trichostatin-a and Nasopharyngeal-Neoplasms

Epithelial-mesenchymal transition (EMT) may be one of the reasons for the failure in some clinical trials regarding histone deacetylase inhibitors (HDACIs)-treated solid tumors. We investigated the effects of a pan-HDACI trichostatin A (TSA) on the proliferation and EMT of nasopharyngeal carcinoma (NPC) cells.. Poorly-differentiated NPC cell line CNE2 and undifferentiated C666-1 were treated with various concentrations of TSA, the cell viability was assessed by CCK-8 assay, the morphology was photographed, and the mRNA level of HDACs was assessed by semiquantitative PCR. After determination the cell cycle distributions, cells were subjected to western blotting analysis of cell cycle and EMT-associated genes expression. And the changes in migration ability were assessed by transwell migration assay and scratch wound healing assay. Finally, histone deacetylases activator ITSA-1 was used to assess the reverse of TSA-induced changes in NPC cells.. TSA inhibited the proliferation of CNE2 and C666-1 cells in a concentration-dependent manner and arrested the cell cycle at G1 phases. TSA reduced PCNA, cyclin D1, cyclin E1, CDK2, p16 and p21 expressions and stimulated CDK6 levels. TSA stimulation for 48 h could effectively induce the EMT in CNE2 and C666-1 cells, which showed an increase of spindle-like cells and promoted expression of Vimentin and Snail1 expression in a concentration-dependent manner. Surprisingly, this short period of TSA treatment that induced EMT also impeded the migration ability of CNE2 and C666-1 cells. Interestingly, ITSA-1 rescued TSA-impeded CNE2 and C666-1 cells' proliferation, migration and HDACs expression, also re-induced the cells to turn into epithelial cell phenotypes.. These results indicate that short-term stimulation of TSA effectively inhibits cell proliferation and induce EMT-like changes in NPC cells but not increase its invasion ability.

Topics: Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Drug Screening Assays, Antitumor; Epithelial-Mesenchymal Transition; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Neoplasm Invasiveness; Time Factors

Trichostatin A (TSA) is a histone deacetylase (HDAC) inhibitor. We here investigated its effects on proliferation and apoptosis of the CNE2 carcinoma cell line, and attempted to establish genome-wide DNA methylation alteration due to differentially histone acetylation status. After cells were treated by TSA, the inhibitory rate of cell proliferation was examined with a CCK8 kit, and cell apoptosis was determined by flow cytometry. Compared to control, TSA inhibited CNE2 cell growth and induced apoptosis. Furthermore, TSA was found to induce genome-wide methylation alteration as assessed by genome-wide methylation array. Overall DNA methylation level of cells treated with TSA was higher than in controls. Function and pathway analysis revealed that many genes with methylation alteration were involved in key biological roles, such as apoptosis and cell proliferation. Three genes (DAP3, HSPB1 and CLDN) were independently confirmed by quantitative real-time PCR. Finally, we conclude that TSA inhibits CNE2 cell growth and induces apoptosis in vitro involving genome-wide DNA methylation alteration, so that it has promising application prospects in treatment of NPC in vivo. Although many unreported hypermethylated/hypomethylated genes should be further analyzed and validated, the pointers to new biomarkers and therapeutic strategies in the treatment of NPC should be stressed.

Topics: Apoptosis; Carcinoma; Cell Cycle; Cell Line, Tumor; Cell Proliferation; DNA Methylation; Genome-Wide Association Study; Humans; Hydroxamic Acids; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms

Protocadherin8 (PCDH8), an integral membrane protein, was reported to be a tumor suppressor involved in tumorigenesis in cancers. We aimed to investigate the epigenetic inactivation of PCDH8 and its tumor-suppressor function in nasopharyngeal carcinoma (NPC). Frequent downregulation/silencing of PCDH8 was found in NPC cell lines using semiquantitative PCR. Promoter methylation of PCDH8 was observed in 100% (5/5) of cell lines and 85.3% (35/41) of primary tumors by methylation-specific PCR, but not in normal nasopharyngeal tissues. Treatment of NPC cells with 5-aza-2'-deoxycytidine and/or trichostatin A could restore PCDH8 expression. Ectopic expression of PCDH8 in silenced NPC cells significantly inhibited cell colony formation and cell migration. For the first time, our study demonstrates the epigenetic inactivation of PCDH8 by promoter methylation and its tumor-suppressive function in NPC. Thus, PCDH8 could be identified as a tumor suppressor in NPC.

Topics: Apoptosis; Azacitidine; Cadherins; Cell Adhesion; Cell Movement; Cell Proliferation; Decitabine; DNA Methylation; Female; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Male; Middle Aged; Nasopharyngeal Neoplasms; Prognosis; Promoter Regions, Genetic; Protocadherins; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured; Tumor Suppressor Proteins; Wound Healing

Human stanniocalcin 1 (STC1) has recently been identified as a putative protein factor involved in cellular apoptosis. The use of histone deacetylase inhibitor (i.e. trichostatin A (TSA)) and doxorubicin (Dox) is one of the common treatment methods to induce apoptosis in human cancer cells. A study on TSA and Dox-mediated apoptosis may shed light on the regulation and function of STC1 in cancer treatment. In this study, TSA and Dox cotreatment in human nasopharyngeal carcinoma cells (CNE2) elicited synergistic effects on STC1 gene expression and cellular apoptosis. An activation of p53 (TP53) transcriptional activity in Dox- or Dox+TSA-treated cells was revealed by the increased expression levels of p53 mRNA/protein as well as p53-driven luciferase activities. To elucidate the possible involvement of p53 in STC1 gene transcription, a vector expressing wild-type or dominant negative (DN) p53 was transiently transfected into the cells. Both STC1 promoter luciferase constructs and chromatin immunoprecipitation assays did not support the direct role of p53 in STC1 gene transactivation. However, the synergistic effects of p53 on the induction of NF-κB phosphorylation and the recruitment of acetylated histone H3 in STC1 promoter were observed in TSA-cotreated cells. The overexpression of exogenous STC1 sensitized apoptosis in Dox-treated cells. Taken together, this study provides data to show the cross talk of NF-κB, p53, and histone protein in the regulation of STC1 expression and function.

Topics: Antibiotics, Antineoplastic; Apoptosis; Carcinoma; Cell Line, Tumor; Doxorubicin; Gene Expression; Glycoproteins; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Tumor Suppressor Protein p53

This study identified LTBP-2 as a pleiotropic tumor suppressor in nasopharyngeal carcinoma, which safeguards against critical malignant behaviors of tumor cells. LTBP-2 expression was significantly decreased or lost in up to 100% of NPC cell lines (7/7) and 80% of biopsies (24/30). Promoter hypermethylation was found to be involved in LTBP-2 silencing. Using a tetracycline-regulated inducible expression system, we unveiled functional roles of LTBP-2 in suppressing colony formation, anchorage-independent growth, cell migration, angiogenesis, VEGF secretion, and tumorigenicity. Three-dimensional culture studies suggested the involvement of LTBP-2 in maintenance of tumor cell dormancy in a growth factor favorable microenvironment.

Topics: Azacitidine; Carcinoma; Cell Line, Tumor; Cell Movement; Cell Transformation, Neoplastic; Decitabine; DNA Methylation; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Latent TGF-beta Binding Proteins; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Neovascularization, Pathologic; Promoter Regions, Genetic; Tumor Microenvironment; Tumor Suppressor Proteins; Vascular Endothelial Growth Factor A

Human disabled-2 (DAB2), is a multi-function signalling molecule that it is frequently down-regulated in human cancers. We aimed to investigate the possible tumour suppressor effect of DAB2 in nasopharyngeal carcinoma (NPC).. We studied the expression of DAB2 in NPC cell lines, xenografts and primary tumour samples. The status of promoter methylation was assessed by methylation specific PCR and bisulfite sequencing. The functional role of DAB2 in NPC was investigated by re-introducing DAB2 expression into NPC cell line C666-1.. Decrease or absent of DAB2 transcript was observed in NPC cell lines and xenografts. Loss of DAB2 protein expression was seen in 72% (33/46) of primary NPC as demonstrated by immunohistochemistry. Aberrant DAB2 promoter methylation was detected in 65.2% (30/46) of primary NPC samples by methylation specific PCR. Treatment of the DAB2 negative NPC cell line C666-1 with 5-aza-2'-deoxycytidine resulted in restoration of DAB2 expression in a dose-dependent manner. Overexpression of DAB2 in NPC cell line C666-1 resulted in reduced growth rate and 35% reduction in anchorage-dependent colony formation, and inhibition of serum-induced c-Fos expression compared to vector-transfected controls. Over expression of DAB2 resulted in alterations of multiple pathways as demonstrated by expression profiling and functional network analysis, which confirmed the role of DAB2 as an adaptor molecule involved in multiple receptor-mediated signalling pathways.. We report the frequent down regulation of DAB2 in NPC and the promoter hypermethylation contributes to the loss of expression of DAB2. This is the first study demonstrating frequent DAB2 promoter hypermethylation in human cancer. Our functional studies support the putative tumour suppressor effect of DAB2 in NPC cells.

Topics: Adaptor Proteins, Signal Transducing; Adolescent; Adult; Aged; Aged, 80 and over; Animals; Apoptosis Regulatory Proteins; Azacitidine; Carcinoma; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Decitabine; DNA Methylation; DNA Modification Methylases; Down-Regulation; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Genes, Tumor Suppressor; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Immunohistochemistry; Male; Mice; Middle Aged; Nasopharyngeal Neoplasms; Neoplasm Transplantation; Polymerase Chain Reaction; Promoter Regions, Genetic; Proto-Oncogene Proteins c-fos; RNA, Messenger; Time Factors; Transfection; Tumor Suppressor Proteins; Young Adult

In about 60% of Epstein-Barr virus (EBV) carrying nasopharyngeal carcinomas (NPC) LMP1 expressing cells can be detected. The frequency of LMP1 positive cells and the expression level varies from cell to cell in the different tumors. Cell lines derived from EBV positive NPCs loose the virus during in vitro culture. The in vitro infected NPC cell line TWO3-EBV used in our study carries the neomycin-resistance gene containing EBV and expresses low level of LMP1. With this cell line it was thus possible to study the regulation of LMP1 expression by modification of chromatin acetylation state.. The TWO-EBV cell line was treated with n -butyrate (NB) or trichostatin A (TSA).. Shown by immunoblotting, the LMP1 level was elevated in the treated samples. Already 2 h after TSA exposure LMP1 expression was higher and it increased up to 24 h. Immunofluorescence staining showed that nearly all cells were LMP1 positive. Neither EBNA2 nor BZLF1 were induced. Tested first 2 h after the treatment, acetylated histone H3 and H4 were already detectable, and their level increased up to 8 h. Chromatin immunoprecipitation (ChIP) verified that the LMP1-promoter (LMP1p) (ED-L1) was acetylated after TSA treatment.. EBV carrying epithelial cells do not express EBNA-2. We showed that LMP1 expression was upregulated by histone deacetylase inhibitors in an in vitro infected, EBV carrier NPC cell line.

Topics: Acetylation; Butyrates; Carcinoma; DNA; Gene Expression Regulation, Viral; Herpesvirus 4, Human; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Nasopharyngeal Neoplasms; Promoter Regions, Genetic; RNA, Messenger; Up-Regulation; Viral Matrix Proteins

Chromatin is a highly dynamic environment playing critical roles in the regulation of gene expression. Modifications to the proteins which make up the nucleosome core have been shown to have profound regulatory effects on gene expression. Of these, the best known modification is acetylation of the histone tails. Two enzymes regulate these processes, histone deacetylases and histone acetyltransferases. Both have been shown to have dysregulated functions in certain tumors. Several classes of histone deacetylase inhibitors have been isolated and are currently undergoing evaluation as potential therapeutic modalities in the treatment of cancer. In this study we examined the effects of three such inhibitors on general gene expression in three tumor cell lines derived from three separate tumor types using microarray gene profiling. Our results show that the patterns of alterations which emerge are similar for each cell type.

Topics: Carcinoma, Hepatocellular; Carcinoma, Renal Cell; Enzyme Inhibitors; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Kidney Neoplasms; Liver Neoplasms; Nasopharyngeal Neoplasms; Neoplasms; Oligonucleotide Array Sequence Analysis; Tumor Cells, Cultured; Vorinostat