RESUMEN
Anaplastic thyroid cancer (ATC), an aggressive malignancy with virtually 100% disease-specific mortality, has long posed a formidable challenge in oncology due to its resistance to conventional treatments and the severe side effects associated with current regimens such as doxorubicin chemotherapy. Consequently, there was urgent need to identify novel candidate compounds that could provide innovative therapeutic strategies for ATC. Ophiopogonin D' (OPD'), a triterpenoid saponin extracted, yet its roles in ATC has not been reported. Our data demonstrated that OPD' potently inhibited proliferation and metastasis of ATC cells, promoting cell cycle arrest and apoptosis. Remarkably, OPD' impeded growth and metastasis of ATC in vitro and in vivo, displaying an encouraging safety profile. Regulator of G-protein signalling 4 (RGS4) expression was significantly up-regulated in ATC compared to normal tissues, and this upregulation was suppressed by OPD' treatment. Mechanistically, we elucidated that the transcription factor JUN bound to the RGS4 promoter, driving its transactivation. However, OPD' interacted with JUN, attenuating its transcriptional activity and thereby disrupting RGS4 overexpression. In summary, our research revealed that OPD' bound with JUN, which in turn resulted in the suppression of transcriptional activation of RGS4, thereby eliciting cell cycle arrest and apoptosis in ATC cells. These findings could offer promise in the development of high-quality candidate compounds for treatment in ATC.
Asunto(s)
Apoptosis , Proliferación Celular , Proteínas RGS , Saponinas , Transducción de Señal , Espirostanos , Carcinoma Anaplásico de Tiroides , Humanos , Carcinoma Anaplásico de Tiroides/tratamiento farmacológico , Carcinoma Anaplásico de Tiroides/metabolismo , Carcinoma Anaplásico de Tiroides/patología , Saponinas/farmacología , Proteínas RGS/metabolismo , Proteínas RGS/genética , Proliferación Celular/efectos de los fármacos , Animales , Línea Celular Tumoral , Transducción de Señal/efectos de los fármacos , Apoptosis/efectos de los fármacos , Espirostanos/farmacología , Ratones , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proteínas Proto-Oncogénicas c-jun/metabolismo , Ratones Desnudos , Neoplasias de la Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/patología , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Metástasis de la NeoplasiaRESUMEN
OBJECTIVE: This study was designed to investigate the regulatory effects of kinesin family member (KIF) 23 on anaplastic thyroid cancer (ATC) cell viability and migration and the underlying mechanism. METHODS: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to analyze the levels of KIF23 in ATC cells. Besides, the effects of KIF23 and sirtuin (SIRT) 7 on the viability and migration of ATC cells were detected using cell counting kit-8, transwell and wound healing assays. The interaction between SIRT7 and KIF23 was evaluated by co-immunoprecipitation (Co-IP) assay. The succinylation (succ) of KIF23 was analyzed by western blot. RESULTS: The KIF23 expression was upregulated in ATC cells. Silencing of KIF23 suppressed the viability and migration of 8505C and BCPAP cells. The KIF23-succ level was decreased in ATC cells. SIRT7 interacted with KIF23 to inhibit the succinylation of KIF23 at K537 site in human embryonic kidney (HEK)-293T cells. Overexpression of SIRT7 enhanced the protein stability of KIF23 in HEK-293T cells. Besides, overexpression of KIF23 promoted the viability and migration of 8505C and BCPAP cells, which was partly blocked by silenced SIRT7. CONCLUSIONS: SIRT7 promoted the proliferation and migration of ATC cells by regulating the desuccinylation of KIF23.
Asunto(s)
Sirtuinas , Carcinoma Anaplásico de Tiroides , Neoplasias de la Tiroides , Humanos , Carcinoma Anaplásico de Tiroides/genética , Carcinoma Anaplásico de Tiroides/metabolismo , Línea Celular Tumoral , Apoptosis , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/metabolismo , Proliferación Celular/genética , Proteínas Asociadas a Microtúbulos , Sirtuinas/genética , Sirtuinas/farmacologíaRESUMEN
Anaplastic thyroid carcinoma (ATC) is a rare but highly aggressive thyroid cancer with poor prognosis. Killing cancer cells by inducing DNA damage or blockage of DNA repair is a promising strategy for chemotherapy. It is reported that aldehyde-reactive alkoxyamines can capture the AP sites, one of the most common DNA lesions, and inhibit apurinic/apyrimidinic endonuclease 1(APE1)-mediated base excision repair (BER), leading to cell death. Whether this strategy can be employed for ATC treatment is rarely investigated. The aim of this study is to exploit GSH-responsive AP site capture reagent (AP probe-net), which responses to the elevated glutathione (GSH) levels in the tumor micro-environment (TME), releasing reactive alkoxyamine to trap AP sites and block the APE1-mediated BER for targeted anti-tumor activity against ATC. In vitro experiments, including MTT andγ-H2AX assays, demonstrate their selective cytotoxicity towards ATC cells over normal thyroid cells. Flow cytometry analysis suggests that AP probe-net arrests the cell cycle in the G2/M phase and induces apoptosis. Western blotting (WB) results show that the expression of apoptotic protein increased with the increased concentration of AP probe-net. Further in vivo experiments reveal that the AP probe-net has a good therapeutic effect on subcutaneous tumors of the ATC cells. In conclusion, taking advantage of the elevated GSH in TME, our study affords a new strategy for targeted chemotherapy of ATC with high selectivity and reduced adverse effects.
Asunto(s)
Apoptosis , Glutatión , Carcinoma Anaplásico de Tiroides , Neoplasias de la Tiroides , Carcinoma Anaplásico de Tiroides/tratamiento farmacológico , Carcinoma Anaplásico de Tiroides/patología , Carcinoma Anaplásico de Tiroides/metabolismo , Humanos , Glutatión/metabolismo , Animales , Ratones , Neoplasias de la Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/patología , Neoplasias de la Tiroides/metabolismo , Línea Celular Tumoral , Apoptosis/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Reparación del ADN/efectos de los fármacos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Daño del ADN/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacosRESUMEN
Pyroptosis is a novel type of proinflammatory programmed cell death that is associated with inflammation, immunity, and cancer. Anaplastic thyroid carcinoma (ATC) has a high fatality rate, and there is no effective or standard treatment. The disease progresses rapidly and these tumors can invade the trachea and esophagus, leading to breathing and swallowing difficulties. Hence, new treatment methods are greatly needed. Ibuprofen is a common drug that can exert antitumor effects in some cancers. In this study, we demonstrated in vitro and in vivo that ibuprofen can induce ATC pyroptosis. Hence, we treated C643 and OCUT-2C ATC cells with ibuprofen and found that several dying cells presented the characteristic morphological features of pyroptosis, such as bubble-like swelling and membrane rupture, accompanied by activation of ASC and NLRP3 and cleavage of GSDMD. Along with the increased release of LDH, ibuprofen treatment promoted apoptosis and inhibited viability, invasion, and migration. However, overexpression of GSDMD significantly inhibited ibuprofen-induced pyroptosis. In vivo, research has demonstrated that thyroid tumor growth in nude mice can be suppressed by ibuprofen-induced pyroptosis in a dose-dependent manner. In this research, we explored a new mechanism by which ibuprofen inhibits ATC growth and progression and highlighted its promise as a therapeutic agent for ATC.
Asunto(s)
Ibuprofeno , Piroptosis , Carcinoma Anaplásico de Tiroides , Neoplasias de la Tiroides , Animales , Ratones , Ibuprofeno/farmacología , Ibuprofeno/uso terapéutico , Ratones Desnudos , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Piroptosis/efectos de los fármacos , Carcinoma Anaplásico de Tiroides/tratamiento farmacológico , Carcinoma Anaplásico de Tiroides/metabolismo , Carcinoma Anaplásico de Tiroides/patología , Neoplasias de la Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patologíaRESUMEN
Anaplastic thyroid carcinoma (ATC) is a deadly disease with a poor prognosis. Thus, there is a pressing need to determine the mechanism of ATC progression. The homeobox D9 (HOXD9) transcription factor has been associated with numerous malignancies but its role in ATC is unclear. In the present study, the carcinogenic potential of HOXD9 in ATC was investigated. We assessed the differential expression of HOXD9 on cell proliferation, migration, invasion, apoptosis, and epithelial-mesenchymal transition (EMT) in ATC and explored the interactions between HOXD9, microRNA-451a (miR-451a), and proteasome 20S subunit beta 8 (PSMB8). In addition, subcutaneous tumorigenesis and lung metastasis in mouse models were established to investigate the role of HOXD9 in ATC progression and metastasis in vivo. HOXD9 expression was enhanced in ATC tissues and cells. Knockdown of HOXD9 inhibited cell proliferation, migration, invasion, and EMT but increased apoptosis in ATC cells. The UCSC Genome Browser and JASPAR database identified HOXD9 as an upstream regulator of miR-451a. The direct binding of miR-451a to the untranslated region (3'-UTR) of PSMB8 was established using a luciferase experiment. Blocking or activation of PI3K by LY294002 or 740Y-P could attenuate the effect of HOXD9 interference or overexpression on ATC progression. The PI3K/AKT signaling pathway was involved in HOXD9-stimulated ATC cell proliferation and EMT. Consistent with in vitro findings, the downregulation of HOXD9 in ATC cells impeded tumor growth and lung metastasis in vivo. Our research suggests that through PI3K/AKT signaling, the HOXD9/miR-451a/PSMB8 axis may have significance in the control of cell proliferation and metastasis in ATC. Thus, HOXD9 could serve as a potential target for the diagnosis of ATC.
Asunto(s)
Neoplasias Pulmonares , MicroARNs , Carcinoma Anaplásico de Tiroides , Neoplasias de la Tiroides , Animales , Humanos , Ratones , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Transición Epitelial-Mesenquimal/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Neoplasias Pulmonares/genética , MicroARNs/genética , MicroARNs/metabolismo , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Carcinoma Anaplásico de Tiroides/genética , Carcinoma Anaplásico de Tiroides/metabolismo , Carcinoma Anaplásico de Tiroides/patología , Neoplasias de la Tiroides/patologíaRESUMEN
Abnormal spindle-like microcephaly-associated (ASPM) protein is crucial to the mitotic spindle function during cell replication and tumor progression in multiple tumor types. However, the effect of ASPM in anaplastic thyroid carcinoma (ATC) has not yet been understood. The present study is to elucidate the function of ASPM in the migration and invasion of ATC. ASPM expression is incrementally upregulated in ATC tissues and cell lines. Knockout (KO) of ASPM pronouncedly attenuates the migration and invasion of ATC cells. ASPM KO significantly reduces the transcript levels of Vimentin, N-cadherin, and Snail and increases E-cadherin and Occludin, thereby inhibiting epithelial-to-mesenchymal transition (EMT). Mechanistically, ASPM regulates the movement of ATC cells by inhibiting the ubiquitin degradation of KIF11 and thus stabilizing it via direct binding to it. Moreover, xenograft tumors in nude mice proved that KO of ASPM could ameliorate tumorigenesis and tumor growth accompanied by a decreased protein expression of KIF11 and an inhibition of EMT. In conclusion, ASPM is a potentially useful therapeutic target for ATC. Our results also reveal a novel mechanism by which ASPM inhibits the ubiquitin process in KIF11.
Asunto(s)
Carcinoma Anaplásico de Tiroides , Neoplasias de la Tiroides , Animales , Ratones , Humanos , Carcinoma Anaplásico de Tiroides/genética , Carcinoma Anaplásico de Tiroides/metabolismo , Carcinoma Anaplásico de Tiroides/patología , Neoplasias de la Tiroides/metabolismo , Ratones Desnudos , Línea Celular Tumoral , Ratones Noqueados , Proteínas del Tejido Nervioso , Ubiquitinas/farmacología , Movimiento Celular , Proliferación Celular , Cinesinas/genéticaRESUMEN
Anaplastic thyroid cancer (ATC) is a rare and lethal form of thyroid cancer that requires urgent investigation of new molecular targets involved in its aggressive biology. In this context, the overactivation of Polycomb Repressive Complex 2/EZH2, which induces chromatin compaction, is frequently observed in aggressive solid tumors, making the EZH2 methyltransferase a potential target for treatment. However, the deregulation of chromatin accessibility is yet not fully investigated in thyroid cancer. In this study, EZH2 expression was modulated by CRISPR/Cas9-mediated gene editing and pharmacologically inhibited with EZH2 inhibitor EPZ6438 alone or in combination with the MAPK inhibitor U0126. The results showed that CRISPR/Cas9-induced EZH2 gene editing reduced cell growth, migration and invasion in vitro and resulted in a 90% reduction in tumor growth when EZH2-edited cells were injected into an immunocompromised mouse model. Immunohistochemistry analysis of the tumors revealed reduced tumor cell proliferation and less recruitment of cancer-associated fibroblasts in the EZH2-edited tumors compared to the control tumors. Moreover, EZH2 inhibition induced thyroid-differentiation genes' expression and mesenchymal-to-epithelial transition (MET) in ATC cells. Thus, this study shows that targeting EZH2 could be a promising neoadjuvant treatment for ATC, as it promotes antitumoral effects in vitro and in vivo and induces cell differentiation.
Asunto(s)
Carcinoma Anaplásico de Tiroides , Neoplasias de la Tiroides , Animales , Ratones , Línea Celular Tumoral , Proliferación Celular , Proteína Potenciadora del Homólogo Zeste 2/genética , Complejo Represivo Polycomb 2/metabolismo , Carcinoma Anaplásico de Tiroides/tratamiento farmacológico , Carcinoma Anaplásico de Tiroides/genética , Carcinoma Anaplásico de Tiroides/metabolismo , Neoplasias de la Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/metabolismo , HumanosRESUMEN
This study aimed to explore the specific effect of miR-200c in anaplastic thyroid cancer (ATC). Hth74 and ARO cell lines were used. Proliferation, invasion, and colony formation activities of Hth74 and ARO cell lines affected by miR-200c were studied. Expression of epithelial-to-mesenchymal transition (EMT) markers (E-cadherin, N-cadherin, Slug, and Snail) in the Hth74 and ARO cell lines were validated by western blot and qRT-PCR. In addition, the regulation of the parathyroid hormone-like hormone (PTHLH) by miR-200c was assessed. Overexpression of miR-200c inhibited the invasion, proliferation, and colony formation of the ATC cell lines, whereas its downregulation achieved the opposite results. PTHLH was found to be regulated negatively by miR-200c through a miR-200c binding site within the 3'-UTR of PTHLH. miR-200c repressed the proliferation, invasion, and EMT process of cells in ATC cell lines by targeting PTHLH post-transcriptionally, which indicates that miR-200c may be a potential target for the treatment of ATC.
Asunto(s)
MicroARNs , Carcinoma Anaplásico de Tiroides , Neoplasias de la Tiroides , Humanos , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , MicroARNs/metabolismo , Invasividad Neoplásica/genética , Hormona Paratiroidea , Carcinoma Anaplásico de Tiroides/genética , Carcinoma Anaplásico de Tiroides/metabolismo , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/metabolismoRESUMEN
BACKGROUND: Hypoxia-induced angiogenesis functions importantly in anaplastic thyroid cancer (ATC) progression. However, the therapeutic potential of broad-spectrum anti-angiogenic agent remains undefined. Anlotinib conventionally targets VEGFR, FGFR and PDGFR. Here, a novel role of anlotinib on ATC angiogenesis was illustrated. METHODS: Molecular expressions were established via tissue microarray. Multiple assays (tubule formation, 3D sprouting and chicken chorioallantoic membrane model) were used for angiogenic evaluation. Panels of molecular screening were achieved by antibody and PCR arrays. The loop binding motif of EGFR for homology modelling was prepared using Maestro. RESULTS: Anlotinib could dose- and time-dependently inhibit cell viability under normoxia and hypoxia and could repress hypoxia-activated angiogenesis more efficiently in vitro and in vivo. CXCL11 and phospho-EGFR were hypoxia-upregulated with a positive correlation. The cancer-endothelium crosstalk could be mediated by the positive CXCL11-EGF-EGFR feedback loop, which could be blocked by anlotinib directly targeting EGFR via a dual mechanism by simultaneous inhibitory effects on cancer and endothelial cells. The AKT-mTOR pathway was involved in this regulatory network. CONCLUSIONS: The newly identified CXCL11-EGF-EGFR signalling provided mechanistic insight into the interaction between cancer and endothelial cells under hypoxia, and EGFR was a novel target. Anlotinib may be the encouraging therapeutic candidate in ATC.
Asunto(s)
Quimiocina CXCL11/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Indoles/administración & dosificación , Inhibidores de Proteínas Quinasas/administración & dosificación , Quinolinas/administración & dosificación , Carcinoma Anaplásico de Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/tratamiento farmacológico , Animales , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Receptores ErbB/metabolismo , Retroalimentación Fisiológica/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Indoles/farmacología , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Quinolinas/farmacología , Transducción de Señal/efectos de los fármacos , Carcinoma Anaplásico de Tiroides/metabolismo , Neoplasias de la Tiroides/metabolismo , Análisis de Matrices Tisulares , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Anaplastic thyroid cancer (ATC) is a rare but highly lethal disease. So far, there is no available established treatment which can prolong its survival. In this regard, effective therapies are urgently needed. Vitamin C widely serves as an anti-cancer agent. However, the potential effects of vitamin C against thyroid tumorigenesis remained unclear. The present study demonstrated that vitamin C could significantly inhibit ATC cells growth through ferroptosis activation, evidenced by the GPX4 inactivation, ROS accumulation and iron-dependent lipid peroxidation. Our results demonstrated that vitamin C treatment induced ferritinophagy and subsequent degradation of ferritin, leading to the release of free iron. Excessive iron further triggered ROS generation via Fenton reaction. The positive feedback mediated by ROS and iron sustained lipid peroxidation and further resulted in ferroptosis of ATC cells. The better understanding of the anti-cancer mechanisms of vitamin C provides a potential strategy for ATC therapy.
Asunto(s)
Ácido Ascórbico/farmacología , Autofagia/efectos de los fármacos , Ferritinas/metabolismo , Ferroptosis/efectos de los fármacos , Carcinoma Anaplásico de Tiroides/patología , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Ácido Ascórbico/uso terapéutico , Carcinogénesis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Peroxidación de Lípido , Estrés Oxidativo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Carcinoma Anaplásico de Tiroides/tratamiento farmacológico , Carcinoma Anaplásico de Tiroides/metabolismoRESUMEN
Anaplastic thyroid carcinomas (ATC) are rare, but represent the most lethal malignancy of the thyroid. Selective molecular markers and drivers distinguishing ATC from other thyroid carcinomas of follicular origin remain largely unknown, limiting advances in diagnosis and treatment. In a retrospective study, we analyzed gene expression in 36 ATC, 18 poorly differentiated, 132 papillary, and 55 follicular thyroid carcinoma, as well as 124 paired and unpaired normal thyroid tissues in three independent cohorts by RNA-sequencing and immunohistochemistry. RNA-sequencing data in the test cohort suggested selective ATC protein biomarkers. Evaluation of these revealed that ATCs are characterized by the de novo expression of various testis antigens, including melanoma-associated antigen A3 (MAGEA3), but most importantly the oncofetal IGF2 mRNA binding protein 1 (IGF2BP1). Shallow whole genome sequencing essentially excluded that IGF2BP1 upregulation results from gene copy number alterations. Immunohistochemical analyses in all three tumor cohorts confirmed the selective de novo expression of IGF2BP1 protein in ATC. In sum, 75% (27/36) of all tested ATC and 0.5% (1/204) of poorly and well-differentiated thyroid carcinoma tissue samples were positive for IGF2BP1 protein. This indicates that IGF2BP1 protein expression identifies ATC with a diagnostic odds ratio of 612 (95% CI: 74.6-5021). In addition, we found that MAGEA3 is exclusively, although less consistently upregulated in ATC, presenting with an odds ratio of 411 (95% CI: 23.8-7098.7). Importantly, we provide confirmatory evidence that IGF2BP1 and MAGEA3 expression distinguishes ATC from poorly differentiated thyroid carcinoma. IGF2BP1 furthermore identified ATC foci within low-grade follicular thyroid carcinoma. In conclusion, IGF2BP1 represents the most promising single-gene marker available for ATC, followed by MAGEA3, improving on current techniques. Robust markers are essential to help distinguish this high-grade malignancy from other thyroid carcinomas, to guide surgical decision making, therapy and post-resection/therapy monitoring strategies.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Proteínas de Unión al ARN/biosíntesis , Carcinoma Anaplásico de Tiroides/diagnóstico , Neoplasias de la Tiroides/diagnóstico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antígenos de Neoplasias/biosíntesis , Niño , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/biosíntesis , Estudios Retrospectivos , Carcinoma Anaplásico de Tiroides/metabolismo , Neoplasias de la Tiroides/metabolismo , Adulto JovenRESUMEN
Abnormal microRNA (miR) expression has frequently been reported to be implicated in cancer-related drug resistance. Herein, we planned to investigate whether miR-381-3p contributes to doxorubicin (DOX) resistance in anaplastic thyroid carcinoma (ATC). DOX-resistant ATC tissues and cell lines were prepared to detect miR-381-3p and homeobox A9 (HOXA9) expression. CCK8, transwell and TUNEL assays were performed to evaluate cell proliferation, migration and invasion, and apoptosis in in vitro experiments. HOXA9 expression is intensively expressed in ATC tissues compared with benign thyroid tissues. Compared with parental ATC cell lines, HOXA9 protein expression is significantly up-regulated in DOX-resistant SW1736 and CAL62 cells. The knockdown of HOXA9 leads to growth inhibition and apoptosis of DOX-resistant SW1736 and CAL62 cells. Our results also indicate a significant decrease in miR-381-3p expression levels in DOX-resistant ATC tissues and cell lines. miR-381-3p may function as a tumour suppressor to impede proliferation, migration and invasion and induce apoptosis of DOX-resistant SW1736 and CAL62 cells by inhibiting HOXA9 protein expression. Our results present a novel signalling axis miR-381-3p/HOXA9 that mediates DOX resistance in ATC. miR-381-3p and HOXA9 may be promising molecular targets for preventing ATC progression and drug resistance.
Asunto(s)
Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Proteínas de Homeodominio/metabolismo , MicroARNs/metabolismo , Carcinoma Anaplásico de Tiroides , Línea Celular Tumoral , Humanos , Transducción de Señal , Carcinoma Anaplásico de Tiroides/tratamiento farmacológico , Carcinoma Anaplásico de Tiroides/metabolismo , Carcinoma Anaplásico de Tiroides/patología , Neoplasias de la Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patologíaRESUMEN
Anaplastic thyroid cancer (ATC) is a rare type of thyroid cancer (TC) with no effective therapeutic strategy. Although surgery, chemotherapy and radiation are all available for ATC treatment, the median survival for ATC patients is less than 6 months. In this study, we aimed to study on resistant mechanisms to B-Raf proto-oncogene serine/threonine kinase (BRAF) inhibitor and identify effective combinational therapy for ATC patients. TC cells were treated with Vemurafenib and cell apoptosis and viability were analyzed by flow cytometry and MTT assay. Monolayer and sphere cells were isolated from ATC cells to detect the mRNA level of stem cell markers and differentiation markers by RT-PCR. Phosphor-STAT3 level in sphere and monolayer cells was tested by Western blotting. The xenotransplantation animal model has established to analyze the anti-tumor effect of Vemurafenib and Stattic combinational therapy. Undifferentiated TC cells were resistant to Vemurafenib treatment. Sphere cells isolated from ATC showed no significant change in cell viability and apoptosis upon Vemurafenib treatment, and expressed a high level of stem cell marker and phosphor-STAT3. STAT3 inhibition enhanced the tumorigenic capacity and increased Vemurafenib sensitivity in ATC cell lines. Stattic significantly enhanced anti-tumor effect of Vemurafenib in mouse model. Our findings demonstrate that the combinational therapy of Vemurafenib and Stattic is an effective therapeutic treatment for ATC patients.
Asunto(s)
Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Óxidos S-Cíclicos/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Factor de Transcripción STAT3/antagonistas & inhibidores , Carcinoma Anaplásico de Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/tratamiento farmacológico , Vemurafenib/uso terapéutico , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Óxidos S-Cíclicos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Humanos , Ratones Desnudos , Factor de Transcripción STAT3/metabolismo , Carcinoma Anaplásico de Tiroides/metabolismo , Carcinoma Anaplásico de Tiroides/patología , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patología , Carga Tumoral/efectos de los fármacos , Vemurafenib/farmacologíaRESUMEN
BACKGROUND: Survival rate of patients affected with anaplastic thyroid carcinoma (ATC) is less than 5% with current treatment. In ATC, BRAFV600E mutation is the major mutation that results in the transformation of normal cells in to an undifferentiated cancer cells via aberrant molecular signaling mechanisms. Although vemurufenib is a selective oral drug for the BRAFV600E mutant kinase with a response rate of nearly 50% in metastatic melanoma, our study has showed resistance to this drug in ATC. Hence the rationale of the study is to explore combinational therapeutic effect to improve the efficacy of vemurafenib along with metformin. Metformin, a diabetic drug is an AMPK activator and has recently proved to be involved in preventing or treating several types of cancer. METHODS AND RESULTS: Using iGEMDock software, a protein-ligand interaction was successful between Metformin and TSHR (receptor present in the thyroid follicular cells). Our study demonstrates that combination of vemurufenib with metformin has synergistic anti-cancer effects which was evaluated through MTT assay (cytotoxicity), colony formation assay (antiproliferation evaluation) and suppressed the progression of ATC cells growth by inducing significant apoptosis, proven by Annexin V-FITC assay (Early Apoptosis Detection). Downregulation of ERK signaling, upregulation of AMPK pathway and precision in epithelial-mesenchymal transition (EMT) pathway which were assessed by RT-PCR and Western blot provide the evidence that the combination of drugs involved in the precision of altered molecular signaling Further our results suggest that Metformin act as a demethylating agent in anaplastic thyroid cancer cells by inducing the expression of NIS and TSHR. Our study for the first time explored cAMP signaling in ATC wherein cAMP signaling is downregulated due to decrease in intracellular cAMP level upon metformin treatment. CONCLUSION: To conclude, our findings demonstrate novel therapeutic targets and treatment strategies for undifferentiated ATC.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica , Proteínas de Neoplasias , Receptores de Tirotropina , Carcinoma Anaplásico de Tiroides , Neoplasias de la Tiroides , Protocolos de Quimioterapia Combinada Antineoplásica/química , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Línea Celular Tumoral , Sinergismo Farmacológico , Humanos , Metformina/química , Metformina/farmacología , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Receptores de Tirotropina/química , Receptores de Tirotropina/metabolismo , Carcinoma Anaplásico de Tiroides/química , Carcinoma Anaplásico de Tiroides/tratamiento farmacológico , Carcinoma Anaplásico de Tiroides/metabolismo , Neoplasias de la Tiroides/química , Neoplasias de la Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/metabolismo , Vemurafenib/química , Vemurafenib/farmacologíaRESUMEN
Thyroid cancer is the most common endocrine malignancy, and the characterization of the genetic alterations in coding-genes that drive thyroid cancer are well consolidated in MAPK signaling. In the context of non-coding RNAs, microRNAs (miRNAs) are small non-coding RNAs that, when deregulated, cooperate to promote tumorigenesis by targeting mRNAs, many of which are proto-oncogenes and tumor suppressors. In thyroid cancer, miR-146b-5p is the most overexpressed miRNA associated with tumor aggressiveness and progression, while the antisense blocking of miR-146b-5p results in anti-tumoral effect. Therefore, inactivating miR-146b has been considered as a promising strategy in thyroid cancer therapy. Here, we applied the CRISPR/Cas9n editing system to target the MIR146B gene in an aggressive anaplastic thyroid cancer (ATC) cell line. For that, we designed two single-guide RNAs cloned into plasmids to direct Cas9 nickase (Cas9n) to the genomic region of the pre-mir-146b structure to target miR-146b-5p and miR-146b-3p sequences. In this plasmidial strategy, we cotransfected pSp-Cas9n-miR-146b-GuideA-puromycin and pSp-Cas9n-miR-146b-GuideB-GFP plasmids in KTC2 cells and selected the puromycin resistant + GFP positive clones (KTC2-Cl). As a result, we observed that the ATC cell line KTC2-Cl1 showed a 60% decrease in the expression of miR-146b-5p compared to the control, also showing reduced cell viability, migration, colony formation, and blockage of tumor development in immunocompromised mice. The analysis of the MIR146B edited sequence shows a 5 nt deletion in the miR-146b-5p region and a 1 nt deletion in the miR-146b-3p region in KTC2-Cl1. Thus, we developed an effective CRISPR/Cas9n system to edit the MIR146B miRNA gene and reduce miR-146b-5p expression which constitutes a potential molecular tool for the investigation of miRNAs function in thyroid cancer.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Marcación de Gen , MicroARNs , ARN Neoplásico , Carcinoma Anaplásico de Tiroides , Neoplasias de la Tiroides , Animales , Línea Celular , Movimiento Celular/genética , Supervivencia Celular/genética , Xenoinjertos , Humanos , Ratones , MicroARNs/biosíntesis , MicroARNs/genética , Trasplante de Neoplasias , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Carcinoma Anaplásico de Tiroides/genética , Carcinoma Anaplásico de Tiroides/metabolismo , Carcinoma Anaplásico de Tiroides/patología , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patologíaRESUMEN
Anaplastic thyroid cancer (ATC) is one of the most lethal malignancies with a median survival time of about 4 months. Currently, there is no effective treatment, and the development of new therapies is an important and urgent issue for ATC patients. YM155 is a small molecule that was identified as the top candidate in a high-throughput screen of small molecule inhibitors performed against a panel of ATC cell lines by the National Cancer Institute. However, there were no follow-up studies investigating YM155 in ATC. Here, we determined the effects of YM155 on ATC and human primary benign thyroid cell (PBTC) survival with alamarBlue assay. Our data show that YM155 inhibited proliferation of ATC cell lines while sparing normal thyroid cells, suggesting a high therapeutic window. YM155-induced DNA damage was detected by measuring phosphorylation of γ-H2AX as a marker for DNA double-strand breaks. The formamidopyrimidine-DNA glycosylase (FPG)-modified alkaline comet assay in conjunction with reactive oxygen species (ROS) assay and glutathione (GSH)/glutathione (GSSG) assay suggests that YM155-mediated oxidative stress contributes to DNA damage. In addition, we provide evidence that YM155 causes cell cycle arrest in S phase and in the G2/M transition and causes apoptosis, as seen with flow cytometry. In this study, we show for the first time the multiple effects of YM155 in ATC cells, furthering a potential therapeutic approach for ATC.
Asunto(s)
Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Imidazoles/farmacología , Naftoquinonas/farmacología , Estrés Oxidativo/efectos de los fármacos , Carcinoma Anaplásico de Tiroides/metabolismo , Neoplasias de la Tiroides/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Carcinoma Anaplásico de Tiroides/patología , Glándula Tiroides/citología , Glándula Tiroides/efectos de los fármacos , Neoplasias de la Tiroides/patologíaRESUMEN
Stem cells that express therapeutic proteins have been identified to have an anticancer effects on various types of cancer. In the present study study, human neural stem cells (hNSCs) that were genetically engineered to express cytosine deaminase (CD) and human interferon-ß (IFN-ß) were used for anaplastic thyroid cancer (ATC) treatment owing to their tumor-tropic properties and therapeutic effects. CD is an enzyme that converts 5-fluorocytosine (5-FC), a prodrug, to 5-fluorouracil (5-FU) which is a medication to suppress tumor growth through DNA synthesis inhibition. Also, IFN-ß suppresses tumor growth by the induction of apoptotic process. In water soluble tetrazolium salt (WST) assay, SNU-80 cells which are human female ATC cells were cocultured with three cell types including engineered hNSCs such as HB1.F3, HB1.F3.CD, and HB1.F3.CD.IFN-ß cells on transwells and treated with 5-FC for 72 hours. Finally, the SNU-80 cell viability was reduced by the coculture with HB1.F3.CD and HB1.F3.CD.IFN-ß cells. In dichlorofluorescein diacetate (DCF-DA) and TdT-mediated dUTP nick-end labeling (TUNEL) assays, the production of reactive oxygen species (ROS) and the number of apoptotic cells were increased by HB1.F3.CD and HB1.F3.CD.IFN-ß cells in the presence of 5-FC. In Western blot assay, ROS, and apoptosis-related genes were increased in SNU-80 cells when they were cocultured with HB1.F3.CD and HB1.F3.CD.IFN-ß cells. In transwell migration assay, hNSCs selectively migrated to SNU-80 cells because hNSCs interacted with chemoattractant factors like SDF-1α, uPAR, and CCR2 secreted by SNU-80 cells. Taken together, engineered hNSCs were revealed to selectively migrate to ATC cells and to inhibit growth as well as to induce apoptosis of ATC cells via ROS production through the actions of transgenes such as CD and IFN-ß. Therefore, these engineered hNSCs can be promising candidates for the treatment of metastatic ATC.
Asunto(s)
Citosina Desaminasa/metabolismo , Flucitosina , Expresión Génica , Células-Madre Neurales/enzimología , Carcinoma Anaplásico de Tiroides , Línea Celular Tumoral , Técnicas de Cocultivo , Citosina Desaminasa/genética , Flucitosina/farmacocinética , Flucitosina/farmacología , Humanos , Profármacos/farmacocinética , Profármacos/farmacología , Carcinoma Anaplásico de Tiroides/metabolismo , Carcinoma Anaplásico de Tiroides/patología , Carcinoma Anaplásico de Tiroides/terapia , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patología , Neoplasias de la Tiroides/terapiaRESUMEN
Anaplastic thyroid cancer (ATC) is a rare neoplasia with a poor prognosis. Proliferation and apoptosis assays were performed on ATC cell lines (8305C, 8505C) exposed to vinorelbine, lenvatinib, as well as to concomitant combinations. ABCB1, ABCG2 and CSF-1 mRNA expression was evaluated by real time PCR. The relative levels of pospho Akt were investigated as part of a human phospho-kinase array analysis, and CSF-1 and VEGFR-2 protein levels were measured by ELISA. The intracellular concentration of lenvatinib in ATC cells was measured by combined reversed-phase liquid chromatography-tandem mass spectrometry. An ATC subcutaneous xenograft tumor model in nude mice was treated with vinorelbine, lenvatinib, or vinorelbine plus lenvatinib. After treatment with vinorelbine, lenvatinib, a significant antiproliferative effect in ATC cell lines was observed. The concomitant treatment of vinorelbine and lenvatinib revealed synergism for all the fractions of affected cells. A decrease in ABCB1 expression was reported in both ATC cell lines treated with the lenvatinib plus vinorelbine combination, as was an increase in the intracellular concentration of lenvatinib. The combination caused a decrease in Akt, GSK3α/ß, PRAS40 and Src phosphorylation, and in both CSF-1 mRNA and protein levels. In the subcutaneous tumor model, the combination reduced the tumor volume during the treatment period. Our results establish the synergistic ATC antitumor activity of a vinorelbine and lenvatinib combination.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Compuestos de Fenilurea/administración & dosificación , Quinolinas/administración & dosificación , Carcinoma Anaplásico de Tiroides/tratamiento farmacológico , Neoplasias de la Tiroides/tratamiento farmacológico , Vinorelbina/administración & dosificación , Animales , Antineoplásicos Fitogénicos/administración & dosificación , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Relación Dosis-Respuesta a Droga , Humanos , Masculino , Ratones , Ratones Desnudos , Ratones Transgénicos , Carcinoma Anaplásico de Tiroides/metabolismo , Carcinoma Anaplásico de Tiroides/patología , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patologíaRESUMEN
Anaplastic cancer constitutes 1% of thyroid cancers, and it is one of the most aggressive cancers. Treatment options are external radiation therapy and/or chemotherapy. The success rate with these treatment modalities is not satisfactory. We aimed to evaluate the effects of metformin (MET) and pioglitazone (PIO) combination on apoptosis and AMP-activated protein kinase/mammalian target of rapamycin (mTOR) signaling pathway in human anaplastic thyroid cancer cells. In this study, we evaluated the effects of MET and PIO individually and the combination of the two drugs on the cellular lines SW1736 and C643 ATC. Genes contained in the mTOR signaling pathway were examined using human mTOR Signalization RT2 Profiler PCR Array. In C643 and SW1736 cell lines, IC50 doses of MET and PIO were found out as 17.69 mM, 11.64 mM, 27.12 µM, and 23.17 µM. Also, the combination of MET and PIO was determined as an additive according to isobologram analyses. We have found the downregulation of the expression levels of oncogenic genes: AKT3, CHUK, CDC42, EIF4E, HIF1A, IKBKB, ILK, MTOR, PIK3CA, PIK3CG, PLD1, PRKCA, and RICTOR genes, in the MET and PIO combination-treated cells. In addition, expression levels of tumor suppressor genes, DDIT4, DDIT4L, EIF4EBP1, EIF4EBP2, FKBP1A, FKBP8, GSK3B, MYO1C, PTEN, ULK1, and ULK2, were found to have increased significantly. The MET + PIO combination was first applied to thyroid cancer cells, and significant reductions in the level of oncogenic genes were detected. The decreases, particularly, in AKT3, DEPTOR, EIF4E, ILK, MTOR, PIK3C, and PRKCA expressions indicate that progression can be prevented in thyroid cancer cells and these genes could be selected as therapeutic targets.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Apoptosis/efectos de los fármacos , Metformina/farmacología , Pioglitazona/farmacología , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Carcinoma Anaplásico de Tiroides/metabolismo , Neoplasias de la Tiroides/metabolismo , Línea Celular Tumoral , Quimioterapia Combinada , Expresión Génica/efectos de los fármacos , Humanos , Metformina/administración & dosificación , Pioglitazona/administración & dosificación , Carcinoma Anaplásico de Tiroides/patología , Neoplasias de la Tiroides/patologíaRESUMEN
We aimed to investigate the effects of Krüppel-like factor 5 (KLF5) on cell biological function and chemotherapy sensitivity of anaplastic thyroid carcinoma (ATC) and explore the underlying mechanism. In this study, we found that KLF5 was expressed higher in ATC cells than that in normal thyroid cells. Knockdown of KLF5 inhibited proliferation, induced apoptosis and restrained invasion and migration abilities of ATC cells. KLF5 overexpression promoted proliferation and inhibited apoptosis of ATC cells in response to doxorubicin (Dox), whereas KLF5 knockdown increased the sensitivity of ATC cells to Dox. Multidrug resistance gene 1/permeability glycoprotein and ATP-binding cassette superfamily G member 2 were heightened in ATC cells with KLF5 overexpression, but the opposite results were found in sh-KLF5-treated cells. Phosphorylation (p)-c-Jun N-terminal kinase (JNK) was upregulated in KLF5 overexpression cells, whereas it was downregulated in the KLF5 knockdown treatment group. Furthermore, KLF5 knockdown inhibited ATC growth and enhanced the Dox sensitivity of ATC by inactivating the JNK signaling pathway. Taken together, our findings concluded that KLF5 knockdown can remarkably inhibit the proliferation, invasion, and migration and induce apoptosis of ATC cells, and increase the chemotherapy sensitivity of ATC, all of which probably through inhibiting the JNK signaling pathway.