trichostatin-a and Adenocarcinoma--Follicular

The purpose of this study was to investigate the effects of treating human thyroid cancer cell lines with demethylating agents and histone deacetylase (HDAC) inhibitors to see if they would downregulate expression and activity of the matrix metalloproteinases (MMP)-2 and MMP-9, resulting in inhibition of growth and invasion.. A total of 1 papillary cancer cell line (TPC-1) and 3 follicular thyroid cancer cell lines (FTC-133, FTC-236, and FTC-238) were treated with the demethylating agent 5-azacytidine (5-AZC) and the HDAC inhibitors trichostatin A (TSA) and valproic acid (VA). The activity of MMP proteins was determined using gelatin zymography, and commercially available assays were used to quantify growth inhibition and thyroid cancer cell invasion.. Treatment with TSA and VA resulted in decreased protein activity of MMP-2 and MMP-9 in all cell lines in a dose-dependent manner after 48 hours of treatment compared with untreated controls. In addition, 5-, TSA, and VA caused inhibition of growth in the range of 25-80% for all cell lines at 24, 48, and 72 hours. VA and TSA significantly decreased cell invasion in the FTC-133 and TPC-1 cell lines.. The HDAC inhibitors TSA and VA decreased the protein activity of MMP-2 and MMP-9 and, in combination with the demethylating agent 5-AZC, inhibited cellular growth in human papillary and follicular thyroid cancer cell lines. These results elucidate our understanding of the pathways affected by the demethylating agents and HDAC inhibitors, and provide further evidence that MMPs are a potentially useful target for molecular therapies in patients with aggressive or refractory thyroid cancers.

Topics: Adenocarcinoma, Follicular; Adenocarcinoma, Papillary; Antimetabolites, Antineoplastic; Azacitidine; Cell Line, Tumor; Drug Screening Assays, Antitumor; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Thyroid Neoplasms; Valproic Acid

Nucleophosmin (NPM) is a multifunctional nucleolar protein that, depending on the context, can act as oncogene or tumour suppressor. Mutations of the NPM1 gene induce delocalization of NPM in acute myeloid leukaemia. Differently, in solid tumours, only NPM overexpression, but not delocalization, has been so far reported. Here, NPM localization in thyroid tumours was investigated. By using immunohistochemistry, we show increase of NPM cytoplasmic localization in follicular adenomas and papillary carcinomas compared to normal thyroid tissue (p = 0.0125 and <0.0001, respectively). NPM1 mutations commonly found in human leukaemia are not present in thyroid tumours. Immunofluorescence in cultured cell lines was utilized to discriminate between nucleolar and nuclear localization. We show that in thyroid cancer cell lines NPM localizes both in the nucleolus and in nucleus, while in non-tumorigenic thyroid cell lines localizes only in nucleolus. Either presence of the histone deacetylase inhibitor trichostatin A or absence of thyroid-stimulating hormone induces NPM nuclear localization in non-tumorigenic thyroid cell lines.

Topics: Adenocarcinoma, Follicular; Adenoma; Biomarkers, Tumor; Carcinoma; Carcinoma, Papillary; Cell Line, Tumor; Cell Nucleolus; Cell Nucleus; DNA Mutational Analysis; DNA, Neoplasm; Humans; Hydroxamic Acids; Nuclear Proteins; Nucleophosmin; Thyroid Neoplasms; Thyrotropin

Increases in Rap activity have been associated with tumor progression. Although activating mutations in Rap have not been described, downregulation of Rap1GAP is frequent in human tumors including thyroid carcinomas. In this study, we explored whether endogenous Rap1GAP expression could be restored to thyroid tumor cells. The effects of deacetylase inhibitors and a demethylating agent, individually and in combination, were examined in four differentiated and six anaplastic thyroid carcinoma (ATC) cell lines. Treatment with the structurally distinct histone deacetylase (HDAC) inhibitors, sodium butyrate and trichostatin A, increased Rap1GAP expression in all the differentiated thyroid carcinoma cell lines and in four of the six ATC cell lines. The demethylating agent, 5-aza-deoxycytidine, restored Rap1GAP expression in one anaplastic cell line and enhanced the effects of HDAC inhibitors in a second anaplastic cell line. Western blotting indicated that Rap2 was highly expressed in human thyroid cancer cells. Importantly, treatment with HDAC inhibitors impaired Rap2 activity in both differentiated and anaplastic tumor cell lines. The mechanism through which Rap activity is repressed appears to entail effects on the expression of multiple Rap regulators, including RapGEFs and RapGAPs. These results suggest that HDAC inhibitors may provide a tractable approach to impair Rap activity in human tumor cells.

Topics: Adenocarcinoma, Follicular; Azacitidine; Butyrates; Carcinoma; Carcinoma, Papillary; Cell Line, Tumor; Decitabine; DNA Methylation; Drug Screening Assays, Antitumor; Drug Synergism; Gene Expression Regulation, Neoplastic; GTPase-Activating Proteins; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Neoplasm Proteins; rap GTP-Binding Proteins; rap1 GTP-Binding Proteins; RNA, Small Interfering; Thyroid Neoplasms; Up-Regulation

The effectiveness rate of all-trans-retinoic acid (RA) is only about 30% in the clinical application of inducing thyroid carcinoma differentiation. In addition, there are severe toxic side effects, which limit its clinical application. Phase I-III clinical studies have been conducted on the combined application of two or more kinds of inductors in tumors. Nevertheless, the combination of RA with histone deacetylase inhibitors is rarely reported. This article studied the effects of differentiation for papillary thyroid carcinoma and follicular thyroid carcinoma cell lines induced by RA combined with trichostatin A (TSA), enhancing the effect of induction, while reducing the toxic side effects of a single drug, to provide a theoretical basis for preclinical trials.. After incubation with RA combined with TSA, K1 and FTC-133 were grouped into Group 1 (RA 10(-4) mol/L plus TSA 1.65 x 10(-7) mol/L), Group 2 (RA 1 x 10(-4) mol/L plus TSA 3.31 x 10(-7) mol/L), Group 3 (RA 10(-5) mol/L plus TSA 1.65 x 10(-7) mol/L), Group 4 (RA 1 x10(-5) mol/L plus TSA 3.31 x 10(-7) mol/L) by four varied concentrations and three time points (12 h, 24 h, and 48 h). The cell proliferation, conformation, toxic effect, and induced differentiation on K1 and FTC-133 cell lines were studied microscopically with hematoxylin-eosin (HE) to observe cell quantity and morphology, methyl-thiazolyl-tetrazolium (MTT) to calculate cell survival rates, and electrochemiluminescence analysis measuring in vitro thyroglobulin (Tg) levels.. The research showed that K1 and FTC-133 cells had cell spacing increases, with an outer edge of smooth, nuclear chromatin condensation after RA combined TSA. Survival rate were assessed by an analysis of variance (ANOVA) by concentration and time point, F values of K1 and FTC-133 were 23.52 and 170.14, and 57.09 and 224.35, respectively. There were significant differences for both cells (P < 0.01). The SNK analysis indicated that survival rates were in the order of Group 2 < Group 1 < Group 4 < Group 3. Tg was also assessed by ANOVA, F values of K1 were 69.63 and 101.07, and F values of FTC-133 were 79.77 and 81.72 (P < 0.01). Group 1 was compared with Group 3 of K1 and FTC-133 by the least significant difference (LSD) method, and there was no statistical difference between the two group (P = 0.06, 0.2, respectively; P > 0.05), yet a significant difference was seen between the other Groups.. Lower concentrations of RA combined with lower concentrations of TSA have both inhibited cell proliferation, decreased toxicity of the drugs, and increased the effect of K1 and FTC-133 cell differentiation. The mechanism of action may be that TSA has pretranscription DNA regulation and that RA has posttranscriptional signal regulation to enhance the effects of inhibited proliferation and differentiation of cells by transcription systems.

Topics: Adenocarcinoma, Follicular; Antineoplastic Agents; Apoptosis; Carcinoma; Carcinoma, Papillary; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Synergism; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Thyroglobulin; Thyroid Cancer, Papillary; Thyroid Neoplasms; Tretinoin