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Hydroalcoholic extracts obtained from buds of P. nigra, P. deltoides and P. trichocarpa were characterized by HPLC-DAD-MS analysis and subsequently evaluated in vitro for their antioxidant and anti-inflammatory activities. ABTS and DPPH assays evidenced that P. nigra showed the best antioxidant activity in line with its highest total phenolic content. The analysis of the anti-inflammatory activity clearly demonstrated that all extracts suppressed the production of key pro-inflammatory cytokines (IL-6, Il-1ß and TNF-α) and HMGB1 inflammatory danger signal. These results show antioxidant and critical anti-inflammatory activities mediated by the extracts, emphasising their potentiality as therapeutic agents.
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Populus , Salicaceae , Antiinflamatorios/farmacología , Antioxidantes/química , Antioxidantes/farmacología , Extractos Vegetales/química , Extractos Vegetales/farmacología , Populus/químicaRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Kiwifruit is native to eastern China and many are the references about the consumption of fruits and fruits extracts of the Actinidia plants in Chinese traditional medicine as therapeutic food supplements to prevent and/or counteract numerous disorders including inflammation-related diseases like cancer. AIM OF THE STUDY: Aim of the present work was to obtain a kiwifruit peel extract, rich in polyphenols, and to explore the anti-inflammatory potential by analyzing its capability to target multiple pathways involved in monocyte-mediated inflammatory response. MATERIALS AND METHODS: The extract was obtained from the fruit peel of Actinidia deliciosa (A.Chev.) C.F.Liang & A.R.Ferguson, cv Hayward and characterized by HPLC-DAD-ESI-MS. Lipopolysaccharide-stimulated THP-1 monocytes were used as a model of human inflammation in vitro. RESULTS: Analytical data evidenced that procyanidins resulted the main polyphenols present in the extract, representing the 92% w/w of the total. The extract inhibited the production of inflammatory molecules such as IL-6, IL-1ß, TNF-α pro-inflammatory cytokines, HMGB1 danger signal and granzyme B serine protease by activated monocytes. In particular, an inhibitory activity of 81%, 68%, 63%, 76% and 60% on the extracellular release of IL-6, IL-1ß, TNF-α, HMGB1 and granzyme B, respectively, was observed by western blot analysis. Moreover, the extract prevented STAT3 activation and promoted autophagy. CONCLUSIONS: The reported findings demonstrated a strong and broad anti-inflammatory profile of the kiwifruit peel extract, which makes it a promising preventive and therapeutic natural ingredient for nutraceutical, cosmetic and pharmaceutical formulations to counteract multiple inflammatory disorders.
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Actinidia , Antiinflamatorios/farmacología , Monocitos/efectos de los fármacos , Extractos Vegetales/farmacología , Autofagia/efectos de los fármacos , Citocinas/metabolismo , Frutas , Granzimas/metabolismo , Proteína HMGB1/metabolismo , Humanos , Lipopolisacáridos/farmacología , Monocitos/metabolismo , Factor de Transcripción STAT3/metabolismo , Células THP-1RESUMEN
In metabolic diseases, like type 2 diabetes (T2D), adipose tissue (AT) is infiltrated by macrophages and other leukocytes - which secrete many bioactive peptides leading to local and systemic low-grade chronic inflammation - and undergoes remodeling and aberrant fibrosis. Granzyme B (GrB) is a serine protease produced by some leukocytes, including cytotoxic lymphocytes and macrophages. It exerts both intracellular apoptotic function and extracellular functions, leading to tissue injury, inflammation and repair. Elevated circulating GrB levels have been found in aging- and inflammation-associated diseases and a role for GrB in the pathogenesis of several chronic inflammatory diseases has been reported. Aims of this study were to investigate circulating GrB levels in T2D patients in relation to their systemic inflammatory profile and to unravel its correlates. For this cross-sectional study, we recruited 51 consecutive T2D patients referring to our diabetes outpatient clinics (Sapienza University, Rome, Italy) for metabolic evaluations, and 29 sex, age and body mass index comparable non-diabetic subjects as control group. Study participants underwent clinical work-up; fasting blood sampling was performed for routine biochemistry and for inflammatory profile (CRP, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α, IFN-γ, GM-CSF, adiponectin, WISP1); serum GrB was measured by Human Granzyme-B Platinum Elisa kit (Affymetrix EBIO). We found that T2D patients had serum levels of GrB significantly higher than the control group (10.17⯱â¯12.6 vs 7.2⯱â¯14.1â¯pg/ml, pâ¯=â¯0.03). Moreover, in T2D patients increased GrB correlated with unfavorable inflammatory profile, as described by elevated levels of validated adipokines such as IL-6 (pâ¯=â¯0.04), TNF-α (pâ¯=â¯0.019) and WISP1 (pâ¯=â¯0.005). Furthermore, multivariate linear regression analysis showed that increased GrB was associated with T2D diagnosis independently from possible confounders. In conclusion, our results show that increased levels of circulating GrB are associated with T2D diagnosis and correlates with markers of AT-linked systemic inflammation, suggesting a potential role for GrB in the inflammatory and reactive processes occurring in metabolic diseases.
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Diabetes Mellitus Tipo 2/sangre , Granzimas/sangre , Inflamación/sangre , Adipoquinas/sangre , Adiponectina/sangre , Tejido Adiposo/metabolismo , Biomarcadores/sangre , Estudios Transversales , Diabetes Mellitus Tipo 2/metabolismo , Femenino , Humanos , Inflamación/metabolismo , Masculino , Persona de Mediana Edad , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Treatment of olive vegetation waste with tyrosinase immobilized on multiwalled carbon nanotubes increased the antioxidant activity as a consequence of the conversion of phenols to corresponding catechol derivatives, as evaluated by DPPH, Comet assay, and micronucleus analyses. During this transformation, 4-hydroxyphenethyl alcohol (tyrosol) was quantitatively converted to bioactive 3,4-dihydroxyphenethyl alcohol (hydroxytyrosol). The hydroxytyrosol-enriched olive vegetation waste also promoted autophagy and inhibited the inflammatory response in human THP-1 monocytes.
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Antioxidantes/farmacología , Autofagia/efectos de los fármacos , Monocitos/citología , Monocitos/inmunología , Olea/química , Alcohol Feniletílico/análogos & derivados , Extractos Vegetales/farmacología , Residuos/análisis , Antioxidantes/química , Biocatálisis , Línea Celular , Humanos , Monocitos/efectos de los fármacos , Monofenol Monooxigenasa/química , Alcohol Feniletílico/química , Alcohol Feniletílico/farmacología , Extractos Vegetales/químicaRESUMEN
Docosahexaenoic acid (DHA), a ω-3 polyunsaturated fatty acid found in fish oil, is a multi-target agent and exerts anti-inflammatory and anticancer activities alone or in combination with chemotherapies. Combinatorial anticancer therapies, which induce immunogenic apoptosis, autophagy and STAT3 inhibition have been proposed for long-term therapeutic success. Here, we found that DHA promoted immunogenic apoptosis in multiple myeloma (MM) cells, with no toxicity on PBMCs and DCs. Immunogenic apoptosis was shown by the emission of specific DAMPs (CRT, HSP90, HMGB1) by apoptotic MM cells and the activation of their pro-apoptotic autophagy. Moreover, immunogenic apoptosis was directly shown by the activation of DCs by DHA-induced apoptotic MM cells. Furthermore, we provided the first evidence that DHA activated autophagy in PBMCs and DCs, thus potentially acting as immune stimulator and enhancing processing and presentation of tumor antigens by DCs. Finally, we found that DHA inhibited STAT3 in MM cells. STAT3 pathway, essential for MM survival, contributed to cancer cell apoptosis by DHA. We also found that DHA inhibited STAT3 in blood immune cells and counteracted STAT3 activation by tumor cell-released factors in PBMCs and DCs, suggesting the potential enhancement of the anti-tumor function of multiple immune cells and, in particular, that of DCs.
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BACKGROUND: Granzyme B (GrB) is a serine protease, traditionally known as expressed by cytotoxic lymphocytes to induce target cell apoptosis. However, it is emerging that GrB, being also produced by a variety of normal and neoplastic cells and potentially acting on multiple targets, might represent a powerful regulator of a wide range of fundamental biological processes. We have previously shown that GrB is expressed in urothelial carcinoma tissues and its expression is associated to both pathological tumor spreading and EMT. We have also shown that docosahexaenoic acid (DHA), a dietary ω-3 polyunsaturated fatty acid with anti-tumor activity, while inhibiting urothelial and pancreatic carcinoma cell invasion also inhibited their GrB expression in vitro. In this study, we characterized a panel of colorectal carcinoma (CRC) cells, with different invasive capabilities, for GrB expression and for the contribution of GrB to their EMT and invasive phenotype. In addition, we investigated the effect of DHA on CRC cell-associated GrB expression, EMT and invasion. METHODS: The expression levels of GrB and EMT-related markers were evaluated by Western blotting. GrB knockdown was performed by Stealth RNAi small interfering RNA silencing and ectopic GrB expression by transfection of human GrB vector. Cell invasion was determined by the BioCoat Matrigel invasion chamber test. RESULTS: GrB was produced in 57.1% CRC cell lines and 100% CRC-derived Cancer Stem Cells. Although GrB was constitutive expressed in both invasive and noninvasive CRC cells, GrB depletion in invasive CRC cells downmodulated their invasion in vitro, suggesting a contribution of GrB to CRC invasiveness. GrB loss or gain of function downmodulated or upmodulated EMT, respectively, according to the analysis of cancer cell expression of three EMT biomarkers (Snail1, E-cadherin, N-cadherin). Moreover, TGF-ß1-driven EMT was associated to the enhancement of GrB expression in CRC cell lines, and GrB depletion led to downmodulation of TGF-ß1-driven EMT. In addition, DHA inhibited GrB expression, EMT and invasion in CRC cells in vitro. CONCLUSIONS: These findings present a novel role for GrB as upmodulator of EMT in CRC cells. Moreover, these results support the use of DHA, a dietary compound without toxic effects, as adjuvant in CRC therapy.
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Antineoplásicos/farmacología , Neoplasias Colorrectales/metabolismo , Ácidos Docosahexaenoicos/farmacología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Granzimas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Granzimas/genética , Humanos , Invasividad Neoplásica , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Cancer is a major disease worldwide. Despite progress in cancer therapy, conventional cytotoxic therapies lead to unsatisfactory long-term survival, mainly related to development of drug resistance by tumor cells and toxicity towards normal cells. n-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), can exert anti-neoplastic activity by inducing apoptotic cell death in human cancer cells either alone or in combination with conventional therapies. Indeed, n-3 PUFAs potentially increase the sensitivity of tumor cells to conventional therapies, possibly improving their efficacy especially against cancers resistant to treatment. Moreover, in contrast to traditional therapies, n-3 PUFAs appear to cause selective cytotoxicity towards cancer cells with little or no toxicity on normal cells. This review focuses on studies investigating the cytotoxic activity of n-3 PUFAs against cancer cells via apoptosis, analyzing the molecular mechanisms underlying this effective and selective activity. Here, we highlight the multiple molecules potentially targeted by n-3 PUFAs to trigger cancer cell apoptosis. This analysis can allow a better comprehension of the potential cytotoxic therapeutic role of n-3 PUFAs against cancer, providing specific information and support to design future pre-clinical and clinical studies for a better use of n-3 PUFAs in cancer therapy, mainly combinational therapy.
RESUMEN
OBJECTIVES: Immunostimulation by anticancer cytotoxic drugs is needed for long-term therapeutic success. Activation of dendritic cells (DCs) is crucial to obtain effective and long-lasting anticancer T-cell mediated immunity. The aim of this study was to explore the effect of capsaicin-mediated cell death of bladder cancer cells on the activation of human monocyte-derived CD1a+ immature DCs. METHODS: Immature DCs (generated from human peripheral blood-derived CD14+ monocytes cultured with granulocyte-macrophage colony stimulating factor and interleukin-4) were cocultured with capsaicin (CPS)-induced apoptotic bladder cancer cells. DC activation was investigated using immunofluorescence and flow cytometric analysis for key surface molecules. In some experiments, CD91 was silenced in immature DCs. RESULTS: We found that capsaicin-mediated cancer cell apoptosis upregulates CD86 and CD83 expression on DCs, indicating the induction of DC activation. Moreover, silencing of CD91 (a common receptor for damage-associated molecular patterns, such as calreticulin and heat-shock protein-90/70) in immature DCs led to the inhibition of DC activation. CONCLUSIONS: Our data show that CPS-mediated cancer cell apoptosis activates DCs via CD91, suggesting CPS as an attractive candidate for cancer therapy.
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Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Capsaicina/farmacología , Capsicum/química , Células Dendríticas/metabolismo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Adyuvantes Inmunológicos/farmacología , Adyuvantes Inmunológicos/uso terapéutico , Antígenos CD/metabolismo , Antígenos CD1/metabolismo , Antineoplásicos Fitogénicos/uso terapéutico , Capsaicina/uso terapéutico , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Interleucina-4/metabolismo , Monocitos/metabolismo , Fitoterapia , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Neoplasias de la Vejiga Urinaria/inmunología , Neoplasias de la Vejiga Urinaria/metabolismoRESUMEN
ω -3 Polyunsaturated fatty acids (PUFAs), mainly present in fish oil, are part of the human diet. Among PUFAs, docosahexaenoic acid (DHA) has received particular attention for its anti-inflammatory, antiproliferative, proapoptotic, antiangiogenetic, anti-invasion, and antimetastatic properties. These data suggest that DHA can exert antitumor activity potentially representing an effective adjuvant in cancer chemotherapy. This review is focused on current knowledge supporting the potential use of DHA for the enhancement of the efficacy of anticancer treatments in relation to its ability to enhance the uptake of anticancer drugs, regulate the oxidative status of tumor cells, and inhibit tumor cell invasion and metastasis.
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Adyuvantes Farmacéuticos/uso terapéutico , Ácidos Docosahexaenoicos/uso terapéutico , Neoplasias/tratamiento farmacológico , Adyuvantes Farmacéuticos/farmacología , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Ácidos Docosahexaenoicos/farmacología , Humanos , Metástasis de la Neoplasia , Neoplasias/patología , Estrés Oxidativo/efectos de los fármacosRESUMEN
Few conventional cytotoxic anticancer therapeutics induce immunogenic cell death (ICD). This means that they induce tumor cells to undergo apoptosis while eliciting the emission of a spatiotemporal-defined combination of damage-associated molecular patterns (DAMPs) decoded by the immune system to activate antitumor immunity effective for long-term therapeutic success. The neurotoxin capsaicin (CPS) can induce both cancer cell apoptosis and immune-mediated tumor regression. In the present study, we investigated whether CPS is capable of eliciting the emission of ICD hallmarks in human bladder cancer cell lines undergoing apoptosis. We demonstrated that CPS induces pre- and early apoptotic cell surface exposure of calreticulin (CRT), HSP90, and HSP70 as well as ATP release. Moreover, CRT exposure was prevented by inhibition of endoplasmic reticulum-Golgi traffic by brefeldin A. Furthermore, high-mobility group box 1, HSP90, and HSP70 were passively released at late apoptotic stages. We provide the first evidence that CPS is an inducer of ICD hallmarks, suggesting CPS as a novel potential immunogenic cytotoxic agent.
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Apoptosis/efectos de los fármacos , Capsaicina/toxicidad , Sistema Inmunológico/metabolismo , Adenosina Trifosfato/metabolismo , Antineoplásicos/toxicidad , Brefeldino A/antagonistas & inhibidores , Brefeldino A/metabolismo , Calreticulina/metabolismo , Línea Celular Tumoral , Proteína HMGB1/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Sistema Inmunológico/efectos de los fármacos , Neoplasias de la Vejiga Urinaria/inmunología , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Failure in cytokinesis, the final step in cell division, by generating tetra- and polyploidization promotes chromosomal instability, a hallmark of cancer. Here we show that HIPK2, a kinase involved in cell fate decisions in development and response to stress, controls cytokinesis and prevents tetraploidization through its effects on histone H2B. HIPK2 binds and phosphorylates histone H2B at S14 (H2B-S14(P)), and the two proteins colocalize at the midbody. HIPK2 depletion by targeted gene disruption or RNA interference results in loss of H2B-S14(P) at the midbody, prevention of cell cleavage, and tetra- and polyploidization. In HIPK2 null cells, restoration of wild-type HIPK2 activity or expression of a phosphomimetic H2B-S14D derivative abolishes cytokinesis defects and rescues cell proliferation, showing that H2B-S14(P) is required for a faithful cytokinesis. Overall, our data uncover mechanisms of a critical HIPK2 function in cytokinesis and in the prevention of tetraploidization.
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Proteínas Portadoras/metabolismo , Citocinesis , Histonas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Western Blotting , Proteínas Portadoras/genética , División Celular , Línea Celular , Línea Celular Tumoral , Embrión de Mamíferos/citología , Fibroblastos/citología , Fibroblastos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Células HeLa , Histonas/genética , Humanos , Ratones , Ratones Noqueados , Microscopía Fluorescente , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , TetraploidíaRESUMEN
Fish oil-derived n-3 polyunsaturated fatty acids (n-3 PUFAs) inhibit invasion of some tumor cell types in vitro and in vivo. The mechanisms underlying this activity are unclear. Here, we examined the capability of n-3 PUFA-docosahexaenoic acid (22:6n-3; DHA) to affect the invasiveness of human RT112 urinary bladder and PT45 pancreatic carcinoma cell lines in vitro and the mechanism underlying this activity; n-6 PUFA-arachidonic acid (20:4n-6; AA) served as control. We showed that, in contrast to AA, 25, 50 and 100 µM DHA significantly inhibited in a dose-dependent manner the invasion through Matrigel of both RT112 and PT45 cells. Then, we analyzed whether the serine proteinase granzyme B (GrB), originally known as cytotoxic molecule of lymphocytes and recently also characterized for its extracellular functions such as invasion promotion of bladder cancer cells, might be involved in the invasion inhibitory activity exerted by DHA. We demonstrated that, accordingly to RT112 bladder cancer cells, PT45 cells expressed GrB and GrB promoted their invasion, since stealth RNA interference-mediated down-regulation of GrB dramatically suppressed PT45 cell invasion. Notably, we also showed that, in contrast to AA, 25, 50 and 100 µM DHA induced a dose-dependent down-modulation of GrB expression in both RT112 and PT45 cells. In conclusion, DHA can reduce the invasive phenotype of bladder and pancreatic carcinoma cells, and we provide the first evidence for a possible causative role of GrB in DHA-induced inhibition of cancer cell invasion. The potential use of fish oil as adjuvant antibladder and antipancreatic cancer agent may be suggested.
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Ácidos Docosahexaenoicos/farmacología , Regulación hacia Abajo , Granzimas/genética , Neoplasias Pancreáticas/patología , Vejiga Urinaria/patología , Ácidos Grasos Insaturados/farmacología , Regulación Neoplásica de la Expresión Génica , Granzimas/metabolismo , Humanos , Invasividad Neoplásica , Neoplasias Pancreáticas/metabolismo , Interferencia de ARN , Células Tumorales Cultivadas , Vejiga Urinaria/metabolismoRESUMEN
Some anticancer chemotherapeutics, such as anthracyclines and oxaliplatin, elicit immunogenic apoptosis, meaning that dying cancer cells are engulfed by dendritic cells and tumor antigens are efficiently presented to CD8+ T cells, which control residual tumor cells. Immunogenic apoptosis is characterized by pre-apoptotic cell surface exposure of calreticulin (CRT), which usually resides into the endoplasmic reticulum. We investigated the ability of the n3-polyunsaturated fatty acid docosahexaenoic acid (22:6n3, DHA) to induce pre-apoptotic CRT exposure on the surface of the human PaCa-44 pancreatic and EJ bladder cancer cell lines. Cells were treated with 150 µM DHA for different time periods, and, by immunoblot and immunofluorescence, we showed that DHA induced CRT exposure, before the apoptosis-associated phosphatidylserine exposure. As for the known immunogenic compounds, CRT exposure was inhibited by the antioxidant GSH, the pan-caspase zVAD-FMK, and caspase-8 IETD-FMK inhibitor. We provide the first evidence that DHA induces CRT exposure, representing thus a novel potential anticancer immunogenic chemotherapeutic agent.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Calreticulina/metabolismo , Ácidos Docosahexaenoicos/farmacología , Antineoplásicos/inmunología , Apoptosis/inmunología , Calreticulina/inmunología , Línea Celular Tumoral , Separación Celular , Ácidos Docosahexaenoicos/inmunología , Citometría de Flujo , Humanos , ImmunoblottingRESUMEN
Granzyme B (GrB) is a serine proteinase known to be expressed by cytotoxic lymphocytes and to induce, in presence of perforin (Pf), apoptosis in target cells. Recently, GrB expression has been shown (often in absence of Pf) in nonlymphoid cells, but its function is not defined. In our study, we investigated GrB and Pf expression in bladder cancer cell lines and in urothelial carcinoma (UC) tissues by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, ELISA, immunofluorescence and immunohistochemistry. We also assessed the function of GrB in UC cells; the in vitro function of GrB was examined by loss-of-function experiments. Our results revealed that GrB is expressed, in absence of Pf, in UC cells. Significant differences were found between GrB expression and both increasing pathological tumor spreading and high-grade vs. low-grade pTa tumors. Notably, GrB in UC tissues was concentrated at the cancer invasion front and was expressed in neoplastic cells undergoing epithelial-mesenchymal transition, a key event in carcinoma invasion. Indeed, GrB-positive cells also expressed Snail, N-cadherin or were negative for E-cadherin. GrB expressed in tumor cell lines was enzymatically active and capable of vitronectin cleavage, implying extracellular matrix (ECM) remodeling by GrB. Inhibition of GrB activity or Stealth RNA interference-mediated GrB gene silencing markedly suppressed bladder cancer cell invasion through matrigel. This data provides the first evidence for a role of GrB in promoting cancer cell invasion. Taken together, our findings suggest that GrB, via ECM degradation, contributes to the establishment of the UC invasive phenotype.
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Granzimas/metabolismo , Invasividad Neoplásica , Neoplasias de la Vejiga Urinaria/enzimología , Secuencia de Bases , Western Blotting , Línea Celular Tumoral , Cartilla de ADN , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunohistoquímica , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
The Polycomb group (PcG) of proteins conveys epigenetic inheritance of repressed transcriptional states. In Drosophila, the Polycomb repressive complex 1 (PRC1) maintains the silent state by inhibiting the transcription machinery and chromatin remodelling at core promoters. Using immunoprecipitation of in vivo formaldehyde-fixed chromatin in phenotypically diverse cultured cell lines, we have mapped PRC1 components, the histone methyl transferase (HMT) Enhancer of zeste (E(z)) and histone H3 modifications in active and inactive PcG-controlled regions. We show that PRC1 components are present in both cases, but at different levels. In particular, active target promoters are nearly devoid of E(z) and Polycomb. Moreover, repressed regions are trimethylated at lysines 9 and 27, suggesting that these histone modifications represent a mark for inactive PcG-controlled regions. These PcG-specific repressive marks are maintained by the action of the E(z) HMT, an enzyme that has an important role not only in establishing but also in maintaining PcG repression.