trichostatin-a and Fibrosarcoma

Chromatin texture patterns of tumour cell nuclei can serve as cancer biomarkers, either to define diagnostic classifications or to obtain relevant prognostic information, in a large number of human tumours. Epigenetic mechanisms, mainly DNA methylation and histone post-translational modification, have been shown to influence chromatin packing states, and therefore nuclear texture. The aim of this study was to analyse effects of these two mechanisms on chromatin texture, and also on correlation with gelatinase expression, in human fibrosarcoma tumour cells.. We investigated effects of DNA hypomethylating agent 5-aza-2'-deoxycytidine (5-azadC) and histone deacetylase inhibitor trichostatin A (TSA) on nuclear textural characteristics of human HT1080 fibrosarcoma cells, evaluated by image cytometry, and expression of gelatinases MMP-2 and MMP-9, two metalloproteinases implicated in cancer progression and metastasis.. 5-azadC induced significant variation in chromatin higher order organization, particularly chromatin decondensation, associated with reduction in global DNA methylation, concomitantly with increase in MMP-9, and to a lesser extent, MMP-2 expression. TSA alone did not have any effect on HT1080 cells, but exhibited differential activity when added to cells treated with 5-azadC. When treated with both drugs, nuclei had higher texture abnormalities. In this setting, reduction in MMP-9 expression was observed, whereas MMP-2 expression remained unaffected.. These data show that hypomethylating drug 5-azadC and histone deacetylase inhibitor TSA were able to induce modulation of higher order chromatin organization and gelatinase expression in human HT1080 fibrosarcoma cells.

Topics: Azacitidine; Biomarkers, Tumor; Cell Line, Tumor; Cell Nucleus; Chromatin; Chromatin Assembly and Disassembly; Decitabine; Disease Progression; DNA Methylation; Epigenesis, Genetic; Fibrosarcoma; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Image Cytometry; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Neoplasm Metastasis; Neoplasm Proteins

The cell nucleus is the largest and stiffest organelle rendering it the limiting compartment during migration of invasive tumor cells through dense connective tissue. We here describe a combined atomic force microscopy (AFM)-confocal microscopy approach for measurement of bulk nuclear stiffness together with simultaneous visualization of the cantilever-nucleus contact and the fate of the cell. Using cantilevers functionalized with either tips or beads and spring constants ranging from 0.06-10 N m(-1), force-deformation curves were generated from nuclear positions of adherent HT1080 fibrosarcoma cell populations at unchallenged integrity, and a nuclear stiffness range of 0.2 to 2.5 kPa was identified depending on cantilever type and the use of extended fitting models. Chromatin-decondensating agent trichostatin A (TSA) induced nuclear softening of up to 50%, demonstrating the feasibility of our approach. Finally, using a stiff bead-functionalized cantilever pushing at maximal system-intrinsic force, the nucleus was deformed to 20% of its original height which after TSA treatment reduced further to 5% remaining height confirming chromatin organization as an important determinant of nuclear stiffness. Thus, combined AFM-confocal microscopy is a feasible approach to study nuclear compressibility to complement concepts of limiting nuclear deformation in cancer cell invasion and other biological processes.

Topics: Cell Line, Tumor; Cell Nucleus; Elasticity; Equipment Design; Fibrosarcoma; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Microscopy, Atomic Force

Histone deacetylase (HDAC) plays a key role in gene expression, by suppressing the transcription of a number of target genes. Identification of such genes is important for deciphering the functional role of HDAC. Here, using cancer gene-focused DNA microarray analysis, we identified plakoglobin as a new target gene of HDAC. Functional inhibition of HDAC by its specific inhibitors induced the expression of plakoglobin by eight-fold in human fibrosarcoma HT1080 cells. However, the expression of beta-catenin, which is closely related to plakoglobin, was not altered, implying the specific function of HDAC in plakoglobin expression. Using antiacetyl-H4 antibody, chromatin immunoprecipitation analysis revealed that the distal region (-945 approximately -646) of the promoter of plakoglobin is responsible for the HDAC-mediated repression of the gene. Moreover, the induced expression of plakoglobin by the inhibition of HDAC activated the Tcf/Lef-dependent luciferase reporter gene, a well-known downstream effector of the Wnt signaling pathway. Furthermore, transient transfection of plakoglobin also activated Tcf/Lef reporter gene expression. Taken together, our results demonstrate that plakoglobin is a new target gene governed by HDAC, and that it acts as an oncogene in HT1080 cells.

Topics: Azacitidine; beta Catenin; Cell Line, Tumor; Chromatin; Chromatin Assembly and Disassembly; Cytoskeletal Proteins; Decitabine; Desmoplakins; DNA-Binding Proteins; Enzyme Inhibitors; Fibrosarcoma; gamma Catenin; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, Reporter; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Lymphoid Enhancer-Binding Factor 1; Oligonucleotide Array Sequence Analysis; Promoter Regions, Genetic; RNA, Messenger; Trans-Activators; Transcription Factors; Transcriptional Activation

Trichostatin A (TSA) is a histone deacetylase (HDAC) inhibitor with potential in cancer therapeutics. In a recent communication, we demonstrated that TSA is a selective, potent inhibitor of gelatinase A in 3T3 fibroblasts. In the present study, we extend these observations and examine the effects of TSA in 3T3 fibroblasts compared to HT-1080 fibrosarcoma cells with respect to gelatinase A expression, cell viability, and apoptosis. We find that while expression of gelatinase A in 3T3 fibroblasts is exquisitely sensitive to inhibition by TSA, expression of this enzyme in HT-1080 cells is minimally affected by this compound. Moreover, we show that TSA is pro-apoptotic in HT-1080 cells, but is anti-apoptotic in 3T3 cells. We propose a two-pronged model for the therapeutic action of TSA. On the one hand TSA selectively decreases cancer cell viability, while enhancing the viability of stromal cells. On the other hand, by selectively decreasing gelatinase A expression in stromal but not cancer cells, TSA acts to control metastatic potential by reducing the ability of metastatic cells to recruit stromal cells to secrete gelatinase A.

Topics: 3T3 Cells; Animals; Antineoplastic Agents; Cell Survival; Enzyme Inhibitors; Fibroblasts; Fibrosarcoma; Gene Expression; Hydroxamic Acids; Matrix Metalloproteinase 2; Mice; Tumor Cells, Cultured