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1.
Life (Basel) ; 13(6)2023 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-37374179

RESUMO

Radiotherapy and immunotherapy have shown promising efficacy for the treatment of solid malignancies. Here, we aim to clarify the potential of a combined application of radiotherapy and programmed cell death-ligand 1 (PD-L1) monoclonal antibody atezolizumab in primary anaplastic thyroid cancer (ATC) cells. The radiation caused a significant reduction in cell proliferation, measured by luminescence, and of the number of colonies. The addition of atezolizumab caused a further reduction in cell proliferation of the irradiated ATC cells. However, the combined treatment did not cause either the exposure of the phosphatidylserine or the necrosis, assessed by luminescence/fluorescence. Additionally, a reduction in both uncleaved and cleaved forms of caspases 8 and 3 proteins was detectable in radiated cells. The DNA damage evidenced the over-expression of TP53, CDKN1A and CDKN1B transcripts detected by RT-qPCR and the increase in the protein level of P-γH2AX and the DNA repair deputed kinases. PD-L1 protein level increased in ATC cells after radiation. Radiotherapy caused the reduction in cell viability and an increase of PD-L1-expression, but not apoptotic cell death in ATC cells. The further combination with the immunotherapeutic atezolizumab could increase the efficacy of radiotherapy in terms of reduction in cell proliferation. Further analysis of the involvement of alternative cell death mechanisms is necessary to clarify their cell demise mechanism of action. Their efficacy represents a promising therapy for patients affected by ATC.

2.
Cancer Invest ; 41(4): 405-421, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-36811581

RESUMO

Anaplastic thyroid carcinoma (ATC) has poor prognosis, high mortality rate and lack of effective therapy. A synergic combination of PD-L1 antibody together with cell death promoting substances like deacetylase inhibitors (DACi) and multi-kinase inhibitors (MKI) could sensitize ATC cells and promote decay by autophagic cell death. The PD-L1-inhibitor atezolizumab synergized with panobinostat (DACi) and sorafenib (MKI) leading to significant reduction of the viability, measured by real time luminescence, of three different patient-derived primary ATC cells, of C643 cells and follicular epithelial thyroid cells too. Solo administration of these compounds caused a significant over-expression of autophagy transcripts; meanwhile autophagy proteins were almost not detectable after the single administration of panobinostat, thus supporting a massive autophagy degradation process. Instead, the administration of atezolizumab caused an accumulation of autophagy proteins and the cleavage of the active caspases 8 and 3. Interestingly, only panobinostat and atezolizumab were able to exacerbate the autophagy process by increasing the synthesis, the maturation and final fusion with the lysosomes of the autophagosome vesicles. Despite ATC cells could be sensitized by atezolizumab via the cleavage of the caspases, no reduction of cell proliferation or promotion of cell death was observed. The apoptosis assay evidenced the ability of panobinostat alone and in combination with atezolizumab to induce the phosphatidil serine exposure (early apoptosis) and further the secondary necrosis. Instead, sorafenib was only able to cause necrosis. The increase of caspases activity induced by atezolizumab, the apoptosis and autophagy processes promoted by panobinostat synergize thus promoting cell death in well-established and primary anaplastic thyroid cancer cells. The combined therapy could represent a future clinical application for the treatment of such lethal and untreatable solid cancer.


Assuntos
Autofagia , Panobinostat , Sorafenibe , Carcinoma Anaplásico da Tireoide , Neoplasias da Glândula Tireoide , Carcinoma Anaplásico da Tireoide/tratamento farmacológico , Carcinoma Anaplásico da Tireoide/patologia , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/patologia , Panobinostat/farmacologia , Sorafenibe/farmacologia , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Humanos , Masculino , Idoso , Idoso de 80 Anos ou mais , Morte Celular , Esferoides Celulares
3.
Cancer Invest ; 40(3): 254-267, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34726962

RESUMO

Adrenocortical carcinoma (ACC) is characterized by poor prognosis and high mortality. The suppression of the long-non-coding RNA H19, counterbalanced by IGF2 over-expression, leads to down-regulation of the autophagy markers, high proliferation rate and metastatic potential in patients affected by ACC. The administration of the deacetylase inhibitors (DACi) panobinostat, trichostatin A (TSA) and SAHA affected the cell viability of H295R monolayer and spheroids and induced the over-expression of H19 and autophagy transcripts. H19 knock down in H295R cells was not able to modulate the expression level of autophagy transcripts. Instead, H19 knock down was able to impede the ability of DACi to modulate the protein level of the autophagy markers. Furthermore, the administration of higher concentration of DACi was able to down-regulate the protein level of Beclin1 and p62 and to induce the conversion of LC3B-I into the active LC3B-II form, thus confirming an active autophagic process. Neither the active protein level nor the activity of caspases 8 and 3 was prompted by the DACi, thus excluding the involvement of the executioners of apoptosis in H295R decay. The DACi restore H19, the autophagy markers and trigger cell death in ACC cells. The re-activation of autophagy would represent a novel strategy for the treatment of patients affected by this severe malignancy.


Assuntos
Neoplasias do Córtex Suprarrenal/genética , Carcinoma Adrenocortical/genética , Autofagia/fisiologia , RNA Longo não Codificante/fisiologia , Adolescente , Neoplasias do Córtex Suprarrenal/tratamento farmacológico , Neoplasias do Córtex Suprarrenal/patologia , Carcinoma Adrenocortical/tratamento farmacológico , Carcinoma Adrenocortical/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/análise , Proteína Beclina-1/análise , Linhagem Celular Tumoral , Feminino , Inibidores de Histona Desacetilases/uso terapêutico , Humanos , Masculino , Pessoa de Meia-Idade , Panobinostat/uso terapêutico , RNA Longo não Codificante/análise , Adulto Jovem
4.
Oncotarget ; 9(68): 32997-33010, 2018 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-30250645

RESUMO

Osteosarcoma is an aggressive cancer with a poor long term prognosis. Neo-adjuvant poly-chemotherapy followed by surgical resection remains the standard treatment, which is restricted by multi-drug resistance. If first-line therapy fails, disease control and patient survival rate drop dramatically. We aimed to identify alternative apoptotic mechanisms induced by the histone deacetylase inhibitor panobinostat in osteosarcoma cells. Saos-2, MG63 and U2-OS osteosarcoma cell lines, the immortalized human osteoblast line hFOB and the mouse embryo osteoblasts (MC3T3-E1) were treated with panobinostat. Real time viability and FACS confirmed the cytotoxicity of panobinostat. Cell stress/death related factors were analysed by RT-qPCR and western blot. Cell morphology was assessed by electron microscopy. 10 nM panobinostat caused cell viability arrest and death in all osteosarcoma and osteoblast cells. P21 up-regulation was observed in osteosarcoma cells, while over-expression of p73 was restricted to Saos-2 (TP53-/-). Survivin and Bcl-2 were suppressed by panobinostat. Endoplasmic reticulum (ER) stress markers BiP, CHOP, ATF4 and ATF6 were induced in osteosarcoma cells. The un-spliced Xbp was no further detectable after treatment. Autophagy players Beclin1, Map1LC3B and UVRAG transcripts over-expressed after 6 hours. Protein levels of Beclin1, Map1LC3B and p62 were up-regulated at 72 hours. DRAM1 was stable. Electron micrographs revealed the fragmentation and the disappearance of the ER and the statistically significant increase of autophagosome vesiculation after treatment. Panobinostat showed a synergistic suppression of survival and promotion of cell death in osteosarcoma cells. Panobinostat offers new perspectives for the treatment of osteosarcoma and other malignant bone tumours.

5.
Neuroendocrinology ; 107(3): 257-266, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30025403

RESUMO

Pancreatic neuroendocrine neoplasias (pNEN) are the most common cause of death in adult patients with multiple endocrine neoplasia type 1 (MEN1). So far, only few chemopreventive strategies (e.g., with somatostatin analogues) have been evaluated for MEN1 associated pNENs. In this experimental study on 75 Men1(+/T) knockout mice, the effect of aspirin (n = 25) and an inhibitor of angiotensin-I converting enzyme (enalapril, n = 25) compared to controls (n = 25) were evaluated as single chemopreventive strategies for pNENs after 6, 9, 12, 15, and 18 months. After each study period, mice were sacrificed and the resected pancreata were evaluated by histopathological analysis, immunostaining, and real-time PCR. PNEN size and number was measured. Aspirin and enalapril lead to a pNEN size reduction of 80% (167,518 vs. 838,876 µm2, p < 0.001) and 79% (174,758 vs. 838,876 µm2, p < 0.001) compared to controls. Furthermore, aspirin and enalapril treatment resulted in a significant reduction of the number of pNENs by 33%, (p = 0.04) and 41% (p = 0.002) respectively. The apoptosis marker caspase 3 revealed a higher positive expression in pNEN of treated Men1(+/T) mice. Immunostaining of VEGF in pNEN detected a downregulation of its expression in treated Men1(+/T) mice compared to the control group. REL A transcript was significantly downregulated in 18-months treated enalapril Men1(+/T) mice, but not in aspirin-treated Men1(+/T) mice. There was no significant difference in the Ki-67 index. Using a transgenic mouse model that imitates human MEN1, this study provides first evidence that aspirin and enalapril are effective chemopreventive agents that aid in the progression of pNENs.


Assuntos
Aspirina/uso terapêutico , Quimioprevenção/métodos , Enalapril/uso terapêutico , Neoplasia Endócrina Múltipla Tipo 1/patologia , Tumores Neuroendócrinos/prevenção & controle , Neoplasias Pancreáticas/prevenção & controle , Proteínas Proto-Oncogênicas/genética , Animais , Camundongos , Camundongos Knockout , Neoplasia Endócrina Múltipla Tipo 1/genética , Tumores Neuroendócrinos/genética , Tumores Neuroendócrinos/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia
6.
Int J Mol Sci ; 19(7)2018 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-30018229

RESUMO

BACKGROUND: The MEK (mitogen-activated protein kinase)⁻inhibitor selumetinib led to increased radioiodine uptake and retention in a subgroup of patients suffering from radioiodine refractory differentiated thyroid cancer (RR-DTC). We aimed to analyse the effect of selumetinib on the expression of sodium iodide symporter (NIS; SLC5A5) and associated miRNAs in thyroid cancer cells. METHODS: Cytotoxicity was assessed by viability assay in TPC1, BCPAP, C643 and 8505C thyroid cancer cell lines. NIS, hsa-let-7f-5p, hsa-miR-146b-5p, and hsa-miR-146b-3p expression was determined by quantitative RT-PCR. NIS protein was detected by Western blot. Radioiodine uptake was performed with a Gamma counter. RESULTS: Selumetinib caused a significant reduction of cell viability in all thyroid cancer cell lines. NIS transcript was restored by selumetinib in all cell lines. Its protein level was found up-regulated in TPC1 and BCPAP cells and down-regulated in C643 and 8505C cells after treatment with selumetinib. Treatment with selumetinib caused a down-regulation of hsa-let-7f-5p, hsa-miR-146b-5p and hsa-miR-146b-3p in TPC1 and BCPAP cells. In 8505C cells, a stable or down-regulated hsa-miR-146b-5p was detected after 1h and 48h of treatment. C643 cells showed stable or up-regulated hsa-let-7f-5p, hsa-miR-146b-5p and hsa-miR-146b-3p. Selumetinib treatment caused an increase of radioiodine uptake, which was significant in TPC1 cells. CONCLUSIONS: The study shows for the first time that selumetinib restores NIS by the inhibition of its related targeting miRNAs. Further studies are needed to clarify the exact mechanism activated by hsa-miR-146b-5p, hsa-miR-146b-3p and hsa-let7f-5p to stabilise NIS. Restoration of NIS could represent a milestone for the treatment of advanced RR-DTC.


Assuntos
Benzimidazóis/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , MicroRNAs/genética , Simportadores/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Relação Dose-Resposta a Droga , Humanos , Radioisótopos do Iodo/farmacocinética , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Simportadores/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia
7.
J Clin Med ; 7(5)2018 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-29762469

RESUMO

The prognosis of anaplastic (ATC) and poorly differentiated thyroid cancer (PDTC) is poor, due to their radioiodine refractoriness (RAI-R), high metastatic potential and current lack of effective treatment strategies. We aimed to examine the efficacy of the tyrosine kinase inhibitors (TKIs) sorafenib and selumetinib and the histone deacetylase inhibitor (HDACI) panobinostat in patient-derived tumor tissue (PDTT) of ATCs/PDTCs, the expression of sodium iodide symporter (NIS) and radioiodine up-take (RAI-U). High Mobility Group AT-Hook 2 (HMGA2) and associated miRNAs expression was correlated with the clinical course of the patients. Inhibitory effects of panobinostat, sorafenib and selumetinib were measured by real time cell analyser xCELLigence in five PDTTs and human foreskin fibroblasts (HF) used as control. Expression of NIS, HMGA2 and associated miRNAs hsa-let-7f-5p, hsa-let-7b-5p, hsa-miR-146b-5p and hsa-miR-146b-3p was performed by RT-qPCR and Western blot. RAI-U was performed by Gamma Counter with I-131. Panobinostat showed the strongest cytotoxic effect (10 nM) in all PDTTs and HF and caused a significant over-expression of NIS transcript. TKIs were able to up-regulate NIS transcript in patient 5 and in HF. RAI-U was up-regulated after 24 h of treatment with TKIs and panobinostat in all PDTT and HF, except in patient 5. Selumetinib caused a significant suppression of HMGA2 in PDTT 1, 2, 4, 5 and HF; whereas sorafenib caused no change of HMGA2 expression. Panobinostat suppressed significantly HMGA2 in PDTT 2, 4 and HF. The expression of miRNAs hsa-let-7f-5p, has-let-7b-5p hsa-miR-146b-5p and hsa-miR-146b-3p was modulated heterogeneously. NIS protein level was over-expressed in three PDTTs (patients 1, 3 and 4) after 24 h of treatment with selumetinib, sorafenib and in particular with panobinostat. HF showed a stable NIS protein level after treatment. Panobinostat showed the strongest cytotoxicity in all treated PDTTs at the lowest dosage in comparison with TKI. All three compounds were able to modulate differently NIS, HMGA2 and related miRNAs. These factors represent valuable markers in PDTT for new treatment strategies for patients suffering from ATC/PDTC. Thus, the establishment of PDTT could be a useful tool to test the efficacy of compounds and to develop new and individualised multimodal treatment options for PDTCs and ATCs.

8.
J Clin Med ; 7(4)2018 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-29561759

RESUMO

Epigenetic modifications have been identified as being responsible for the de-differentiation of thyroid tissue and its malignant transformation. Cell proliferation inhibitory effects of the pan-deacetylase inhibitors panobinostat, SAHA and Trichostatin A (TSA), the modulation of the sodium iodide symporter (NIS; SLC5A5), thyroid transcription factor 1 (TTF1), high mobility group A2 (HMGA2), and H19 and their putative targeting miRNAs have been evaluated in vitro. The cell viability was measured in five thyroid cancer cell lines (FTC133, TPC1, BCPAP, 8505C, C643) by real time cell analyzer xCELLigence. Expression of the above mentioned markers was performed by RT-qPCR and Western Blot. Radioiodine up-take was detected by Gamma Counter with I131. Cell viability decreased after treatment in all five cell lines. 10 nM panobinostat; 1 µM TSA or 10 µM SAHA caused a significant over-expression of NIS transcript in all five cell lines, whereas NIS protein was up-regulated in FTC133, BCPAP, and C643 cell lines only. Radioiodine up-take increased in FTC133 and C643 cells after 48 h of treatment with 10 nM panobinostat and 1 µM TSA. A significant down-regulation of the oncogene HMGA2 was detected in all five cell lines; except for TPC1 cells that were treated with 1 µM TSA. In accordance, hsa-let-7b-5p and hsa-let-7f-5p were stable or significantly over-expressed in all of the cell lines, except for TPC1 cells that were treated with 10 µM SAHA. TTF1 was significantly down-regulated in FTC133, BCPAP, and 8505C cells; whereas, TPC1 and C643 showed an up-regulated or stable expression. TTF1 was over-expressed in samples of human anaplastic thyroid cancer; whereas, it was down-regulated in follicular and undetectable in papillary thyroid cancer. H19 was over-expressed after 48 h treatment, except for BCPAP cells that were treated with panobinostat and SAHA. H19 was differently expressed in human anaplastic, follicular and papillary thyroid tumor samples. Deacetylase inhibitors reduced cell viability, restored NIS and H19, and suppressed the oncogenes HMGA2 and TTF1 in thyroid cancer cells.

9.
Tumour Biol ; 39(10): 1010428317728417, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29017393

RESUMO

Typical and atypical carcinoid tumors belong to the neuroendocrine lung tumors. They have low recurrence and proliferation rate, lymph node, and distant metastases. Nevertheless, these tumors have shown a more aggressive behavior. In the last years, microRNAs were screened as new tumor markers for their potential diagnostic and therapeutic relevance. The expression of hsa-let-7b-5p, hsa-let-7f-5p, hsa-miR-222-3p, and their targets HMGA2 (high-mobility group A2) and CDKN1B (cyclin-dependent kynase inhibitor 1B, p27kip1) was evaluated in this rare small group of patients. We analyzed the clinical data of all typical and atypical carcinoid tumors of patients who underwent surgical operation at Marburg University Hospital (n = 18) from 2000. Quantitative reverse transcription polymerase chain reaction was performed in formalin-fixed paraffin-embedded tumor tissue versus four tumor-free lung tissue samples. HMGA2 was stable or downregulated; only one patient showed a significant overexpression. CDKN1B showed a significant overexpression or a stable level; it was downregulated in two samples only. Hsa-miR-222-3p resulted almost stable or overexpressed except for two samples (significantly downregulated). Hsa-let-7f-5p was stable or overexpressed in the majority of analyzed samples, whereas hsa-let-7b-5p was significantly downregulated. HMGA2 and CDKN1B are differently expressed between atypical and typical carcinoid tumors, thus representing valid biomarkers for the classification of the two tumor groups. Hsa-let-7f-5p and HMGA2 are inversely correlated. Hsa-miR-222-3p does not correlate with its predicted target CDKN1B.


Assuntos
Biomarcadores Tumorais/análise , Tumor Carcinoide/classificação , Tumor Carcinoide/patologia , Neoplasias Pulmonares/classificação , Neoplasias Pulmonares/patologia , Adulto , Idoso , Inibidor de Quinase Dependente de Ciclina p27/análise , Inibidor de Quinase Dependente de Ciclina p27/biossíntese , Feminino , Proteína HMGA2/análise , Proteína HMGA2/biossíntese , Humanos , Masculino , MicroRNAs/análise , MicroRNAs/biossíntese , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase
10.
J Cancer Res Clin Oncol ; 142(6): 1213-20, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26960757

RESUMO

PURPOSE: Thyroid cancer (TC), the most common endocrine malignancy, increases its incidence worldwide. MicroRNAs have been shown to be abnormally expressed in tumors and could represent valid diagnostic markers for patients affected by TC. Our aim was to analyze the expression of tumorsuppressor hsa-let7b-5p and hsa-let7f-5p, together with their predicted targets SLC5A5 (NIS) and HMGA2, in papillary (PTC), follicular (FTC) and anaplastic (ATC). METHODS: 8 FTC, 14 PTC, 12 ATC and three normal thyroid tissue samples were analyzed for the expression of pre-let7b, hsa-let7b-5p and hsa-let7f-5p as SLC5A5 and HMGA2 by RT-qPCR. Data were analyzed by REST 2008. RESULTS: FTC patients showed a significant down-regulation of hsa-let7b-5p and its precursor. hsa-let7f-5p was overexpressed, and SLC5A5 was strongly suppressed. HMGA2 was overexpressed, reflecting no correlation with its regulatory let7 miRNAs. PTC samples were characterized by up-regulation of hsa-let7b-5p, its precursor and hsa-let7f-5p. SLC5A5 was strongly suppressed in comparison with normal thyroid tissue. HMGA2 was overexpressed, as shown in FTC, also. ATC samples showed a similar miRNAs profile as PTC. In contrast with FTC and PTC, these patients showed a stable or up-regulated SLC5A5 and HMGA2. CONCLUSIONS: Expression of HMGA2 is not correlated with the regulatory let7 miRNAs. Interestingly, SLC5A5 was down-regulated in FTC and PTC. Its expression could be modulated by hsa-let-7f-5p. ATC showed a loss of SLC5A5/hsa-let7f-5p correlation. SLC5A5, in ATC, needs further investigation to clarify the genetic/epigenetic mechanism altering its expression.


Assuntos
Proteína HMGA2/metabolismo , MicroRNAs/genética , Simportadores/metabolismo , Neoplasias da Glândula Tireoide/genética , Humanos , Neoplasias da Glândula Tireoide/metabolismo
11.
Oncotarget ; 7(15): 20312-23, 2016 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-26967385

RESUMO

HBV represents the most common chronic viral infection and major cause of hepatocellular carcinoma (HCC), although its exact role in liver tumorigenesis is unclear. Massive storage of the small (SHBs), middle (MHBs) and large surface (LHBs) HBV envelope proteins leads to cell stress and sustained inflammatory responses. Cannabinoid (CB) system is involved in the pathogenesis of liver diseases, stimulating acute and chronic inflammation, liver damage and fibrogenesis; it triggers endoplasmic reticulum (ER) stress response. The aim of our work was to investigate the activation of ER stress pathway after ectopic HBV envelope proteins expression, in liver cancer cells, and the role exerted by CB receptors. PCR, immunofluorescence and western blotting showed that exogenous LHBs and MHBs induce a clear ER stress response in Huh-7 cells expressing CB1 receptor. Up-regulation of the chaperone BiP/GRP78 (Binding Immunoglobulin Protein/Glucose-Regulated Protein 78) and of the transcription factor CHOP/GADD153 (C/EBP Homologous Protein/Growth Arrest and DNA Damage inducible gene 153), phosphorylation of PERK (PKR-like ER Kinase) and eIF2α (Eukaryotic Initiation Factor 2α) and splicing of XBP1 (X-box binding protein 1) was observed. CB1-/- HepG2 cells did not show any ER stress activation. Inhibition of CB1 receptor counteracted BiP expression in transfected Huh-7 and in HBV+ PLC/PRF/5 cells; whereas no effect was observed in HBV- HLF cells. These results suggest that HBV envelope proteins are able to induce the ER stress pathway. CB1 expression is directly correlated with ER stress function. Further investigations are needed to clarify the involvement of cannabinoid in HCC progression after HBV infection.


Assuntos
Carcinoma Hepatocelular/patologia , Estresse do Retículo Endoplasmático , Neoplasias Hepáticas/patologia , Receptor CB1 de Canabinoide/metabolismo , Proteínas do Envelope Viral/metabolismo , Apoptose , Canabinoides/farmacologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/virologia , Proliferação de Células , Chaperona BiP do Retículo Endoplasmático , Hepatite B/complicações , Hepatite B/virologia , Vírus da Hepatite B/fisiologia , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/virologia , Fosforilação , Transdução de Sinais , Células Tumorais Cultivadas
12.
Endocrine ; 45(3): 439-47, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23860623

RESUMO

The purpose of this work is to study the activation of the hedgehog signalling pathway is associated with tumour progression in various types of cancer, hence the development of specific antagonists raises hope for new therapeutic strategies. Therefore, the expression of hedgehog pathway components in anaplastic thyroid cancer (ATC) and effects of the hedgehog inhibitor Cyclopamine on ATC cells were investigated in this study. Expression of the ligand Sonic Hedgehog (SHh), the transmembrane protein Smoothened (Smo), the receptor Patched (Ptc) and the target gene Gli-1 was evaluated in two ATC cell lines (Hth 74, C643) by RT-PCR and in tumour specimens by immunohistochemistry. The corresponding gene products were examined by western blotting analysis. After treatment with different concentrations of Cyclopamine the time-dependent course of cell viability in ATC cell lines was evaluated by MTT assay. SHh, Smo, Ptc and Gli were clearly expressed on mRNA and protein levels in both cell lines and in tumour samples (41 %SHh, 65 %Smo, 65 %Ptc and 65 %Gli). Treatment with Cyclopamine showed a time- and dose-dependent inhibition of cell numbers with IC50 values between 1 and 4 µM in both cell lines, comparable to other types of cancer. In conclusion, we believe that the hedgehog pathway is expressed in anaplastic thyroid carcinoma specimens and proliferation of ATC cell lines can be influenced by the Hh inhibitor Cyclopamine. Aberrant activation of this pathway might be involved in the aggressive biology of anaplastic cancer and further evaluation regarding a possible clinical impact of pathway inhibition is warranted.


Assuntos
Biomarcadores Tumorais , Proteínas Hedgehog/metabolismo , Transdução de Sinais/fisiologia , Carcinoma Anaplásico da Tireoide/metabolismo , Alcaloides de Veratrum/farmacologia , Linhagem Celular Tumoral , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Receptores Patched , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Proteína GLI1 em Dedos de Zinco
13.
J Surg Res ; 185(2): 676-83, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23845866

RESUMO

BACKGROUND: Despite various attempts at modifying usual treatment modalities, anaplastic thyroid cancer (ATC) is still associated with unfavorable prognosis. Results of preclinical investigations are often of limited transferability to clinical tumor biology. Individualized multimodal treatment regimens, including novel growth-inhibiting drugs, might be a future option. METHODS: Tumor tissue, freshly prepared from a patient operated for ATC, was xenotransplanted to nude mice. While the patient obtained a hyperfractionated external beam radiation, mice carrying xenotransplanted tumors were randomized (n = 6) and treated by multikinase inhibitors (sorafenib [S]: vascular endothelial growth factor receptor [VEGF-R], platelet derived growth factor receptor, RET; vandetanib [V]: VEGF-R, endothelial growth factor receptor [EGF-R]; and MLN8054 [M]: Aurora kinases [AK]). Antiproliferative, antiangiogenic, and proapoptotic effects were evaluated. RESULTS: Treatment of successfully xenotransplanted fresh ATC tumor tissue by multikinase inhibitors and aurora kinase inhibitor reduced the tumor volume up to 61% depending on the drug and time of application (3 wk of treatment: 46% [M], 34% [V], 30% [S]; 5 wk of treatment: 61% [S]). Tumor cell proliferation (BrdU) was reduced between 34% and 58% [S] and [V]. Reduction of tumor vascularity was between 67% [V] and 33% [S] and was accompanied by decreased EGF-R/VEGF-R2 receptor activity [V/V,S]. Tumor cell apoptosis (caspase 3 activity) increased up to 2.4-fold [S]. CONCLUSIONS: Successful in vivo evaluation of novel drugs in xenotransplanted fresh tumor tissue allows in-time (while patient receives standard treatment) prospective analysis for possible additional clinical application. However, technical specifications have to be taken into account to obtain stable in vivo tumor growth. Based on the individual results, a tailored clinical drug application seems possible.


Assuntos
Benzazepinas/farmacologia , Niacinamida/análogos & derivados , Compostos de Fenilureia/farmacologia , Piperidinas/farmacologia , Quinazolinas/farmacologia , Neoplasias da Glândula Tireoide/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Neovascularização Patológica/tratamento farmacológico , Niacinamida/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Distribuição Aleatória , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Sorafenibe , Carcinoma Anaplásico da Tireoide , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/cirurgia , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Endocrine ; 42(3): 637-46, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22477151

RESUMO

Conventional treatment by surgery, radioiodine, and thyroxin-suppressive therapy often fails to cure anaplastic thyroid cancer (ATC). Therefore several attempts have been made to evaluate new therapy options by use of "small molecule inhibitors". ATC was shown to respond to monotherapeutic proteasome and Aurora kinase inhibition in vitro as well as in xenotransplanted tumor cells. Aim of this study was to evaluate the effect of combined treatment targeting the ubiquitin-proteasome system by bortezomib and Aurora kinases by use of MLN8054. Three ATC cell lines (Hth74, C643, and Kat4.1) were used. The antiproliferative effect of combined treatment with bortezomib and MLN8054 was assessed by MTT-assay and cell cycle analysis (FACS). Proapoptotic effects were evaluated by measurement of Caspase-3 activity, and effects on VEGF secretion were analyzed by ELISA. Compared to mono-application combined treatment with bortezomib and MLN8054 resulted in a further decrease of cell density, whereas antagonizing effects were found regarding cell cycle progression. Caspase-3 activity was increased up to 2.7- and 14-fold by mono-application of MLN8054 and bortezomib, respectively. When the two drugs were used in combination, a further enhancement of Caspase-3 activity was achieved, depending on the cell line. VEGF secretion was decreased following bortezomib treatment and remained unchanged by MLN8054. Only in C643 cells, the bortezomib-induced down-regulation was enhanced when MLN8054 was applied simultaneously. In conclusion, our data demonstrate that targeting the proteasome and Aurora kinases simultaneously results in additional antitumoral effects in vitro, especially regarding cell growth and induction of apoptosis. The efficacy of this therapeutic approach remains to be revised by in vivo and clinical application.


Assuntos
Carcinoma/tratamento farmacológico , Carcinoma/fisiopatologia , Transdução de Sinais/fisiologia , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/fisiopatologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Aurora Quinases , Benzazepinas/farmacologia , Benzazepinas/uso terapêutico , Ácidos Borônicos/farmacologia , Ácidos Borônicos/uso terapêutico , Bortezomib , Caspase 3/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Interações Medicamentosas , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Indicadores e Reagentes , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazinas/farmacologia , Pirazinas/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/metabolismo
15.
J Surg Oncol ; 105(4): 357-64, 2012 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-22006286

RESUMO

BACKGROUND AND OBJECTIVES: Targeting the ubiquitin-proteasome system by using proteasome inhibitors represents a novel approach for cancer therapy. Anaplastic thyroid cancer (ATC), a subtype of thyroid cancer (TC), fails to respond to conventional TC treatment. Here we investigated the effects of bortezomib on TC in vitro. Further, the study aimed to evaluate its potential for TC treatment in vivo. METHODS: Three anaplastic (Hth74, C643, Kat4), one follicular (FTC133), and one papillary (TPC1) TC cell lines were used. Antiproliferative, proapoptotic, and transcriptional effects of bortezomib treatment were analyzed in vitro and growth inhibition of ATC xenografts in vivo. Tumor samples were analyzed by Ki67, CD31, caspase-3, and NF-κB immunohistochemistry. RESULTS: In vitro, bortezomib inhibited proliferation of TC cells (IC(50) 4-10 nM), increased caspase-3 activity and induced cell cycle arrest. NF-κB activity was affected differently. In vivo, bortezomib treatment was effective in reducing tumor volume (up to 74%), accompanied by reduced proliferation (Ki67) and 57% reduced tumor vascularity. CONCLUSION: Proteasome inhibition is effective in reducing cell growth and inducing apoptosis of ATC in vitro and inhibiting tumor growth and vascularity in vivo. However, the impact on nuclear transcription remains controversial. Clinical evaluation of bortezomib treatment in ATC is warranted.


Assuntos
Apoptose/efeitos dos fármacos , Ácidos Borônicos/farmacologia , Inibidores de Proteases/farmacologia , Inibidores de Proteassoma , Pirazinas/farmacologia , Neoplasias da Glândula Tireoide/tratamento farmacológico , Adenocarcinoma Folicular/tratamento farmacológico , Adenocarcinoma Folicular/metabolismo , Adenocarcinoma Folicular/patologia , Animais , Western Blotting , Bortezomib , Carcinoma/tratamento farmacológico , Carcinoma/metabolismo , Carcinoma/patologia , Carcinoma Papilar/tratamento farmacológico , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patologia , Caspase 3/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Nus , NF-kappa B/genética , NF-kappa B/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Células Tumorais Cultivadas
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