FRO Cells Research Articles

TIM3 activates the ERK1/2 pathway to promote invasion and migration of thyroid tumors.

This study aims to study the possible action mechanism of T-cell immunoglobulin and mucin domain 3 (TIM3) on the migratory and invasive abilities of thyroid carcinoma (TC) cells. GSE104005 and GSE138198 datasets were downloaded from the GEO database for identifying differentially expressed genes (DEGs). Functional enrichment analysis and protein-protein interaction (PPI) analysis were performed on the common DEGs in GSE104005 and GSE138198 datasets. Subsequently, in order to understand the effect of a common DEG (TIM3) on TC cells, we performed in vitro experiments using FRO cells. The migratory and invasive abilities of FRO cells were detected by wound scratch assay and Transwell assay. Proteins expression levels of the phosphorylated (p)-extracellular signal-regulated kinase (ERK)1/2, matrix metalloproteinase-2 (MMP-2) and MMP-9 were determined via Western blotting after ERK1/2 inhibition in TIM3-NC group and TIM3-mimic group. 316 common DEGs were identified in GSE104005 and GSE138198 datasets. These DEGs were involved in the biological process of ERK1 and ERK2 cascade. TIM3 was significantly up-regulated in TC. In vitro cell experiments showed that TIM3 could promote migration and invasion of TC cells. Moreover, TIM3 may affect the migration, invasive abilities of TC cells by activating the ERK1/2 pathway. The above results indicate that TIM3 may affect the migratory and invasive of TC cells by activating the ERK1/2 pathway.

Mechanisms of vemurafenib-induced anti-tumor effects in ATC FRO cells

BackgroundAnaplastic Thyroid Carcinoma (ATC) is a rare and deadly malignant tumor in humans. It is prone to developing resistance to radiotherapy and chemotherapy. Molecular targeted therapy offers a novel way to treat ATC. The BRAF mutation is closely associated with many cancers, including thyroid carcinoma. Vemurafenib, a small-molecule inhibitor, is specifically designed to target the mutant serine/threonine kinase BRAF. The objective of this study is to elucidate the regulatory mechanisms underlying the effects of vemurafenib on human anaplastic thyroid carcinoma cell line FRO and to assess its potential therapeutic role. MethodsThe effects of vemurafenib on the proliferation of FRO cells were assessed by the CCK-8 method and Colony-forming assay. Transwell chambers and scratch tests were employed to examine the impact of vemurafenib on the invasion and migration of FRO cells. Apoptosis and cycle distribution of FRO cells were analyzed by tunel assay and flow cytometry. The effects of vemurafenib on the expression of BRAF-activated non-protein coding RNA (BANCR), Bax, Bcl2, and E-cadherin were evaluated by qRT-PCR. Furthermore, the effects of vemurafenib on the expression of phosphoinositol-3-kinase (PI3K)/phosphoinositol-3-kinase (AKT) pathway-related proteins, BRAF, CyclinD1, Bcl-2, Bax, and E-cadherin proteins in FRO cells were investigated through the western-blot method. All experiments were conducted in three replicates. ResultsVemurafenib was observed to inhibit proliferation and induce apoptosis in a dose- and time-dependent manner (P < 0.05). The formation of FRO cell colonies, as well as migration and invasion, all showed a dose-dependent reduction (P < 0.05). Flow cytometric analysis indicated G0/G1 cell cycle arrest (P < 0.05). QRT-PCR revealed that vemurafenib could suppress the expression of BANCR and Bcl2 while increasing the expression of Bax and E-cadherin in a dose-dependent manner (P < 0.05). The protein expression levels of Bax and E-cadherin were up-regulated significantly, and the expression levels of BRAF, CyclinD1, Bcl-2, p-PI3K, p-AKT, and p-mTOR were markedly down-regulated with increasing concentrations of vemurafenib (P < 0.05). ConclusionsThe proliferation and metastasis of FRO cells can be suppressed by vemurafenib through the silencing of BRAF and BANCR expression, inhibition of PI3K/AKT signaling pathway activation, induction of apoptosis, and cell cycle arrest.

A-Kinase Interacting Protein 1 Knockdown Restores Chemosensitivity via Inactivating PI3K/AKT and β-Catenin Pathways in Anaplastic Thyroid Carcinoma.

BackgroundA-kinase interacting protein 1 (AKIP1) promotes tumor progression and chemoresistance in several malignancies; meanwhile, it is related to higher tumor size and recurrence risk of papillary thyroid carcinoma, while the role of AKIP1 in anaplastic thyroid carcinoma (ATC) is unclear. The aim of this study is to explore the effect of AKIP1 knockdown on cell malignant behaviors and doxorubicin resistance in ATC.MethodsAKIP1 knockdown was conducted in ATC cell lines (8505C and CAL-62 cells) by siRNA; then, cell viability, apoptosis, invasion, PI3K/AKT and β-catenin pathways, and doxorubicin sensitivity were detected. Subsequently, doxorubicin-resistant 8505C cells (8505C/Dox) were established. Additionally, AKIP1 was modified in 8505C and 8505C/Dox cells that underwent doxorubicin treatment by siRNA or overexpression plasmid, followed by cellular function and pathway detection.ResultsAKIP1 was elevated in FRO, 8505C, CAL-62, and KHM-5M cells compared to control cells (all p < 0.05). Subsequently, AKIP1 knockdown elevated apoptosis, inhibited viability and invasion, and inactivated PI3K/AKT and β-catenin pathways in 8505C and CAL-62 cells (all p < 0.05). AKIP1 knockdown decreased relative cell viability in doxorubicin-treated 8505C and CAL-62 cells; then, AKIP1 was elevated in 8505C/Dox cells compared to 8505C cells (all p < 0.05). Furthermore, AKIP1 knockdown restored doxorubicin sensitivity (reflected by decreased cell viability and invasion, and increased apoptosis), but inactivated PI3K/AKT and β-catenin pathways in doxorubicin-treated 8505C/Dox cells. However, AKIP1 overexpression presented an opposite effect on these functions and pathways in doxorubicin-treated 8505C cells.ConclusionAKIP1 knockdown decreases cell survival and invasion while promoting sensitivity to doxorubicin via inactivating PI3K/AKT and β-catenin pathways in ATC.

TBX3 activating PVT1 accelerates proliferation, migration, and invasion by modulating the miR-30a/LOX axis in anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma (ATC) is a nearly chemo-resistant malignancy with high invasion and mortality. Long non-coding RNAs (lncRNAs) have been demonstrated to be dysregulated and play a crucial role in the development and process of ATC. The present study aimed to explore the mechanism of PVT1 dysregulation in ATC. The mRNA levels of PVT1 and T-box3 (TBX3), and the protein levels of TBX3 in ATC and paracancerous tissues, and FRO and Nthy-ori 3-1 cells were determined by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and western blot, respectively. The transcriptional factor binding site was predicted and validated between TBX3 and PVT1 promoter through the JASPAR website, and ChIP and luciferase analysis. The proliferation, migration, and invasion of FRO cells were assessed by MTT, colony formation, and transwell assays. PVT1 expression was upregulated in ATC, which was positively correlative with the level of transcription factor TBX3. Downregulation of PVT1 inhibited the proliferation, migration, and invasion of FRO cells. Moreover, TBX3 targeting the promoter region of PVT1 promoted the expression level of PVT1 and modulated the downstream signalling axis of PVT1, miR-30a/LOX. Also, interference of PVT1 reversed the stimulative role of overexpression of TBX3 in the progress of FRO cells. TBX3 enhanced proliferation, migration, and invasion of ATC cells via activation of PVT1 and modulation of the miR-30a/LOX signalling axis.

Preparation of fluorescent bimodal probe coupled with ultra-small superparamagnetic iron oxide particles

To prepare USPIO-PEG-Cy5.5 targeted bimodal probe for noninvasive diagnosis of thyroid cancer. The characterization was verified, and the physical and chemical properties were discussed. Fe3O4 was used as the carrier. Fluorescent Cy5.5 was linked with polyethylene glycol (PEG). A small amount of Cy5.5-NHS fluorescent powder were dissolved in dimethyl sulfoxide solution. PEG-Fe3O4 nanoparticles were centrifuged and ultra-filtrated. The nanoparticles were resuspended with phosphate buffer to change the pH. The Cy5.5 fluorescence was added to the Fe3O4 particles and incubated at 37 °C for 2 h. Characterization test of Cy5.5-PEG-Fe3O4 and relaxation rate measurement were performed. Fro cells were used to perform cell imaging experiment. CCK-8 assay was carried out to detect the cytotoxicity. The bimodal molecular probe Cy5.5-PEG-Fe3O4 was synthesized successfully. Transmission electron microscopy showed that the particle size of the probe was 18.8 ± 3.5 nm, the water and particle size of the nanoparticles was 35.7 ± 18.6 nm, and the zeta potential was −18.0 mv. The relaxation rate was 0.044 and the slope was 0.97. The cytotoxicity test showed that the bimodal molecular probe had slight toxicity to fro cells with a concentration-dependent manner. The synthesized Cy5.5-PEG-Fe3O4 probe has low toxicity, good biocompatibility, stable particle size, which can be used for cell imaging for noninvasive diagnosis of thyroid cancer.

MicroRNA-144-3p Represses the Growth and EMT of Thyroid Cancer via the E2F2/TNIK Axis in Cells and Male BALB/c Nude Mice.

microRNA (miR/miRNA)-144-3p has been implicated in thyroid cancer (TC) progression with poorly identified mechanisms. Furthermore, E2F2 has been documented to assume a role in the development of various cancers. This research sought to ascertain the role of miR-144-3p in growth and epithelial-mesenchymal transition (EMT) in TC in cells and male BALB/c nude mice. In the obtained TC cells, miR-144-3p expression was detected by quantitative reverse transcription polymerase chain reaction, and E2F2 and TNIK expression by Western blot analysis. After gain- and loss-of-function assays, cell viability, clone formation, migration, and invasion were assessed by cell counting kit-8, clone formation, scratch, and Transwell assays. The expression of EMT-related proteins (Snail, Vimentin, N-cadherin, and E-cadherin) was tested by Western blot analysis. The targeting relationship between miR-144-3p and E2F2 was evaluated by dual-luciferase reporter and radioimmunoprecipitation assays, and the binding relationship between E2F2 and TNIK by dual-luciferase reporter and chromatin immunoprecipitation assays. TC cell growth in vivo was determined by subcutaneous tumorigenesis assays in nude mice. miR-144-3p was downregulated, whereas E2F2 and TNIK were upregulated in TC cells. Mechanistically, miR-144-3p inversely targeted E2F2, which increased TNIK expression by binding to TNIK promoter in TC cells. Overexpression of miR-144-3p reduced proliferation, migration, invasion, and EMT of FRO and KTC3 cells, which was nullified by overexpressing E2F2 or TNIK expression. Upregulation of miR-144-3p diminished FRO cell growth and EMT in nude mice, which was abrogated by overexpressing TNIK. miR-144-3p inhibits cell growth and EMT in TC through E2F2/TNIK axis inactivation in cells and male BALB/c nude mice.

Capsaicin inhibits the stemness of anaplastic thyroid carcinoma cells by triggering autophagy-lysosome mediated OCT4A degradation.

Capsaicin (CAP) is a well-known anti-cancer agent. Recently, we reported capsaicin-induced apoptosis in anaplastic thyroid cancer (ATC) cells. It is well accepted that the generation of cancer stem cells (CSCs) is responsible for the dedifferentiation of ATC, the most lethal subtype of thyroid cancer with highly dedifferentiation status. Whether CAP inhibited the ATC growth through targeting CSCs needed further investigation. In the present study, CAP was found to induce autophagy in ATC cells through TRPV1 activation and subsequent calcium influx. Meanwhile, CAP dose-dependently decreased the sphere formation capacity of ATC cells. The stemness-inhibitory effect of CAP was further by extreme limiting dilution analysis (ELDA). CAP significantly decreased the protein level of OCT4A in both 8505C and FRO cells. Furthermore, CAP-induced OCT4A degradation was reversed by autophagy inhibitors 3-MA and chloroquine, BAPTA-AM and capsazepine, but not proteasome inhibitor MG132. Collectively, our study firstly showed CAP suppressed the stemness of ATC cells partially via calcium-dependent autophagic degradation of OCT4A. Our study lent credence to the feasible application of capsaicin in limiting ATC stemness.

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.

Exosome-mediated delivery of SCD-1 siRNA promoted the death of anaplastic thyroid carcinoma cells via regulating ROS level.

Anaplastic thyroid carcinoma (ATC) is one of the most aggressive cancers in the world. Stearoyl-CoA desaturase-1 (SCD-1) is one of major enzymes in the de novo synthesis of fatty acids and is related to cancer aggressiveness and poor patient prognosis. The study aimed to construct exosomes loaded SCD-1 interference, investigate its effects and mechanisms on the cell proliferation and apoptosis of ATC cells. The expressions of SCD-1 in normal thyroid cell line and ATC cell lines were determined by qRT-PCR and western blotting, respectively. Exosomes were prepared and purification then loaded with SCD-1 siRNA by electroporation and observed by transmission electron microscopy. Higher SCD-1 mRNA and protein levels were found in ATC cell lines compared than normal thyroid cell line (P < 0.05), and both Hth-7 and FRO cells could uptake PKH67-labeled exosomes. The effects of exosomes loaded SCD-1 siRNA on ATC cells were measured by CCK8 assay and apoptosis detection kit. When compared with control group, the cell viability significantly decreased in both two ATC cell lines taken up exosomes loaded SCD-1 siRNA (P < 0.001), and apoptotic and necrotic cells obviously increased (P < 0.05). In order to explore the mechanism of exosomes loaded SCD-1 on ATC, the ROS level was detected by fluorescence reagent. It was found that exosomes loaded SCD-1 siRNA significantly increased intracellular ROS level of ATC cells (P < 0.05). Exosomes loaded SCD-1 siRNA inhibited ATC cellular proliferation and promoted cellular apoptosis, and the mechanisms involved maybe the regulation of fatty acids metabolism and ROS level. Our study provides a promising therapeutic strategy for ATC.

Abstract 2693: Macrophage-derived endotrophin supports tumor migration potentials in thyroid cancer

Abstract Endotrophin (ETP), a cleaved fragment of the C5 domain of the Type VI collagen α3 (Col6α3), has been shown to play pro-tumorigenic roles in breast and liver cancers. However, the ETP actions in tumor microenvironment (TME) is still undetermined. This study aimed to investigate the role and the mechanism of ETP in macrophage-enriched thyroid cancer TMEs. First, the expression of ETP on various human thyroid tissues was studied. Immunohistochemical staining showed that the ETP was expressed in papillary (PTC) or anaplastic (ATC) thyroid cancer tissues but not in benign adenoma or autoimmune thyroiditis. Among 146 PTCs, tumors were divided into two groups according to their ETP expression, ETPlow and ETPHigh. Tumor size was bigger and LN metastasis rates were higher in ETPHigh than ETPlow group. Interestingly, the ETP expression showed positive correlations with the CD163-positive macrophages in thyroid tumors. To clarify the origin of ETP in TME of thyroid cancer, xenograft tumor from FRO (ATC) cells was co-stained with anti-ETP and anti-CD11b antibodies. Immunofluorescent staining showed that the expression of ETP and CD11b-postive macrophages were co-localized in the peritumoral area. Moreover, CD163+ cells, as a marker of M2-type macrophage, were co-localized with ETP+ cells. Additionally, human monocytic THP-1 cells were treated with conditioned media (CM) from FRO or BCPAP cells (designated FRO-CM or BCPAP-CM) and demonstrated that ETP expression was more up-regulated in the FRO-CM or BCPAP-CM treated group than in the control group. Collectively, ETP produced from M2-type macrophages in thyroid cancer microenvironments. Next, the pro-tumorigenic functions of ETP has been studied. CMs from co-cultures of FRO or BCPAP cells with THP-1 cells were harvested and treated to FRO or BCPAP cells. Treatment of CMs from co-cultures increased cell migration potentials than that of the single cell alone, and these effects were reduced by anti-ETP neutralizing antibody, indicating that ETPs play essential role in macrophage-induced tumor cell migration potentials. Moreover, MMP-9 and MMP-14, the candidates of Col6α3 protease to produce ETP were increased in THP-1 cells by treatment of CM. Additionally, treatment of CMs from MMP-9 or MMP-14 inhibitor-treated co-cultures of FRO and THP-1 cells decreased cancer cells migration potentials than the control CMs, and these effects were recovered by treatment of ETP. Finally, the RNA-seq dataset of human thyroid cancer showed that higher expressions of ETP positively correlated with macrophage-related genes such as CD163 and MMPs including MMP-9 and MMP-14. In conclusion, macrophage contributes ETP productions by modulating MMP expressions and ETP supports pro-metastatic potentials of human thyroid cancer cells in TMEs. Thus ETP can be a good therapeutic target for macrophage-enriched advanced thyroid cancers Citation Format: Hyo Shik Shin, Hana Kim, Young Shin Song, Hyun Jin Sun, Young Mi Whang, Jiyoung Park, Do Joon Park, Young Joo Park, Sun Wook Cho. Macrophage-derived endotrophin supports tumor migration potentials in thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2693.

Macrophage-Derived Endotrophin Supports Tumor Migration Potentials in Thyroid Caner

Abstract Endotrophin (ETP), a cleaved fragment of the C5 domain of the Type VI collagen α3 (Col6α3), has been shown to play pro-tumorigenic roles in breast and liver cancers. However, the ETP actions in tumor microenvironment (TME) is still undetermined. This study aimed to investigate the role and the mechanism of ETP in macrophage-enriched thyroid cancer TMEs. First, the expression of ETP on various human thyroid tissues was studied. Immunohistochemical staining showed that the ETP was expressed in papillary (PTC) or anaplastic (ATC) thyroid cancer tissues but not in benign adenoma or autoimmune thyroiditis. Among 146 PTCs, tumors were divided into two groups according to their ETP expression, ETPlow and ETPHigh. Tumor size was bigger and LN metastasis rates were higher in ETPHigh than ETPlow group. Interestingly, the ETP expression showed positive correlations with the CD163-positive macrophages in thyroid tumors. To clarify the origin of ETP in TME of thyroid cancer, xenograft tumor from FRO (ATC) cells was co-stained with anti-ETP and anti-F4/80 antibodies. Immunofluorescent staining showed that the expression of ETP and F4/80-postive macrophages were co-localized in the peritumoral area. Additionally, human monocytic THP-1 cells were treated with conditioned media (CM) from FRO or BCPAP cells (designated FRO-CM or BCPAP-CM) and demonstrated that ETP expression was more up-regulated in the FRO-CM or BCPAP-CM treated group than in the control group. Collectively, ETP produced from macrophages in thyroid cancer microenvironments. Next, the pro-tumorigenic functions of ETP has been studied. CMs from co-cultures of FRO or BCPAP cells with THP-1 cells were harvested and treated to FRO or BCPAP cells. Treatment of CMs from co-cultures increased cell migration potentials than that of the single cell alone, and these effects were reduced by anti-ETP neutralizing antibody, indicating that ETPs play essential role in macrophage-induced tumor cell migration potentials. Moreover, MMP-9 and MMP-14, the candidates of Col6α3 protease to produce ETP were increased in THP-1 cells by treatment of CM. Additionally, treatment of CMs from MMP-9 inhibitor-treated co-cultures of FRO and THP-1 cells decreased cancer cells migration potentials than the control CMs. Finally, the RNA-seq dataset of human thyroid cancer showed that higher expressions of ETP positively correlated with macrophage-related genes such as CD163 and MMPs including MMP-9 and MMP-14. In conclusion, macrophage contributes ETP productions by modulating MMP expressions and ETP supports pro-metastatic potentials of human thyroid cancer cells in TMEs. Thus ETP can be a good therapeutic target for macrophage-enriched advanced thyroid cancers.

Growth Differentiation Factor 15 is a Cancer Cell-Induced Mitokine That Primes Thyroid Cancer Cells for Invasiveness.

Background: Mitochondrial stress is known to activate the mitochondrial unfolded protein response (UPRmt). The UPRmt results in the secretion of mitochondrial cytokines (mitokines), which can promote a hormetic response cell nonautonomously, and has been reported to be protumorigenic. Growth differentiation factor 15 (GDF15) is a well-characterized mitokine, which is reported to have a mitohormetic effect. Thus, we investigated whether GDF15 induction could prime a subpopulation of thyroid cancer cells to provide invasive advantages. Methods: The UPRmt, including mitokine expression, was assessed in the context of thyroid cancer in vitro and in vivo. GDF15 expression in 266 patients with papillary thyroid carcinoma (PTC) was determined by immunohistochemistry. The serum levels of GDF15 were measured in healthy subjects and PTC patients. In addition, our own and The Cancer Genome Atlas data were analyzed to determine the expression level of GDF15 in thyroid cancers. The role of GDF15 in tumor aggressiveness was investigated by observing the effects of GDF15 knockdown in BCPAP, TPC-1, 8505C, and FRO cells. Results: Pharmacological inhibition of mitochondrial oxidative phosphorylation function in thyroid cancer cells robustly increased GDF15 expression. The expression of GDF15 was associated with activation of the mitochondrial integrated stress response pathway in PTC patients. Circulating GDF15 levels were significantly higher in PTC patients than in the controls, and tumor expression of GDF15 was related to tumor aggressiveness. In vitro and in vivo knockdown of GDF15 in a thyroid cancer model showed decreased viability, migration, and invasion compared with the control cells via regulation of STAT3. Conclusions: In this study, we demonstrated that GDF15 is a mitokine induced in thyroid cancer cells upon mitochondrial stress. GDF15-induced STAT3 activation determined tumor progression in thyroid cancer. The GDF15-STAT3 signaling axis may be a target in aggressiveness of thyroid cancer.

Exploiting GRK2 Inhibition as a Therapeutic Option in Experimental Cancer Treatment: Role of p53-Induced Mitochondrial Apoptosis.

Simple SummaryThe involvement of GRK2 in cancer growth and an inverse correlation with p53 levels were suggested in breast cancer. Furthermore, increased GRK2 expression and activity were detected in thyroid cancer, but its effects and mechanisms of action were not investigated yet. This study aimed to explore the role of GRK2 in thyroid cancer both in vitro and in vivo and its crosstalk with p53. We demonstrated that thyroid cancer cells bearing a mutant form of p53 but not p53 null cells rely on GRK2 as a mechanism of proliferation by regulating p53 levels. Indeed, GRK2 indirectly induces p53 degradation through means of its catalytic activity. The pharmacological inhibition of the kinase effectively inhibits cancer growth by inducing p53-dependent mitochondrial pathways of apoptosis. Our results demonstrate a p53-dependent effect of GRK2 in cancer and suggest kinase inhibition as a potential therapeutic strategy for thyroid cancer.The involvement of GRK2 in cancer cell proliferation and its counter-regulation of p53 have been suggested in breast cancer even if the underlying mechanism has not yet been elucidated. Furthermore, the possibility to pharmacologically inhibit GRK2 to delay cancer cell proliferation has never been explored. We investigated this possibility by setting up a study that combined in vitro and in vivo models to underpin the crosstalk between GRK2 and p53. To reach this aim, we took advantage of the different expression of p53 in cell lines of thyroid cancer (BHT 101 expressing p53 and FRO cells, which are p53-null) in which we overexpressed or silenced GRK2. The pharmacological inhibition of GRK2 was achieved using the specific inhibitor KRX-C7. The in vivo study was performed in Balb/c nude mice, where we treated BHT-101 or FRO-derived tumors with KRX-C7. In our in vitro model, FRO cells were unaffected by GRK2 expression levels, whereas BHT-101 cells were sensitive, thus suggesting a role for p53. The regulation of p53 by GRK2 is due to phosphorylative events in Thr-55, which induce the degradation of p53. In BHT-101 cells, the pharmacologic inhibition of GRK2 by KRX-C7 increased p53 levels and activated apoptosis through the mitochondrial release of cytochrome c. These KRX-C7-mediated events were also confirmed in cancer allograft models in nude mice. In conclusion, our data showed that GRK2 counter-regulates p53 expression in cancer cells through a kinase-dependent activity. Our results further corroborate the anti-proliferative role of GRK2 inhibitors in p53-sensitive tumors and propose GRK2 as a therapeutic target in selected cancers.

Relationship between thyroid tumor radiosensitivity and nuclear localization of DNA-dependent protein kinase catalytic subunit.

Thyroid tumors are the most common types of endocrine malignancies and are commonly treated with radioactive iodine (RAI) to destroy remaining cancer cells following surgical intervention. We previously reported that the expression levels of double-stranded DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which plays a key role in non-homologous end joining, are correlated with the radiosensitivity of cancer cells. Specifically, cells expressing high levels of DNA-PKcs exhibited radiation resistance, whereas cells expressing low levels were sensitive to radiation treatment. In this study, we observed full-length native DNA-PKcs (460 kDa) in radiation-resistant FRO and KTC-2 cells through western blot analysis using an antibody against the C-terminus of DNA-PKcs. In contrast, cleaved DNA-PKcs (175 kDa) were observed in radiation-sensitive TPC-1 and KTC-1 cells. Almost equal amounts of DNA-PKcs were observed in moderately radiation-sensitive WRO cells. We also describe a simple method for the prediction of radiation therapy efficacy in individual cases of thyroid cancers based on staining for DNA-PKcs in human cancer cell lines. Immunofluorescent staining showed that native DNA-PKcs was localized largely in the cytoplasm and only rarely localized in the nuclei of radiation-resistant thyroid cancer cells, whereas in radiation-sensitive cancer cells a 175-kDa cleaved C-terminal fragment of DNA-PKcs was localized mainly inside the nuclei. Therefore, DNA-PKcs moved to the nucleus after γ-ray irradiation. Our results suggest a new method for classifying human thyroid tumors based on their cellular distribution patterns of DNA-PKcs in combination with their radiosensitivity.

Effect of mesoporous silica nanoparticles co‑loading with 17‑AAG and Torin2 on anaplastic thyroid carcinoma by targeting VEGFR2.

Anaplastic thyroid carcinoma (ATC) is a highly aggressive tumor with a poor prognosis and a low median survival rate because of insufficient effective therapeutic modalities. Recently, mesoporous silica nanoparticles (MSNs) as a green non-toxic and safe nanomaterial have shown advantages to be a drug carrier and to modify the targeting group to the targeted therapy. To aim of the study was to explore the effects of MSNs co-loading with 17-allylamino-17-demethoxy-geldanamycin (17-AAG; HSP90 inhibitor) and 9-(6-aminopyridin-3-yl)-1-(3-(trifluoromethyl)phenyl)benzo[h][1,6]naphthyridin-2(1H)-one (Torin2; mTOR inhibitor) by targeting vascular endothelial growth factor receptor 2 (VEGFR2) on the viability of human anaplastic thyroid carcinoma FRO cells. The cytotoxicity of 17-AAG and Torin2 were analyzed by MTT assay. The possible synergistic antitumor effects between 17-AAG and Torin2 were evaluated by CompuSyn software. Flow cytometry was performed to assess the VEGFR2 targeting of (17-AAG+Torin2)@MSNs-anti-VEGFR2 ab and uptake by FRO cells. An ATC xenograft mouse model was established to assess the antitumor effect of (17-AAG+Torin2)@MSNs-anti-VEGFR2 ab in vivo. The results revealed that the combination of 17-AAG and Torin2 inhibited the growth of FRO cells more effectively compared with single use of these agents. Additionally, the synergistic antitumor effect appeared when concentration ratio of the two drugs was 1:1 along with total drug concentration greater than 0.52 µM. Furthermore, in an ATC animal model, it was revealed that the (17-AAG+Torin2)@MSNs-anti-VEGFR2 ab therapy modality could most effectively prolong the median survival time [39.5 days vs. 33.0 days (non-targeted) or 27.5 days (control)]. Compared to (17-AAG+Torin2)@MSNs, the (17-AAG+Torin2)@MSNs-anti-VEGFR2 ab could not only inhibit ATC cell growth but also prolong the median survival time of tumor-bearing mice in vivo and vitro more effectively, which may provide a new promising therapy for ATC.

SRY‑related HMG box‑2 role in anaplastic thyroid cancer aggressiveness is related to the fibronectin 1 and PI3K/AKT pathway.

Anaplastic thyroid cancer (ATC) is a rare thyroid tumor associated with high mortality rates; thus, the identification of novel molecular targets and the development of therapeutic strategies are urgently required. The present study aimed to investigate the role of SRY-related HMG box-2 (SOX2) in ATC cells and explore whether the underlying mechanism was associated with fibronectin 1 (FN1). The proliferative, migratory and invasive ability of ATC cell lines was investigated using Cell Counting Kit-8, colony formation, wound-healing and Transwell assays, respectively; SOX2 expression in FRO cells was knocked down using small interfering RNA and SOX2 overexpression in FRO cells was achieved using cDNA constructs; and reverse transcription-quantitative PCR and western blotting were used to identify the mechanism of action underlying the SOX-2 mediated increased in cell aggressive phenotypes. Increased protein expression levels of SOX2 protein were observed in ATC tissue, and FRO and 8505c ATC cell lines. SOX2 overexpression increased the cell viability, and proliferative, migratory and invasive abilities of FRO cell lines. SOX2 overexpression increased FN1, p65, phosphorylated PI3K and AKT expression levels, whereas the knockdown of SOX2 promoted the opposite effects. In conclusion, the present study suggested a possible model of SOX2-mediated gene regulation in ATC cells, in which the overexpression of SOX2 promoted FN1 expression via the PI3K/AKT signaling pathway to induce the aggressive phenotype of ATC. These findings may provide crucial molecular insights into ATC pathogenesis and may demonstrate potential to develop into novel therapeutic interventions for patients with ATC.

MiR-155 promotes anaplastic thyroid cancer progression by directly targeting SOCS1

BackgroundAnaplastic thyroid cancer (ATC) is considered to be a rare type of thyroid cancer but takes up the most important proportion of thyroid cancer-related deaths. Therefore, the development of molecular targeted therapy is an exciting strategy in the management of ATC.MethodsmiR-155 and SOCS1 expression were measured by qRT-PCR as well as western blot analysis. 8305c and FRO cells were transfected and cultured for apoptosis assays, transwell, MTT on miR-155 or SOCS1 suppression and overexpression. Dual-luciferase reporter assays and SOCS1 restoration experimentswas implemented for define the relation between SOCS1 and miR-155. In addition, the correlation between miR-155 expression and patients’ clinicopathological features were also explored.ResultsAberrant miR-155 and SOCS1 expression and inverse correlation were found in ATC samples. In addition, it indicated that miR-155 expression correlated with cervical metastasis as well as extrathyroidal invasion. Moreover, we demonstrated that miR-155 inhibited 8305c and FRO cells apoptosis, promoted proliferation, invasion and migration. Furthermore, miR-155 inhibition was associated with a significant overexpression of SOCS1. Additionally, luciferase reporter assays presented that miR-155 could bind to SOCS1 3′-UTR, influencing its stability negatively and finally lowering SOCS1 levels. Moreover, it was illustrated that the impacts of miR-155 suppression were reversed by the inhibition of SOCS1 on cell proliferation, apoptosis as well as invasion.ConclusionsAberrant miR-155/SOCS1 expression has been included in ATC progression: miR-155 overexpression leads to SOCS1 suppression and develops ATC progression. Thus, miR-155 has been considered to be an underlying therapeutic target for ATC.

JAK/STAT3 and NF-κB Signaling Pathways Regulate Cancer Stem-Cell Properties in Anaplastic Thyroid Cancer Cells.

Background: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive and refractory cancers, and a therapy with a new concept needs to be developed. Recently, research on cancer stem cells (CSCs) has progressed, and CSCs have been suggested to be responsible for metastasis, recurrence, and therapy resistance. In ATC-CSCs, aldehyde dehydrogenase (ALDH) activity is the most reliable marker to enrich CSCs. However, it is just a marker and is not involved in CSC properties. The present study therefore aimed to identify key signaling pathways specific for ATC-CSCs. Methods: A small interfering RNA library targeting 719 kinases was used in a sphere formation assay and cell survival assay using ATC cell lines to select target molecules specific for CSC properties. The functions of the selected candidates were confirmed by sphere formation, cell survival, soft agar, and nude mice xenograft assays using small compound inhibitors. Results: The study focused on PDGFR, JAK, and PIM, whose small interfering RNAs had a higher inhibitory effect on sphere formation, as well as a lower or no effect on regular cell growth in both FRO and KTC3 cells. Next, inhibitors of PDGFR, JAK, STAT3, PIM and NF-κB were used, and all of them successfully suppressed sphere formation in a dose-dependent manner but not regular cell growth, confirming the screening results. Inhibition of the JAK/STAT3 and NF-κB pathways also reduced anchorage-independent growth in soft agar and tumor growth in nude mice. Conclusions: These results suggest that JAK/STAT3 and NF-κB signals play important roles in ATC-CSCs. Targeting these signaling pathways may be a promising approach to treat ATC.

Anticancer Effects of Baicalein in FRO Thyroid Cancer Cells Through the Up-regulation of ERK/p38 MAPK and Akt Pathway

The aim of the study was to evaluate the anticancer effects of baicalein in FRO anaplastic thyroid cancer (ATC) cells. FRO cells were treated with baicalein and viability was measured by the MTT assay. Cell apoptosis was observed by staining with Hoechst dye. The expression of apoptotic proteins (Bax, Bcl-2, PARP, cytochrome c, and caspase-3) and the inflammatory protein Cox-2 and the phosphorylation of MAPKs and Akt were determined by western blot. Treatment with baicalein inhibited cell proliferation in a time-dependent manner and increased DNA fragmentation and apoptosis in FRO cells. Baicalein at 50 and 100 μM inhibited the expression of Bax, PARP, cytochrome c, cleaved caspase-3, and Cox-2, and increased the expression of Bcl-2. Baicalein increased the phosphorylation of ERK, p38 MAPK, and Akt and decreased JNK phosphorylation. Baicalein caused anticancer effects in FRO ATC cells through induction of apoptosis and regulation of the MAPK and Akt pathway.

Cytotoxic evaluation and concurrent analysis of two diterpenes in the chloroform extract of Plectranthus barbatus using a validated HPTLC-UV method

The objective of this study was to develop a validated high-performance thin layer chromatography (HPTLC) method for the concurrent analysis of two diterpenes Sugiol (compound 1) and 11,14-dihydroxy-8,11,13-abietatrien-7-one (compound 2) in Plectranthus barbatus chloroform extract (PBCE) and to investigate cytotoxicity of both compounds. The chromatographic estimations were carried out using toluene : ethyl acetate : formic acid (8.2:1.3:0.5 v/v/v) as mobile phase. The compact spots of biomarkers were scanned at λmax= 275 nm. Cytotoxic evaluation of both compounds was performed using follicular thyroid cancer cells (FRO cells). A well resolved, compact and intense peaks of compound 1 (Rf = 0.550 ± 0.001) and compound 2 (Rf = 0.700 ± 0.002) were recorded. The proposed method for both compounds was recorded as simple, linear, precise (% RSD = 1.02-1.25), accurate (98-100%), robust and sensitive for the analysis of both compounds. Cytotoxicity evaluation showed significant cell cytotoxicity at 100 μg/mL concentration against the FRO cancer cells after 72 h of incubation. Both compounds were recorded as cytotoxic, however compound 1 showed significant cytotoxic effects. The proposed HPTLC method was found to be suitable for routine analysis of these two biomarkers in chloroform extract of P. barbatus. Both compounds were found to be cytotoxic against FRO cancer cells. KEY WORDS: Diterpenes, Sugiol, Plectranthus barbatus, Cytotoxicity, HPTLC Bull. Chem. Soc. Ethiop. 2018, 32(3), 407-419.DOI: https://dx.doi.org/10.4314/bcse.v32i3.1