metallothionein and Thyroid-Neoplasms

Metallothioneins (MTs) are involved in numerous cell processes such as binding and transport of zinc and copper ions, differentiation, proliferation and apoptosis, therefore contributing to carcinogenesis. Scarce data exist on their expression in benign and malignant lesions of the thyroid.. mRNA expression of functional isoforms of MT genes (MT1A, MT1B, MT1E, MT1F, MT1G, MT1H, MT1X, MT2A, MT4) was studied in 17 nodular goiters (NG), 12 follicular adenomas (FA) and 26 papillary thyroid carcinomas (PTC).. One-way ANOVA revealed significant differences in mRNA expression levels of MT1A (p<0.05), MT1E (p<0.005), MT1F (p<0.0001), MT1G (p<0.005), MT1X (p<0.0005) and MT2A (p<0.005) in the analyzed samples. Post hoc analysis confirmed a significantly lower expression of MT1A mRNA in PTC compared to NG (p<0.05). Significant down-regulation was also noted for other MT isoforms in PTC in comparison to NG: MT1E (p<0.05), MT1F (p<0.0001), MT1G (p<0.005), MT1X (p<0.0005) and MT2A (p<0.05). In addition, significant down-regulation of MT1F and MT1G in FA compared to NG was observed (p<0.005 and p<0.05, respectively).. Expression of functional MT isoforms may contribute to thyroid carcinogenesis and potentially serve as a diagnostic marker in distinguishing benign and malignant lesions.

Topics: Adenoma; Adult; Aged; Aged, 80 and over; Carcinoma; Carcinoma, Papillary; Female; Gene Expression Regulation, Neoplastic; Goiter, Nodular; Humans; Male; Metallothionein; Middle Aged; Protein Isoforms; RNA, Messenger; Thyroid Cancer, Papillary; Thyroid Neoplasms; Young Adult

Hypoxia inducible factor-1α (HIF-1α) is upregulated by hypoxia, and involved in tumor growth and metastasis in many malignant tumors including papillary thyroid carcinoma (PTC). Metallothionein (MT) is a group of small molecular weight cysteine-rich proteins with a broad variety of functions. SLUG is a member of SNAIL superfamily of zinc finger transcriptional factors implicated in epithelial-mesenchymal transition (EMT). The purpose of this study was to examine HIF-1α, MT and SLUG expression in PTC and assess association of their expression with clinicopathological indicators. HIF-1α, MT and SLUG protein expression in 129 PTCs, 61 nodular hyperplasia and 118 normal thyroid tissue specimens were analyzed using immunohistochemistry. The protein expression levels of these three molecules were up-regulated in PTCs. High protein expression of HIF-1α, MT and SLUG was significantly correlated with high TNM stage (P=0.003, 0.002, 0.024, respectively) and lymph node metastasis (LNM) (P<0.001 for all three molecules). Furthermore, HIF-1α, MT and SLUG protein expression were correlated with one another. Concomitant high expression of any two of these three molecules had stronger correlation with high TNM stage (P≤0.001 for HIF-1α/MT, MT/SLUG and HIF-1α/SLUG) and LNM (P=0.008, 0.002, 0.019 for HIF-1α/MT, MT/SLUG and HIF-1α/SLUG, respectively) than did each alone, and concomitant high expression of all these three molecules is significantly associated with high TNM stage and LNM as compared with cases not showing such expression (P<0.001). These results demonstrated that the evaluation of HIF-1α, MT and SLUG expression in PTC may be useful in predicting the risk of LNM and high TNM stage.

Topics: Biomarkers, Tumor; Carcinoma; Carcinoma, Papillary; Female; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Lymphatic Metastasis; Male; Metallothionein; Middle Aged; Neoplasm Staging; Snail Family Transcription Factors; Thyroid Cancer, Papillary; Thyroid Neoplasms; Tissue Array Analysis; Transcription Factors; Up-Regulation

MT1G inactivation mediated by promoter methylation has been reported in thyroid cancer. However, the role of MT1G in thyroid carcinogenesis remains unclear. The aim of this study is to examine the biological functions and related molecular mechanisms of MT1G in thyroid cancer.. Methylation-specific PCR (MSP) was performed to analyze promoter methylation of MT1G and its relationship with clinicopathological characteristics of papillary thyroid cancer (PTC) patients. Conventional and real-time quantitative RT-PCR assays were used to evaluate mRNA expression. The functions of ectopic MT1G expression were determined by cell proliferation and colony formation, cell cycle and apoptosis, as well as cell migration and invasion assays.. MT1G expression was frequently silenced or down-regulated in thyroid cancer cell lines, and was also significantly decreased in primary thyroid cancer tissues compared with non-malignant thyroid tissues. Promoter methylation, along with histone modification, contributes to MT1G inactivation in thyroid tumorigenesis. Moreover, our data showed that MT1G hypermethylation was significantly positively associated with lymph node metastasis in PTC patients. Importantly, restoring MT1G expression in thyroid cancer cells dramatically suppressed cell growth and invasiveness, and induced cell cycle arrest and apoptosis through inhibiting phosphorylation of Akt and Rb.. We have for the first time revealed that MT1G appears to be functional tumor suppressor involved in thyroid carcinogenesis mainly through modulating the phosphatidylinositol-3-kinase (PI3K)/Akt pathway and partially through regulating the activity of Rb/E2F pathway in this study.

Topics: Adult; Aged; Aged, 80 and over; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Movement; Cell Proliferation; DNA Methylation; Epigenesis, Genetic; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Lymphatic Metastasis; Male; Metallothionein; Middle Aged; Models, Biological; Neoplasm Staging; Phosphatidylinositol 3-Kinase; Promoter Regions, Genetic; Proto-Oncogene Proteins c-akt; Signal Transduction; Thyroid Neoplasms; Tumor Burden; Tumor Suppressor Proteins; Young Adult

Thyroid carcinomas are the most frequently occurring tumours in the endocrine system. Metallothioneins (MT) and Ki-67 proteins are present in intensely proliferating cells, and their expression has been observed in numerous tumours, including thyroid tumours. The purpose of this study was to analyse the relationship between intensity of MT expression and Ki-67 antigen on one hand and histological features of the examined thyroid tumours on the other. The investigated material included 186 archival paraffin blocks with samples of various thyroid tissues, obtained from the Chair and Department of Pathomorphology, Medical University of Wroclaw. In paraffin sections, immunohistochemical reactions were performed with the use of monoclonal anti-MT (I/II) and anti-Ki-67 antibodies. Intensity of MT and Ki-67 antigen expression was evaluated using a light microscope using the semi-quantitative method of Remmele. A significant difference in MT expression was noted between different tumours of the thyroid: the highest expression was detected in follicular carcinoma and the lowest was detected in medullary carcinoma. Expression of MT was also significantly elevated in follicular carcinoma as compared to follicular adenoma. On the other hand, no significant differences were seen between expression of Ki-67 antigen in follicular adenoma and follicular carcinoma. Moreover, these investigations detected no correlation between the expression of MT and Ki-67 antigen in follicular adenoma and follicular carcinoma. In view of the obtained results, the expression of MT can be considered as a potential marker of differentiation between the two types of thyroid tumours, which are otherwise difficult to differentiate.

Topics: Adenocarcinoma, Follicular; Adenoma; Cell Nucleus; Cytoplasm; Humans; Immunohistochemistry; Ki-67 Antigen; Metallothionein; Paraffin Embedding; Thyroid Neoplasms

Metallothionein (MT) isoforms have not been studied in papillary thyroid cancer. We examined how the functional MT1 and MT2 isoforms were expressed in papillary thyroid cancer (KAT5) cells. We demonstrated that KAT5 cells expressed eight functional MT1 and MT2 isoforms induced by cadmium. Elevated calcium and activated ERK1/2 predated MT expression. The inhibition of either calcium or ERK1/2 significantly blocked the isoform expression. The induction of these isoforms accompanied an increased progression of cell cycle from G0/G1 to G2-M. The alternation in cell cycle disappeared when the expression of MT isoforms was blocked by calcium inhibitor or ERK1/2 inhibitor. Collectively, KAT5 cells express eight functional MT1 and MT2 isoforms in a pathway controlled by calcium and ERK1/2. The elevation of the MT isoforms contributes to the decreased G0/G1 but increased G2-M phase. These results reveal a novel pathway for the expression of the functional MT in papillary thyroid cancer.

Topics: Blotting, Western; Carcinoma, Papillary; Cell Cycle; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Metallothionein; Protein Isoforms; Receptor, Melatonin, MT1; Receptor, Melatonin, MT2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thyroid Neoplasms

To investigate the role of copper (Cu)-related cellular responses on thyroid carcinogenesis, the expression of ceruloplasmin (Cp) and metallothionein (MT)-1/2 were examined in relation to the activities of cell proliferation/apoptosis in the thyroid of rats at an early stage of tumor promotion under different dietary Cu levels. Male F344 rats were initiated with N-bis(2-hydroxypropyl)nitrosamine by single subcutaneous injection at 2800 mg/kg body weight, and 1 week later promoted with 6-propyl-2-thiouracil at 12 ppm in the drinking water for 4 weeks. Animals were fed a diet containing Cu at 0.6, 6 or 60 ppm from the time point of initiator-treatment to create marginally deficient, normal, or non-toxic supplementary levels of Cu. At both 0.6 and 60 pm, the multiplicity of preneoplastic focal follicular cell hyperplasias (FFCHs) was decreased as compared with 6 ppm Cu, while adenomas also decreased at 0. 6 ppm Cu. Both 0.6 and 60 ppm Cu levels revealed decreased Ki-67-immunoreactive proliferating cells in both FFCHs and surrounding follicles accompanied by mRNA downregulation of Cdc2a and Ccnb1, while TUNEL-positive apoptotic cells were unaltered with change of dietary Cu. Both Cp and MT-1/2 were immunolocalized in FFCHs and adenomas, with higher distribution in the latter. At both 0.6 and 60 ppm, the immunoreactivities and/or thyroidal mRNA levels of Cp and MT-1/2 were also decreased. Transcript levels of several antioxidant enzymes were up- or downregulated in the same direction at both Cu levels. Serum levels of thyroid-related hormones were unaltered at both Cu levels, except for non-significant reduction of thyroid-stimulating hormone at 0.6 ppm. These results suggest an involvement of Cp and MT-1/2 on the thyroid tumor promotion that can be suppressed by dietary Cu level through inhibition of cell proliferation associated with altered redox balance.

Topics: Animals; Ceruloplasmin; Copper; Diet; Dose-Response Relationship, Drug; Iron; Liver; Male; Metallothionein; Organ Size; Oxidative Stress; Propylthiouracil; Rats; Thyroid Gland; Thyroid Neoplasms; Thyrotropin; Thyroxine; Triiodothyronine

The molecular pathogenesis of tumors arising from the thyroid follicular epithelial cells, including papillary (PTC) and follicular thyroid carcinoma (FTC), is only partially understood, and the role of tumor suppressor genes has not yet been assessed. The metallothionein (MT) gene family encodes a class of metal-binding proteins involved in several cellular processes, and their expression is often deregulated in human tumors. Recently, downregulation of MT gene expression in PTC has been reported, suggesting a possible oncosuppressor role of this gene family in the pathogenesis of thyroid tumors. To further explore this possibility, we performed expression and functional studies. Analysis of microarray data of thyroid tumors of different histologic types showed that several MT genes were downregulated with respect to normal tissue. The microarray data were corroborated by quantitative PCR experiments, showing downregulation of MTs in PTC and FTC, but to a greater extent in papillary carcinoma. The expression of MTs was also investigated at the protein level by immunohistochemistry; the results were consistent with the microarray data, showing general downregulation in tumor samples, which was more evident in PTC. The functional consequence of MT downregulation was addressed employing an experimental model made of the PTC-derived K1 cell line in which MT1G expression is repressed by promoter methylation. Restoration of MT1G expression by cDNA transfection affected growth rate and in vivo tumorigenicity of K1 cells, indicating an oncosuppressor role for MT1G in thyroid papillary tumorigenesis.

Topics: Carcinoma; Carcinoma, Papillary; Cell Line, Tumor; DNA Methylation; DNA, Complementary; Down-Regulation; Gene Expression; Humans; Immunohistochemistry; Metallothionein; Microarray Analysis; Promoter Regions, Genetic; Thyroid Neoplasms; Transfection; Tumor Suppressor Proteins

Metallothionein (MT) expression in carcinogenesis of thyrocytes is unknown. We demonstrated that cadmium induced transcription of all functional MT-1 and MT-2 isoforms and promoted the cell cycle from the G1 to the S phase in thyroid cancer cells, which can be suppressed by the ERK inhibitor. Cadmium exposure stimulated intracellular calcium and the phosphorylation of ERK1/2. Therefore, a common pathway initiated by a rapid rise in calcium and followed by calcium-mediated activation of ERK is involved in the transcriptional induction of functional MT1 and MT2 isoforms and in the progression of the cell cycle in thyroid cancer cells exposed to cadmium.

Topics: Calcium; Cell Cycle; Cell Line, Tumor; DNA Primers; Humans; Metallothionein; Polymerase Chain Reaction; Protein Isoforms; RNA, Neoplasm; Thyroid Neoplasms

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

High levels of metallothionein (MT) are present in the developing mammalian liver; however, a remarkable decrease is observed during postnatal life after weaning. This developmental profile is similar to that of certain oncofetal gene products such as alpha-fetoprotein, which is used as a tumor marker. This study deals with the reexpression of MT genes in thyroid tumors. With an immunohistochemical method, the presence of MT was investigated in tissue sections of normal and neoplastic human thyroid glands. Tissue sections of 34 thyroid tumors and 10 normal human thyroid glands were studied by means of the peroxidase-antiperoxidase method. MT was localized in 31 of the thyroid gland tumors. MT was also present in two of the normal thyroid glands. These findings indicate that although high levels of MT are mainly found in the fetal liver, it may also be expressed actively in certain human thyroid neoplastic tissues, and occasionally in normal thyroid tissue.

Topics: Adenocarcinoma; Adenocarcinoma, Papillary; Adenoma; Adolescent; Adult; Aged; Aged, 80 and over; Cell Nucleus; Cytoplasm; Female; Humans; Immunohistochemistry; Male; Metallothionein; Middle Aged; Thyroid Neoplasms