Human Thyroid Carcinoma Research Articles

Diallyl trisulfide, a H2 S donor, inhibits cell growth of human papillary thyroid carcinoma KTC-1 cells through a positive feedback loop between H2 S and cystathionine-gamma-lyase.

Diallyl trisulfide (DATS), derived from garlic, is a well-known hydrogen sulfide (H2 S) donor. H2 S has recently emerged as a novel gasotransmitter involved in the regulation of cancer progression. The present study demonstrated that DATS along with other two H2 S donors, NaHS and GYY4137, significantly inhibited papillary thyroid carcinoma KTC-1 cells growth. DATS treatment triggered a rapid H2 S generation within 5 min in KTC-1 cells. Iodoacetamide, a potent thiol blocker reagent, partially rescued the cell membrane damage and ultimate cell death induced by DATS, indicating H2 S contributed to the apoptosis-inducing efficacy of DATS on thyroid cancer cells. Specifically, DATS treatment significantly upregulated the expression and enzymatic activity of cystathionine gamma-lyase (CTH), one of H2 S-producing enzymes, which was responsible for endogenous H2 S generation. After DATS treatment, H2 S quickly permeated cell membranes and activated NF-κΒ/p65 signaling pathway in KTC-1 cells. Nuclear translocated NF-κB bound to the promoter of CTH to enhance its transcription. These evidences proved that exogenous H2 S elevated CTH expression. CTH, in turn, catalytically generated a much higher level of endogenous H2 S. This positive feedback sustained excess H2 S production, which resulted in PTC cells growth inhibition. These findings may shed light on the development of novel H2 S-based antitumor agents.

Expression of the ret Proto-oncogene in Human Medullary Thyroid Carcinomas and Pheochromocytomas of MEN 2A

We studied the expression of the ret proto-oncogene (proto-ret) in human medullary thyroid carcinomas (MTCs) and pheochromocytomas of multiple endocrine neoplasia type 2A (MEN 2A) by Northern blot analysis. Expression of the normal-sized transcripts was detected in all 12 MTCs and in 6 of 8 pheochromocytomas. In situ localization of proto-ret mRNA revealed that the signal was confined to the cytoplasm of MTC cells. By Southern blot analysis neither amplification nor gross genetic changes of proto-ret were found in the tumors. Although no transcripts were detected in the normal portion of the thyroid from one MEN 2A patient, faint signals were detected in normal adrenal glands by Northern blot analysis, probably due to minor populations of C-cells and chromaffin cells in specimens from which MTC and pheochromocytoma might later develop. Proto-ret may play an important role in differentiation of a specific cell lineage from neuroectoderm, and it may be involved in development of MEN 2A tumors.

Precise engineering of Gemcitabine prodrug cocktails into single polymeric nanoparticles delivery for metastatic thyroid cancer cells

GLOBOCAN estimates 36 types of cancers in 185 countries based on the incidence, mortality, and prevalence in the year 2019. Nowadays, chemotherapy is the most widely used cancer treatment among immune, radio, hormone, and gene therapies. Here, we describe a very simple yet cost-effective approach that synergistically combines drug reconstitution, supramolecular nano-assembly, and tumor-specific targeting to address the multiple challenges posed by the delivery of the chemotherapeutic Gemcitabine (GEM) drug. The GEM prodrugs were gifted to impulsively self-assemble into excellent steady nanoparticles size on covalent conjugation of linoleic acid hydrophobic through amide group with ∼100 nm. Newly synthesized GEM-NPs morphology was confirmed by various electron microscopic techniques. After successful synthesis, we have evaluated the anticancer property of GEM and GEM-NPs against B-CPAP (papillary thyroid carcinoma) and FTC-133 (human follicular thyroid carcinoma) cancer cell lines. Further studies such as AO-EB (acridine orange-ethidium bromide), nuclear staining and flow cytometry analyses on cell death mechanism signified that the cytotoxicity was associated with apoptosis in thyroid cancer cells. GEM-NPs show excellent biocompatibility compared to GEM. The present study explained that GEM-NPs as a safe and hopeful strategy for chemotherapeutics of thyroid cancer therapy and deserve for further clinical evaluations.

NIR triggered PLGA coated Au-TiO2 core loaded CPT-11 nanoparticles for human papillary thyroid carcinoma therapy

MDR (multi-drug resistance) is one of the significant deterrents of effective chemotherapy for malignant growth. One of the powerful ways to deal with defeat of the MDR is to utilize inorganic nanoparticle-intervened tranquilize conveyance to build the medication aggregations in cancerous growth cells. In this work, we have developed the presentation that is accurately made of medication conveyance framework dependent on the TiO2 nanoparticles stacked CPT-11 to defeat the thyroid malignancy cells. The synthesized nanoparticles are characterized by spectroscopy methods (UV–vis, XPS, SEM, TEM, and DLS). The TEM results suggested that the shape of PLGA-Au-TiO2@CPT-11 of nanoparticles is ∼250 nm. After successful synthesis, we have evaluated the MTT of PLGA-Au-TiO2@CPT-11 nanoparticles with and without NIR radiations. Further, the morphological changes were observed using various biochemical stainings, such as acridine orange and ethidium bromide (AO–EB) and nuclear staining through Hoechst-33258. Also, migration and cell invasion were examined. The results show that these PLGA-Au-TiO2@CPT-11 and PLGA-Au-TiO2@CPT-11 + NIR nanoparticles exhibited promising antimetastatic property and reduced the cell invasion activity in B-CPAP and FTC-133 thyroid cancer cell lines. Based on the above findings, these PLGA-Au-TiO2@CPT-11 and PLGA-Au-TiO2@CPT-11 + NIR nanoparticles can be used as a promising candidate for the malignant thyroid cells.

1,25-Dihydroxyvitamin D3 Induces N-Myc Downstream Regulated Gene-2 Expression In Papillary Thyroid Carcinoma Cells

Purpose: In addition to its role in serum calcium homeostasis, the anti-tumor function of 1,25-dihydroxyvitamin D3 calcitriol in cancer development is well established. N-myc Downstream Regulated Gene 2 which functions as a tumor suppressor gene has recently been shown to be downregulated in various cancer leading to increased tumor incidence, progression and metastasis. The goal of this study was to investigate the possible effects of calcitriol treatment on NDRG2 expression in BCPAP papillary thyroid carcinoma cells.Methods: The experiments were carried on human primary thyroid follicular epithelial cells , and human papillary thyroid carcinoma cells BCPAP . The half maximal inhibitory concentration IC50 of calcitriol on BCPAP cells was determined by WST-1 assay. BCPAP cells were treated with 15 and 30µM calcitriol for 24, 48, and 72 hours, respectively. Basal NDGR2 expression in Nthy-ori-3–1 and BCPAP cells as well as the alterations on NDRG2 expression in calcitriol treated BCPAP cells were evaluated with western blot.Results: A significant downregulation of NDRG2 was observed in BCPAP cells when compared to Nthy-ori-3–1 cells p

The correlation and role analysis of KCNK2/4/5/15 in Human Papillary Thyroid Carcinoma microenvironment.

Background: KCNKs, potassium two pore domain channel family K members, can maintain the resting potential, regulate the amplitude and duration of the plateau of the action potential, and change the membrane potential and membrane excitability. Evidence from many studies indicates that KCNKs is abnormally expressed in many solid tumors and plays a regulatory role in the development and malignant progression of cancer. However, the expression pattern and prognostic value of KCNK factors in papillary thyroid carcinoma have not been reported.Methods: In this study, we used the data from databases such as ONCOMINE, GEPIA, Kaplan-Meier Plotter, and cBioPortal to perform bioinformatics analysis of KCNK factors in patients with thyroid cancer.Results: We found that the mRNA expression of KCNK1, KCNK5, KCNK6, KCNK7, and KCNK15 were significantly higher in thyroid cancer tissues than that in normal tissues, while KCNK2, KCNK4, KCNK9, KCNK16 and KCNK17 mRNA levels were decreased compared to normal tissues. And the expression levels of KCNK1/2/4/5/6/7/15 were correlated with the tumor stage. Survival analysis using the Kaplan-Meier Plotter database revealed that KCNK2/3/4/5/12/15 were associated with overall survival (OS) in patients with thyroid cancer.Conclusion: Finally, the results of ROC curves, immunohistochemical staining, immune cell infiltration and kinase / miRNA / transcription factor regulation showed that KCNK2, KCNK4, KCNK5 and KCNK15 levels could be used as biomarkers for PTC diagnosis. This study implied that KCNK2, KCNK4, KCNK5 and KCNK15 are potential targets of precision therapy for patients with thyroid cancer and these genes are new biomarkers for the therapeutic target for thyroid cancer.

Effect of miR‑449a‑mediated Notch signaling pathway on the proliferation, apoptosis and invasion of papillary thyroid carcinoma cells.

The present study aimed to investigate the effect of miR-449a-mediated Notch signaling pathway on the proliferation, apoptosis and invasion of papillary thyroid carcinoma cells. Human papillary thyroid carcinoma cell line TPC-1 was selected, and cells were grouped and transfected: Control group (without any treatment), negative control (NC) group (transfection with NC plasmid), miR-449a mimic group (transfection with miR-449a mimic), miR-449a inhibitor group (transfection with miR-449a inhibitor), DAPT group (addition of γ-secretase inhibitor DAPT to inhibit the Notch signaling pathway), and miR-449a inhibitor + DAPT group (transfection with miR-449a inhibitor and addition of DAPT). The target relationship between miR-449a and Notch1 was detected by dual-luciferase reporter assay. qRT-PCR and western blotting were used to assess the expression of miR-449a, Notch1 and Jagged1 in cells. Cell proliferation was detected using EdU; the cell cycle and apoptosis were detected by flow cytometry; cell invasion ability was detected by Transwell assay. PCNA, MMP-2, MMP-9, Bcl-2 and Bax mRNA and protein expression were assessed by qRT-PCR and western blotting. The results revealed that miR-449a negatively regulated Notch1. Compared with the control group, there was significantly increased miR-449a expression in the miR-449a mimic group, and there was significantly decreased expression of Notch1, Jagged1, PCNA, MMP-2, MMP-9 and Bcl-2, increased Bax, reduced cell proliferation, increased G1-phase cell fraction, decreased S-phase cell fraction, an increased apoptosis rate, and decreased invasion ability in the miR-449a mimic group and DAPT group (all P<0.05). However, the results in the miR-449a inhibitor group were the opposite of those in miR-449a mimic group (all P<0.05). There was no significant difference in cell proliferation, apoptosis and invasion in the NC group and miR-449a inhibitor + DAPT group compared to the control group (all P>0.05). miR-449a overexpression can inhibit Notch signaling pathway, thereby inhibiting the proliferation and invasion of papillary thyroid carcinoma cells and promoting cell apoptosis.

MicroRNA-92b suppresses proliferation of papillary thyroid cancer cells by regulating ilent information regulator 1 gene expression

Objective To investigate the regulating effects of microRNA (miRNA, miR)-122 on the expression of ilent information regulator 1 (SIRT1), and the proliferation abilities of papillary thyroid cancer cells. Methods Real-time quantitative polymerase chain reaction (Real-time PCR) was used to observe the expression of miR-122 in papillary thyroid carcinoma and adjacent tissues and to analyze its correlation with SIRT1 expression. After transfecting miR-122 mimics into human papillary thyroid carcinoma cell line TPC-1, the dual luciferase reporter gene assay was used to observe the regulation of miR-122 on SIRT1 gene. The effect of miR-122 on the proliferation of thyroid cancer cells was observed by CCK-8 assay. At the same time, the overexpressed SIRT1 plasmid was co-transfected into TPC-1 cells, and then the effect of miR-122 and SIRT1 expression on the proliferation of papillary thyroid carcinoma cells was detected by CCK-8 assay. Analysis was performed using GraphPad Prism 7.0 statistical software. Correlation analysis was performed using Spearman rank correlation analysis. Student’s t test was used to compare the two sample means. Results Compared with adjacent tissues (1.20±0.15), the expression level of miR-122 (3.06±0.26) in papillary thyroid carcinoma was significantly down-regulated, and the difference was statistically significant (t=5.972, P<0.01). Correlation analysis showed that miR-122 was negatively correlated with SIRT1 mRNA expression (r=-0.621, P<0.01). The relative luciferase activity of SIRT1 in miR-122 mimics transfection group was significantly lower than that in the control group, and the difference was statistically significant (t=17.730, P<0.01). The cell proliferation ability of miR-122 mimics group was significantly lower than that of the control group, and the difference was statistically significant (t=3.991, P<0.01). After co-transfection of the SIRT1 overexpression plasmid, the inhibition of the proliferation of thyroid papillary carcinoma cells by miR-122 mimics was reversed. Conclusion miR-122 can inhibit the proliferation of papillary thyroid cancer cells, and its mechanism may be related to the targeted inhibition of SIRT1 expression. Key words: MicroRNA-122; Ilent information regulator 1; Papillary thyroid cancer; Proliferation

GPX7 promotes the growth of human papillary thyroid carcinoma via enhancement of cell proliferation and inhibition of cell apoptosis

Background: Abnormal expression of glutathione peroxidase 7 (GPX7) has been linked to the occurrence and development of a variety of tumors. However, the role of GPX7 in the progression of papillary thyroid carcinoma (PTC) has not been elucidated. This study investigated the role of GPX7 in the progression of PTC. Methods: The methods employed included immunohistochemistry, Western blotting, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), MTT assay, Celigo cell counting, flow cytometric analysis, caspase activity assay, cell clone formation assay, and GPX7 knockdown. Results: The data showed that GPX7 protein was localized in the cytoplasm of thyroid cells. The level of GPX7 expression was higher in PTC tissues than in the nodular goiter. The positive rate for GPX7 was also higher in the PTC group than in the nodular goiter group (100.0% vs. 35.7%). The maximum tumor diameter in the group highly expressing GXP7 was significantly greater than that in the group with low expression of GXP7 (1.56±0.56 vs. 0.56±0.13 cm, P<0.001). The GPX7 mRNA level was higher in K1 cells. Knockdown of GPX7 decreased the number of cells, cell clone formation ability, and cell proliferation rate and increased the activity of caspase 3/7 and cell apoptosis in PTC K1 cells. Conclusions: These results indicate that high expression of GPX7 increases the proliferation and reduces the apoptosis of PTC cells, and thus, promotes the growth and progression of human PTC.

Silencing of FoxM1 blocks growth, migration and invasion of papillary thyroid carcinoma cells.

The aim of the present study was to investigate the effect of Forkhead box transcription factor M1 (FoxM1)-silencing on the growth, migration and invasion of K1 human papillary thyroid carcinoma (PTC) cells. The effect of FoxM1-small interfering RNA (siRNA) in K1 cells was detected by western blot analysis. FoxM1-siRNA and control siRNA were transfected into K1 cells using Lipofectamine® 2000 (transfection group, T) and the non-meaning sequence group (NM). K1 cells exposed to PBS solution comprised the blank control group (CON). Cell proliferation ability was detected using an MTT assay. Cell migration and invasion was detected by the single cell scratch test and Transwell invasion assay, respectively. Western blot analysis indicated that FoxM1 siRNA downregulated the expression of FoxM1 protein. Cell proliferation, migration and invasion were significantly lower in the T group compared with the NM and CON groups (P<0.05). These results indicated that silencing of FoxM1 expression could block growth, invasion and migration of K1 cells. This study may provide a novel target gene for targeted therapy of PTC.

A ceRNA Circuitry Involving the Long Noncoding RNA Klhl14-AS, Pax8, and Bcl2 Drives Thyroid Carcinogenesis.

Klhl14-AS is a long noncoding RNA expressed since early specification of thyroid bud and is the most enriched gene in the mouse thyroid primordium at E10.5. Here, we studied its involvement in thyroid carcinogenesis by analyzing its expression in cancer tissues and different models of neoplastic transformation. Compared with normal thyroid tissue and cells, Klhl14-AS was significantly downregulated in human thyroid carcinoma tissue specimens, particularly the anaplastic histotype, thyroid cancer cell lines, and rodent models of thyroid cancer. Downregulating the expression of Klhl14-AS in normal thyroid cells decreased the expression of thyroid differentiation markers and cell death and increased cell viability. These effects were mediated by the binding of Klhl14-AS to two miRNAs, Mir182-5p and Mir20a-5p, which silenced Pax8 and Bcl2, both essential players of thyroid differentiation. MIR182-5p and MIR20a-5p were upregulated in human thyroid cancer and thyroid cancer experimental models and their effects on Pax8 and Bcl2 were rescued by Klhl14-AS overexpression, confirming Klhl14-AS as a ceRNA for both Pax8 and Bcl2. This work connects deregulation of differentiation with increased proliferation and survival in thyroid neoplastic cells and highlights a novel ceRNA circuitry involving key regulators of thyroid physiology. SIGNIFICANCE: This study describes a new ceRNA with potential tumor suppression activity and helps us better understand the regulatory mechanisms during thyroid differentiation and carcinogenesis.

GPX7 promotes the growth of human papillary thyroid carcinoma via enhancement of cell proliferation and inhibition of cell apoptosis.

BackgroundAbnormal expression of glutathione peroxidase 7 (GPX7) has been linked to the occurrence and development of a variety of tumors. However, the role of GPX7 in the progression of papillary thyroid carcinoma (PTC) has not been elucidated. This study investigated the role of GPX7 in the progression of PTC.MethodsThe methods employed included immunohistochemistry, Western blotting, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), MTT assay, Celigo cell counting, flow cytometric analysis, caspase activity assay, cell clone formation assay, and GPX7 knockdown.ResultsThe data showed that GPX7 protein was localized in the cytoplasm of thyroid cells. The level of GPX7 expression was higher in PTC tissues than in the nodular goiter. The positive rate for GPX7 was also higher in the PTC group than in the nodular goiter group (100.0% vs. 35.7%). The maximum tumor diameter in the group highly expressing GXP7 was significantly greater than that in the group with low expression of GXP7 (1.56±0.56 vs. 0.56±0.13 cm, P<0.001). The GPX7 mRNA level was higher in K1 cells. Knockdown of GPX7 decreased the number of cells, cell clone formation ability, and cell proliferation rate and increased the activity of caspase 3/7 and cell apoptosis in PTC K1 cells.ConclusionsThese results indicate that high expression of GPX7 increases the proliferation and reduces the apoptosis of PTC cells, and thus, promotes the growth and progression of human PTC.

Tripartite motif containing 14: An oncogene in papillary thyroid carcinoma

Tripartite motif (TRIM)-containing protein 14 (TRIM14) is implicated in many malignancies. Presently, we studied whether TRIM14 played a role in human papillary thyroid carcinoma (PTC). Herein, TRIM14 was up-regulated in tumor tissues when compared with normal thyroid samples. Among PTC patients, enhanced TRIM14 predicted short recurrence free survival (RFS) time. Then, we found that knockdown of TRIM14 in human PTC K1 cells inhibited cell proliferation, induced cell apoptosis and prevented the tumorigenicity in nude mice. On the contrary, TRIM14 overexpression in human PTC TPC1 cells promoted cell proliferation while inhibited cell apoptosis. TRIM14 exerted its oncogenic activities via promoting the activation of signal transducer and activator of transcription 3 (STAT3). Further, TRIM14 interacted with the suppressor of cytokine-signaling-1 (SOCS1), a negative regulator of STAT3 activation, and knockdown of TRIM14 inhibited SOCS1 ubiquitination. In conclusion, TRIM14 may be a prognostic factor and oncogene in PTC, and may be a potential target for PTC intervention.

Validation of Reference Genes for Normalization of Relative qRT-PCR Studies in Papillary Thyroid Carcinoma

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) in thyroid tumors require accurate data normalization, however, there are no sufficient studies addressing the suitable reference genes for gene expression analysis in malignant and normal thyroid tissue specimens. The purpose of this study was to identify valid internal control genes for normalization of relative qRT-PCR studies in human papillary thyroid carcinoma tissue samples. The expression characteristics of 12 candidate reference genes (GAPDH, ACTB, HPRT1, TBP, B2M, PPIA, 18SrRNA, HMBS, GUSB, PGK1, RPLP0, and PGM1) were assessed by qRT-PCR in 45 thyroid tissue samples (15 papillary thyroid carcinoma, 15 paired normal tissues and 15 multinodular goiters). These twelve candidate reference genes were selected by a systematic literature search. GeNorm, NormFinder, and BestKeeper statistical algorithms were applied to determine the most stable reference genes. The three algorithms were in agreement in identifying GUSB and HPRT1 as the most stably expressed genes in all thyroid tumors investigated. According to the NormFinder software, the pair of genes including ‘GUSB and HPRT1’ or ‘GUSB and HMBS’ or ‘GUSB and PGM1’ were the best combinations for selection of pair reference genes. The optimal number of genes required for reliable normalization of qPCR data in thyroid tissues would be three according to calculations made by GeNorm algorithm. These results suggest that GUSB and HPRT1 are promising reference genes for normalization of relative qRT-PCR studies in papillary thyroid carcinoma.

Ku80 gene knockdown by the CRISPR/Cas9 technique affects the biological functions of human thyroid carcinoma cells.

In the present study, to evaluate the role of Ku80 in thyroid carcinoma (TC), 86 thyroid tissue samples from patients with a spectrum of thyroid disorders were examined for protein levels of Ku80, nuclear factor-κB (NF-κB) and RET/TC by immunohistochemistry. Furthermore, in TC cells, Ku80 mRNA was detected by reverse transcription-quantitative PCR analysis and silenced using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technique to assess its role. An antibody array was used to identify Ku80-related regulatory genes. The protein levels of Ku80 in the TC tissues were significantly higher than those in non-neoplastic adjacent tissue samples (P<0.01). The activation of NF-kB and expression of RET/TC in the TC group were significantly increased (P<0.05) and were correlated with the protein expression of Ku80 (P<0.05). In papillary TC cells, the mRNA levels of Ku80 were high; Ku80 knockdown resulted in reductions in proliferation, invasion and colony formation, increased apoptosis, and reduced levels of proteins involved in MAPK signaling, cell proliferation and apoptosis. The high expression of Ku80 in TC was found to be associated with the expression of RET/TC and activation of NF-κB, and Ku80 knockdown decreased the malignancy of TC cells.

Promoter Methylation of Four Tumor Suppressor Genes in Human Papillary Thyroid Carcinoma.

Background & Objective:Papillary thyroid cancer (PTC) is considered to be the most common type of thyroid malignancies. Epigenetic alteration, in which the chromatin conformation and gene expression change without changing the sequence of DNA, can occur in some tumor suppressor genes and oncogenes. Methylation is the most common type of epigenetic alterations that can be an excellent indicator of PTC invasive behavior.Methods:In this research, we determined the promoter methylation status of four tumor suppressor genes (SLC5A8, RASSF1, MGMT, and DNMT1) and compared the results of 55 PTC cases with 40 goiter patients. For methylation, we used the methylation-sensitive high resolution melting (MS-HRM) assay technique. The resulting graphs of each run were compared with those of 0%, 50%, and 100% methylated controls.Results:Our data showed that the promoter methylation of SLC5A8, Ras association domain family member 1(RASSF1), and MGMT were significantly different between PTC tissue and goiter with P-value less than 0.05. The most significant differences were observed in RASSF1; 77.2% of hyper-methylated PTC patients versus 15.6% hyper-methylated goiter samples (P<0.001). Conclusion:RASSF1 promoter methylation can be a PTC genetic marker. RASSF1 promoter methylation is under the impact of the methyltransferase genes (DNMT1 and MGMT), protein expression, and promoter methylation.

BRAF Inhibitors in Thyroid Cancer: Clinical Impact, Mechanisms of Resistance and Future Perspectives.

The Kirsten rat sarcoma viral oncogene homolog (RAS)/v-raf-1 murine leukemia viral oncogene homolog 1 (RAF)/mitogen-activated protein kinase 1 (MAPK) signaling cascade is the most important oncogenic pathway in human cancers. Tumors leading mutations in the gene encoding for v-raf murine sarcoma viral oncogene homolog B (BRAF) serine-threonine kinase are reliant on the MAPK signaling pathway for their growth and survival. Indeed, the constitutive activation of MAPK pathway results in continuous stimulation of cell proliferation, enhancement of the apoptotic threshold and induction of a migratory and metastatic phenotype. In a clinical perspective, this scenario opens to the possibility of targeting BRAF pathway for therapy. Thyroid carcinomas (TCs) bearing BRAF mutations represent approximately 29–83% of human thyroid malignancies and, differently from melanomas, are less sensitive to BRAF inhibitors and develop primary or acquired resistance due to mutational events or activation of alternative signaling pathways able to reactivate ERK signaling. In this review, we provide an overview on the current knowledge concerning the mechanisms leading to resistance to BRAF inhibitors in human thyroid carcinomas and discuss the potential therapeutic strategies, including combinations of BRAF inhibitors with other targeted agents, which might be employed to overcome drug resistance and potentiate the activity of single agent BRAF inhibitors.

Interactions of Vascular Endothelial Growth Factor and p53 with miR-195 in Thyroid Carcinoma: Possible Therapeutic Targets in Aggressive Thyroid Cancers.

The clinical pathological features, as well as the cellular mechanisms of miR-195, have not been investigated in thyroid carcinoma. The aim of this study is to identify the interactions of vascular endothelial growth factor (VEGF), p53 and miR-195 in thyroid carcinoma. The clinical and pathological features of miR-195 were also investigated. The expression levels of miR-195 were identified in 123 primary thyroid carcinomas, 40 lymph nodes with metastatic papillary thyroid carcinomas and seven non-neoplastic thyroid tissues (controls) as well as two thyroid carcinoma cell lines, B-CPAP (from metastasizing human papillary thyroid carcinoma) and MB-1 (from anaplastic thyroid carcinoma), by the real-time polymerase chain reaction. Using Western blot and immunofluorescence, the effects of exogenous miR-195 on VEGF-A and p53 protein expression levels were examined. Then, cell cycle and apoptosis assays were performed to evaluate the roles of miR-195 in cell cycle progression and apoptosis. The expression of miR-195 was downregulated in majority of the papillary thyroid carcinoma tissue as well as in cells. Introduction of exogenous miR-195 resulted in downregulation of VEGF-A and upregulation of p53 protein expressions. Upregulation of miR-195 in thyroid carcinoma cells resulted in cell cycle arrest. Moreover, we demonstrated that miR-195 inhibits cell cycle progression by induction of apoptosis in the thyroid carcinoma cells. Our findings showed for the first time that miR-195 acts as a tumour suppressor and regulates cell cycle progression and apoptosis by targeting VEGF-A and p53 in thyroid carcinoma. The current study exhibited that miR-195 might represent a potential therapeutic target for patients with thyroid carcinomas having aggressive clinical behaviour.

Validation of molecular biomarkers for preoperative diagnostics of human papillary thyroid carcinoma in fine needle aspirates.

Despite substantial efforts, reliable preoperative diagnostic for human thyroid malignancies in case of cytologically indeterminate nodules is still missing, resulting in high number of unnecessary thyroidectomies. In an attempt to increase precision of existing preoperative diagnostics, we aimed at validating the panel of molecular biomarkers predictive for papillary thyroid carcinoma (PTC) in preoperative fine needle aspirate (FNA) samples. In this prospective study conducted in preoperative thyroid FNA from 44 thyroid nodules, expression levels of 11 molecular biomarkers previously validated on the postoperative samples of PTCs were measured by Cell-to-CT and QuantiGene Plex methods and correlated with final diagnosis. The QuantiGene Plex resulted in reliable gene expression measurements for FNA and core-needle biopsy (CNB) samples, however this method was less sensitive than pre-amplification based Cell-to-CT. Measurements conducted on the same samples by the two methods significantly correlated for most of the genes. Expression levels of TIMP1, c-MET and ARNTL were upregulated in PTC nodules as compared to benign counterparts, supporting previous post-operative studies. Strong correlation was observed between these biomarker alterations in the same samples. Within the sub-group of 15 indeterminate nodules (Bethesda II-V), TIMP1 had 100% specificity and 83% sensitivity for PTC cases. Assessment of TIMP1, c-MET and core-clock gene ARNTL expression levels by QuantiGene Plex assay in FNA samples holds promise as an ancillary method to the cytological preoperative diagnostics.

Parthenolide Inhibits the Proliferation of MDA-T32 Papillary Thyroid Carcinoma Cells in Vitro and in Mouse Tumor Xenografts and Activates Autophagy and Apoptosis by Downregulation of the Mammalian Target of Rapamycin (mTOR)/PI3K/AKT Signaling Pathway.

BackgroundThis study aimed to examine the effects of the sesquiterpene lactone, parthenolide, on migration, autophagy, and apoptosis of MDA-T32 human papillary thyroid carcinoma cells in vitro and in mouse tumor xenografts.Material/MethodsCell proliferation and viability of MDA-T32 human papillary thyroid carcinoma cells were determined by MTT assay, and cell migration was studied using a transwell assay. Fluorescence microscopy using acridine orange (AO) and ethidium bromide (EB) staining evaluated apoptosis. Transmission electron microscopy was used to study the effects of parthenolide on autophagy, and Western blot examined the levels of autophagy-associated proteins, including Bax, Bcl-2, and LC3-ll. Mice (n=10) were injected with 5×106 MDA-T32 cells subcutaneously into the left flank, and xenograft tumors were grown for six weeks. Control untreated mice (n=5) were compared with treated mice (n=5) given parthenolide three times per week.ResultsParthenolide resulted in a dose-dependent reduction in viability and cell migration of MDA-T32 cells, with a half-maximal inhibitory concentration (IC50) of 12 μM. AO and EB staining showed that parthenolide induced cell apoptosis and electron microscopy identified autophagosomes in MDA-T32 cells. Parthenolide induced increased expression of the autophagocytic proteins, LC3-II and beclin-1, had a dose-dependent inhibitory effect on the mTOR/PI3K/AKT cascade in MDA-T32 cells and inhibited the growth of the mouse xenograft tumors in vivo.ConclusionsParthenolide inhibited the growth and migration of MDA-T32 human papillary thyroid carcinoma cells in vitro and mouse tumor xenografts and activated autophagy and apoptosis by downregulation of the mTOR/PI3K/AKT signaling pathway.