trichostatin-a and Thyroid-Neoplasms

The influence of the heat shock protein 90 (hsp90) inhibitor SNX5422 alone or in combination with the histone deacetylase (HDAC) inhibitors PXD101, suberoylanilide hydroxamic acid (SAHA), and trichostatin A (TSA) on survival of anaplastic thyroid carcinoma (ATC) cells was investigated. In 8505C and CAL62 cells, SNX5422 caused cell death with concomitant changes in the expression of hsp90 client proteins. After treatment of both SNX5422 and PXD101, SAHA and TSA, compared with treatment of SNX5422 alone, cell viability was diminished, whereas inhibition rate and cytotoxic activity were enhanced. All of the combination index values were lower than 1.0, suggesting the synergism between SNX5422 and PXD101, SAHA and TSA in induction of cell death. In cells treated with both SNX5422 and PXD101, SAHA and TSA, compared with cells treated with SNX5422 alone, the protein levels of Akt, phospho-4EBP1, phospho-S6 K, and survivin were diminished, while those of γH2AX, acetyl. histone H3, acetyl. histone H4, cleaved PARP, and cleaved caspase-3 were enhanced. In conclusion, these results demonstrate that SNX5422 has a cytotoxic activity in conjunction with alterations in the expression of hsp90 client proteins in ATC cells. Moreover, SNX5422 synergizes with HDAC inhibitors in induction of cytotoxicity accompanied by the suppression of PI3K/Akt/mTOR signaling and survivin, and the overexpression of DNA damage-related proteins in ATC cells.

Topics: Antineoplastic Agents; Apoptosis; Benzamides; Caspase 3; Cell Line, Tumor; Cell Survival; Drug Synergism; Glycine; Histone Deacetylase Inhibitors; HSP90 Heat-Shock Proteins; Humans; Hydroxamic Acids; Indazoles; Phosphorylation; Proto-Oncogene Proteins c-akt; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms; Vorinostat

In thyroid cancer, the lack of response to specific treatment, for example, radioactive iodine, can be caused by a loss of differentiation characteristics of tumor cells. It is hypothesized that this loss is due to epigenetic modifications. Therefore, drugs releasing epigenetic repression have been proposed to reverse this silencing.. We investigated which genes were reinduced in dedifferentiated human thyroid cancer cell lines when treated with the demethylating agent 5-aza-2'-deoxycytidine (5-AzadC) and the histone deacetylase inhibitors trichostatin A (TSA) and suberoylanilide hydroxamic acid, by using reverse transcriptase-polymerase chain reaction and microarrays. These results were compared to the expression patterns in in vitro human differentiated thyrocytes and in in vivo dedifferentiated thyroid cancers. In addition, the effects of 5-AzadC on DNA quantities and cell viability were investigated.. Among the canonical thyroid differentiation markers, most were not, or only to a minor extent, re-expressed by 5-AzadC, whether or not combined with TSA or forskolin, an inducer of differentiation in normal thyrocytes. Furthermore, 5-AzadC-modulated overall mRNA expression profiles showed only few commonly regulated genes compared to differentiated cultured primary thyrocytes. In addition, most of the commonly strongly 5-AzadC-induced genes in cell lines were either not regulated or upregulated in anaplastic thyroid carcinomas. Further analysis of which genes were induced by 5-AzadC showed that they were involved in pathways such as apoptosis, antigen presentation, defense response, and cell migration. A number of these genes had similar expression responses in 5-AzadC-treated nonthyroid cell lines.. Our results suggest that 5-AzadC is not a strong inducer of differentiation in thyroid cancer cell lines. Under the studied conditions and with the model used, 5-AzadC treatment does not appear to be a potential redifferentiation treatment for dedifferentiated thyroid cancer. However, this may reflect primarily the inadequacy of the model rather than that of the treatment. Moreover, the observation that 5-AzadC negatively affected cell viability in cell lines could still suggest a therapeutic opportunity. Some of the genes that were modulated by 5-AzadC were also induced in nonthyroid cancer cell lines, which might be explained by an epigenetic modification resulting in the adaptation of the cell lines to their culture conditions.

Topics: Antimetabolites, Antineoplastic; Apoptosis; Azacitidine; Carcinoma; Cell Differentiation; Cell Line, Tumor; Cell Movement; Cell Survival; Decitabine; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Oligonucleotide Array Sequence Analysis; Thyroid Gland; Thyroid Neoplasms; Vorinostat

Anaplastic thyroid cancer cells are characterized by a mesenchymal phenotype, as revealed by spindle-shaped cells and absent or reduced levels of E-cadherin. Epigenetic silencing is considered one of the leading mechanisms of E-cadherin impairment, which causes the acquisition of the invasive and metastatic phenotype of anaplastic thyroid cancer.. In this study we investigated the effects of histone deacetylase inhibition on E-cadherin expression, cell motility, and invasion in anaplastic thyroid cancer cell cultures.. Three stabilized cell lines and primary cultures of anaplastic thyroid cancer were treated with various histone deacetylase inhibitors. After treatment, we evaluated histone acetylation by Western blotting and E-cadherin expression by RT-real time PCR. The proper localization of E-cadherin/β-catenin complex was assessed by immunofluorescence and Western blot. Transcription activity of β-catenin was measured by luciferase reporter gene and cyclin D1 expression. The effect on cell motility and invasion was studied both in vitro using scratch-wound and transwell invasion assays and in anaplastic thyroid carcinomas tumor xenografts in mice in vivo.. Histone deacetylase inhibition induced the E-cadherin expression and the proper membrane localization of the E-cadherin/β-catenin complex, leading to reduced cancer cell migration and invasion.. We here demonstrate an additional molecular mechanism for the anticancer effect of histone deacetylase inhibition. The antiinvasive effect in addition to the cytotoxic activity of histone deacetylase inhibitors opens up therapeutic perspectives for the anaplastic thyroid tumor that does not respond to conventional therapy.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Benzamides; beta Catenin; Cadherins; Cell Movement; Down-Regulation; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Indoles; Mice; Mice, SCID; Neoplasm Invasiveness; Panobinostat; Protein Transport; Pyridines; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

More than 95% of the thyroid carcinomas are well differentiated types showing favorable prognosis. However, only a few therapeutic options are available to treat the patients with undifferentiated thyroid carcinomas, especially with refractory thyroid carcinomas that are not amenable to surgery or radioiodine ablation. We investigated the anticancer effects of 20 chemotherapy and hormonal therapy drugs on 8 thyroid carcinoma cell lines. In vitro chemosensitivity was tested using the adenosine-triphosphate-based chemotherapy response assay (ATP-CRA). The tumor inhibition rate (TIR; or cell death rate) or half maximal inhibitory concentration (IC(50)) was analyzed to interpret the results. Of the 12 chemotherapy drugs, etoposide (178.9 index value in follicular carcinoma cell line) and vincristine (211.7 in Hürthle cell carcinoma cell line) were the most active drugs showing the highest chemosensitivity, and of the 8 additional drugs, trichostatin A (0.03 µg/mL IC(50) in follicular carcinoma cell line) showed favorable outcome having the anticancer effect. In our study, the result of etoposide and vincristine show evidence as active anticancer drugs in thyroid carcinoma cell lines and trichostatin A seems be the next promising drug. These drugs may become an innovative therapy for refractory thyroid carcinomas in near future.

Topics: Adenosine Triphosphate; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Etoposide; Humans; Hydroxamic Acids; Thyroid Neoplasms; Vincristine

Prohibitin (PHB) is a ubiquitous protein with a number of different molecular functions. PHB is involved in tumorigenesis by exerting either a permissive or blocking action on tumor growth, depending on the cell context. In the present study, we investigated the effects of the histone deacetylase inhibitors (HDACis), trichostatin A (TSA) and sodium butyrate (NaB), on PHB expression in the thyroid tumor cell lines, TPC-1 and FRO. Both TSA and NaB increased PHB mRNA levels. Transfection experiments showed that the overexpression of HDAC1 or 2, but not 3, inhibited PHB promoter activity. The effects of TSA and NaB on the two major PHB mRNA splicing isoforms, were also investigated. Both TSA and NaB decreased the mRNA levels of the shorter isoform, but increased those of the longer isoform. Only the latter isoform contains a 3'UTR, which has been reported to exert a growth suppressive action. Thus, our data demonstrate that HDACis control both PHB transcription and alternative splicing. The effect of HDACis on PHB alternative splicing was not due to the modification of the expression of the ASF/SF2 splicing factor.

Topics: Alternative Splicing; Carcinoma, Papillary, Follicular; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Models, Biological; Prohibitins; Promoter Regions, Genetic; Repressor Proteins; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms; Transcription, Genetic; Transfection

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

Periostin expression is a feature of the epithelial-mesenchymal transition, which occurs during cancer progression. Previous reports indicate that periostin expression is related to tumour aggressiveness.. In order to identify mechanisms regulating periostin expression in thyroid cancer, a panel of continuous thyroid cancer cell lines was investigated. Levels of posttranslational modifications of the H3 histone were investigated by chromatin immunoprecipitation. Moreover, treatment of cell lines with deacetylase inhibitors and transfection experiments were performed.. Our insights show that levels of H3 histone acetylated at lysines 9 and 14 (which are epigenetic marks of active transcription) are not related to periostin mRNA levels. Moreover, treatment of WRO and FRO thyroid cancer cell lines with the deacetylase inhibitor tricostatin A (TSA) or suberoylanilide hydroxamic acid (SAHA) increases levels of acetylated H3 histone to periostin promoter however, unpredictably, reduces periostin mRNA levels. Interestingly, treatment of WRO cells with either TSA or SAHA increases levels of the H3 histone trimethylated at lysine 4, which is a different epigenetic mark of active transcription. Instead, data obtained by cell transfection indicate that ΔNp73, a member of p53 family selectively expressed in thyroid carcinomas, plays a role in activating periostin gene expression.. Levels of epigenetic marks of active transcription do not contribute to regulation of periostin gene expression. The ΔNp73 effects suggest a novel molecular mechanism involved in thyroid cancer progression.

Topics: Acetylation; Blotting, Western; Cell Adhesion Molecules; Cell Line, Tumor; DNA-Binding Proteins; Epigenomics; Forkhead Transcription Factors; Gene Expression Regulation, Neoplastic; HeLa Cells; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Lysine; Mutation; Nuclear Proteins; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thyroid Neoplasms; Transcription Factors; Tumor Protein p73; Tumor Suppressor Proteins

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

Thyroid transcription factor-1 (TTF-1), also known as NKX2-1, is a homeodomain containing transcriptional factor identified in thyroid, lung and central nervous system. In the thyroid, TTF-1 is essential for thyroid organogenesis and governs thyroid functions by regulating various thyroid-specific genes. We previously demonstrated that most differentiated thyroid neoplasms, including follicular adenomas/carcinomas and papillary carcinomas, express TTF-1 at both protein and mRNA levels. However, certain subtypes of thyroid cancers have shown low or negative expression of TTF-1. The aim of our study was to investigate the function of epigenetic modification in dysregulation of TTF-1 in thyroid carcinoma cells. We evaluated the expression of TTF-1 in primary thyroid tissues (normal thyroid, papillary carcinoma and undifferentiated carcinoma) and in thyroid carcinoma cell lines using immunohistochemistry and RT-PCR. Methylation-specific PCR targeting CpG islands of TTF-1 and chromatin immunoprecipitation (ChIP) for histone H3 lysine 9 (H3-lys9) were applied to clarify the correlation of the TTF-1 expression profile and epigenetic status. We also explored whether epigenetic modifiers, including 5-aza-deoxycytidine, could restore TTF-1 expression in thyroid carcinoma cells. In our current study, immunohistochemistry and RT-PCR showed positive expression of TTF-1 in normal thyroids and papillary carcinomas. Meanwhile, most of the undifferentiated carcinomas and the cell lines lost TTF-1 expression. No methylation in the CpG of TTF-1 promoter was detected in normal thyroids or papillary carcinomas. In contrast, DNA methylation was identified in 60% of the undifferentiated carcinomas (6/10) and 50% of the cell lines (4/8). ChIP assay demonstrated that acetylation of H3-lys9 was positively correlated with TTF-1 expression in thyroid carcinoma cells. Finally, DNA demethylating agents could restore TTF-1 gene expression in the thyroid carcinoma cell lines. Our data suggest that epigenetics is involved with inactivation of TTF-1 in thyroid carcinomas, and provide a possible means of using TTF-1 as a target for differentiation-inducing therapy through epigenetic modification.

Topics: Azacitidine; Base Sequence; Carcinoma, Papillary; Cell Line, Tumor; Chromatin Immunoprecipitation; CpG Islands; Decitabine; DNA Methylation; DNA Primers; Epigenesis, Genetic; Gene Expression Profiling; Gene Silencing; Histones; Humans; Hydroxamic Acids; Nuclear Proteins; RNA, Messenger; RNA, Neoplasm; Thyroid Gland; Thyroid Neoplasms; Thyroid Nuclear Factor 1; Transcription Factors

Treatment with retinoic acid (RA) is effective to restore radioactive iodine uptake in metastases of a small fraction of thyroid cancer patients. In order to find predictive markers of response, we took advantage of two thyroid cancer cell lines, FTC133 and FTC238, with low RA-receptor (RAR)beta expression but differing in their response to RA. We report that in both cell lines, RA signalling pathways are functional, as transactivation of an exogenous RARbeta2 promoter is effective in the presence of pharmacological concentrations of all-trans RA, and enhanced in RA-resistant FTC238 cells after ectopical expression of RARbeta, suggesting a defective endogenous RARbeta2 promoter in these cells. Further analyses show that whereas the RARbeta2 promoter is in an unmethylated permissive status in both FTC133 and FTC238 cells, it failed to be associated with acetylated forms of histones H3 or H4 in FTC238 cells upon RA treatment. Incubation with a histone deacetylase inhibitor, alone or in combination with RA, restored histone acetylation levels and reactivated RARbeta and differentiation marker Na+/I- symporter gene expression. Thus, histone modification patterns may explain RA-refractoriness in differentiated thyroid cancer patients and suggest a potential benefit of combined transcriptional and differentiation therapies.

Topics: Acetylation; Antineoplastic Agents; Blotting, Western; Cell Line, Tumor; DNA Methylation; Drug Resistance, Neoplasm; Epigenesis, Genetic; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histones; Humans; Hydroxamic Acids; Promoter Regions, Genetic; Receptors, Retinoic Acid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thyroid Neoplasms; Transcriptional Activation; Tretinoin

The aim of this study was to comparatively investigate the effects of 5-azacytidine-C (5-Aza), trichostatin-A (TSA), and all-trans retinoic acid (ATRA) on mRNA expressions of Na/I symporter (NIS), thyroglobulin (Tg), thyroid peroxidase (TPO), and thyroid stimulating hormone receptor (TSH-R), and radioiodine (RAI) uptake in cancer (B-CPAP) and normal (Nthy-ori 3-1) thyroid cell lines. Cell lines were treated with 10 ng/mL of TSA, 5 microM of 5-Aza, and 1 microM of ATRA, according to the MTT (methyl-thiazol-tetrazolium) test results. Additionally, recombinant thyroid stimulating hormone (rTSH) was also applied, with a selected dose of 100 ng/mL. Following the treatment, NIS, Tg, TPO, and TSH-R mRNA levels were detected by real-time-polymerase chain reaction (RT-PCR) and RAI uptakes were measured by using a well counter as the counts/cell number. 5-Aza increased TSH-R mRNA expression in both of the cell lines and decreased TPO, NIS, and Tg mRNA levels in the cancer cell line. In the normal thyroid cell line, 5-Aza increased TPO mRNA levels 2-fold and made no differences in NIS and Tg mRNA levels. TSA treatment repressed NIS and Tg mRNA levels, and made no differences on other thyroid specific genes investigated in the cancer cell line. In the normal thyroid cell line, TSA increased TSH-R mRNA levels in 72 hours and created no important differences in other genes. ATRA repressed the TSH-R mRNA levels in the normal thyroid cell line and increased the TPO and Tg mRNA levels slightly in both cell lines. Furthermore, in short-term treatment, ATRA repressed NIS gene expression slightly, but in the long term, this repression turned to basal levels. 5-Aza, TSA, and ATRA did not make any differences in RAI uptake in the cancer cell line, but rTSH increased RAI uptake significantly. In the normal thyroid cell line, TSA and ATRA decreased RAI uptake (to 1/10 and 1/2, respectively), but 5-Aza and rTSH increased RAI uptake significantly (2- and 4-fold, respectively). We have shown an increase in TSH-R gene expression and radioiodine uptake with 5-Aza. Further in vitro and in vivo studies are needed to support our findings and the potential clinical use of this agent.

Topics: Aged; Apoptosis; Azacitidine; Cell Line, Tumor; Gene Expression Regulation; Humans; Hydroxamic Acids; Iodine Radioisotopes; Proteins; RNA, Messenger; Thyroid Gland; Thyroid Neoplasms; Time Factors; Tretinoin

Doxorubicin is known to be the most effective single cytotoxic drug against anaplastic thyroid carcinoma (ATC). Although doxorubicin has been shown to cause cell death, at least partly, by inducing apoptosis in ATC cells, the mechanism underlying its pharmacological efficacy has not been fully delineated. We, in this study, revealed that doxorubicin induced apoptosis in ATC cells by altering the acetylation state of histone. Doxorubicin reduced histone deacetylase activity and induced hyperacetylation of histone 3. Noticeably, ladder-like DNA fragments from their genomic DNA on agarose gel were not detected irrespective of several lines of evidence supporting the induction of apoptosis. Pulse field electrophoresis showed disintegration of nuclear DNA into giant fragments of 1-2 Mbp and high molecular-weight fragments of 100-1000 kbp. We next examined whether a histone deacetylase inhibitor trichostatin A (TsA) augmented doxorubicin-induced apoptosis in ATC cells. TSA potentiated doxorubicin-induced stage I apoptosis in ATC cells. Our study sheds light on the development of a new combination therapy strategy for more effective responses for ATC treatment.

Topics: Acetylation; Antibiotics, Antineoplastic; Apoptosis; Apoptosis Inducing Factor; Cell Line, Tumor; Cell Nucleus; Cell Survival; DNA Fragmentation; Dose-Response Relationship, Drug; Doxorubicin; Drug Synergism; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; Humans; Hydroxamic Acids; Mitochondria; Protein Transport; Thyroid Neoplasms; Time Factors

We previously obtained gene expression profiles of 8 matched papillary thyroid carcinoma (PTC) and normal tissues using DNA microarrays. To identify novel tumor suppressor genes involved in thyroid carcinogenesis, we here analyze genes showing lower expression in PTC tumors than in normal thyroid tissues. A search for loss of heterozygosity (LOH) in 49 regions that harbor consistently down-regulated genes revealed LOH in only 4 regions and in just a very small number of tumors. To determine whether the underexpression might be due to promoter methylation, we used combined bisulfite restriction analysis and bisulfite sequencing to study 7 underexpressed genes. Loss of expression of MT1G and CRABP1 is accompanied by hypermethylation in the 5' regions of these genes, but methylation was not seen in other genes tested. Combined treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-Aza-dC) and the histone deacetylase inhibitor trichostatin A (TSA) resulted in demethylation and re-expression of the MT1G gene in the cell line K2. Treatment with 5-Aza-dC alone restored CRABP1 expression in a colorectal cancer cell line, SW48. In conclusion, LOH is a remarkably rare mechanism of loss of gene function in PTC. In contrast, hypermethylation of promoter CpG islands seems to occur at higher frequency. MT1G and CRABP1 are novel genes that are likely involved in the pathogenesis of sporadic PTC.

Topics: Adenocarcinoma, Papillary; Adult; Aged; Azacitidine; Base Sequence; CpG Islands; DNA Methylation; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Genes, Tumor Suppressor; Humans; Hydroxamic Acids; Loss of Heterozygosity; Male; Metallothionein; Microsatellite Repeats; Middle Aged; Molecular Sequence Data; O(6)-Methylguanine-DNA Methyltransferase; Polymerase Chain Reaction; Promoter Regions, Genetic; Receptors, Retinoic Acid; Thyroid Neoplasms; Tumor Cells, Cultured

Radioactive iodine is used to identify and treat recurrent and metastatic thyroid cancer of follicular cell origin. Between 30% and 40% of thyroid cancers are either resistant or become resistant to radioactive iodine. Increased sodium-iodide symporter (NIS) and decreased Pendrin (PDS) activity may be associated with increased radioactive iodine effectiveness. In this investigation the effects of Trichostatin A (TSA), a histone deacetylating inhibitor, on human thyroid NIS and PDS gene expression was investigated.. Cell lines from papillary, Hürthle, and follicular cell carcinomas were treated with TSA for 72 hours at concentrations up to 100 ng/mL. NIS and PDS gene expression was determined using quantitative RT-polymerase chain reaction.. . NIS messenger RNA expression in cell carcinomas was increased 107- (1.8-307) and 217- (5.7-408) fold in papillary, 39- (20-63) and 58- (37-80) fold in Hürthle, and 459- (178-810) and 781- (412-1229) fold in follicular after treatment with 50 and 100 ng/mL of TSA, respectively. PDS messenger RNA expression in cell carcinomas was decreased 0.22- (0.05-0.45) and 0.27- (0.09-0.47) fold in papillary, 0.53- (0.46-0.60) and 0.54- (0.44-0.64) fold in Hürthle, and 0.32- (0.26-0.39) and 0.56- (0.47-0.64) fold in follicular, after the same treatment.. In thyroid cancer cell lines, TSA dramatically increased NIS gene expression and reduced PDS expression. The increased NIS expression and reduced PDS expression may make radioiodine therapy more effective in patients with thyroid cancer, especially when the tumors have no or low uptake of radioiodine.

Topics: Carcinoma, Papillary; Carrier Proteins; Cell Survival; Colonic Neoplasms; Enzyme Inhibitors; Gene Expression; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; In Vitro Techniques; Iodine Radioisotopes; Membrane Transport Proteins; Radiopharmaceuticals; Sulfate Transporters; Symporters; Thyroid Neoplasms; Tumor Cells, Cultured

Little information exists concerning the response of anaplastic thyroid carcinoma (ATC) cells to histone deacetylase inhibitors (HDAIs). In this study, the cellular response to the histone deacetylase inhibitors, sodium butyrate and trichostatin A, was analyzed in cell lines derived from primary anaplastic thyroid carcinomas. HDAIs repress the growth (proliferation) of ATC cell lines, independent of p53 status, through the induction of apoptosis and differential cell cycle arrest (arrested in G1 and G2/M). Apoptosis increases in response to drug treatment and is associated with the appearance of the cleaved form of the caspase substrate, poly-(ADP-ribose) polymerase (PARP). Cell cycle arrest is associated with the reduced expression of cyclins A and B, the increased expression of the cyclin-dependent kinase inhibitors, p21(Cip1/WAF1) and p27Kip1, the reduced phosphorylation of the retinoblastoma protein (pRb), and a reduction in cdk2 and cdk1-associated kinase activities. In ATC cells overexpressing cyclin E, drug treatment failed to replicate these events. These results suggest that growth inhibition of ATC cells by HDAIs is due to the promotion of apoptosis through the activation of the caspase cascade and the induction of cell cycle arrest via a reduction in cdk2- and cdk1-associated kinase activities.

Topics: Apoptosis; Butyrates; Cell Cycle; Cyclin E; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Inhibitors; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Thyroid Neoplasms; Tumor Cells, Cultured; Tumor Suppressor Protein p53