Human Anaplastic Thyroid Cancer Cell Research Articles

Honokiol Suppresses Cell Proliferation and Tumor Migration through ROS in Human Anaplastic Thyroid Cancer Cells.

Honokiol is a natural polyphenolic compound extracted from Magnolia officinali, which is commonly used material in Chinese herbal medicine, has a variety of biological functions, including anti-tumor, anti-oxidant, anti-inflammation, anti-microbial and anti-allergy. Although honokiol has numerous beneficial effects on human diseases, the underlying mechanisms of tumor metastasis are still unclear. Previously, we reported that honokiol suppresses thyroid cancer cell proliferation with cytotoxicity through cell cycle arrest, apoptosis, and dysregulation of intracellular hemostasis. Herein, we hypothesized that the antioxidant effect of honokiol might play a critical role in thyroid cancer cell proliferation and migration. The cell viability assays, cellular reactive oxygen species (ROS) activity, cell migration, and immunoblotting were performed after cells were treated with honokiol. Based on this hypothesis, we first demonstrated that honokiol suppresses cell proliferation in two human anaplastic thyroid carcinoma (ATC) cell lines, KMH-2 and ASH-3, within a dosage- and time-dependent manner by cell counting kit-8 (CCK-8) assay. Next, we examined that honokiol induced ROS activation and could be suppressed by pre-treated with an antioxidant agent, N-acetyl-l-cysteine (NAC). Furthermore, the honokiol suppressed cell proliferation can be rescued by pre-treated with NAC. Finally, we demonstrated that honokiol inhibited ATC cell migration by modulating epithelial-mesenchymal transition (EMT)-related markers by Western blotting. Taken together, we provided the potential mechanism for treating ATC cells with honokiol, which significantly suppresses tumor proliferation and inhibits tumor metastasis in vitro through reactive oxygen species (ROS) induction.

Abstract 3576: CRISPR Cas9 mediated MADD deletion can induce autophagy via downregulation of MAPK and PI3K/AKT/mTOR signaling in anaplastic thyroid cancer

Abstract Thyroid cancer is a highly prevalent endocrine malignancy with one of the fastest growing incidences of all cancers in the United States. Anaplastic thyroid cancer (ATC) has an incidence of about 1.7% of all thyroid cancers, but it causes over 40% of thyroid cancer-related deaths with a median survival period of less than one year. This is why agents that inhibit the aberrant signaling pathways which drive ATC aggressiveness are of significant interest. MAPK-activating Death Domain-activating protein (MADD) plays a key role in cancer cell survival and proliferation. MADD is constitutively expressed at higher levels in most tumor tissues and cancer cells relative to normal tissues. Studies using shRNA suggest that MADD prevents apoptosis in ATC cells. For a more definitive and mechanistic analysis of MADD in ATC, we used conditional CRISPR-Cas9 gene editing to target MADD. We observed that MADD deletion in ATC cells induced spontaneous apoptosis and also activated the autophagy pathway in human ATC cells through downregulation of ERK and mTOR signals involving the MAPK and PI3K/Akt/mTOR signaling pathways. Transcriptomic analysis of publicly available TCGA data showed several PI3K/AKT/mTOR pathway genes were positively correlated with MADD levels. RNA sequencing data of ATC lines demonstrated that enhanced activity of the PI3K/AKT/mTOR signaling pathway was correlated with a worse prognosis in ATC. Targeted MADD deletion in ATC cells combined with treatment using ERK/PI3K/Akt/mTOR/autophagy inhibitors resulted in enhanced cell death compared with MADD deletion alone. Further, MADD deleted 8505C-ATC cells showed significantly delayed tumor formation in an orthotopic nude mouse model. These findings suggest that combining MADD deletion with inhibitors of PI3K/Akt/mTOR pathway could provide an attractive new therapeutic option for ATC. Citation Format: Velavan Bakthavachalam, Manikannan Madhayan, Mark Sanborn, Jalees Rehman, Prabhakar Bellur. CRISPR Cas9 mediated MADD deletion can induce autophagy via downregulation of MAPK and PI3K/AKT/mTOR signaling in anaplastic thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3576.

Anaplastic thyroid cancer spheroids as preclinical models to test therapeutics

Anaplastic thyroid cancer (ATC) is the most aggressive thyroid cancer. Despite advances in tissue culture techniques, a robust model for ATC spheroid culture is yet to be developed. In this study, we created an efficient and cost-effective 3D tumor spheroids culture system from human ATC cells and existing cell lines that better mimic patient tumors and that can enhance our understanding of in vivo treatment response. We found that patient-derived ATC cells and cell lines can readily form spheroids in culture with a unique morphology, size, and cytoskeletal organization. We observed both cohesive (dense and solid structures) and discohesive (irregularly shaped structures) spheroids within the same culture condition across different cell lines. BRAFWT ATC spheroids grew in a cohesive pattern, while BRAFV600E-mutant ATC spheroids had a discohesive organization. In the patient-derived BRAFV600E-mutant ATC spheroids, we observed both growth patterns, but mostly the discohesive type. Histologically, ATC spheroids had a similar morphology to the patient’s tumor through H&E staining and proliferation marker staining. Moreover, RNA sequencing analysis revealed that the gene expression profile of tumor cells derived from the spheroids closely matched parental patient tumor-derived cells in comparison to monolayer cultures. In addition, treatment response to combined BRAF and MEK inhibition in BRAFV600E-mutant ATC spheroids exhibited a similar sensitivity to the patient clinical response. Our study provides a robust and novel ex vivo spheroid model system that can be used in both established ATC cell lines and patient-derived tumor samples to better understand the biology of ATC and to test therapeutics.

Inhibition of MEK Signaling Attenuates Cancer Stem Cell Activity in Anaplastic Thyroid Cancer.

Background: Anaplastic thyroid cancer (ATC) is highly aggressive and has very limited treatment options. Recent studies suggest that cancer stem cell (CSC) activity in ATC could underlie this recurrence and resistance to treatment. The recent approval by the U.S. Food and Drug Administration of the combined treatment of BRAF and MEK inhibitors for ATC patients has shown some efficacy in patients harboring the BRAFV600E mutation. However, it was unknown whether the combined treatment could affect the CSC activity. This study explores the effects of the BRAF and MEK inhibitors on CSC activity in human ATC cells. Methods: Using three human ATC cells, THJ-11T, THJ-16T, and 8505C cells, we evaluated the effects of dabrafenib (a BRAF kinase inhibitor), trametinib (an MEK inhibitor), or a combined treatment of the two drugs on the CSC activity by tumorsphere formation, Aldefluor assays, expression profiles of key CSC markers, immunohistochemistry, and in vivo xenograft mouse models. Furthermore, we also used confocal imaging to directly visualize the effects on drugs on CSCs by the SORE6-mCherry reporter in cultured cells and xenograft tumor cells. Results: The BRAF inhibitor, dabrafenib, had weak efficacy, while the MEK inhibitor, trametinib, showed strong efficacy in attenuating the CSC activity, as evidenced by suppression of CSC marker expression, tumorsphere formation, and Aldefluor assays. Using ATC cells expressing a fluorescent CSC SORE6 reporter, we showed reduction of CSC activity in the rank order of combined > trametinib > dabrafenib through in vitro and in vivo xenograft models. Molecular analyses showed that suppression of CSC activity by these drugs was, in part, mediated by attenuation of the transcription by dampening the RNA polymerase II activity. Conclusions: Our analyses demonstrated the presence of CSCs in ATC cells. The inhibition of CSC activity by the MEK signaling could partially account for the efficacy of the combined treatment shown in ATC patients. However, our studies also showed that not all CSC activity was totally abolished, which may account for the recurrence observed in ATC patients. Our findings have provided new insights into the molecular basis of efficacy and limitations of these drugs in ATC patients.

Thyroid hormone receptor α1: a novel regulator of thyroid cancer cell differentiation.

Thyroid hormone receptor α1 (TRα1) mediates the genomic actions of thyroid hormone (T3). The biology of TRα1 in growth and development has been well studied, but the functional role of TRα1 in cancers remains to be elucidated. Analysis of the human thyroid cancer database of The Cancer Genome Atlas (TCGA) showed that THRA gene expression is lost in highly dedifferentiated anaplastic thyroid cancer (ATC). We, therefore, explored the effects of TRα1 on the progression of ATC. We stably expressed TRα1 in two human ATC cell lines, THJ-11T (11T-TRα1 #2, #7, and #8) and THJ-16T (16T-TRα1 #3, #4, and #8) cells. We found that the expressed TRα1 inhibited ATC cell proliferation and induced apoptosis. TCGA data showed that THRA gene expression was best correlated with the paired box gene 8 (PAX8). Consistently, we found that the PAX8 expression was barely detectable in parental 11T and 16T cells. However, PAX8 gene expression was elevated in 11T- and 16T-TRα1-expressing cells at the mRNA and protein levels. Using various molecular analyses, we found that TRα1 directly regulated the expression of the PAX8 gene. Single-cell transcriptomic analyses (scRNA-seq) demonstrated that TRα1 functions as a transcription factor through multiple signaling pathways to suppress tumor growth. Importantly, scRNA-seq analysis showed that TRα1-induced PAX8, via its transcription program, shifts the cell landscape of ATC toward a differentiated state. The present studies suggest that TRα1 is a newly identified regulator of thyroid differentiation and could be considered as a potential therapeutic target to improve the outcome of ATC patients.

ICAM1 antibody drug conjugates exert potent antitumor activity in papillary and anaplastic thyroid carcinoma

Treatment options for anaplastic thyroid cancer (ATC) and refractory papillary thyroid carcinoma (PTC) are limited and outcomes remain poor. In this study, we determined via bioinformatic expression analyses and immunohistochemistry staining that intercellular adhesion molecule-1(ICAM1) is an attractive target for ATC and PTC. We designed and engineered two ICAM1-directed antibody-drug conjugate (I1-MMAE and I1-DXd), both of which potently and selectively ablate multiple human ATC and PTC cell lines without affecting non-plastic cells invitro. Furthermore, I1-MMAE and I1-DXd mediated a potent tumor regression in ATC and PTC xenograft models. To develop a precision medicine, we also explored magnetic resonance imaging (MRI) as a non-invasive biomarker detection method to quantitatively map ICAM1 antigen expression in heterogeneous thyroid tumors. Taken together, this study provides a strong rationale for the further development of I1-MMAE and I1-DXd as promising therapeutic candidates to treat advanced PTC and ATC.

Rhodamine B-Quinoline based schiff base as fluorescent ‘turn on’ sensor of Al3+, Cr3+, HSO4− and its cytotoxicity and cell imaging application on TPC-1 and HtH-7 cell lines

A designed Rhodamine-B-quinoline based Schiff base chemosensor (RhBQ) has been synthesized and characterized using various spectroscopic techniques. It shows ‘turn on’ fluorescence emission and also colorimetric sensitivity and selectivity towards trivalent metal ions (Al3+ and Cr3+) and anion (HSO4−) present in aqueous medium. Stoichiometry of both metal (Al3+ & Cr3+) - RhBQ complexes are found to be 1:1 using Job’s plot. A notable enhancement in emission intensity for RhBQ-Al3+ (∼341 fold), RhBQ-Cr3+ (∼292 fold), and RhBQ-HSO4− (∼136 fold) complexes compare to RhBQ are observed. Sensing mechanism i.e. opening of spirolactam ring of RhBQ has been confirmed through various spectroscopic techniques such as; 1H NMR, 13C NMR, ESI-MS, TRPL, FT-IR, and DFT based computational study. The limit of detection (LOD) for Al3+, Cr3+, and HSO4− are found to be 2.2 × 10−8 M, 2.12 × 10−8 M and 8.63 × 10−7 M respectively. An advanced level 3 input OR–INHIBIT logic gate are constructed using chelating ligand EDTA. For onsite detection, TLC paper strip spot of the probe, RhBQ is prepared and the detection of Al3+ in few antacid drug are presented. In addition, cell-imaging analysis of Al3+, Cr3+, and HSO4− ions in the Human papillary thyroid carcinoma cell line (TPC-1) & Human anaplastic thyroid cancer cell lines (HtH-7) are presented and shows promising concentration and time-dependent detection results.

Antineoplastic Activity of Pazopanib in Anaplastic Thyroid Cancer in Primary Culture.

Anaplastic thyroid cancer (ATC) is a rare and rapidly fatal human cancer. Its usual treatment includes the combination of surgery, external hyperfractionated radiation therapy, and chemotherapy. These treatments permit achieving about 6-10 months of median survival. For this reason, it is challenging to predict the ATC patient clinical therapy responsiveness. Pazopanib is a multitarget tyrosine kinase inhibitor of VEGF receptors, PDGF, and c-Kit. Until now, the effect of pazopanib in primary human ATC cells (pATC) has not been reported in the literature. The aim of our study was to evaluate in vitro the antineoplastic effect of pazopanib in pATC. Surgical thyroidal tissues were collected from five patients with ATC, from thyroid biopsy at the moment of first surgical operation. An inhibition of proliferation, migration, and invasion, and an increase in apoptosis were demonstrated upon treating pATC cells with pazopanib (p < 0.05). Moreover, pazopanib was able to significantly decrease the VEGF expression in pATC cells (p < 0.05). To conclude, in this study, we demonstrate the antineoplastic activity of the antiangiogenic inhibitor, pazopanib, in human pATC in vitro.

Nuclear-targeted EGF receptor enhances proliferation and migration of human anaplastic thyroid cancer cells.

Epidermal growth factor (EGF) has various important physiological functions, which it exerts by binding to the epidermal growth factor receptor (EGFR). Reports show that EGF expression is strongly correlated with the occurrence and development of many types of tumour. To date, however, the relationship between EGF/EGFR and the occurrence and development of thyroid carcinoma remains unclear. In the current study, we investigated this phenomenon using human anaplastic thyroid carcinoma cell lines (SUN-80). The results indicated that EGF triggered the EGFR-mediated intracellular signalling pathway, including signal transducers and activators of transcription 1/3/5 (STAT1/3/5) and protein kinase B (AKT) in a time- and dose-dependent manner. In addition, results from EGF-induced EGFR internalization and co-localization analyses showed that clathrin, Rab5/7, and EEA1 play critical roles in the intracellular trafficking of EGF/EGFR. Interestingly, EGF triggered EGFR translocation into the nucleus, while nuclear-localized EGFR affected cell cycle distribution, thereby significantly promoting the ration of S phase. Overall, these findings indicated that nuclear EGFR exerts biological activity and physiological functions, including changing cell cycle, which in turn promotes proliferation and migration of SUN-80 cells. These findings lay a foundation for further explorations seeking to understand the biological effects of the EGF/EGFR system on the occurrence and development of thyroid cancer.

RNA-Seq transcriptome analysis reveals Maackia amurensis leukoagglutinin has antitumor activity in human anaplastic thyroid cancer cells.

BackgroundLectins are carbohydrate-binding molecules that can bind specifically to the sugar residues of glycoconjugates and are found in almost all organisms. Plant lectins subjected to many studies reported exhibiting anti-cancer activity. This study aimed to investigate the possible molecular mechanisms of Maackia amurensis leukoagglutinin II (MAL-II) treated ATCCs.Methods and resultsWe tested the effects of MAL-II, which is isolated from Amur seeds, on cancerous features of 8505C human anaplastic thyroid cancer cells (ATCCs) on a large scale using RNA-Seq. Transcriptome analysis was performed using Illumina next-generation sequencing technology by using cDNA libraries obtained from total RNA isolates of ATCCs treated with 0.25 µM MAL-II for 24 h. Gene ontology and pathway enrichment analysis were performed for the systematic analysis of gene functions. Moreover, we validated RNA-Seq findings using qPCR. Our results showed that many cancer-related genes such as TENM4, STIM2, SYT12, PIEZO2, ABCG1, SPNS2, ARRB1, and IRX5 were downregulated and many anticancer genes such as HSPA6, G0S2, TNFAIP3, GEM, GADD45G, RND1, SERPINB2, and IL24 were upregulated. Also, pathway enrichment analysis showed that differentially expressed genes were found to be associated with Ras, p53, and apoptosis signaling pathways, which are some important signal transduction pathways in development, proliferation, stem cell control, and carcinogenesis.ConclusionCollectively, our results show that MAL-II treatment reveals significant antitumor activity by changing the expression of many cancer-related genes and implies that MAL-II treatment might be a potential candidate molecule to inhibit the malignancy of human anaplastic thyroid cancer.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11033-022-07759-6.

Regulation of cancer stem cell activity by thyroid hormone receptor β.

Increasing numbers of cancer stem cell markers have been recently identified. It is not known, however, whether a member of the nuclear receptor superfamily, thyroid hormone receptor β (TRβ), can function to regulate cancer stem cell (CSC) activity. Using anaplastic thyroid cancer cells (ATC) as a model, we highlight the role of TRβ in CSC activity. ATC is one of the most aggressive solid cancers in humans and is resistant to currently available therapeutics. Recent studies provide evidence that CSC activity underlies aggressiveness and therapeutic resistance of ATC. Here we show that TRβ inhibits CSC activity by suppressing tumor-sphere formation of human ATC cells and their tumor-initiating capacity. TRβ suppresses the expression of CSC regulators, including ALDH, KLF2, SOX2, b-catenin, and ABCG2, in ATC cell-induced xenograft tumors. Single-cell transcriptomic analysis shows that TRβ reduces CSC population in ATC-induced xenograft tumors. Analysis of The Cancer Genome Atlas (TCGA) database demonstrates that the inhibition of CSC capacity by TRβ contributes to favorable clinical outcomes in human cancer. Our studies show that TRβ is a newly identified transcription regulator that acts to suppress CSC activity and that TRβ could be considered as a molecular target for therapeutic intervention of ATC.

In vitro antitumor activity of nano-pulse stimulation on human anaplastic thyroid cancer cells through nitric oxide-dependent mechanisms

Although most thyroid cancer patients have good clinical outcomes, there are still no effective therapies for patients with anaplastic thyroid cancers (ATC). Nano-pulse stimulation (NPS) is increasingly recognized as a non-thermal ablation technology that selectively targeting cancer cells. The aim of this study was to investigate the in vitro antitumor mechanism of NPS on human ATC cells. The cell line 8305C were treated with NPS and the CCK-8 assay, clonogenic assay, apoptosis assay and nitric oxide synthase (NOS) activity assay were used to test cell proliferation, apoptosis and NOS changes respectively. Our results showed that NPS can significantly (P < 0.05) inhibit ATC cell proliferation and induce apoptosis in a field strength-dependent manner. Moreover, the NOS activity in NPS treated groups obviously increased (P < 0.05). Further analysis indicated that NPS induced an inducible nitric oxide synthase (iNOS)-dependent killing in ATC cells. Finally, our research reveals for the first time that NPS is an effective non-thermal candidate for the treatment of ATC and supports a new possible calcium independent mechanism involvement in NPS treatment.

Betel leaf derived multicolor emitting carbon dots as a fluorescent probe for imaging mouse normal fibroblast and human thyroid cancer cells

A facile and eco-friendly approach was adapted for the preparation of multicolor emitting carbon dots (BL-MECDs) by hydrothermal method using betel leaves. BL-MECDs were synthesized at various reaction times-12 h, 24 h, 36 h, and 48 h at 200 °C. The reaction times were varied to find the optimum reaction condition to obtain BL-MECDs with excellent optical- and chemical properties. BL-MECDs were characterized by various analytical techniques. The surface morphology, degree of graphitization, and their composition were examined by field emission scanning electron microscope, Raman spectrum, and attenuated total reflection-Fourier transform infrared spectroscopy, respectively. The optical properties were evaluated via ultraviolet–visible and fluorescence spectroscopy techniques. The result suggests that BL-MECDs prepared at 36 h have enriched functional groups and excellent fluorescence emission with acceptable quantum yield (15%). Thus, BL-MECDs prepared at 36 h were utilized for further studies. X-ray photoelectron spectroscopy confirmed the existence of functional groups such as carboxyl, carbonyl, and hydroxyl groups. Moreover, the obtained BL-MECDs exhibited excellent prolonged stability and photostability even after 150 days of storage in atmospheric condition and by continuous irradiation of about 100 min with 365 nm UV-lamb, respectively. Further, BL-MECDs showed excellent biocompatibility and negligible cytotoxicity and thus explored for cell imaging for normal mouse fibroblast cell line (L929) and human anaplastic thyroid cancer cells line (SW1736). Cellular fluorescence imaging suggesting that BL-MECDs have great potential in bioimaging applications.

Anticancer Effects of Ursi Fel Extract and Its Active Compound, Ursodeoxycholic Acid, in FRO Anaplastic Thyroid Cancer Cells.

Anaplastic thyroid cancer (ATC) is one of the most fatal human malignancies. Ursi Fel (UF) is the bile of a brown bear that has been traditionally used for heat clearance and toxin relief in Korean and Chinese medicines. In this study, we determined the anticancer effects of a UF extract and its active compound, ursodeoxycholic acid (UDCA), in FRO human ATC cells. FRO cells were treated with UF extract and UDCA at different concentrations for various durations. Cell viability was measured using an MTT assay. Cell apoptosis was investigated by flow cytometric analysis following Annexin V and propidium iodide (PI) staining, and Hoechst staining was used to observe nuclear fragmentation. The expression of pro-apoptotic (Bax, caspase-3, cytochrome c, and PARP), anti-apoptotic (Bcl-2), and angiogenetic (TGF-β, VEGF, N-cadherin, and sirtuin-1) proteins and the phosphorylation of Akt and mechanistic target of rapamycin (mTOR) were determined by western blot analysis. Treatment with UF extract at 10, 25, and 50 μg/mL and UDCA at 25, 50, and 100 μM/mL significantly inhibited the growth of FRO cells in a dose-dependent manner. Flow cytometry and Hoechst staining revealed an increase in the apoptosis of FRO cells mediated by UF extract and UDCA in a dose-dependent manner. UF extract (25 and 50 μg) and UDCA (50 and 100 μM) significantly increased the expression of Bax, caspase-3, cytochrome c, and PARP and inhibited the expression of Bcl-2, TGF-β, VEGF, N-cadherin, and sirtuin-1 in FRO cells. Furthermore, UF extract and UDCA treatment stimulated Akt phosphorylation and inhibited mTOR phosphorylation in these cells. These results indicate that UF extract and UDCA exert anticancer properties in FRO cells by inducing apoptosis and inhibiting angiogenesis via regulating the Akt/mTOR signaling pathway.

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 20‑50% of thyroid cancer‑associated deaths. The absence of response to conventional treatments makes the search for novel therapeutics a clinical challenge. In the present study, the effects of 15,16‑dihydrotanshinoneI (DHT), a tanshinone extracted from Salviamiltiorrhiza 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 colony‑forming ability and proliferation of these cells in soft agar and downregulated the expression of epithelial‑to‑mesenchymal transition‑related 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 invivo studies are required to assess this phytochemical compound as a potential adjuvant for the treatment of ATC.

Effects of melatonin on the toxicity and proliferation of human anaplastic thyroid cancer cell line

BackgroundThyroid carcinoma is the most common endocrine malignancy and anaplastic thyroid carcinoma (ATC) is a rare but most aggressive cancer. Melatonin has enhanced or induced apoptosis in many different cancer cells, however, there has not been any study on the effects of melatonin in the treatment of ATC. In this study, we examined the effect of melatonin on cytotoxicity in the human ATC cell line. Materials and methodsCultured ATC cells were treated at melatonin concentrations 0.6, 1, 4, 16, 28 mM for 24 h. The MTT assay was performed to examine cell viability. Cytotoxicity was assayed with the determination of lactic dehydrogenase (LDH) activity. Apoptosis was detected by acridine orange/ethidium bromide and Hoechst 33342 staining. Giemsa staining is considered for evaluating the morphological changes of ATC cells. The reproductive ability of cells to form a colony was evaluated by the clonogenic assay. ResultsResults showed that melatonin could significantly decrease cell viability and the lowest cell viability was observed at 28 mM, 10.26 % ± 0.858 versus control. Similar results were obtained when analyzing LDH activity. The highest LDH levels were observed at 16 and 28 mM (546.08 ± 4.66, 577.82 ± 3.14 munit/mL versus control) that confirmed the occurrence of late apoptosis. The clonogenic assay showed that cells at the high concentration of melatonin (16 and 28 mM) don't enable to form the colony that approved the occurrence of reproductive death. ConclusionOur results showed a dose-dependent cytotoxic effect of melatonin on ATC cells that significantly decreased cell viability and induced cell reproductive death at the concentration greater than 1 mM and findings suggested that MLT might be useful as an adjuvant in ATC therapy.

Synergistic Effects of Lenvatinib (E7080) and MEK Inhibitors against Anaplastic Thyroid Cancer in Preclinical Models.

Simple SummaryLenvatinib has been found to be effective against radioiodine-refractory thyroid cancer. However, compliance with lenvatinib therapy is poor due to cancer progression and adverse effects. To improve the success rate of lenvatinib treatment, we propose a combination of lenvatinib and another drug class that has a different target than lenvatinib. We tested the potential of this combination in preclinical models and found that it is effective in our models. The combination also does not cause significant adverse effects in our mouse model. Thus, our results suggest that the combination we propose in this study is a potential therapeutic approach for thyroid cancer that is not responsive to radiotherapy.E7080, known as lenvatinib, is an oral multitargeted tyrosine kinase inhibitor that has been shown to improve the survival rate of patients with radioiodine-refractory thyroid cancer. However, a majority of patients do not continue lenvatinib intake due to disease progression or significant toxicity. To improve treatment success rates, we propose the combination of lenvatinib with mitogen-activated protein kinase (MEK) inhibitors. To test this hypothesis, we tested the effects of lenvatinib with the MEK inhibitor U0126 in vitro using two human anaplastic thyroid cancer (ATC) cell lines, 8505C and TCO1, and with another MEK inhibitor, selumetinib (AZD6244), in an ATC mouse model. We found that the combination of lenvatinib with MEK inhibitors enhanced the antitumor effects of monotherapy with either agent in vitro and in vivo, and these effects may be through the AKT (Protein Kinase B) and extracellular signal-regulated kinase (ERK) signaling pathways. Furthermore, the combination does not have significant adverse effects in the ATC mouse models in terms of body weight, blood biochemical parameters, and histopathology. In conclusion, the combination of lenvatinib with an MEK inhibitor is a potentially viable therapeutic approach for ATC treatment.

Inhibitor of apoptosis protein Livin promotes tumor progression and chemoradioresistance in human anaplastic thyroid cancer

Anaplastic thyroid cancer (ATC) is characterized by a rapid and aggressive course of progression. Despite significant advances in surgery, radiotherapy and chemotherapy, the disease‑specific mortality due to ATC is approximately100%. New strategies, such as molecular targeted therapies, are imperative for improving survival. Livin, a member of the human inhibitor of apoptosis protein family, has been found to be associated with tumor progression and poor prognosis in various human cancers. The aim of the present study was to evaluate the role of Livin in cancer progression and chemoradioresistance of ATC and to investigate its potential as a therapeutic target. Endogenous Livin expression in the human BHT101 ATC cell line was silenced by Livin‑specific small interfering RNA. To assess the impact of Livin on cancer cell behavior in human ATC cells, various methods such as cell invasion, cell viability and cell apoptosis assays were applied. To assess the expression of Livin and the change of apoptosis‑related proteins associated with Livin expression, reverse transcription‑quantitative PCR and western blotting were performed. Immunohistochemistry was performed to detect Livin protein expression in human ATC tissues. The association between Livin expression and apoptotic/proliferation index was analyzed in human ATC cells. Livin‑knockdown suppressed tumor cell invasion; and conversely, it enhanced cell apoptosis, with elevated expression levels of cleaved caspase‑3 and ‑7 and cleaved PARP. Livin‑knockdown enhanced radiation‑induced apoptosis, while reducing cell viability following radiotherapy, as well as lenvatinib treatment. In addition, human ATC tissues with high Livin‑expression exhibited a high Ki‑67 labeling index and low apoptotic index. In summary, these findings indicate the contribution of Livin to tumor progression and chemoradioresistance in ATC.

Integrin αvβ3-dependent thyroid hormone effects on tumour proliferation and vascularisation

Thyroid hormones are emerging as critical regulators of tumour growth and progression. To assess the contribution of thyroid hormone signalling via integrin αvβ3, expressed on many tumour cells, endothelial cells, and stromal cells, to tumour growth, we compared the effects of thyroid hormones vs tetrac, a specific inhibitor of thyroid hormone action at integrin αvβ3, in two murine xenograft tumour models with and without integrin αvβ3 expression. Integrin αvβ3-positive human anaplastic thyroid cancer cells SW1736 and integrin αvβ3-negative human hepatocellular carcinoma cells HuH7 were injected into the flanks of nude mice. Tumour growth was monitored in euthyroid, hyperthyroid, hypothyroid, and euthyroid tetrac-treated mice. In SW1736 xenografts, hyperthyroidism led to a significantly increased tumour growth resulting in a decreased survival compared to euthyroid mice, while tumour growth was significantly reduced and, hence, survival prolonged in hypothyroid and tetrac-treated mice. Both proliferation and vascularisation, as determined by Ki67 and CD31 immunofluorescence staining, respectively, were significantly increased in tumours from hyperthyroid mice as compared to hypothyroid and tetrac-treated mice. No differences in tumour growth, survival, or Ki67 staining were observed between the different groups in integrin αvβ3-negative HuH7 xenografts. Vascularisation, however, was significantly decreased in hypothyroid and tetrac-treated mice compared to euthyroid and hyperthyroid mice. Apoptosis was not affected in either tumour model, nor were cell proliferation or apoptosis in vitro. Tumour growth regulation by thyroid hormones in αvβ3-positive tumours has important implications for cancer patients, especially those with thyroid dysfunctions and thyroid cancer patients treated with thyrotropin-suppressive L-thyroxine doses.

Real-time, high-resolution imaging of tumor cells in genetically engineered and orthotopic models of thyroid cancer.

Genetically engineered and orthotopic xenograft mouse models have been instrumental for increasing our understanding of thyroid cancer progression and for the development of novel therapeutic approaches in a setting that is more physiologically relevant than the classical subcutaneous flank implants. However, the anatomical location of the thyroid gland precludes a non-invasive analysis at the cellular level of the interactions between tumor cells and the surrounding microenvironment and does not allow a real-time evaluation of the response of tumor cells to drug treatments. As a consequence, such studies have generally only relied on endpoint approaches, limiting the amount and depth of the information that could be gathered. Here we describe the development of an innovative approach to imaging specific aspects of thyroid cancer biology, based on the implantation of a permanent, minimally invasive optical window that allows high-resolution, multi-day, intravital imaging of the behavior and cellular dynamics of thyroid tumors in the mouse. We show that this technology allows visualization of fluorescently tagged tumor cells both in immunocompetent, genetically engineered mouse models of anaplastic thyroid cancer (ATC) and in immunocompromised mice carrying orthotopic implanted human or mouse ATC cells. Furthermore, the use of recipient mice in which endothelial cells and macrophages are fluorescently labeled allows the detection of the spatial and functional relationship between tumor cells and their microenvironment. Finally, we show that ATC cells expressing a fluorescent biosensor for caspase 3 activity can be effectively utilized to evaluate, in real-time, the efficacy and kinetics of action of novel small molecule therapeutics. This novel approach to intravital imaging of thyroid cancer represents a platform that will allow, for the first time, the longitudinal, in situ analysis of tumor cell responses to therapy and of their interaction with the microenvironment.