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Malignant pleural mesothelioma (MPM) is an aggressive cancer associated with asbestos exposure. MPM is often diagnosed late, at a point where limited treatment options are available, but early intervention could the chances of successful treatment for MPM patients. Biomarkers to detect MPM in at-risk individuals are needed to implement early diagnosis technologies. Volatile organic compounds (VOCs) have previously shown diagnostic potential as biomarkers when analysed in MPM patient breath. In this study, chorioallantoic membrane (CAM) xenografts of MPM cell lines were used as models of MPM tumour development for VOC biomarker discovery with the aim of generating targets for investigation in breath, biopsies or other complex matrices. VOC headspace analysis of biphasic MESO-7T, epithelioid MESO-8T and MESO-12T MPM CAM xenografts was performed using solid-phase microextraction and gas chromatography-mass spectrometry. We successfully demonstrated the capture, analysis and separation of VOC signatures from CAM xenografts and controls. A panel of VOCs was identified that showed discrimination between MPM xenografts generated from biphasic and epithelioid cells and CAM controls. This is the first application of the CAM xenograft model for the discovery of VOC biomarkers associated with MPM histological subtypes. These findings support the potential utility of non-invasive VOC profiling from breath or headspace analysis of tissues for detection and monitoring of MPM.
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A multimodal mass spectrometry imaging (MSI) approach was used to investigate the chemotherapy drug-induced response of a Multicellular Tumour Spheroid (MCTS) 3D cell culture model of osteosarcoma (OS). The work addresses the critical demand for enhanced translatable early drug discovery approaches by demonstrating a robust spatially resolved molecular distribution analysis in tumour models following chemotherapeutic intervention. Advanced high-resolution techniques were employed, including desorption electrospray ionisation (DESI) mass spectrometry imaging (MSI), to assess the interplay between metabolic and cellular pathways in response to chemotherapeutic intervention. Endogenous metabolite distributions of the human OS tumour models were complemented with subcellularly resolved protein localisation by the detection of metal-tagged antibodies using Imaging Mass Cytometry (IMC). The first application of matrix-assisted laser desorption ionization-immunohistochemistry (MALDI-IHC) of 3D cell culture models is reported here. Protein localisation and expression following an acute dosage of the chemotherapy drug doxorubicin demonstrated novel indications for mechanisms of region-specific tumour survival and cell-cycle-specific drug-induced responses. Previously unknown doxorubicin-induced metabolite upregulation was revealed by DESI-MSI of MCTSs, which may be used to inform mechanisms of chemotherapeutic resistance. The demonstration of specific tumour survival mechanisms that are characteristic of those reported for in vivo tumours has underscored the increasing value of this approach as a tool to investigate drug resistance.
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Porphyromonas gingivalis contributes to the chronic oral disease periodontitis, triggering the activation of host inflammatory responses, inducing cellular stresses such as oxidation. During stress, host cells can activate the Integrated Stress Response (ISR), a pathway which determines cellular fate, by either downregulating protein synthesis and initiating a stress-response gene expression program, or by initiating programmed cell death. Recent studies have implicated the ISR within both host antimicrobial defenses and the pathomechanism of certain microbes. In this study, using a combination of immunofluorescence confocal microscopy and immunoblotting, the molecular mechanisms by which P. gingivalis infection alters translation attenuation during oxidative stress-induced activation of the ISR in oral epithelial cells were investigated. P. gingivalis infection alone did not result in ISR activation. In contrast, infection coupled with stress caused differential stress granule formation and composition. Infection heightened stress-induced translational repression independently of core ISR mediators. Heightened translational repression during stress was observed with both P. gingivalis-conditioned media and outer membrane vesicles, implicating a secretory factor in this exacerbated translational repression. The effects of gingipain inhibitors and gingipain-deficient P. gingivalis mutants confirmed these pathogen-specific proteases as the effector of exacerbated translational repression. Gingipains are known to degrade the mammalian target of rapamycin (mTOR) and the findings of this study implicate the gingipain-mTOR axis as the effector of host translational dysregulation during stress.
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Apigenin is a dietary polyphenol found abundantly in fruit and vegetables, which sensitizes leukaemia cells to topoisomerase inhibitor agents (e.g., etoposide), and alkylating agents (e.g., cyclophosphamide), reducing ATP levels and inducing apoptosis; whilst being protective to control haematopoietic stem cells. This study analysed the expression profiles of intrinsic and extrinsic apoptosis-related genes and proteins to help elucidate the mechanisms of action of apigenin when used in combination with etoposide or cyclophosphamide in lymphoid and myeloid leukaemia cell lines (Jurkat and THP-1). Expression of apoptosis-related genes were measured using a TaqMan® Human Apoptosis Array and the StepOne Plus RT-qPCR System, whilst apoptosis-related proteins were determined using a protein profiler™-human apoptosis array and the LI-COR OdysseyR Infrared Imaging System. Apigenin when combined with etoposide or cyclophosphamide-induced apoptosis via the mitochondrial pathway, increasing the expression of pro-apoptotic cytochrome c, SMAC/DIABLO, and HTRA2/OMI, which promoted caspase-9 and -3 activation. Targeting anti-apoptotic and/or pro-apoptotic members of the apoptotic pathways is a promising strategy to induce cancer cell death and improve sensitivity to chemotherapy agents. Here the apoptotic pathways induced by apigenin in combination with etoposide or cyclophosphamide were identified within human leukaemia cell lines, such applications could provide combination therapies for the treatment of leukaemia.
Asunto(s)
Apigenina , Proteínas Reguladoras de la Apoptosis , Apoptosis , Leucemia , Apigenina/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/genética , Línea Celular/efectos de los fármacos , Línea Celular/metabolismo , Ciclofosfamida/farmacología , Quimioterapia Combinada , Etopósido/farmacología , Humanos , Leucemia/tratamiento farmacológico , Leucemia/genética , Proteínas Mitocondriales/metabolismoRESUMEN
Osteosarcoma (OS) is the most common primary bone malignancy and largely effects adolescents and young adults, with 60% of patients under the age of 25. There are multiple cell models of OS described in vitro that express the specific genetic alterations of the sarcoma. In the work reported here, multiple mass spectrometry imaging (MSI) modalities were employed to characterise two aggregated cellular models of OS models formed using the MG63 and SAOS-2 cell lines. Phenotyping of the metabolite activity within the two OS aggregoid models was achieved and a comparison of the metabolite data with OS human tissue samples revealed relevant fatty acid and phospholipid markers. Although, annotations of these species require MS/MS analysis for confident identification of the metabolites. From the putative assignments however, it was suggested that the MG63 aggregoids are an aggressive tumour model that exhibited metastatic-like potential. Alternatively, the SAOS-2 aggregoids are more mature osteoblast-like phenotype that expressed characteristics of cellular differentiation and bone development. It was determined the two OS aggregoid models shared similarities of metabolic behaviour with different regions of OS human tissues, specifically of the higher metastatic grade.
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Mass spectrometry imaging (MSI) is an established analytical tool capable of defining and understanding complex tissues by determining the spatial distribution of biological molecules. Three-dimensional (3D) cell culture models mimic the pathophysiological environment of in vivo tumors and are rapidly emerging as a valuable research tool. Here, multimodal MSI techniques were employed to characterize a novel aggregated 3D lung adenocarcinoma model, developed by the group to mimic the in vivo tissue. Regions of tumor heterogeneity and the hypoxic microenvironment were observed based on the spatial distribution of a variety of endogenous molecules. Desorption electrospray ionization (DESI)-MSI defined regions of a hypoxic core and a proliferative outer layer from metabolite distribution. Targeted metabolites (e.g., lactate, glutamine, and citrate) were mapped to pathways of glycolysis and the TCA cycle demonstrating tumor metabolic behavior. The first application of imaging mass cytometry (IMC) with 3D cell culture enabled single-cell phenotyping at 1 µm spatial resolution. Protein markers of proliferation (Ki-67) and hypoxia (glucose transporter 1) defined metabolic signaling in the aggregoid model, which complemented the metabolite data. Laser ablation inductively coupled plasma (LA-ICP)-MSI analysis localized endogenous elements including magnesium and copper, further differentiating the hypoxia gradient and validating the protein expression. Obtaining a large amount of molecular information on a complementary nature enabled an in-depth understanding of the biological processes within the novel tumor model. Combining powerful imaging techniques to characterize the aggregated 3D culture highlighted a future methodology with potential applications in cancer research and drug development.
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Adenocarcinoma del Pulmón/diagnóstico , Ácido Cítrico/análisis , Glutamina/análisis , Ácido Láctico/análisis , Neoplasias Pulmonares/diagnóstico , Adenocarcinoma del Pulmón/metabolismo , Ácido Cítrico/metabolismo , Glutamina/metabolismo , Humanos , Ácido Láctico/metabolismo , Neoplasias Pulmonares/metabolismo , Imagen Multimodal , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Células Tumorales CultivadasRESUMEN
Three-dimensional (3D) cell culture is a rapidly emerging field, which mimics some of the physiological conditions of human tissues. In cancer biology, it is considered a useful tool in predicting in vivo chemotherapy responses, compared with conventional two-dimensional (2D) cell culture. We have developed a novel 3D cell culture model of osteosarcoma composed of aggregated proliferative tumour spheroids, which shows regions of tumour heterogeneity formed by aggregated spheroids of polyclonal tumour cells. Aggregated spheroids show local necrotic and apoptotic regions and have sizes suitable for the study of spatial distribution of metabolites by mass spectrometry imaging (MSI). We have used this model to perform a proof-of-principle study showing a heterogeneous distribution of endogenous metabolites that colocalise with the necrotic core and apoptotic regions in this model. Cytotoxic chemotherapy (doxorubicin) responses were significantly attenuated in our 3D cell culture model compared with those of standard cell culture, as determined by resazurin assay, despite sufficient doxorubicin diffusion demonstrated by localisation throughout the 3D constructs. Finally, changes to the distribution of endogenous metabolites in response to doxorubicin were readily detected by MSI. Principal component analysis identified 50 metabolites which differed most in their abundance between treatment groups, and of these, 10 were identified by both in-software t test and mixed-effects analysis of variance (ANOVA). Subsequent independent MSIs of identified species were consistent with principle component analysis findings. This proof-of-principle study shows for the first time that chemotherapy-induced changes in metabolite abundance and distribution may be determined in 3D cell culture by MSI, highlighting this method as a potentially useful tool in the elucidation of chemotherapy responses as an alternative to in vivo testing.
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Neoplasias Óseas/tratamiento farmacológico , Doxorrubicina/farmacología , Osteosarcoma/tratamiento farmacológico , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Antibióticos Antineoplásicos/farmacología , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Humanos , Imagen Molecular/métodos , Osteosarcoma/metabolismo , Osteosarcoma/patología , Análisis de Componente Principal , Prueba de Estudio Conceptual , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/patologíaRESUMEN
Polyphenols have been shown to sensitize solid tumours to alkylating agents such as cisplatin, and induce apoptosis and/or cell-cycle arrest. Here, we assess the effects of five polyphenols alone and in combination with three alkylating agents: cisplatin, cyclophosphamide and chlorambucil in lymphoid and myeloid leukaemia cells lines, and non-tumour control cells. In lymphoid leukaemia cell lines there was a synergistic reduction in ATP and glutathione levels, an induction of cell cycle arrest, DNA damage and apoptosis when quercetin, apigenin, emodin and rhein were combined with cisplatin and cyclophosphamide; and when apigenin and rhein were combined with chlorambucil. In myeloid leukaemia cells quercetin, apigenin and emodin showed a similar synergistic effect with all alkylating agents; however antagonistic effects were observed with some or all alkylating agents when combined with emodin, rhein and cis-stilbene. All synergistic effects were associated with reduced glutathione levels, DNA damage and apoptosis; whilst during antagonism the reverse effects were observed. The combination of alkylating agents, particularly cisplatin with polyphenols could be promising for the treatment of lymphoid leukaemias, with apigenin showing the greatest effects. Likewise in myeloid cells apigenin also synergised the action of all alkylating agents, suggesting that apigenin may also be beneficial in myeloid leukaemias.
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Purpose: Uveal melanoma (UM) is the most common primary intraocular malignancy in adults and approximately half of those diagnosed will die of metastasis. This study investigates whether UM progression is driven by a subpopulation of stem-like cells, termed "cancer stem cells" (CSCs). Methods: Expression of postulated stem cell markers aldehyde dehydrogenase (ALDH), CD44, and CD133 was analyzed in UM cell lines and primary UM short-term cultures (STCs) established from tumor samples. Additionally, the notion of a "cellular hierarchy" within UM was investigated. Finally, the phenomenon of phenotypic plasticity in response to environmental factors was explored. Results: We demonstrate that expression of ALDH, CD44, and CD133 does not select for a subpopulation of stem-like cells in either UM cell lines or UM STCs. Furthermore, there is an absence of a cellular hierarchy in cell lines and all cells in culture are able to drive tumor progression. Last, we show that established UM cell lines and UM STCs are plastic in nature and switch their phenotype in response to environmental stimuli. Conclusions: We hypothesize that this capacity to undergo phenotypic plasticity may be a consequence of neural crest lineage and renders the exploration of the CSC hypothesis extremely challenging in UM.
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Plasticidad de la Célula , Melanoma/patología , Células Madre Neoplásicas/patología , Neoplasias de la Úvea/patología , Antígeno AC133/metabolismo , Aldehído Deshidrogenasa/metabolismo , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Citometría de Flujo , Humanos , Receptores de Hialuranos/metabolismo , Melanoma/metabolismo , Células Madre Neoplásicas/metabolismo , Fenotipo , Ensayo de Tumor de Célula Madre , Neoplasias de la Úvea/metabolismoRESUMEN
The ability of phosphonium cations to act as intracellular transport vectors is well-established. Phosphonioalkylthiosulfate zwitterions, and ω-thioacetylalkylphosphonium salts, which act as 'masked thiolate' ligands, are useful precursors for the formation of phosphonium-functionalised gold nanoparticles, enabling the nanoparticles to be transported into cells for diagnostic and therapeutic purposes. In this study we have completed cytotoxicity studies of ω-thioacetylpropylphosphonium salts derived from triphenylphosphine and tri(4-fluorophenyl)phosphine, which show that the compounds are only toxic towards PC3 prostate cancer cells at high concentrations and at prolonged incubation periods and display IC50 values of 67 µM and 252 µM respectively, significantly higher than those of other phosphonium salts. MALDI-TOF-MS has been used to investigate the uptake of the compounds by PC3 cells and to quantify detectable levels of the compounds inside the cells. The structures of ω-thioacetylpropyl(tri-4-fluorophenyl) phosphonium bromide and the corresponding tri(4-fluorophenyl)phosphoniopropylthiosulfate zwitterion have been investigated by single crystal X-ray crystallography. The results show that molecules of the zwitterion are held together through an extensive array of electrostatic and non-covalent interactions. The unit cell of ω-thioacetylpropyl(tri-4-fluorophenyl)phosphonium bromide contains eight cations together with eight bromide anions and two waters of crystallisation, all held together through a complex network of hydrogen bonds. The differences in the molecular packing of the two compounds may account for the lower solubility of the zwitterion in aqueous solutions, compared with that of the phosphonium salt.
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Compuestos Organofosforados/química , Compuestos Organofosforados/toxicidad , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Enlace de Hidrógeno , Concentración 50 Inhibidora , Masculino , Modelos Moleculares , Estructura Molecular , Compuestos Organofosforados/análisis , Compuestos Organofosforados/farmacocinética , Neoplasias de la Próstata , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Two new triphenylarsonium alkylthiolate precursors, a thiosulfate zwitterion and a thioacetate salt, have been structurally characterised and their cytotoxicity evaluated against PC3 cells. The arsonium compounds have been used to prepare gold nanoparticles decorated with triphenylarsonium groups.
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Arsenicales/química , Arsenicales/farmacología , Portadores de Fármacos/química , Oro/química , Espacio Intracelular/efectos de los fármacos , Nanopartículas del Metal/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Modelos Moleculares , Conformación Molecular , Sulfatos/químicaRESUMEN
BACKGROUND: Mortality rates for leukemia are high despite considerable improvements in treatment. Since polyphenols exert pro-apoptotic effects in solid tumors, our study investigated the effects of polyphenols in haematological malignancies. The effect of eight polyphenols (quercetin, chrysin, apigenin, emodin, aloe-emodin, rhein, cis-stilbene and trans-stilbene) were studied on cell proliferation, cell cycle and apoptosis in four lymphoid and four myeloid leukemic cells lines, together with normal haematopoietic control cells. METHODS: Cellular proliferation was measured by CellTiter-Glo(®) luminescent assay; and cell cycle arrest was assessed using flow cytometry of propidium iodide stained cells. Apoptosis was investigated by caspase-3 activity assay using flow cytometry and apoptotic morphology was confirmed by Hoescht 33342 staining. RESULTS: Emodin, quercetin, and cis-stilbene were the most effective polyphenols at decreasing cell viability (IC50 values of 5-22 µM, 8-33 µM, and 25-85 µM respectively) and inducing apoptosis (AP50 values (the concentration which 50% of cells undergo apoptosis) of 2-27 µM, 19-50 µM, and 8-50 µM respectively). Generally, lymphoid cell lines were more sensitive to polyphenol treatment compared to myeloid cell lines, however the most resistant myeloid (KG-1a and K562) cell lines were still found to respond to emodin and quercetin treatment at low micromolar levels. Non-tumor cells were less sensitive to all polyphenols compared to the leukemia cells. CONCLUSIONS: These findings suggest that polyphenols have anti-tumor activity against leukemia cells with differential effects. Importantly, the differential sensitivity of emodin, quercetin, and cis-stilbene between leukemia and normal cells suggests that polyphenols are potential therapeutic agents for leukemia.
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Antineoplásicos Fitogénicos/farmacología , Emodina/farmacología , Linfocitos/efectos de los fármacos , Células Mieloides/efectos de los fármacos , Quercetina/farmacología , Estilbenos/farmacología , Antineoplásicos Fitogénicos/química , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Emodina/química , Humanos , Concentración 50 Inhibidora , Linfocitos/patología , Estructura Molecular , Células Mieloides/patología , Especificidad de Órganos , Quercetina/química , Estereoisomerismo , Estilbenos/química , Relación Estructura-ActividadRESUMEN
Uveal melanomas (UM) are aggressive ocular tumours that spread to the liver. They are characterised by alterations of chromosome 3 and 8, which are highly predictive of a poor prognosis. Unfortunately, being able to identify those patients with aggressive disease has not, as yet, translated into improved survival. Recently, mutations of guanine nucleotide-binding protein G(q) subunit alpha (GNAQ, or G-alpha-q), which effectively turn it into a dominantly acting oncogene, have been identified in approximately half of UM. These mutations are specific to UM and other non-cutaneous melanomas, and are not found in normal tissues, thus making them potential therapeutic targets. Here, the authors review the background to GNAQ in UM and explore what makes it such an interesting target for the future treatment of patients.
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Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Mutación/genética , Proteínas de Neoplasias/genética , Progresión de la Enfermedad , Humanos , Neoplasias Hepáticas/secundario , Melanocitos/patología , Melanoma/genética , Melanoma/patología , Melanoma/secundario , Pronóstico , Transducción de Señal/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Neoplasias de la Úvea/genética , Neoplasias de la Úvea/patología , Neoplasias de la Úvea/secundarioRESUMEN
BACKGROUND: Chlamydia trachomatis is an important pathogen, being the commonest sexually transmitted bacterial disease in the Western world and is also implicated in a number of acute and chronic diseases. Persistent infections of C. trachomatis are particularly associated with chronic infections, which although eliciting an immune response, result in tissue damage leading to complications such as pelvic inflammatory disease. Interferon (IFN)-gamma is known to induce persistent infections of C. trachomatis both in vitro and in vivo. METHODS: A model of IFN-gamma-induced persistence containing aberrant inclusions of C. trachomatis was developed in the HEp-2 cell line. Morphological changes to inclusions were assessed by fluorescence immunocytochemistry and transcript levels determined by Real-Time RT-PCR. To assess infectivity of C. trachomatis in an IFN-gamma-induced persistent state, cultures containing aberrant inclusions were inoculated onto fresh HEp-2 monolayers. RESULTS: IFN-gamma induced aberrant inclusion formation at 0.01 ng/ml. Doses from 0.05 to 100 ng/ml did not significantly increase numbers of aberrant inclusions, and some normal inclusions were observed at the highest dose of IFN-gamma. Transfer of IFN-gamma-treated C. trachomatis onto fresh cultures confirmed the infectivity of these cultures. Real-Time RT-PCR identified apparent increased expression of the C. trachomatis heat-shock response genes ct604 and ct755 at 96-h post-infection. However comparisons with control cultures suggest that this more likely reflects a failure to down regulate gene expression as observed in untreated cultures. CONCLUSIONS: These data show that whereas IFN-gamma induces aberrant inclusion formation, many normal inclusions are still observed at high doses of IFN-gamma, and that the infectivity of such cultures is presumably from these. Transcriptional changes observed in response to IFN-gamma suggest a failure of the C. trachomatis life cycle in response to IFN-gamma, however IFN-gamma-induced transcriptional changes may be masked by the presence of normal inclusions. The implications of these observations in relation to models of persistence of C. trachomatis are discussed.
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Chlamydia trachomatis/inmunología , Chlamydia trachomatis/patogenicidad , Perfilación de la Expresión Génica , Hepatocitos/inmunología , Hepatocitos/microbiología , Interacciones Huésped-Patógeno , Interferón gamma/inmunología , Línea Celular , Regulación Bacteriana de la Expresión Génica , Humanos , Cuerpos de Inclusión/microbiología , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
BACKGROUND: According to the cancer stem cell hypothesis, tumor growth is sustained by a subpopulation of cancer stem/progenitor-like cells. Self-renewal and high clonogenic potential are characteristics shared by normal stem and neoplastic stem/progenitor-like cells. We investigated whether human prostate cancer specimens contain cells with these properties. METHODS: Self-renewal and clonogenic potential were assessed by serial passaging of spheres and colony formation, respectively. Gene expression was analyzed by real time PCR. Protein expression was detected by immunocytochemistry. The neoplastic nature of the cells was verified by detection of the TMPRSS2/ERG gene fusion expression. RESULTS: The epithelial fraction isolated from surgical specimens generated colonies in 68% (19/28) of the patients. Laminin adhesion selected for cells with high clonogenic potential. The epithelial fraction from 85% (42/49) of the patients generated primary prostaspheres. Serial passaging of prostaspheres demonstrated their self-renewal capacity, which is also supported by their expression of the stem cell markers Oct-4, Nanog, Bmi-1, and Jagged-1 mRNA. Cells derived from prostaspheres were more clonogenic than the parental epithelial fraction. The pattern of mRNA expression in prostaspheres resembled that of the basal compartment of the prostate (CK5(+)/CK14(+)/CK19(high)/CK18(-/low)/c-met(+)/AR(-/low)/PSA(-/low)), but also included stem cell markers (CD49b(+)/CD49f(+)/CD44(+)/DeltaNp63(+)/Nestin(+)/CD133(+)). The distribution of marker expression in prostaspheres suggests their heterogeneous cell composition. Prostaspheres expressed significantly higher PSCA mRNA levels than the epithelial fraction. CONCLUSION: Human prostate cancer specimens contain neoplastic cells with self-renewal and clonogenic potential, which can be enriched and perpetuated in prostaspheres. Prostaspheres should prove valuable for the identification of prostate cancer stem/progenitor-like cells.
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Células Madre Neoplásicas/patología , Neoplasias de la Próstata/patología , Antígenos CD/biosíntesis , Antígenos CD/genética , Antígenos de Neoplasias , Proteínas de Unión al Calcio/biosíntesis , Proteínas de Unión al Calcio/genética , Procesos de Crecimiento Celular/fisiología , Células Clonales/patología , Proteínas Ligadas a GPI , Perfilación de la Expresión Génica , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/genética , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas de Filamentos Intermediarios/biosíntesis , Proteínas de Filamentos Intermediarios/genética , Proteína Jagged-1 , Queratina-14/biosíntesis , Queratina-14/genética , Masculino , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Proteína Homeótica Nanog , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Nestina , Factor 3 de Transcripción de Unión a Octámeros/biosíntesis , Factor 3 de Transcripción de Unión a Octámeros/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-met/biosíntesis , Proteínas Proto-Oncogénicas c-met/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Estudios Retrospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Serrate-JaggedRESUMEN
Tubulin-binding vascular-disrupting agents (VDA) are currently in clinical trials for cancer therapy but the factors that influence tumor susceptibility to these agents are poorly understood. We evaluated the consequences of modifying tumor vascular morphology and function on vascular and therapeutic response to combretastatin-A4 3-O-phosphate (CA-4-P), which was chosen as a model VDA. Mouse fibrosarcoma cell lines that are capable of expressing all vascular endothelial growth factor (VEGF) isoforms (control) or only single isoforms of VEGF (VEGF120, VEGF164, or VEGF188) were developed under endogenous VEGF promoter control. Once tumors were established, VEGF isoform expression did not affect growth or blood flow rate. However, VEGF188 was uniquely associated with tumor vascular maturity, resistance to hemorrhage, and resistance to CA-4-P. Pericyte staining was much greater in VEGF188 and control tumors than in VEGF120 and VEGF164 tumors. Vascular volume was highest in VEGF120 and control tumors (CD31 staining) but total vascular length was highest in VEGF188 tumors, reflecting very narrow vessels forming complex vascular networks. I.v. administered 40 kDa FITC-dextran leaked slowly from the vasculature of VEGF188 tumors compared with VEGF120 tumors. Intravital microscopy measurements of vascular length and RBC velocity showed that CA-4-P produced significantly more vascular damage in VEGF120 and VEGF164 tumors than in VEGF188 and control tumors. Importantly, this translated into a similar differential in therapeutic response, as determined by tumor growth delay. Results imply differences in signaling pathways between VEGF isoforms and suggest that VEGF isoforms might be useful in vascular-disrupting cancer therapy to predict tumor susceptibility to VDAs.
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Inhibidores de la Angiogénesis/farmacología , Fibrosarcoma/irrigación sanguínea , Fibrosarcoma/tratamiento farmacológico , Estilbenos/farmacología , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Animales , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Femenino , Fibrosarcoma/metabolismo , Inmunohistoquímica , Ratones , Ratones SCID , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Isoformas de Proteínas , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
A commonly used monoclonal antibody targeting osteoprotegerin (OPG), MAB8051, detects a truncated protein species in breast and prostate cancer cell lysates. OPG expression has been reported to contribute to cell survival of both of these cancers. We hypothesised that the truncated protein represented a unique tumour-associated OPG isoform. However, here we show that the truncated protein identified by MAB8051 in cancer cell lines is carbonic anhydrase II (CA II), also implicated in tumour biology. We clearly demonstrate cross-reactivity of this OPG antibody in western blots. OPG and CA II RNA-interference studies confirmed the identity of the bands. We show almost identical staining patterns between MAB8051 and CA II immunohistochemistry of different human tissue types and human tumour types using serial sections. We conclude that care should be exercised using this antibody for immunohistochemistry studies, without additional in situ hybridisation, or parallel use of other OPG-specific antibodies.
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Anticuerpos Monoclonales/inmunología , Anhidrasa Carbónica II/inmunología , Anhidrasa Carbónica II/metabolismo , Neoplasias/inmunología , Neoplasias/metabolismo , Osteoprotegerina/inmunología , Secuencia de Aminoácidos , Anhidrasa Carbónica II/química , Anhidrasa Carbónica II/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Datos de Secuencia Molecular , Peso Molecular , Neoplasias/genética , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Procesamiento Proteico-Postraduccional , ARN Mensajero/genética , ARN Interferente Pequeño/genéticaRESUMEN
TGFbeta 1 and FGF2 are autocrine growth factors in prostatic stroma and are elevated in benign prostatic hyperplasia (BPH), a disease characterized by enlargement of the stromal compartment of the prostate. TGFbeta1 has a biphasic effect on proliferation of prostatic stromal cells, inducing proliferation at low doses (< 1 ng/ml), but inhibiting growth above 1 ng/ml. This study investigated the role of TGFP 1 and FGF2 on growth factor bioavailability and extracellular matrix (ECM) accumulation synthesis in cultured prostatic stromal cells. Real-Time-PCR showed that TGFbeta1 expression is auto-inductive, whereas FGF2 is auto-repressive. FGF2 also induced TGFbeta1 secretion in the absence of increased TGFbeta1 mRNA expression. TGFbeta1 and FGF2 have opposing actions on Type 1 collagen expression, a finding confirmed by Western blotting. The bioavailability of TGFbeta1 regulated by FGF2 may represent part of a negative feedback mechanism controlling stromal growth, differentiation and ECM. Dysregulation of this pathway in favour of TGFbeta1 bioactivity may exacerbate BPH.
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Matriz Extracelular/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/farmacología , Próstata/efectos de los fármacos , Células del Estroma/efectos de los fármacos , Factor de Crecimiento Transformador beta1/farmacología , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Matriz Extracelular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Humanos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Masculino , Modelos Biológicos , Datos de Secuencia Molecular , Próstata/citología , Hiperplasia Prostática , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
In addition to its role in bone turnover, osteoprotegerin (OPG) has been reported to bind to and inhibit Tumour necrosis factor-related apoptosis inducing ligand (TRAIL). TRAIL is produced in tumours by invading monocytes, inducing apoptosis in neoplastic cells sensitive to this cytokine. OPG production by tumour cells would therefore be a novel mechanism whereby cancer cells evade host defences and gain a growth advantage. In this study we show that OPG produced by breast cancer cells enhances tumour cell survival by inhibiting TRAIL-induced apoptosis. OPG expression by breast cancer cells (MDA-MB 436/231) grown in vitro was examined using PCR and ELISA, and the sensitivity of these cells to TRAIL was determined. The effects of OPG on TRAIL induced apoptosis was investigated by exposing MDA-MB 436 cells to TRAIL, in the presence or absence of OPG, followed by assessment of nuclear morphology. We found that the levels of OPG produced were sufficient to inhibit TRAIL-induced apoptosis, suggesting that OPG may play a role in tumour cell survival. We also examined the expression pattern of OPG in a selection of breast tumours (n=400) by immunohistochemistry, and related OPG expression to the clinico-pathological data for each tumour. OPG expression was found to be negatively correlated with increasing tumour grade. To our knowledge these results are the first to demonstrate that OPG can act as an endocrine survival factor for breast cancer cells, as well as reporting the expression patterns of OPG in a large cohort of human breast tumours.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Supervivencia Celular , Glicoproteínas/metabolismo , Glicoproteínas de Membrana/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Apoptosis , Línea Celular Tumoral , Femenino , Humanos , Inmunohistoquímica , Osteoprotegerina , Ligando Inductor de Apoptosis Relacionado con TNFRESUMEN
Tumour cell cultures are often highly heterogeneous, containing sub-populations of cells with differing characteristics. To identify chromosome abnormalities that are associated with the invasive phenotype, we isolated highly invasive uveal melanoma cell populations using the Transwell assay. Using this invasion assay, invasive sub-populations of primary uveal melanoma short-term cultures, and an established cell line, were specifically isolated. A series of sequential assays were undertaken to enrich the invasive population, and the enhanced invasive ability was confirmed by Transwell invasion assay. Chromosome abnormalities in invasive and parental cells were identified by karyotyping and confirmed by comparative genome hybridisation. Invasive sub-populations of uveal melanoma cells were isolated from 3 uveal melanoma short term cultures and a uveal melanoma cell line. In all cases, invasive sub-populations had either acquired additional chromosome abnormalities to those present in the parental cell line, or other abnormalities present in the parental lines were lost. In the established cell line (SOM 157), invasive cells were characterised by widespread chromosomal instability, frequent telomere associations and additional copies of chromosome 20. The invasive phenotype of SOM 196 associated with the presence of a derivative chromosome 5, der(5)t(5;11)(q35;q12) whilst a translocation t(17;20)(q12;q13) was predominant amongst non-invasive cells. In two additional cultures, deletions on chromosome 6q were associated with reduced invasive ability. In conclusion, highly invasive populations of uveal melanoma cells demonstrate chromosomal abnormalities that differ from non-invasive cells. These include chromosome instability and abnormalities of chromosome 20, observations echoing those seen in metastatic uveal melanoma.