RESUMO
A causal link between overexpression of aryl hydrocarbon receptor (AHR) and its target cytochrome P450 1A1 (CYP1A1) and metastatic outgrowth of various cancer entities has been established. Nevertheless, the mechanism how AHR/CYP1A1 support metastasis formation is still little understood. In vitro we discovered a potential mechanism facilitating tumour dissemination based on the production of 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). Utilising a three-dimensional lymph endothelial cell (LEC) monolayer & MDA-MB231 breast cancer cell spheroid co-culture model in combination with knock-down approach allowed elucidation of the molecular/biochemical basis of AHR/CYP1A1-induced tumour breaching through the LEC barrier. Enzyme immunoassay evidenced the potential of recombinant CYP1A1 to synthesise 12(S)-HETE in vitro and qPCR and Western blotting measured gene and protein expression in specific experimental settings. In detail, AHR induced CYP1A1 expression and 12(S)-HETE secretion in tumour spheroids, which caused LEC junction retraction thereby forming large discontinuities allowing transmigration of the tumour. This was enforced by the activating AHR ligand 6-formylindolo (3,3-b)carbazole (FICZ), or inhibited by the AHR antagonist 3,3'-diindolylmethane (DIM) as well as by siRNA against AHR and CYP1A1. AHR and NF-κB were negatively cross talking and therefore, the inhibition of AHR (but not CYP1A1) induced RELA, RELB, NFKB1, NFKB2 and the NF-κB target MMP1, which itself promotes tumour intravasation by a mechanism that is different from 12(S)-HETE. Conversely, the inhibition of NFKB2 induced AHR, CYP1A1 and 12(S)-HETE synthesis. The approved clinical drugs guanfacine and vinpocetine, which inhibit CYP1A1 and NF-κB, respectively, significantly inhibited LEC barrier breaching in vitro indicating an option to reduce metastatic dissemination.
Assuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias da Mama/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Neoplasias da Mama/patologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Técnicas de Silenciamento de Genes , Humanos , Metástase Linfática , Linfócitos/metabolismo , Células MCF-7 , Metaloproteinase 1 da Matriz/metabolismo , NF-kappa B/metabolismo , Metástase Neoplásica , Transdução de Sinais , Esferoides Celulares , Células Tumorais CultivadasRESUMO
Retraction of mesenchymal stromal cells supports the invasion of colorectal cancer cells (CRC) into the adjacent compartment. CRC-secreted 12(S)-HETE enhances the retraction of cancer-associated fibroblasts (CAFs) and therefore, 12(S)-HETE may enforce invasivity of CRC. Understanding the mechanisms of metastatic CRC is crucial for successful intervention. Therefore, we studied pro-invasive contributions of stromal cells in physiologically relevant three-dimensional in vitro assays consisting of CRC spheroids, CAFs, extracellular matrix and endothelial cells, as well as in reductionist models. In order to elucidate how CAFs support CRC invasion, tumour spheroid-induced CAF retraction and free intracellular Ca2+ levels were measured and pharmacological- or siRNA-based inhibition of selected signalling cascades was performed. CRC spheroids caused the retraction of CAFs, generating entry gates in the adjacent surrogate stroma. The responsible trigger factor 12(S)-HETE provoked a signal, which was transduced by PLC, IP3, free intracellular Ca2+, Ca2+-calmodulin-kinase-II, RHO/ROCK and MYLK which led to the activation of myosin light chain 2, and subsequent CAF mobility. RHO activity was observed downstream as well as upstream of Ca2+ release. Thus, Ca2+ signalling served as central signal amplifier. Treatment with the FDA-approved drugs carbamazepine, cinnarizine, nifedipine and bepridil HCl, which reportedly interfere with cellular calcium availability, inhibited CAF-retraction. The elucidation of signalling pathways and identification of approved inhibitory drugs warrant development of intervention strategies targeting tumour-stroma interaction.
Assuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Fibroblastos Associados a Câncer/patologia , Colo/patologia , Neoplasias Colorretais/patologia , Reto/patologia , Transdução de Sinais , Cálcio/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Miosinas Cardíacas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Colo/metabolismo , Neoplasias Colorretais/metabolismo , Humanos , Cadeias Leves de Miosina/metabolismo , Invasividade Neoplásica/patologia , Reto/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
Invasive colorectal cancer is associated with poor prognosis requiring treatment with systemic chemotherapies usually including 5-fluorouracil. A consequence of prolonged treatment is the acquisition of resistance eventually resulting in the recurrence of highly metastatic cancer cells. To address the relationship between drug resistance and increased lymphatic metastatic potential, we used a 3D co-culture model of colon tumour cell spheroids of parent CCL227 cells and subclones with gradually increasing resistance against 5-fluorouracil. From each investigated cell line, homogeneous tumour spheroids were generated in the presence of methylcellulose yielding emboli of â¼700 µm diameter. When invasive, tumour spheroids disrupt the continuous lymphendothelial cell (LEC) layer and generate a 'circular chemorepellent-induced defect' (CCID), reminiscent of the entry gates through which tumour emboli intravasate lymphatic vasculature. Here we provide evidence that increasingly chemoresistant colon cancer spheroids were strongly associated with enhanced intravasative properties. In naïve CCL227 spheroids, miR-200 family members were released into exosomes thereby repressing the epithelial to mesenchymal transition-regulating transcription factors ZEB1 and SLUG in LEC. As a consequence of attenuated plasticity and migration of LEC, CCID formation was impaired. Loss of exosomal transferred miR-200c in resistant colon cells rendered LEC more susceptible to pro-migratory signals that were generated and directly transmitted by colon cancer spheroids. This observation indicates a common molecular axis in colon cancer and LEC where miR-200 family members act as regulators of ZEB proteins. The data support the notion that horizontal miR-200 signalling prevents the permeation of cells into adjacent epithelia and contributes to organ integrity.
Assuntos
Neoplasias do Colo/metabolismo , Células Endoteliais/metabolismo , Fluoruracila/farmacologia , MicroRNAs/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Resistencia a Medicamentos Antineoplásicos , Células Endoteliais/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Metástase Linfática , MicroRNAs/genética , Família Multigênica , Invasividade Neoplásica , Fatores de Transcrição da Família Snail , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
BACKGROUND: The arachidonic acid metabolite 12(S)-HETE is suspected to enhance metastatic spread by inducing cancer cell- and lymph endothelial cell (LEC) motility. However, the molecular mechanisms leading to 12(S)-HETE-triggered cell migration are still elusive. METHODS: To delineate the signalling pathways involved in 12(S)-HETE-mediated migration, inhibitors against RHO and ROCK, and specific siRNAs downregulating 12(S)-HETE receptor (12-HETER) and myosin light chain 2 (MLC2) were used. The breaching of the endothelial barrier was investigated by an assay measuring tumour spheroid-triggered 'circular chemorepellent-induced defects' (CCIDs), and respective signal transduction was elucidated by western blotting. RESULTS: We provide evidence that 12(S)-HETE phosphorylated (and activated) MLC2, which regulates actin/myosin-based contraction. MLC2 activation was found to be essential for LEC retraction and CCID formation. Furthermore, we show that 12(S)-HETE activated a 12-HETER-RHO-ROCK-MYPT signalling cascade to induce MLC2 function. CONCLUSIONS: Signalling via this pathway is described for this metabolite for the first time. This may provide potential targets for the intervention of metastatic colonisation.
Assuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Endotélio Linfático/metabolismo , Cadeias Leves de Miosina/metabolismo , Receptores Eicosanoides/metabolismo , Fator Rho/metabolismo , Transdução de Sinais , Quinases Associadas a rho/metabolismo , Linhagem Celular Tumoral , Humanos , Permeabilidade , FosforilaçãoRESUMO
Organic anion transporting polypeptides 1B1 and 1B3 are transporters selectively expressed on the basolateral membrane of the hepatocyte. Several studies reveal that they are involved in drug-drug interactions, cancer, and hyperbilirubinemia. In this study, we developed a set of classification models for OATP1B1 and 1B3 inhibition based on more than 1700 carefully curated compounds from literature, which were validated via cross-validation and by use of an external test set. After combining several sets of descriptors and classifiers, the 6 best models were selected according to their statistical performance and were used for virtual screening of DrugBank. Consensus scoring of the screened compounds resulted in the selection and purchase of nine compounds as potential dual inhibitors and of one compound as potential selective OATP1B3 inhibitor. Biological testing of the compounds confirmed the validity of the models, yielding an accuracy of 90% for OATP1B1 and 80% for OATP1B3, respectively. Moreover, at least half of the new identified inhibitors are associated with hyperbilirubinemia or hepatotoxicity, implying a relationship between OATP inhibition and these severe side effects.
Assuntos
Transportadores de Ânions Orgânicos/antagonistas & inibidores , Peptídeos/antagonistas & inibidores , Preparações Farmacêuticas/administração & dosagem , Animais , Transporte Biológico/efeitos dos fármacos , Células CHO , Linhagem Celular , Consenso , Cricetulus , Relação Dose-Resposta a Droga , Interações Medicamentosas , Hepatócitos/metabolismo , Fígado/metabolismo , Proteínas de Membrana Transportadoras/metabolismoRESUMO
Many endogenous and xenobiotic substances and their metabolites are substrates for drug metabolizing enzymes and cellular transporters. These proteins may not only contribute to bioavailability of molecules but also to uptake into organs and, consequently, to overall elimination. The coordinated action of uptake transporters, metabolizing enzymes, and efflux pumps, therefore, is a precondition for detoxification and elimination of drugs. As the understanding of the underlying mechanisms is important to predict alterations in drug disposal, adverse drug reactions and, finally, drug-drug interactions, this review illustrates the interplay between selected uptake/efflux transporters and phase I/II metabolizing enzymes.
Assuntos
Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Transporte Biológico/fisiologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/enzimologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Enzimas/sangue , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/enzimologia , Animais , Antineoplásicos/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Transporte Biológico/genética , Resistencia a Medicamentos Antineoplásicos/genética , Enzimas/genética , Humanos , Neoplasias Hepáticas/genética , Ratos , Ratos WistarRESUMO
The COVID-19 pandemic has severely affected the lives of people worldwide, and consequently, it has dominated world news since March 2020. Thus, it is no surprise that it has also been the topic of a massive amount of misinformation, which was most likely amplified by the fact that many details about the virus were not known at the start of the pandemic. While a large amount of this misinformation was harmless, some narratives spread quickly and had a dramatic real-world effect. Such events are called digital wildfires. In this paper we study a specific digital wildfire: the idea that the COVID-19 outbreak is somehow connected to the introduction of 5G wireless technology, which caused real-world harm in April 2020 and beyond. By analyzing early social media contents we investigate the origin of this digital wildfire and the developments that lead to its wide spread. We show how the initial idea was derived from existing opposition to wireless networks, how videos rather than tweets played a crucial role in its propagation, and how commercial interests can partially explain the wide distribution of this particular piece of misinformation. We then illustrate how the initial events in the UK were echoed several months later in different countries around the world.
RESUMO
The COVID-19 pandemic has been accompanied by a surge of misinformation on social media which covered a wide range of different topics and contained many competing narratives, including conspiracy theories. To study such conspiracy theories, we created a dataset of 3495 tweets with manual labeling of the stance of each tweet w.r.t. 12 different conspiracy topics. The dataset thus contains almost 42,000 labels, each of which determined by majority among three expert annotators. The dataset was selected from COVID-19 related Twitter data spanning from January 2020 to June 2021 using a list of 54 keywords. The dataset can be used to train machine learning based classifiers for both stance and topic detection, either individually or simultaneously. BERT was used successfully for the combined task. The dataset can also be used to further study the prevalence of different conspiracy narratives. To this end we qualitatively analyze the tweets, discussing the structure of conspiracy narratives that are frequently found in the dataset. Furthermore, we illustrate the interconnection between the conspiracy categories as well as the keywords.
RESUMO
Metastatic cancer cells cross endothelial barriers and travel through the blood or lymphatic fluid to premetastatic niches, leading to their colonisation. 'S' stereoisomer 12Shydroxy5Z,8Z,10E,14Zeicosatetraenoic acid [12(S)HETE] is secreted by a variety of cancer cell types and has been indicated to open up these barriers. In the present study, another aspect of the endothelial unlocking mechanism was elucidated. This was achieved by investigating 12(S)HETEtreated lymph endothelial cells (LECs) with regard to their expression and mutual interaction with vrel avian reticuloendotheliosis viral oncogene homolog A (RELA), intercellular adhesion molecule 1, SRYbox transcription factor 18 (SOX18), prospero homeobox 1 (PROX1) and focal adhesion kinase (FAK). These key players of LEC retraction, which is a prerequisite for cancer cell transit into vasculature, were analysed using western blot analysis, reverse transcriptionquantitative PCR and transfection with small interfering (si)RNA. The silencing of a combination of these signalling and executing molecules using siRNA, or pharmacological inhibition with defactinib and Bay117082, extended the monoculture experiments to coculture settings using HCT116 colon cancer cell spheroids that were placed on top of LEC monolayers to measure their retraction using the validated 'circular chemorepellentinduced defect' assay. 12(S)HETE was indicated to induce the upregulation of the RELA/SOX18 feedback loop causing the subsequent phosphorylation of FAK, which fed back to RELA/SOX18. Therefore, 12(S)HETE was demonstrated to be associated with circuits involving RELA, SOX18 and FAK, which transduced signals causing the retraction of LECs. The FAKinhibitor defactinib and the NFκB inhibitor Bay117082 attenuated LEC retraction additively, which was similar to the suppression of FAK and PROX1 (the target of SOX18) by the transfection of respective siRNAs. FAK is an effector molecule at the distal end of a prometastatic signalling cascade. Therefore, targeting the endothelialspecific activity of FAK through the pathway demonstrated herein may provide a potential therapeutic method to combat cancer dissemination via vascular routes.
Assuntos
Movimento Celular , Endotélio Linfático/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Ácidos Hidroxieicosatetraenoicos/farmacologia , Neoplasias/patologia , Fatores de Transcrição SOXF/metabolismo , Fator de Transcrição RelA/metabolismo , Linhagem Celular Tumoral , Endotélio Linfático/efeitos dos fármacos , Endotélio Linfático/patologia , Retroalimentação Fisiológica , Quinase 1 de Adesão Focal/genética , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Metástase Neoplásica , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Fatores de Transcrição SOXF/genética , Transdução de Sinais , Fator de Transcrição RelA/genéticaRESUMO
Mechanisms how colorectal cancer (CRC) cells penetrate blood micro-vessel endothelia and metastasise is poorly understood. To study blood endothelial cell (BEC) barrier breaching by CRC emboli, an in vitro assay measuring BEC-free areas underneath SW620 cell spheroids, so called "circular chemorepellent induced defects" (CCIDs, appearing in consequence of endothelial retraction), was adapted and supported by Western blotting, EIA-, EROD- and luciferase reporter assays. Inhibition of ALOX12 or NF-κB in SW620 cells or BECs, respectively, caused attenuation of CCIDs. The FDA approved drugs vinpocetine [inhibiting ALOX12-dependent 12(S)-HETE synthesis], ketotifen [inhibiting NF-κB], carbamazepine and fenofibrate [inhibiting 12(S)-HETE and NF-κB] significantly attenuated CCID formation at low µM concentrations. In the 5-FU-resistant SW620-R/BEC model guanfacine, nifedipine and proadifen inhibited CCIDs stronger than in the naïve SW620/BEC model. This indicated that in SW620-R cells formerly silent (yet unidentified) genes became expressed and targetable by these drugs in course of resistance acquisition. Fenofibrate, and the flavonoids hispidulin and apigenin, which are present in medicinal plants, spices, herbs and fruits, attenuated CCID formation in both, naïve- and resistant models. As FDA-approved drugs and food-flavonoids inhibited established and acquired intravasative pathways and attenuated BEC barrier-breaching in vitro, this warrants testing of these compounds in CRC models in vivo.
Assuntos
Neoplasias Colorretais/patologia , Células Endoteliais/fisiologia , Endotélio Vascular/fisiologia , Flavonoides/farmacologia , Esferoides Celulares/fisiologia , Araquidonato 12-Lipoxigenase/genética , Araquidonato 12-Lipoxigenase/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , NF-kappa B/genética , NF-kappa B/metabolismo , Metástase Neoplásica/fisiopatologia , Preparações FarmacêuticasRESUMO
Resveratrol is a naturally occurring polyphenolic compound with various pharmacological activities. It is unknown whether the expression of metabolizing enzymes correlates with resveratrol levels in organs and tissues. Therefore, we investigated the metabolism and tissue distribution of resveratrol in mice and assessed its association with the expression of UDP-glucuronosyltransferase (Ugt) and sulfotransferase (Sult) genes. Plasma, urine, feces, and various organs were analyzed using high-performance liquid chromatography at up to 8 h after intragastric resveratrol administration. The metabolism of resveratrol was pronounced, leading to the formation of resveratrol glucuronides and sulfates. Concentrations of resveratrol and its metabolites were high in the gastrointestinal organs, urine, and feces, but low in the liver and kidneys. In lung, heart, thymus, and brain tissues, parent resveratrol levels exceeded the sulfate and glucuronide concentrations. The formation of resveratrol conjugates correlated with the expression of certain Ugt and Sult genes. Reverse transcription quantitative PCR (RT-qPCR) analysis revealed high mRNA expression of Ugt1a1 and Ugt1a6a in the liver, duodenum, jejunum, ileum, and colon, leading to high concentrations of resveratrol-3-O-glucuronide in these organs. Strong correlations of resveratrol-3-O-sulfate and resveratrol-3-O-4'-O-disulfate formation with Sult1a1 mRNA expression were also observed, particularly in the liver and colon. In summary, our data revealed organ-specific expression of Sults and Ugts in mice that strongly affects resveratrol concentrations; this may also be predictive in humans following oral uptake of dietary resveratrol.
Assuntos
Glucuronídeos/síntese química , Glucuronosiltransferase/metabolismo , Estilbenos/síntese química , Estilbenos/farmacocinética , Sulfotransferases/metabolismo , Animais , Camundongos , Resveratrol , Distribuição TecidualRESUMO
Lymph node metastasis of breast cancer is a clinical marker of poor prognosis. Yet, there exist no therapies targeting mechanisms of intravasation into lymphatics. Herein we report on an effect of the antidyslipidemic drug fenofibrate with vasoprotective activity, which attenuates breast cancer intravasation in vitro, and describe the potential mechanisms. To measure intravasation in a 3-dimensional co-culture model MDA-MB231 and MCF-7 breast cancer spheroids were placed on immortalised lymphendothelial cell (LEC) monolayers. This provokes the formation of circular chemorepellent induced defects (CCIDs) in the LEC barrier resembling entry ports for the intravasating tumour. Furthermore, the expression of adhesion molecules ICAM-1, CD31 and FAK was investigated in LECs by western blotting as well as cell-cell adhesion and NF-κB activity by respective assays. In MDA-MB231 cells the activity of CYP1A1 was measured by EROD assay. Fenofibrate inhibited CCID formation in the MDA-MB231/LEC- and MCF-7/LEC models and the activity of NF-κB, which in turn downregulated ICAM-1 in LECs and the adhesion of cancer cells to LECs. Furthermore, CD31 and the activity of FAK were inhibited. In MDA-MB231 cells, fenofibrate attenuated CYP1A1 activity. Combinations with other FDA-approved drugs, which reportedly inhibit different ion channels, attenuated CCID formation additively or synergistically. In summary, fenofibrate inhibited NF-κB and ICAM-1, and inactivated FAK, thereby attenuating tumour intravasation in vitro. A combination with other FDA-approved drugs further improved this effect. Our new concept may lead to a novel therapy for cancer patients.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Técnicas de Cocultura , Fenofibrato/administração & dosagem , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Citocromo P-450 CYP1A1/genética , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Feminino , Humanos , Molécula 1 de Adesão Intercelular/genética , Metástase Linfática , Células MCF-7 , NF-kappa B/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Castration-resistant prostate cancer (CRPC) has greater intratumoral testosterone concentrations than similar tumors from eugonadal men; simple diffusion does not account for this observation. This study was undertaken to ascertain the androgen uptake kinetics, functional, and clinical relevance of de novo expression of the steroid hormone transporter OATP1B3 (SLCO1B3). Experiments testing the cellular uptake of androgens suggest that testosterone is an excellent substrate of OATP1B3 (Km = 23.2 µmol/L; Vmax = 321.6 pmol/mg/minute), and cells expressing a doxycycline-inducible SLCO1B3 construct had greater uptake of a clinically relevant concentration of 3H-testosterone (50 nmol/L; 1.6-fold, P = 0.0027). When compared with Slco1b2 (-/-) mice, Slco1b2 (-/-)/hSLCO1B3 knockins had greater hepatic uptake (15% greater AUC, P = 0.0040) and lower plasma exposure to 3H-testosterone (17% lower AUC, P = 0.0030). Of 82 transporters genes, SLCO1B3 is the second-most differentially expressed transporter in CRPC cell lines (116-fold vs. androgen-sensitive cells), with a differentially spliced cancer-type ct-SLCO1B3 making up the majority of SLCO1B3 expression. Overexpression of SLCO1B3 in androgen-responsive cells results in 1.5- to 2-fold greater testosterone uptake, whereas siRNA knockdown of SLCO1B3 in CRPC cells did not change intracellular testosterone concentration. Primary human prostate tumors express SLCO1B3 to a greater extent than ct-SLCO1B3 (26% of total SLCO1B3 expression vs. 0.08%), suggesting that androgen uptake in these tumor cells also is greater. Non-liver tumors do not differentially express SLCO1B3.Implications: This study suggests that de novo OATP1B3 expression in prostate cancer drives greater androgen uptake and is consistent with previous observations that greater OATP1B3 activity results in the development of androgen deprivation therapy resistance and shorter overall survival. Mol Cancer Res; 15(8); 1096-105. ©2017 AACR.
Assuntos
Androgênios/metabolismo , Neoplasias de Próstata Resistentes à Castração/genética , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de Soluto/genética , Testosterona/metabolismo , Animais , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos Knockout , Próstata/metabolismo , Próstata/patologia , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , RNA Interferente Pequeno/genética , Testosterona/administração & dosagemRESUMO
Secretion of 12(S)-HETE by breast cancer emboli provokes "circular chemorepellent induced defects" (CCIDs) in the adjacent lymphatic vasculature facilitating their intravasation and lymph node metastasis which determines prognosis. Therefore, elucidating the mechanism of lymph endothelial cell (LEC) wall disintegration may provide cues for anti-metastatic intervention. The role of intracellular free Ca(2+) for CCID formation was investigated in LECs using MCF-7 or MDA-MB231 breast cancer cell spheroids in a three-dimensional cell co-culture model. 12(S)-HETE elevated the Ca(2+) level in LEC by activating PLC/IP3. Downstream, the Ca(2+)-calmodulin kinase MYLK contributed to the phosphorylation of Ser19-MLC2, LEC contraction and CCID formation. Approved clinical drugs, lidoflazine, ketotifen, epiandrosterone and cyclosporine, which reportedly disturb cellular calcium supply, inhibited 12(S)-HETE-induced Ca(2+) increase, Ser19-MLC2 phosphorylation and CCID formation. This treatment strategy may reduce spreading of breast cancer through lymphatics.
Assuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Neoplasias da Mama/patologia , Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Movimento Celular , Células Endoteliais/efeitos dos fármacos , Vasos Linfáticos/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Quelantes de Cálcio/farmacologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Miosinas Cardíacas/metabolismo , Técnicas de Cocultura , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Feminino , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Metástase Linfática , Vasos Linfáticos/metabolismo , Células MCF-7 , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/genética , Quinase de Cadeia Leve de Miosina/metabolismo , Permeabilidade , Fosforilação , Interferência de RNA , Serina , Esferoides Celulares , Fatores de Tempo , Transfecção , Fosfolipases Tipo C/metabolismoRESUMO
Anthocyans (anthocyanins and their aglycones anthocyanidins) are colorful pigments, naturally occurring in fruits. They exhibit many biological effects and have potent health benefits. Anthocyans are widely used as dietary supplements and the safety of products containing them is of great importance. To investigate whether anthocyans influence the expression of hepatic uptake transporters from the organic anion transporting polypeptide (SLCO gene/OATP protein) family, we carried out studies on primary cultures of human hepatocytes. The hepato-cellular accumulation of widely used drugs such as statins and some anticancer drugs is mediated by the liver-specific OATP1B1 and OATP1B3, thus any interference with expression of these particular transporters might influence therapeutic outcomes. We evaluated the effects of 21 anthocyanins and their corresponding 6 anthocyanidins on the expression levels of SLCO1B1/SLCO1B3 by RT-qPCR. Changes in OATP protein levels were confirmed by western blotting. Our data show that OATP1B1 responds differently to anthocyans compared with OATP1B3. We observed the induction of SLCO1B1 gene and OATP1B1 protein in four hepatocyte samples by the anthocyanins malvin chloride, malvidin-3-O-galactoside chloride and cyanidin-3-O-sophoroside chloride. For SLCO1B3, a reduction in the expression levels was seen with delphin chloride and the anthocyanidin pelargonidin. Although the values varied considerably between primary hepatocyte isolates from different individuals, a mean induction of SLCO1B1 (up to 60%) and reduction of SLCO1B3 (by less than 25%) were detected. We propose that the effects of anthocyans derived from high dose dietary supplements may have to be taken into account in patients undergoing a therapy with drugs transported by OATP1B1 and OATP1B3.
Assuntos
Antocianinas/metabolismo , Anticarcinógenos/metabolismo , Suplementos Nutricionais , Regulação da Expressão Gênica , Hepatócitos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Transportadores de Ânions Orgânicos/agonistas , Adulto , Antocianinas/química , Anticarcinógenos/química , Células Cultivadas , Frutas/química , Galactosídeos/química , Galactosídeos/metabolismo , Glucosídeos/química , Glucosídeos/metabolismo , Hepatócitos/citologia , Humanos , Transportador 1 de Ânion Orgânico Específico do Fígado , Masculino , Pessoa de Meia-Idade , Estrutura Molecular , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de SolutoRESUMO
The contribution of organic anion transporting polypeptides (OATPs) to the cellular uptake of flavopiridol was investigated in OATP1B1-, OATP1B3- and OATP2B1-expressing Chinese hamster ovary (CHO) cells. Uptake of flavopiridol into these cells showed typical Michaelis-Menten kinetics with much higher transport capacity for OATP1B3 compared to OATP1B1 and OATP2B1 (Vmax/Km, 33.9 vs. 8.84 and 2.41 µl/mg/min, respectively). The predominant role of OATPs was further supported by a dramatic inhibition of flavopiridol uptake in the presence of the OATP substrate rifampicin. Uptake of flavopiridol by OATPs also seems to be an important determinant in breast cancer cells. The much higher mRNA level for OATP1B1 found in wild-type compared to ZR-75-1 OATP1B1 knockdown cells correlated with higher flavopiridol initial uptake leading to 4.6-fold decreased IC50 values in the cytotoxicity assay (IC50, 1.45 vs. 6.64 µM). Cell cycle profile also showed a clear incidence for a stronger cell cycle arrest in the G2/M phase for ZR-75-1 wild-type cells compared to OATP1B1 knockdown cells, further indicating an active uptake via OATP1B1. In conclusion, our results revealed OATP1B1, OATP1B3 and OATP2B1 as uptake transporters for flavopiridol in cancer cells, which may also apply in patients during cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Flavonoides/farmacologia , Transportadores de Ânions Orgânicos/fisiologia , Piperidinas/farmacologia , Animais , Antineoplásicos/farmacocinética , Neoplasias da Mama/metabolismo , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Feminino , Flavonoides/farmacocinética , Humanos , Transportador 1 de Ânion Orgânico Específico do Fígado , Transportadores de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/fisiologia , Piperidinas/farmacocinética , Membro 1B3 da Família de Transportadores de Ânion Orgânico Carreador de SolutoRESUMO
The expression of organic anion transporting polypeptides (OATPs) was elucidated in cell lines from small cell lung cancer (SCLC) and lung carcinoids and in paraffin-embedded samples from primary and metastatic SCLCs. We found a strong relationship between OATP expression and the origin of the cells, as cells from primary or metastatic SCLC and carcinoid tumors differ with respect to OATP levels. OATP4A1 is most prominent in non-malignant lung tissue and in all SCLC and carcinoid cell lines and tissues, OATP5A1 is most prominent in metastatic cells, and OATP6A1 is most prominent in SCLC cell lines and tumors. Treatment with topotecan, etoposide and cisplatin caused significant changes in the expression patterns of OATP4A1, OATP5A1, OATP6A1, chromogranin and synaptophysin. This effect was also evident in GLC-14 cells from an untreated SCLC patient before chemotherapy compared to GLC-16/-19 chemoresistant tumor cells from this patient after therapy. mRNA expression of OATP4A1, 5A1 and 6A1 correlates with protein expression as confirmed by quantitative microscopic image analysis and Western blots. OATPs might be novel biomarkers for tumor progression and the development of metastasis in SCLC patients.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Pulmonares/genética , Transportadores de Ânions Orgânicos/metabolismo , Carcinoma de Pequenas Células do Pulmão/genética , Biomarcadores Tumorais/genética , Western Blotting , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/secundário , Cromograninas/genética , Cromograninas/metabolismo , Cisplatino/administração & dosagem , Resistencia a Medicamentos Antineoplásicos , Etoposídeo/administração & dosagem , Imunofluorescência , Humanos , Técnicas Imunoenzimáticas , Pulmão/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Transportadores de Ânions Orgânicos/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/secundário , Sinaptofisina/genética , Sinaptofisina/metabolismo , Topotecan/administração & dosagem , Células Tumorais CultivadasRESUMO
BACKGROUND: The t(2;5)(p23;q35) chromosomal translocation results in the expression of the fusion protein NPM/ALK that when expressed in T-lymphocytes gives rise to anaplastic large cell lymphomas (ALCL). In search of new therapy options the dichloromethane extract of the ethnomedicinal plant Neurolaena lobata (L.) R.Br. ex Cass was shown to inhibit NPM/ALK expression. PURPOSE: Therefore, we analysed whether the active principles that were recently isolated and found to inhibit inflammatory responses specifically inhibit growth of NPM/ALK+ ALCL, leukaemia and breast cancer cells, but not of normal cells, and the intravasation through the lymphendothelial barrier. METHODS: ALCL, leukaemia and breast cancer cells, and normal peripheral blood mononuclear cells (PBMCs) were treated with isolated sesquiterpene lactones and analysed for cell cycle progression, proliferation, mitochondrial activity, apoptosis, protein and mRNA expression, NF-κB and cytochrome P450 activity, 12(S)-HETE production and lymphendothelial intravasation. RESULTS: In vitro treatment of ALCL by neurolenin B suppressed NPM/ALK, JunB and PDGF-Rß expression, inhibited the growth of ALCL cells late in M phase, and induced apoptosis via caspase 3 without compromising mitochondrial activity (as a measure of general exogenic toxicity). Moreover, neurolenin B attenuated tumour spheroid intravasation probably through inhibition of NF-κB and CYP1A1. CONCLUSION: Neurolenin B specifically decreased pro-carcinogenic NPM/ALK expression in ALK+ ALCL cells and, via the inhibition of NF-kB signalling, attenuated tumour intra/extravasation into the lymphatics. Hence, neurolenin B may open new options to treat ALCL and to manage early metastatic processes to which no other therapies exist.
Assuntos
Asteraceae/química , Lactonas/farmacologia , Linfoma Anaplásico de Células Grandes/patologia , NF-kappa B/metabolismo , Proteínas Tirosina Quinases/metabolismo , Sesquiterpenos de Germacrano/farmacologia , Sesquiterpenos/farmacologia , Apoptose , Ciclo Celular , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Estrutura Molecular , Plantas Medicinais/química , Transdução de SinaisRESUMO
An apolar extract of the traditional medicinal plant Neurolaena lobata inhibited the expression of the NPM/ALK chimera, which is causal for the majority of anaplastic large cell lymphomas (ALCLs). Therefore, an active principle of the extract, the furanoheliangolide sesquiterpene lactone lobatin B, was isolated and tested regarding the inhibition of ALCL expansion and tumour cell intravasation through the lymphendothelium. ALCL cell lines, HL-60 cells and PBMCs were treated with plant compounds and the ALK inhibitor TAE-684 to measure mitochondrial activity, proliferation and cell cycle progression and to correlate the results with protein- and mRNA-expression of selected gene products. Several endpoints indicative for cell death were analysed after lobatin B treatment. Tumour cell intravasation through lymphendothelial monolayers was measured and potential causal mechanisms were investigated analysing NF-κB- and cytochrome P450 activity, and 12(S)-HETE production. Lobatin B inhibited the expression of NPM/ALK, JunB and PDGF-Rß, and attenuated proliferation of ALCL cells by arresting them in late M phase. Mitochondrial activity remained largely unaffected upon lobatin B treatment. Nevertheless, caspase 3 became activated in ALCL cells. Also HL-60 cell proliferation was attenuated whereas PBMCs of healthy donors were not affected by lobatin B. Additionally, tumour cell intravasation, which partly depends on NF-κB, was significantly suppressed by lobatin B most likely due to its NF-κB-inhibitory property. Lobatin B, which was isolated from a plant used in ethnomedicine, targets malignant cells by at least two properties: I) inhibition of NPM/ALK, thereby providing high specificity in combating this most prevalent fusion protein occurring in ALCL; II) inhibition of NF-κB, thereby not affecting normal cells with low constitutive NF-κB activity. This property also inhibits tumour cell intravasation into the lymphatic system and may provide an option to manage this early step of metastatic progression.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Asteraceae/química , Endotélio Linfático/efeitos dos fármacos , Linfoma Anaplásico de Células Grandes/tratamento farmacológico , Linfoma Anaplásico de Células Grandes/patologia , NF-kappa B/antagonistas & inibidores , Extratos Vegetais/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Sesquiterpenos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Caspases/genética , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Endotélio Linfático/patologia , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Linfoma Anaplásico de Células Grandes/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , NF-kappa B/genética , NF-kappa B/metabolismo , Invasividade Neoplásica , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
SCOPE: Resveratrol is a naturally occurring polyphenolic compound with various pharmacological activities. These effects are observed despite its low bioavailability, which is particularly caused by extensive phase II metabolism. It is unknown whether resveratrol and its metabolites can accumulate to bioactive levels in organs and tissues through protein-mediated transport mechanisms. Because organic anion transporting polypeptides (OATPs) mediate the uptake of many clinically important drugs, we investigated their role in the cellular transport of resveratrol and its major glucuronides and sulfates. METHODS AND RESULTS: Uptake experiments were performed with resveratrol and its glucuronides and sulfates in OATP-expressing Chinese hamster ovary (CHO) and breast cancer (ZR-75-1) cells. The uptake rates for resveratrol in OATP1B1-, OATP1B3-, and OATP2B1-transfected Chinese hamster ovary cells were four- to sixfold higher compared to wild-type cells. Resveratrol-3-O-4'-O-disulfate was transported by OATP1B1 and OATP1B3, while resveratrol-3-O-sulfate was exclusively transported by OATP1B3. However, resveratrol-4'-O-sulfate, resveratrol-3-O-glucuronide, and resveratrol-4'-O-glucuronide did not show any affinity for these OATPs. OATP-dependent uptake of resveratrol was also confirmed in ZR-75-1 cells. CONCLUSION: Our data revealed that OATPs act as cellular uptake transporters for resveratrol and its major sulfates, which must be considered in humans following oral uptake of dietary resveratrol.