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Molecularly imprinted polymers (MIPs) come with the promise to be highly versatile, useful artificial receptors for sensing a wide variety of analytes. Despite a very large body of literature on imprinting, the number of papers addressing real-life biological samples and analytes is somewhat limited. Furthermore, the topic of MIP-based sensor design is still, rather, in the research stage and lacks wide-spread commercialization. This review summarizes recent advances of MIP-based sensors targeting biological species. It covers systems that are potentially interesting in medical applications/diagnostics, in detecting illicit substances, environmental analysis, and in the quality control of food. The main emphasis is placed on work that demonstrates application in real-life matrices, including those that are diluted in a reasonable manner. Hence, it does not restrict itself to the transducer type, but focusses on both materials and analytical tasks.
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Impresión Molecular , Biomimética , Polímeros Impresos Molecularmente , PolímerosRESUMEN
WNT2 acts as a pro-angiogenic factor in placental vascularization and increases angiogenesis in liver sinusoidal endothelial cells (ECs) and other ECs. Increased WNT2 expression is detectable in many carcinomas and participates in tumor progression. In human colorectal cancer (CRC), WNT2 is selectively elevated in cancer-associated fibroblasts (CAFs), leading to increased invasion and metastasis. However, if there is a role for WNT2 in colon cancer, angiogenesis was not addressed so far. We demonstrate that WNT2 enhances EC migration/invasion, while it induces canonical WNT signaling in a small subset of cells. Knockdown of WNT2 in CAFs significantly reduced angiogenesis in a physiologically relevant assay, which allows precise assessment of key angiogenic properties. In line with these results, expression of WNT2 in otherwise WNT2-devoid skin fibroblasts led to increased angiogenesis. In CRC xenografts, WNT2 overexpression resulted in enhanced vessel density and tumor volume. Moreover, WNT2 expression correlates with vessel markers in human CRC. Secretome profiling of CAFs by mass spectrometry and cytokine arrays revealed that proteins associated with pro-angiogenic functions are elevated by WNT2. These included extracellular matrix molecules, ANG-2, IL-6, G-CSF, and PGF. The latter three increased angiogenesis. Thus, stromal-derived WNT2 elevates angiogenesis in CRC by shifting the balance towards pro-angiogenic signals.
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Fibroblastos Asociados al Cáncer/metabolismo , Neoplasias del Colon/irrigación sanguínea , Neoplasias del Colon/patología , Neovascularización Patológica/inducido químicamente , Proteína wnt2/metabolismo , Proteína wnt2/farmacología , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Medios de Cultivo Condicionados/farmacología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Microambiente Tumoral/fisiologíaRESUMEN
Three-dimensional (3D) cancer models are used as preclinical systems to mimic physiologic drug responses. We provide evidence for strong changes of proliferation and metabolic capacity in three dimensions by systematically analyzing spheroids of colon cancer cell lines. Spheroids showed relative lower activities in the AKT, mammalian target of rapamycin (mTOR) and S6K (also known as RPS6KB1) signaling pathway compared to cells cultured in two dimensions. We identified spatial alterations in signaling, as the level of phosphorylated RPS6 decreased from the spheroid surface towards the center, which closely coordinated with the tumor areas around vessels in vivo These 3D models displayed augmented anti-tumor responses to AKT-mTOR-S6K or mitogen-activated protein kinase (MAPK) pathway inhibition compared to those in 2D models. Inhibition of AKT-mTOR-S6K resulted in elevated ERK phosphorylation in 2D culture, whereas under these conditions, ERK signaling was reduced in spheroids. Inhibition of MEK1 (also known as MAP2K1) led to decreased AKT-mTOR-S6K signaling in 3D but not in 2D culture. These data indicate a distinct rewiring of signaling in 3D culture and during treatment. Detached tumor-cell clusters in vessels, in addition to circulating single tumor cells, play a putative role in metastasis in human cancers. Hence, the understanding of signaling in spheroids and the responses in the 3D models upon drug treatment might be beneficial for anti-cancer therapies.
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Técnicas de Cultivo de Célula/métodos , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Forma de la Célula/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Fenotipo , Fosforilación/efectos de los fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologíaRESUMEN
Solid cancers are not simple accumulations of malignant tumor cells but rather represent complex organ-like structures. Despite a more chaotic general appearance as compared to the highly organized setup of healthy tissues, cancers still show highly differentiated structures and a close interaction with and dependency on the interwoven connective tissue. This complexity within cancers is not known in detail at the molecular level so far. The first part of this article will shortly describe the technology and strategies to quantify and dissect the heterogeneity in human solid cancers. Moreover, there is urgent need to better understand human cancer biology since the development of novel anti-cancer drugs is far from being efficient, predominantly due to the scarcity of predictive preclinical models. Hence, in vivo and in vitro models were developed, which better recapitulate the complexity of human cancers, by their intrinsic three-dimensional nature and the cellular heterogeneity and allow functional intervention for hypothesis testing. Therefore, in the second part 3D in vitro cancer models are presented that analyze and depict the heterogeneity in human cancers. Advantages and drawbacks of each model are highlighted and their suitability to preclinical drug testing is discussed.
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Carcinoma/metabolismo , Carcinoma/patología , Comunicación Celular , Modelos Biológicos , Microambiente Tumoral , Animales , Carcinoma/etiología , Comunicación Celular/genética , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Esferoides Celulares , Células del Estroma/metabolismo , Células del Estroma/patología , Técnicas de Cultivo de Tejidos , Células Tumorales Cultivadas , Microambiente Tumoral/genéticaRESUMEN
BACKGROUND: Hypoxic and necrotic regions that accrue within solid tumors in vivo are known to be associated with metastasis formation, radio- and chemotherapy resistance, and drug metabolism. Therefore, integration of these tumor characteristics into in vitro drug screening models is advantageous for any reliable investigation of the anticancer activity of novel drug candidates. In general, usage of cell culture models with in vivo like characteristics has become essential in preclinical drug studies and allows evaluation of complex problems such as tumor selectivity and anti-invasive properties of the drug candidates. MATERIALS AND METHODS: In this study, we investigated the anticancer activity of clinically approved, investigational and experimental drugs based on platinum (cisplatin, oxaliplatin and KP1537), gallium (KP46), ruthenium (KP1339) and lanthanum (KP772) in different cell culture models such as monolayers, multicellular spheroids, as well as invasion and metastasis models. Results Application of the Alamar Blue assay to multicellular spheroids and a spheroid-based invasion assay resulted in an altered rating of compounds with regard to their cytotoxicity and ability to inhibit invasion when compared with monolayer-based cytotoxicity and transwell assays. For example, the gallium-based drug candidate KP46 showed in spheroid cultures significantly enhanced properties to inhibit protrusion formation and fibroblast mediated invasiveness, and improved cancer cell selectivity. CONCLUSION: Taken together, our results demonstrate the advantages of spheroid-based assays and underline the necessity of using different experimental models for reliable preclinical investigations assessing and better predicting the anticancer potential of new compounds.
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Antineoplásicos/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Ciclo Celular/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Cisplatino/farmacología , Técnicas de Cocultivo , Humanos , Hipoxia , Compuestos Organometálicos/farmacología , Compuestos Organoplatinos/farmacología , Oxaliplatino , Oxiquinolina/análogos & derivados , Oxiquinolina/farmacología , Fenantrolinas/farmacología , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/fisiología , Células Tumorales CultivadasRESUMEN
Determining the migratory and invasive capacity of tumor and stromal cells and clarifying the underlying mechanisms is most relevant for novel strategies in cancer diagnosis, prognosis, drug development and treatment. Here we shortly summarize the different modes of cell travelling and review in vitro methods, which can be used to evaluate migration and invasion. We provide a concise summary of established migration/invasion assays described in the literature, list advantages, limitations and drawbacks, give a tabular overview for convenience and depict the basic principles of the assays graphically. In many cases particular research problems and specific cell types do not leave a choice for a broad variety of usable assays. However, for most standard applications using adherent cells, based on our experience we suggest to use exclusion zone assays to evaluate migration/invasion. We substantiate our choice by demonstrating that the advantages outbalance the drawbacks e.g. the simple setup, the easy readout, the kinetic analysis, the evaluation of cell morphology and the feasibility to perform the assay with standard laboratory equipment. Finally, innovative 3D migration and invasion models including heterotypic cell interactions are discussed. These methods recapitulate the in vivo situation most closely. Results obtained with these assays have already shed new light on cancer cell spreading and potentially will uncover unknown mechanisms.
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Ensayos de Migración Celular , Invasividad Neoplásica , Ensayos de Migración de Leucocitos , Movimiento Celular , Humanos , Esferoides CelularesRESUMEN
Introduction. Several studies demonstrated that anti-inflammatory remedies exhibit excellent anti-neoplastic properties. An extract of Pluchea odorata (Asteraceae), which is used for wound healing and against inflammatory conditions, was fractionated and properties correlating to anti-neoplastic and wound healing effects were separated. Methods. Up to six fractionation steps using silica gel, Sephadex columns, and distinct solvent systems were used, and eluted fractions were analysed by thin layer chromatography, apoptosis, and proliferation assays. The expression of oncogenes and proteins regulating cell migration was investigated by immunoblotting after treating HL60 cells with the most active fractions. Results. Sequential fractionations enriched anti-neoplastic activities which suppressed oncogene expression of JunB, c-Jun, c-Myc, and Stat3. Furthermore, a fraction (F4.6.3) inducing or keeping up expression of the mobility markers MYPT, ROCK1, and paxillin could be separated from another fraction (F4.3.7), which inhibited these markers. Conclusions. Wound healing builds up scar or specific tissue, and hence, compounds enhancing cell migration support this process. In contrast, successful anti-neoplastic therapy combats tumour progression, and thus, suppression of cell migration is mandatory.
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Tumour cell invasiveness is crucial for cancer metastasis and is not yet understood. Here we describe two functional screens for proteins required for the invasion of fibrosarcoma cells that identified the molecular chaperone heat shock protein 90 (hsp90). The hsp90 alpha isoform, but not hsp90 beta, is expressed extracellularly where it interacts with the matrix metalloproteinase 2 (MMP2). Inhibition of extracellular hsp90 alpha decreases both MMP2 activity and invasiveness. This role for extracellular hsp90 alpha in MMP2 activation indicates that cell-impermeant anti-hsp90 drugs might decrease invasiveness without the concerns inherent in inhibiting intracellular hsp90.
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Membrana Celular/metabolismo , Matriz Extracelular/metabolismo , Fibrosarcoma/fisiopatología , Proteínas HSP90 de Choque Térmico/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Invasividad Neoplásica/fisiopatología , Membrana Basal/metabolismo , Sitios de Unión/fisiología , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Fibrosarcoma/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Humanos , Unión Proteica/fisiología , Mapeo de Interacción de Proteínas , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína/fisiología , ProteómicaRESUMEN
Many cell lines derived from solid cancers can form spheroids, which recapitulate tumor cell clusters and are more representative of the in vivo situation than 2D cultures. During spheroid formation, a small proportion of a variety of different colon cancer cell lines did not integrate into the sphere and lost cell-cell adhesion properties. An enrichment protocol was developed to augment the proportion of these cells to 100% purity. The basis for the separation of spheroids from non-spheroid forming (NSF) cells is simple gravity-sedimentation. This protocol gives rise to sub-populations of colon cancer cells with stable loss of cell-cell adhesion. SW620 cells lacked E-cadherin, DLD-1 cells lost α-catenin and HCT116 cells lacked P-cadherin in the NSF state. Knockdown of these molecules in the corresponding spheroid-forming cells demonstrated that loss of the respective proteins were indeed responsible for the NSF phenotypes. Loss of the spheroid forming phenotype was associated with increased migration and invasion properties in all cell lines tested. Hence, we identified critical molecules involved in spheroid formation in different cancer cell lines. We present here a simple, powerful and broadly applicable method to generate new sublines of tumor cell lines to study loss of cell-cell adhesion in cancer progression.
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Antígenos CD/genética , Cadherinas/genética , Adhesión Celular/genética , Regulación Neoplásica de la Expresión Génica , Esferoides Celulares/metabolismo , alfa Catenina/genética , Actinas/genética , Actinas/metabolismo , Cadherinas/deficiencia , Comunicación Celular , Línea Celular Tumoral , Movimiento Celular , Molécula de Adhesión Celular Epitelial/genética , Molécula de Adhesión Celular Epitelial/metabolismo , Células HCT116 , Humanos , Cariotipificación , Fenotipo , Transducción de Señal , Esferoides Celulares/patología , alfa Catenina/deficienciaRESUMEN
Apoptotic evasion is a hallmark of cancer and its resistance to chemotherapeutic drugs. Identification of cellular proteins that mediate apoptotic programs is a critical step toward the development of therapeutics aimed at overcoming apoptosis resistance. We developed an innovative high-throughput screen to identify proteins that modulate Fas ligand-mediated apoptosis using fluorophore-assisted light inactivation (HTS-FALIpop). The FALI protein knockdown strategy was coupled to a caspase activity assay with the ability to detect both proapoptotic and antiapoptotic surface molecules expressed by HT-1080 human fibrosarcoma cells. FALI of the Fas receptor (Fas/CD95) using a fluorescein-conjugated anti-Fas antibody abrogated Fas ligand-mediated caspase activation. Ninety-six single-chain variable fragment antibodies (scFv), selected for binding to the surface of HT-1080 cells, were screened by HTS-FALIpop. Three of the scFvs caused decreases in caspase induction after FALI of their protein targets. One of the targets of these positive scFvs was identified as CD44 and was validated by performing FALI using a CD44-specific monoclonal antibody, which resulted in similar protection from Fas apoptosis. CD44-targeted FALI was antiapoptotic in multiple human cancer cell lines, including both Fas signaling type I and II cells, and was also protective against other ligands of the tumor necrosis factor death receptor family. FALI of CD44 inhibited formation and activation of the death-inducing signaling complex, suggesting that CD44 regulates Fas at the cell surface. This mechanism of death receptor regulation represents a novel means of apoptosis modulation that could be exploited by pharmacologic agents.
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Apoptosis , Receptores de Hialuranos/metabolismo , Fragmentos de Inmunoglobulinas/inmunología , Proteómica , Receptores del Factor de Necrosis Tumoral/metabolismo , Receptor fas/metabolismo , Animales , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Caspasas/metabolismo , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte , Activación Enzimática , Fibrosarcoma/metabolismo , Fibrosarcoma/patología , Humanos , Receptores de Hialuranos/genética , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Linfoma de Células T/metabolismo , Linfoma de Células T/patología , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Espectrometría de Masas , Ratones , Células Tumorales Cultivadas , Receptor fas/genéticaRESUMEN
Prostate cancer (PCa) is the most prevalent cancer in men. Hyperactive STAT3 is thought to be oncogenic in PCa. However, targeting of the IL-6/STAT3 axis in PCa patients has failed to provide therapeutic benefit. Here we show that genetic inactivation of Stat3 or IL-6 signalling in a Pten-deficient PCa mouse model accelerates cancer progression leading to metastasis. Mechanistically, we identify p19(ARF) as a direct Stat3 target. Loss of Stat3 signalling disrupts the ARF-Mdm2-p53 tumour suppressor axis bypassing senescence. Strikingly, we also identify STAT3 and CDKN2A mutations in primary human PCa. STAT3 and CDKN2A deletions co-occurred with high frequency in PCa metastases. In accordance, loss of STAT3 and p14(ARF) expression in patient tumours correlates with increased risk of disease recurrence and metastatic PCa. Thus, STAT3 and ARF may be prognostic markers to stratify high from low risk PCa patients. Our findings challenge the current discussion on therapeutic benefit or risk of IL-6/STAT3 inhibition.
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Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Interleucina-6/metabolismo , Neoplasias de la Próstata/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Línea Celular , Progresión de la Enfermedad , Genes p16 , Humanos , Interleucina-6/genética , Masculino , Ratones , Ratones Transgénicos , Neoplasias Experimentales , Fosfohidrolasa PTEN/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Factor de Transcripción STAT3/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
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.
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Asteraceae/química , Lactonas/farmacología , Linfoma Anaplásico de Células Grandes/patología , FN-kappa B/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Sesquiterpenos de Germacrano/farmacología , Sesquiterpenos/farmacología , Apoptosis , Ciclo Celular , Línea Celular Tumoral/efectos de los fármacos , Proliferación Celular , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Estructura Molecular , Plantas Medicinales/química , Transducción de SeñalRESUMEN
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.
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Antineoplásicos Fitogénicos/farmacología , Asteraceae/química , Endotelio Linfático/efectos de los fármacos , Linfoma Anaplásico de Células Grandes/tratamiento farmacológico , Linfoma Anaplásico de Células Grandes/patología , FN-kappa B/antagonistas & inhibidores , Extractos Vegetales/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Sesquiterpenos/farmacología , Apoptosis/efectos de los fármacos , Western Blotting , Caspasas/genética , Caspasas/metabolismo , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Endotelio Linfático/patología , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/patología , Linfoma Anaplásico de Células Grandes/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , FN-kappa B/genética , FN-kappa B/metabolismo , Invasividad Neoplásica , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
BACKGROUND: Invasion is an important early step of cancer metastasis that is not well understood. Developing therapeutics to limit metastasis requires the identification and validation of candidate proteins necessary for invasion and migration. METHODS: We developed a functional proteomic screen to identify mediators of tumor cell invasion. This screen couples Fluorophore Assisted Light Inactivation (FALI) to a scFv antibody library to systematically inactivate surface proteins expressed by human fibrosarcoma cells followed by a high-throughput assessment of transwell invasion. RESULTS: Using this screen, we have identified CD155 (the poliovirus receptor) as a mediator of tumor cell invasion through its role in migration. Knockdown of CD155 by FALI or by RNAi resulted in a significant decrease in transwell migration of HT1080 fibrosarcoma cells towards a serum chemoattractant. CD155 was found to be highly expressed in multiple cancer cell lines and primary tumors including glioblastoma (GBM). Knockdown of CD155 also decreased migration of U87MG GBM cells. CD155 is recruited to the leading edge of migrating cells where it colocalizes with actin and alphav-integrin, known mediators of motility and adhesion. Knockdown of CD155 also altered cellular morphology, resulting in cells that were larger and more elongated than controls when plated on a Matrigel substrate. CONCLUSION: These results implicate a role for CD155 in mediating tumor cell invasion and migration and suggest that CD155 may contribute to tumorigenesis.
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Movimiento Celular , Proteínas de la Membrana/análisis , Invasividad Neoplásica , Proteínas de Neoplasias/análisis , Receptores Virales/análisis , Línea Celular Tumoral , Fibrosarcoma/metabolismo , Fibrosarcoma/patología , Fibrosarcoma/secundario , Glioblastoma/metabolismo , Glioblastoma/patología , Glioblastoma/secundario , Humanos , Inmunoprecipitación/métodos , Proteínas de la Membrana/fisiología , Proteínas de Neoplasias/fisiología , Proteómica/métodos , Receptores Virales/fisiologíaRESUMEN
Anti-cancer drug development is inefficient, mostly due to lack of efficacy in human patients. The high fail rate is partly due to the lack of predictive models or the inadequate use of existing preclinical test systems. However, progress has been made and preclinical models were improved or newly developed, which all account for basic features of solid cancers, three-dimensionality and heterotypic cell interaction. Here we give an overview of available in vivo and in vitro models of cancer, which meet the criteria of being 3D and mirroring human tumor-stroma interactions. We only focus on drug response models without touching models for pharmacokinetic and dynamic, toxicity or delivery aspects.
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Antineoplásicos/farmacología , Modelos Biológicos , Neoplasias/tratamiento farmacológico , Animales , Técnicas de Cultivo de Célula , Diseño de Fármacos , Evaluación Preclínica de Medicamentos/métodos , Humanos , Neoplasias/patologíaRESUMEN
Spheroid-based cellular screening approaches represent a highly physiologic experimental setup to identify novel anticancer drugs and an innovative preclinical model to reduce the high failure rate of anticancer compounds in clinical trials. The resazurin reduction (RR) assay, known as the alamarBlue or CellTiter-Blue assay, is frequently used to determine cell viability/proliferation capacity in eukaryotic cells. Whether this assay is applicable to assess viability in multicellular spheroids has not been evaluated. We analyzed the RR assay to measure cytotoxic and/or cytostatic responses in tumor cell spheroids compared with conventional 2D cultures. We found that tight cell-cell interactions in compact spheroids hamper resazurin uptake and its subsequent reduction to resorufin, leading to lowered reduction activity in relation to the actual cellular health/cell number. Treatment with staurosporine disrupted close cell-cell contacts, which increased resazurin reduction compared with untreated controls. Loss of tight junctions by trypsinization or addition of EGTA or EDTA restored high resazurin reduction rates in untreated spheroids. In conclusion, the RR assay is unsuited to quantitatively measure cellular health/cell number in compact spheroids. However, it can be used to distinguish between cytotoxic versus cytostatic compounds in spheroids. Restoration of the correlation of cell viability/number to resazurin reduction capacity can be achieved by disruption of tight junctions.
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Antineoplásicos/química , Ensayos de Selección de Medicamentos Antitumorales/métodos , Neoplasias/tratamiento farmacológico , Oxazinas/química , Xantenos/química , Apoptosis , Bioensayo , Comunicación Celular , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Ácido Edético/química , Ácido Egtácico/química , Células HT29 , Humanos , Microscopía Confocal , Esferoides Celulares , Estaurosporina/químicaRESUMEN
The present study investigates extracts of Neuolaena lobata, an anti-protozoan ethnomedicinal plant of the Maya, regarding its anti-neoplastic properties. Firstly, extracts of increasing polarity were tested in HL-60 cells analyzing inhibition of cell proliferation and apoptosis induction. Secondly, the most active extract was further tested in anaplastic large cell lymphoma (ALCL) cell lines of human and mouse origin. The dichloromethane extract inhibited proliferation of HL-60, human and mouse ALCL cells with an IC50 of ~2.5, 3.7 and 2.4 µg/ml, respectively and arrested cells in the G2/M phase. The extract induced the checkpoint kinases Chk1 and Chk2 and perturbed the orchestrated expression of the Cdc25 family of cell cycle phosphatases which was paralleled by the activation of p53, p21 and downregulation of c-Myc. Importantly, the expression of NPM/ALK and its effector JunB were drastically decreased, which correlated with the activation of caspase 3. Subsequently also platelet derived growth factor receptor ß was downregulated, which was recently shown to be transcriptionally controlled by JunB synergizing with ALK in ALCL development. We show that a traditional healing plant extract downregulates various oncogenes, induces tumor suppressors, inhibits cell proliferation and triggers apoptosis of malignant cells. The discovery of the 'Active Principle(s)' is warranted.
Asunto(s)
Asteraceae/química , Linfoma Anaplásico de Células Grandes/prevención & control , Cloruro de Metileno/química , Fitoterapia , Extractos Vegetales/farmacología , Plantas Medicinales , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Ciclo Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Técnicas para Inmunoenzimas , Linfoma Anaplásico de Células Grandes/metabolismo , Linfoma Anaplásico de Células Grandes/patología , Ratones , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Células Tumorales CultivadasRESUMEN
Plants have been the source of several effective drugs for the treatment of cancer and over 60% of anticancer drugs originate from natural sources. Therefore, extracts of the rhizome of Smilax spinosa, an ethnomedicinal plant from Guatemala which is used for the treatment of inflammatory conditions, were investigated regarding their anti-neoplastic activities. By using several solvents the methanol extract was by far the most potent against HL60 cell proliferation (50% inhibition at 60 µg/ml). Furthermore, fractionation of this extract yielded fraction F2, which exhibited enforced pro-apoptotic activity, and activated CYP1A1. Proteins that are relevant for cell cycle progression and apoptosis, as well as proto-oncogenes were investigated by western blotting. This revealed that the methanol extract increased the levels of p21 and this may have caused cell cycle attenuation. The derivative fraction F2 induced apoptosis through the intrinsic pathway, which correlated with the inhibition of Stat3 phosphorylation and concomitant induction of caspase 9, then caspase 8 and caspase 3. In summary, the methanol extract and the derivative fraction F2 of S. spinosa showed anti-neoplastic effects in HL-60 cells and CYP1A1 activation in estrogen receptor-positive MCF-7 breast cancer cells but not in estrogen-negative MDA-MB231 breast cancer cells. Based on our data Smilax spinosa may be a promising source for novel anticancer agents.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Fitoterapia , Extractos Vegetales/farmacología , Smilax , Antiinflamatorios no Esteroideos/farmacología , Apoptosis/efectos de los fármacos , Caspasa 3/biosíntesis , Caspasa 8/biosíntesis , Caspasa 9/biosíntesis , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citocromo P-450 CYP1A1/metabolismo , Femenino , Células HL-60 , Humanos , Fosforilación/efectos de los fármacos , Factor de Transcripción STAT3/metabolismo , Quinasas p21 Activadas/metabolismoRESUMEN
Investigating the bioactivity of traditional medical remedies under the controlled conditions of a laboratory is an option to find additional applications, novel formulations or lead structures for the development of new drugs. The present work analysed the antineoplastic activity of increasing polar extracts of the rainforest plant Critonia morifolia (Asteraceae) that has been successfully used as traditional remedy to treat various inflammatory conditions in the long-lasting medical tradition of the Central American Maya, which was here also confirmed in vitro. The apolar petroleum ether extract exhibited the most potent antiproliferative and proapoptotic effects in HL60 cells and triggered down-regulation of Cdc25C and cyclin D1 within 30 min followed by the inhibition of c-Myc expression and the onset of caspase-3 activation within 2 h. Subsequent to these very rapid molecular responses Chk2 and H2AX became phosphorylated (γH2AX) after 4 h. Analysis of the cell cycle distribution showed an accumulation of cells in the G2-M phase within 8 h and after 24 h in S-phase. This was temporally paralleled by the down-regulation of Cdc25A, Cdc25B, Wee1 and Akt. Therefore, the attenuation of cell cycle progression in the G2-M phase was consistent with the known role of Chk2 for G2-M arrest and with the role of Cdc25B in S-phase progression. These findings suggest the presence of two distinct active principles in the petroleum ether extract of C. moriflia. These facilitated the strong apoptotic response evidenced by the rapid activation of caspase-3 that was later enforced by the inhibition of the survival kinase Akt. Importantly, the efficient down-regulation of Akt, which is successfully tested in current clinical trials, is a unique property of C. morifolia.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Asteraceae/química , Proteínas de Ciclo Celular/metabolismo , Extractos Vegetales/farmacología , Alcanos/química , Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular , Proteínas de Ciclo Celular/genética , Proliferación Celular/efectos de los fármacos , Ciclina D1/genética , Ciclina D1/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HL-60 , Humanos , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Solventes/química , Fosfatasas cdc25/genética , Fosfatasas cdc25/metabolismoRESUMEN
Natural products continue to represent the main source for therapeutics, and ethnopharmacological remedies from high biodiversity regions are a rich source for the development of novel drugs. Hence, in our attempt to find new anti-neoplastic activities we focused on ethno-medicinal plants of the Maya, who live in the world's third richest area in vascular plant species. Pluchea odorata (Asteraceae) is traditionally used for the treatment of various inflammatory disorders and recently, the in vitro anti-cancer activities of different extracts of this plant were described. Here, we present the results of bioassay-guided fractionations of the dichloromethane extract of P. odorata that aimed to enrich the active principles. The separation resulted in fractions which showed the dissociation of two distinct anti-neoplastic mechanisms; firstly, a genotoxic effect that was accompanied by tubulin polymerization, cell cycle arrest, and apoptosis (fraction F2/11), and secondly, an effect that interfered with the orchestrated expression of Cyclin D1, Cdc25A, and Cdc2 and that also led to cell cycle arrest and apoptosis (fraction F3/4). Thus, the elimination of generally toxic properties and beyond that the development of active principles of P. odorata, which disturb cancer cell cycle progression, are of interest for potential future therapeutic concepts against proliferative diseases.