-deoxycytidine, resulted in demethylation and activation of the GSTP1 gene. In contrast, TSA treatment failed to demethylate or activate the GSTP1 gene. Fully methylated but not unmethylated GSTP1 promoter fragment was shown to bind to a complex similar to methyl cytosine-binding protein complex 1 that contains methyl-CpG-binding domain 2 protein (MBD2) in electrophoretic mobility shift assays using LNCaP cell nuclear extracts. These data demonstrate that cytosine methylation can repress GSTP1 gene expression in LNCaP prostate cancer cells and that this effect is possibly mediated by a methyl cytosine-binding protein complex 1-like complex. Furthermore, these data also support the notion of the dominance of methylation over TSA-sensitive histone deacetylation in silencing genes with a high CpG density in the promoter region.

Topics: Azacitidine; Base Sequence; Cytosine; Decitabine; DNA Methylation; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Gene Silencing; Glutathione S-Transferase pi; Glutathione Transferase; HeLa Cells; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Isoenzymes; Male; Molecular Sequence Data; Plasmids; Promoter Regions, Genetic; Prostatic Neoplasms; Transfection; Tumor Cells, Cultured

Expression of the KAI1 gene, a metastasis-suppressor for prostate cancer, is reduced in all foci of prostatic metastasis. The altered regulatory mechanism is not strongly related to mutations or allelic losses of the KAI1 gene in prostate tumors. Since transcriptional silencing of genes has been found to be caused by epigenetic mechanisms, we have investigated the involvement of this epigenetic regulation of KAI1 expression in prostate cancers. The methylation status of the KAI1 promoter region was examined by restriction-enzyme digestion and sequencing, after amplifying a 331-bp fragment in the GC-rich promoter region from 4 human prostate cancer cell lines treated with bisulfite. The same 4 cell lines were also exposed to various concentrations of the demethylating agent, 5-aza-2'-deoxycytidine (5-AzaC) and / or the histone deacetylase inhibitor, trichostatin A (TSA). To clarify the influence of epigenetic modification on reduced KAI1 mRNA expression in the tumor cells, RT-PCR and northern-blot analyses were performed. Bisulfite-sequencing data showed a few methylated CpG islands in the promoter. RT-PCR analysis of 5-AzaC and / or TSA-treated cells indicated reversal of suppression of KAI1 transcription in two cell lines (PC-3 and DU-145), although the expression could not be detected by northern blots. From these results, it is suggested that epigenetic change is not the main mechanism of KAI1 down-regulation, though there remains a possibility that methylation in a more upstream region might be associated with this regulation.

Topics: Adenocarcinoma; Antigens, CD; Azacitidine; Base Sequence; Decitabine; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, Tumor Suppressor; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Kangai-1 Protein; Male; Membrane Glycoproteins; Molecular Sequence Data; Neoplasm Metastasis; Neoplasm Proteins; Polymerase Chain Reaction; Promoter Regions, Genetic; Prostatic Hyperplasia; Prostatic Neoplasms; Proto-Oncogene Proteins; Sulfites; Tumor Cells, Cultured

Epigenetic mechanisms including DNA methylation and histone deacetylation are thought to play important roles in gene transcriptional inactivation. Heterogenous expression of androgen receptor (AR), which appears to be related to variable responses to endocrine therapy in prostate cancer (PCa) may also be due to epigenetic factors. The methylation status of the 5' CpG island of the AR in 3 prostate cancer cell lines and 10 primary and 14 hormone-refractory PCa samples was determined using the bisulfite PCR methods. In DU145, CpG-rich regions of the AR were hypermethylated. By an immunohistochemical analysis, only one PCa sample had no AR expression, the others being heterogenous. Bisulfite sequencing and methylation-specific PCR analysis showed aberrant methylation of AR 5'-regulatory region in 20% of 10 primary and 28% of 14 hormone-refractory PCa samples. To clarify the effect of epigenetic regulation on AR expression, we treated three prostate cancer cell lines with a demethylating agent, 5-aza-2'-deoxycytidine (azaC), and a histone deacetylase inhibitor, Trichostatin A (TSA). In DU145, re-expression of AR mRNA was detected after treatment with azaC and/or TSA. Our results suggest that epigenetic regulations including CpG methylation and histone acetylation may play important roles in the regulation of the AR.

Topics: Antimetabolites, Antineoplastic; Azacitidine; Base Sequence; Binding Sites; Cyclic AMP Response Element-Binding Protein; Decitabine; Dinucleoside Phosphates; DNA Methylation; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Male; Polymerase Chain Reaction; Promoter Regions, Genetic; Prostatic Neoplasms; Receptors, Androgen; Tumor Cells, Cultured