Dihydrotanshinone I exhibits antitumor effects via ß-catenin downregulation in papillary thyroid cancer cell lines.

Thyroid cancer is the most common endocrine carcinoma and, among its different subtypes, the papillary subtype (PTC) is the most frequent. Generally, PTCs are well differentiated, but a minor percentage of PTCs are characterized by a worse prognosis and more aggressive behavior. Phytochemicals, naturally found in plant products, represent a heterogeneous group of bioactive compounds that can interfere with cell proliferation and the regulation of the cell cycle, taking part in multiple signaling pathways that are often disrupted in tumor initiation, proliferation, and progression. In this work, we focused on 15,16-dihydrotanshinone I (DHT), a tanshinone isolated from Salvia miltiorrhiza Bunge (Danshen). We first evaluated DHT biological effect on PTC cells regarding cell viability, colony formation ability, and migration capacity. All of these parameters were downregulated by DHT treatment. We then investigated gene expression changes after DHT treatment by performing RNA-seq. The analysis revealed that DHT significantly reduced the Wnt signaling pathway, which plays a role in various diseases, including cancer. Finally, we demonstrate that DHT treatment decreases protein levels of ß-catenin, a final effector of canonical Wnt signaling pathway. Overall, our data suggest a possible use of this nutraceutical as an adjuvant in the treatment of aggressive papillary thyroid carcinoma.

NK2 homeobox gene cluster: Functions and roles in human diseases.

NK2 genes (NKX2 gene cluster in humans) encode for homeodomain-containing transcription factors that are conserved along the phylogeny. According to the most detailed classifications, vertebrate NKX2 genes are classified into two distinct families, NK2.1 and NK2.2. The former is constituted by NKX2-1 and NKX2-4 genes, which are homologous to the Drosophila scro gene; the latter includes NKX2-2 and NKX2-8 genes, which are homologous to the Drosophila vnd gene. Conservation of these genes is not only related to molecular structure and expression, but also to biological functions. In Drosophila and vertebrates, NK2 genes share roles in the development of ventral regions of the central nervous system. In vertebrates, NKX2 genes have a relevant role in the development of several other organs such as the thyroid, lung, and pancreas. Loss-of-function mutations in NKX2-1 and NKX2-2 are the monogenic cause of the brain-lung-thyroid syndrome and neonatal diabetes, respectively. Alterations in NKX2-4 and NKX2-8 genes may play a role in multifactorial diseases, autism spectrum disorder, and neural tube defects, respectively. NKX2-1, NKX2-2, and NKX2-8 are expressed in various cancer types as either oncogenes or tumor suppressor genes. Several data indicate that evaluation of their expression in tumors has diagnostic and/or prognostic value.

GSK2801 Reverses Paclitaxel Resistance in Anaplastic Thyroid Cancer Cell Lines through MYCN Downregulation.

Anaplastic thyroid cancer (ATC) is a very rare, but extremely aggressive form of thyroid malignancy, responsible for the highest mortality rate registered for thyroid cancer. Treatment with taxanes (such as paclitaxel) is an important approach in counteracting ATC or slowing its progression in tumors without known genetic aberrations or those which are unresponsive to other treatments. Unfortunately, resistance often develops and, for this reason, new therapies that overcome taxane resistance are needed. In this study, effects of inhibition of several bromodomain proteins in paclitaxel-resistant ATC cell lines were investigated. GSK2801, a specific inhibitor of BAZ2A, BAZ2B and BRD9, was effective in resensitizing cells to paclitaxel. In fact, when used in combination with paclitaxel, it was able to reduce cell viability, block the ability to form colonies in an anchor-independent manner, and strongly decrease cell motility. After RNA-seq following treatment with GSK2801, we focused our attention on MYCN. Based on the hypothesis that MYCN was a major downstream player in the biological effects of GSK2801, we tested a specific inhibitor, VPC-70619, which showed effective biological effects when used in association with paclitaxel. This suggests that the functional deficiency of MYCN determines a partial resensitization of the cells examined and, ultimately, that a substantial part of the effect of GSK2801 results from inhibition of MYCN expression.

Role of m6A RNA Methylation in Thyroid Cancer Cell Lines.

N6-methyladenosine (m6A) is the most abundant internal modification of RNA in eukaryotic cells, and, in recent years, it has gained increasing attention. A good amount of data support the involvement of m6A modification in tumorigenesis, tumor progression, and metastatic dissemination. However, the role of this RNA modification in thyroid cancer still remains poorly investigated. In this study, m6A-related RNA methylation profiles are compared between a normal thyroid cell line and different thyroid cancer cell lines. With this approach, it was possible to identify the different patterns of m6A modification in different thyroid cancer models. Furthermore, by silencing METTL3, which is the main player in the RNA methylation machinery, it was possible to evaluate the impact of m6A modification on gene expression in an anaplastic thyroid cancer model. This experimental approach allowed us to identify DDI2 as a gene specifically controlled by the m6A modification in anaplastic thyroid cancer cell lines. Altogether, these data are a proof of concept that RNA methylation widely occurs in thyroid cancer cell models and open a way forward in the search for new molecular patterns for diagnostic discrimination between benign and malignant lesions.

Enantioselective Cytotoxicity of Chiral Diphosphine Ruthenium(II) Complexes Against Cancer Cells.

The chiral cationic complex [Ru(η1 -OAc)(CO)((R,R)-Skewphos)(phen)]OAc (2R ), isolated from reaction of [Ru(η1 -OAc)(η2 -OAc)(R,R)-Skewphos)(CO)] (1R ) with phen, reacts with NaOPiv and KSAc affording [RuX(CO)((R,R)-Skewphos)(phen)]Y (X=Y=OPiv 3R ; X=SAc, Y=OAc 4R ). The corresponding enantiomers 2S -4S have been obtained from 1S containing (S,S)-Skewphos. Reaction of 2R and 2S with (S)-cysteine and NaPF6 at pH=9 gives the diastereoisomers [Ru((S)-Cys)(CO)(PP)(phen)]PF6 (PP=(R,R)-Skewphos 2R -Cys; (S,S)-Skewphos 2S -Cys). The DFT energetic profile for 2R with (S)-cysteine in H2 O indicates that aquo and hydroxo species are involved in formation of 2R -Cys. The stability of the ruthenium complexes in 0.9 % w/v NaCl solution, PBS and complete DMEM medium, as well as their n-octanol/water partition coefficient (logP), have been evaluated. The chiral complexes show high cytotoxic activity against SW1736, 8505 C, HCT-116 and A549 cell lines with EC50 values of 2.8-0.04 µM. The (R,R)-Skewphos derivatives show higher cytotoxicity compared to their enantiomers, 4R (EC50 =0.04 µM) being 14 times more cytotoxic than 4S against the anaplastic thyroid cancer 8505 C cell line.

Effects of Dihydrotanshinone I on Proliferation and Invasiveness of Paclitaxel-Resistant Anaplastic Thyroid Cancer Cells.

ATC is a very rare, but extremely aggressive form of thyroid malignancy, responsible for the highest mortality rate registered for thyroid cancer. In patients without known genetic aberrations, the current treatment is still represented by palliative surgery and systemic mono- or combined chemotherapy, which is often not fully effective for the appearance of drug resistance. Comprehension of the mechanisms involved in the development of the resistance is therefore an urgent issue to suggest novel therapeutic approaches for this very aggressive malignancy. In this study, we created a model of anaplastic thyroid cancer (ATC) cells resistant to paclitaxel and investigated the characteristics of these cells by analyzing the profile of gene expression and comparing it with that of paclitaxel-sensitive original ATC cell lines. In addition, we evaluated the effects of Dihydrotanshinone I (DHT) on the viability and invasiveness of paclitaxel-resistant cells. ATC paclitaxel-resistant cells highlighted an overexpression of ABCB1 and a hyper-activation of the NF-κB compared to sensitive cells. DHT treatment resulted in a reduction of viability and clonogenic ability of resistant cells. Moreover, DHT induces a decrement of NF-κB activity in SW1736-PTX and 8505C-PTX cells. In conclusion, to the best of our knowledge, the results of the present study are the first to demonstrate the antitumor effects of DHT on ATC cells resistant to Paclitaxel in vitro.

Dihydrotanshinone exerts antitumor effects and improves the effects of cisplatin in anaplastic thyroid cancer cells.

Anaplastic thyroid cancer (ATC) is the most aggressive type of thyroid cancer and is responsible for 20­50% of thyroid cancer­associated deaths. The absence of response to conventional treatments makes the search for novel therapeutics a clinical challenge. In the present study, the effects of 15,16­dihydrotanshinone I (DHT), a tanshinone extracted from Salvia miltiorrhiza Bunge (Danshen), which has previously been shown to possess anticancer activity, were examined in two human ATC cell lines. DHT significantly reduced cell viability, which was coupled with an increase in apoptosis. DHT administration also reduced the colony­forming ability and proliferation of these cells in soft agar and downregulated the expression of epithelial­to­mesenchymal transition­related genes. In addition, DHT significantly reduced MAD2 expression, a target of HuR with a relevant role in ATC. Finally, cotreatment with cisplatin and DHT has a greater effect on cell viability than each compound alone. In conclusion, to the best of our knowledge, the present study is the first to demonstrate that DHT exerts antitumor effects on ATC cells by reducing MAD2 expression levels. Moreover, a synergistic effect of DHT with cisplatin was shown. Further in vivo studies are required to assess this phytochemical compound as a potential adjuvant for the treatment of ATC.

Spontaneous coronary artery dissection: Role of prognostic markers and relationship with genetic analysis.

Spontaneous coronary artery dissection (SCAD) is increasingly recognized as an important cause of myocardial infarction (MI). Currently there is little knowledge about prognostic factors for unfavorable outcome at long term follow-up; furthermore, there is also little knowledge about the genetics of these patients. AIMS: This observational and retrospective study describes long-term cardiovascular outcomes of a population affected by SCAD and assesses predictors of recurrent de novo SCAD and major adverse cardiovascular events (MACE). Furthermore, a correlation between genotype and adverse events at follow-up was sought. METHODS: Baseline characteristics, angiographic features, use of medication and long-term cardiovascular events were systematically ascertained between 2000 and 2019. Next generation sequencing was performed with a panel consisting of twenty genes of interest. Variants found were filtered based on their frequency and only frequencies <1% in the general population were considered as "positive". RESULTS: Seventy patients were enrolled and followed for a median time of 39.1 months. Median age was 52 years and the majority were women (86%). Use of hormone therapy (HT) (OR 3.64, p = 0.041) and presence of malignant ventricular arrhythmias (VAs) at onset (OR 7.03, p = 0.0073) were associated with a greater risk of recurrent de novo SCAD. Proximal type SCAD (OR 8.47, p < 0.0001) and presence of VAs at onset (OR 9.97, p = 0.047) were associated with a greater risk of MACE. A potential SCAD-associated mutation was detected in 27 patients (44%); 6 patients (22%) defined as genetically "positive" developed MACE vs. 2 patients (6%) defined as "negative" (p = 0.06 at univariate analysis). MACE at follow-up is reached earlier in genetically positive patients (7.9 vs. 42.5 months). CONCLUSION: use of HT and VAs at SCAD onset are prognostic factors for recurrent de novo SCAD. Proximal SCAD site and VAs at SCAD onset were prognostic factors for MACE. Analysis by molecular genetics seems to be a promising tool for the possible additional role it could play in MACE prediction.

Critical Involvement of Calcium-Dependent Cytosolic Phospholipase A2α in Aortic Valve Interstitial Cell Calcification.

The involvement of calcium-dependent cytosolic phospholipase A2α (cPLA2α) in aortic valve calcification is not exhaustively elucidated. Here, cPLA2α expression in aortic valve interstitial cell (AVIC) pro-calcific cultures simulating either metastatic or dystrophic calcification was estimated by qPCR, Western blotting, and counting of cPLA2α-immunoreactive cells, with parallel ultrastructural examination of AVIC calcific degeneration. These evaluations also involved pro-calcific AVIC cultures treated with cPLA2α inhibitor dexamethasone. cPLA2α over-expression resulted for both types of pro-calcific AVIC cultures. Compared to controls, enzyme content was found to increase by up to 300% and 186% in metastatic and dystrophic calcification-like cultures, respectively. Increases in mRNA amounts were also observed, although they were not as striking as those in enzyme content. Moreover, cPLA2α increases were time-dependent and strictly associated with mineralization progression. Conversely, drastically lower levels of enzyme content resulted for the pro-calcific AVIC cultures supplemented with dexamethasone. In particular, cPLA2α amounts were found to decrease by almost 88% and 48% in metastatic and dystrophic calcification-like cultures, respectively, with mRNA amounts showing a similar trend. Interestingly, these drastic decreases in cPLA2α amounts were paralleled by drastic decreases in mineralization degrees, as revealed ultrastructurally. In conclusion, cPLA2α may be regarded as a crucial co-factor contributing to AVIC mineralization in vitro, thus being an attractive potential target for designing novel therapeutic strategies aimed to counteract onset or progression of calcific aortic valve diseases.

Cationic carboxylate and thioacetate ruthenium(ii) complexes: synthesis and cytotoxic activity against anaplastic thyroid cancer cells.

The cationic acetate ruthenium complex [Ru(η1-OAc)(CO)(dppb)(phen)]OAc (1) is easily prepared in 83% yield from [Ru(η1-OAc)(η2-OAc)(CO)(dppb)] (dppb = 1,4-bis(diphenylphosphino)butane) and 1,10-phenanthroline (phen) in MeOH. The derivative 1 undergoes easy substitution of the coordinated acetate by reaction with NaOPiv, KSAc, and KSCN in MeOH, affording the corresponding complexes [RuX(CO)(dppb)(phen)]X (X = OPiv, 2; SAc, 3; and NCS, 4), whereas its reaction with NaCl and NH4PF6 affords [RuCl(CO)(dppb)(phen)]PF6 (5). Carboxylate complexes 1 and 2 show high solubility in water, enabling easy exchange of the coordinated carboxylate by water and other ligands (CH3CN, glutathione). Cationic complexes 1-5, compared to Cisplatin, display a strong cell viability decrease in two human anaplastic thyroid cancer cell lines (SW1736 and 8505C), ranging from 3.10 µM to 0.09 µM EC50 values. The most active compounds 1-3 show a marked increment of apoptosis and decrease of cancer cell aggressiveness, making them promising candidates for further evaluation studies.

Marked epithelial to mesenchymal transition in surgical margins of oral cancer-an in vitro study.

Epithelial to mesenchymal transition (EMT) is a feature of several types of human cancer, including oral squamous cell carcinoma (OSCC). In the present study, tumor and margin cell cultures obtained from patients with OSCC were used to determine the expression patterns of certain EMT-associated markers, including vimentin, α-smooth muscle actin, SLUG and SNAIL. In addition, other EMT-associated features, including clonal, proliferative and migratory potential were compared between the two cell types. Cell cultures were generated from tumor and margin tissue samples from 6 patients and cultured up to the fifth passage. EMT marker expression was assessed by reverse transcription-quantitative PCR. Cell proliferation, colony formation and scratch wound healing assays were conducted to characterize the two cell types in terms of proliferation rates, clonality and motility. All of the studied markers were expressed in tumor and margin cells. Although no significant differences were noted with regard to the aforementioned markers, their expression tended to be higher in margin cultures than in tumor cultures. The expressions of the EMT markers were also higher in the fifth passage compared with those noted at the first with a few exceptions. The rates of proliferation and cell migration were decreased during passages, while the number of colonies was increased in both types of cell culture. Tumor and margin cells indicated certain similarities with regard to EMT transition characteristics.

The HuR CMLD-2 inhibitor exhibits antitumor effects via MAD2 downregulation in thyroid cancer cells.

Hu antigen R (HuR) is indeed one of the most studied RNA-binding protein (RBP) since its fundamental role both in tumorigenesis and cancer progression. For this reason, downregulation in HuR protein levels or inhibition of HuR biological function are, nowadays, attractive goals in cancer research. Here, we examined the antitumor effects of CMLD-2 in four thyroid cancer cell lines (SW1736, 8505 C, BCPAP and K1). Indeed, CMLD-2 competitively binds HuR protein disrupting its interaction with RNA-targets. 35 µM CLMD-2 produced a significant downregulation in thyroid cancer cell viability, coupled to an increase in apoptosis. Moreover, CMLD-2 treatment hindered both migration and colony formation ability. MAD2 is a microtubules-associated protein known to be greatly overexpressed in cancer and correlating with tumor aggressiveness. Furthermore, MAD2 is known to be a HuR target. CMLD-2 treatment induced a strong MAD2 downregulation and rescue experiments depicted it as a key effector in HuR-mediated in cancer. Altogether, these data contributed to foster HuR inhibition as valid antineoplastic treatment in thyroid cancer, highlighting MAD2 as a novel therapeutic target.

Biological and molecular effects of bromodomain and extra-terminal (BET) inhibitors JQ1, IBET-151, and IBET-762 in OSCC cells.

BACKGROUND: Despite improvements in oral squamous cell carcinoma (OSCC) management, survival rates remain relatively low and novel anti-neoplastic agents are needed. Bromodomain and extra-terminal (BET) inhibitors proved to be promising agents for cancer treatment. We investigated the effects of three BET inhibitors (JQ1, IBET-151, IBET-762) on SCC-25 cell line and primary oral cancer cell culture. METHODS: Cell viability was evaluated by MTT. Protein levels of MCM5 and cleaved-PARP were estimated by Western blot. Clonogenic and migratory abilities were determined by colony forming and scratch assays. BET inhibitors effects on mRNA levels of E-Cadherin, Vimentin, SNAI1, SNAI2, CLU, SERPINI1, MCM5, c-Myc, E2F, IL7R, and PPARg were analyzed by qPCR. RESULTS: BET inhibitors significantly reduced oral cancer cell viability. JQ1 showed the greatest effect reducing cell viability to 10%, both in SCC-25 and primary OSCC cultures (P < 0.001), compared to control cells. Cells treated with BET inhibitors displayed a reduction to 50% in colony forming capacity compared to control cells (P < 0.0001) and the colonies were smaller; they also had a 50%-60% reduction in migratory capacity (P < 0.05) compared to untreated cells. BET inhibitors had a significant impact on genes related to epithelial to mesenchymal transition and other cancer cell markers, notably on MCM5, a gene related to cell cycle control. CONCLUSIONS: BET inhibitors induce both OSCC cell death and reduction of tumor aggressiveness. Molecular mechanisms of BET inhibition involve among others, MCM5 downregulation. Importantly, this study demonstrates for the first time the anti-tumoral effect of IBET-151 and IBET-762 in oral cancer.

Human telomerase reverse transcriptase in papillary thyroid cancer: gene expression, effects of silencing and regulation by BET inhibitors in thyroid cancer cells.

PURPOSE: Mutations in TERT promoter have been detected in the more aggressive papillary thyroid cancers (PTCs). To elucidate the role of TERT as an eligible molecular target in these tumors, the expression of hTERT was analyzed in a series of PTCs and the effects of both pharmacological and RNA-interference-induced hTERT silencing were investigated in two human PTC cell lines (K1 and BCPAP). METHODS: The expression levels of hTERT mRNA and protein were evaluated by real-time PCR and western blot assays, respectively. Effects of hTERT silencing on PTC cell lines were analyzed by MTT, migration and western blot assays. Pharmacological inhibition of hTERT was performed using two bromodomain and extra-terminal (BET) inhibitors, JQ1 and I-BET762. RESULTS: hTERT expression results increased in 20 out of 48 PTCs, including tumors either positive or negative for the presence of hTERT promoter and/or BRAF mutations. In K1 and BCPAP cells, hTERT silencing determined a reduction in cell viability (~50% for K1 and ~70%, for BCPAP, vs control) and migration properties that were associated with a decrease of AKT phosphorylation and ß-Catenin expression. Moreover, hTERT mRNA levels were down-regulated by two BET inhibitors, JQ1 and I-BET762, which at the same dosage (0.5 and 5 µM) reduced the growth of these thyroid cancer cells. CONCLUSIONS: These findings demonstrate that hTERT may represent an excellent therapeutic target in subgroups of aggressive PTCs.

Anti-hTERT siRNA-Loaded Nanoparticles Block the Growth of Anaplastic Thyroid Cancer Xenograft.

The high frequency of hTERT-promoting mutations and the increased expression of hTERT mRNA in anaplastic thyroid cancer (ATC) make TERT a suitable molecular target for the treatment of this lethal neoplasm. In this study, we encapsulated an anti-hTERT oligonucleotide in biocompatible nanoparticles and analyzed the effects of this novel pharmaceutical preparation in preclinical models of ATC. Biocompatible nanoparticles were obtained in an acidified aqueous solution containing chitosan, anti-hTERT oligoRNAs, and poloxamer 188 as a stabilizer. The effects of these anti-hTERT nanoparticles (Na-siTERT) were tested in vitro on ATC cell lines (CAL-62 and 8505C) and in vivo on xenograft tumors obtained by flank injection of CAL-62 cells into SCID mice. The Na-siTERT reduced the viability and migration of CAL-62 and 8505C cells after 48-hour incubation. Intravenous administration (every 48 hours for 13 days) of this encapsulated drug in mice hosting a xenograft thyroid cancer determined a great reduction in the growth of the neoplasm (about 50% vs. untreated animals or mice receiving empty nanoparticles), and decreased levels of Ki67 associated with lower hTERT expression. Moreover, the treatment resulted in minimal invasion of nearby tissues and reduced the vascularity of the xenograft tumor. No signs of toxicity appeared following this treatment. Telomere length was not modified by the Na-siTERT, indicating that the inhibitory effects of neoplasm growth were independent from the enzymatic telomerase function. These findings demonstrate the potential suitability of this anti-TERT nanoparticle formulation as a novel tool for ATC treatment. Mol Cancer Ther; 17(6); 1187-95. ©2018 AACR.

Evaluation of somatic genomic imbalances in thyroid carcinomas of follicular origin by CGH-based approaches.

Application of distinct technologies of cancer genome analysis has provided important information for the molecular characterization of several human neoplasia, including follicular cell-derived thyroid carcinoma. Among them, comparative genomic hybridization (CGH)-based procedures have been extensively applied to evaluate genomic imbalances present in these tumours, obtaining data leading to an increase in the understanding of their complexity and diversity. In this review, after a brief overview of the most commonly used CGH-based technichs, we will describe the major results deriving from the most influential studies in the literature which used this approach to investigate the genomic aberrations of thyroid cancer cells. In most studies a small number of patients have been analyzed. Deletions and duplications at different chromosomal regions were detected in all investigated cohorts. A higher number of genomic imbalances has been detected in anaplastic or poorly differentiated thyroid carcinomas compared to well differentiated ones. Limitations in the interpretation of the results, as well the potential impact in the clinical practice are discussed. Though a quite heterogeneous picture arises from results so far available, CGH array, combined with other methodologies as well as an accurate clinical management, may offer novel opportunities for a better stratification of thyroid cancer patients.

BET bromodomain inhibitor JQ1 modulates microRNA expression in thyroid cancer cells.

Anaplastic thyroid carcinoma (ATC) represents the most lethal thyroid cancer sub-type, currently unresponsive to standard treatments. Recently, bromodomain and extra-terminal (BET) proteins have emerged as attractive therapeutic targets in several diseases, including cancer. In different cancer models, the anti-neoplastic activity of BET inhibitors such as JQ1, I-BET762 and I-BET151 have already been established, due to both direct and indirect effects. miRNAs are 20-22 nucleotide transcriptional regulators which play important roles in proliferation, differentiation and apoptosis. Hitherto, the relationship between JQ1 and miRNAs has not been explored. The goal of this study was to delineate JQ1-associated miRNA regulation in ATC cells. Two ATC-derived cell lines (SW1736 and 8505c) were treated with either 5 µM JQ1 or vehicle for 48 or 72 h. A non-tumorigenic thyroid cell line (Nthy-ori 3-1) was used as a control. miRNome analysis displayed a JQ1-related dysregulation of several miRNAs, 7 of which turned out to be commonly dysregulated in both cell lines at both time-points. Furthermore, miR-4516 turned out to be downregulated in both ATC cell lines, when compared to the non-tumorigenic ones and notably, JQ1 treatment in both ATC cell lines induced its upregulation, restoring, in some way, its basal expression levels. We, therefore, focused on miR-4516 expression and STAT3 levels, since it was previously predicted to be a putative target of this microRNA. Consistently, phospho-STAT3 and its target p21Waf1/Cip1 turned out to be downregulated and upregulated in both JQ1-treated ATC cell lines, respectively. Thus, our data revealed that modulation of miRNA expression is one of the multiple mechanisms of the effect of JQ1 in thyroid cancer cells.

Effects of HuR downregulation on anaplastic thyroid cancer cells.

Anaplastic thyroid cancer (ATC) constitutes one of the most aggressive types of human solid cancer, and is characterized by the absence of thyroid differentiation features and a marked degree of invasiveness. We have previously demonstrated that the RNA-binding protein Hu antigen R (HuR) is overexpressed in thyroid carcinoma; thus, the biological role of this RNA-binding protein was investigated in the present study using the ATC cell lines SW1736 and 8505C. In both cell lines, HuR protein levels were higher compared with in the non-tumorigenic thyroid cell line Nthy-ori-3.1. HuR silencing by RNA interference in both ATC cell lines decreased cell viability, increased apoptosis rates and reduced the capability to form colonies in soft agar. Thus, HuR plays an important role in the proliferation and aggressiveness of ATC cells. The histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) was able to reduce the viability of ATC cells. The results demonstrated that SAHA was able to decrease HuR expression in SW1736 and 8505C cells. Furthermore, since it is known that the transcription factor nuclear factor (NF)-κB modulates HuR expression, whether SAHA affects the nuclear (active) fraction of NF-κB in ATC cells was investigated. The data suggested that SAHA decreases ATC cell viability by reducing the active form of NF-κB, which, in turn, modulates HuR expression.

Effects of nutraceuticals on anaplastic thyroid cancer cells.

PURPOSE: The anaplastic thyroid carcinoma (ATC) is the most aggressive thyroid cancer with a high mortality rate. Since nutraceuticals may exert beneficial effects on tumor biology, here, effects of four of these compounds [resveratrol, genistein, curcumin and epigallocatechin-3-gallate (EGCG)] on ATC cell lines were investigated. METHODS: Two ATC-derived cell lines were used: SW1736 and 8505C. Cell viability and in vitro aggressiveness was tested by MTT and soft agar assays. Apoptosis was investigated by Western Blot, using an anti-cleaved-PARP antibody. mRNA and miRNA levels were quantified by real-time PCR. RESULTS: All tested nutraceuticals caused in both cell lines decrease of cell viability and increase of apoptosis. In contrast, only curcumin reduced in vitro aggressiveness in both SW1736 and 8505C cell lines, while genistein and EGCG determined a reduction of colony formation only in 8505C cells. Effects on genes related to the thyroid-differentiated phenotype were also tested: resveratrol and genistein administration determined the increment of almost all tested mRNAs in both cell lines. Instead curcumin and EGCG treatments had opposite effects in the two cell lines, causing the increment of almost all the mRNAs in 8505C cells and their reduction in SW1736. Finally, effects of nutraceuticals on levels of several miRNAs, known as important in thyroid cancer progression (hsa-miR-221, hsa-miR-222, hsa-miR-21, hsa-miR-146b, hsa-miR-204), were tested. Curcumin induced a strong and significant reduction of all miR analyzed, except for has-miR-204, in both cell lines. CONCLUSIONS: Altogether, our results clearly indicate the anti-cancer proprieties of curcumin, suggesting the promising use of this nutraceutical in ATC treatment. Resveratrol, genistein and EGCG have heterogeneous effects on molecular features of ATC cells.