RESUMO
BACKGROUND: Nutritional deficit and oral iron gastrointestinal intolerance may be a common cause of iron deficiency, which can be managed by pharmacists. AIM: To understand the prevalence of iron deficiency in women of childbearing age with a self-reported history of intolerance to oral iron and the tolerability of three doses of an iron-whey-protein formulation in the care of these women. METHOD: Ferritin and haemoglobin levels were documented in women of childbearing age with oral iron gastrointestinal intolerance. In those with iron deficiency (ferritin < 30 µg/L), adherence, gastrointestinal tolerability, ferritin, transferrin saturation and haemoglobin levels were compared between their prior oral iron product and iron-whey-protein microspheres randomised to three doses (14 mg daily, 25 mg daily and 50 mg daily) for 12 weeks. RESULTS: Most screened women had low iron stores (128 (62.7%); ferritin < 30 µg/L), 65 (31.9%) had moderate to severe iron deficiency (ferritin < 12 µg/L) and 33 (16.2%) had iron deficiency anaemia (ferritin < 30 µg/L, haemoglobin < 12 g/dL). Amongst the 59 women who participated in the prospective clinical study of iron-whey-protein microspheres over 12 weeks, 48 (81.4%) were classified as adherent/persistent and fewer instances of gastrointestinal intolerance were reported (0.59 ± 0.91) when compared to 12 (20.3%) and (4.0 ± 2.2) respectively while taking the prior oral iron (Fisher's Exact and T-test respectively, both p < 0.001). There was no difference in adherence or tolerability of different iron-whey-protein formulation doses. Ferritin, haemoglobin and energy levels increased significantly over 12 weeks. CONCLUSION: Undiagnosed iron deficiency is common in women of childbearing age with a history of intolerance to oral iron and iron-whey-protein microspheres can improve adherence, GI tolerability, iron stores, haemoglobin and energy levels in these women. CLINICAL TRIAL REGISTRATION: Clinicaltrials.gov identifier (registration includes full trial protocol): NCT04778072.
Assuntos
Anemia Ferropriva , Deficiências de Ferro , Feminino , Humanos , Ferro/efeitos adversos , Estudos Prospectivos , Soro do Leite/metabolismo , Anemia Ferropriva/diagnóstico , Anemia Ferropriva/tratamento farmacológico , Anemia Ferropriva/epidemiologia , Ferritinas , Hemoglobinas/metabolismoRESUMO
Multiple myeloma (MM) is a hematological malignancy originated in the bone marrow and characterized by unhindered plasma cell proliferation that results in several clinical manifestations. Although the main role of blood platelets lies in hemostasis and thrombosis, platelets also play a pivotal role in a number of other pathological conditions. Platelets are the less-explored components from the tumor microenvironment in MM. Although some studies have recently revealed that MM cells have the ability to activate platelets even in the premalignant stage, this phenomenon has not been widely investigated in MM. Moreover, thrombocytopenia, along with bleeding, is commonly observed in those patients. In this review, we discuss the hemostatic disturbances observed in MM patients and the dynamic interaction between platelets and myeloma cells, along with present and future potential avenues for the use of platelets for diagnostic and therapeutic purposes.
Assuntos
Mieloma Múltiplo , Trombose , Humanos , Plaquetas/fisiologia , Mieloma Múltiplo/complicações , Mieloma Múltiplo/tratamento farmacológico , Hemorragia , Hemostasia , Trombose/etiologia , Comunicação Celular , Sistemas de Liberação de Medicamentos , Microambiente TumoralRESUMO
The main cause of subretinal neovascularisation in wet age-related macular degeneration (AMD) is an abnormal expression in the retinal pigment epithelium (RPE) of the vascular endothelial growth factor (VEGF). Current approaches for the treatment of AMD present considerable issues that could be overcome by encapsulating anti-VEGF drugs in suitable nanocarriers, thus providing better penetration, higher retention times, and sustained release. In this work, the ability of large pore mesoporous silica nanoparticles (LP-MSNs) to transport and protect nucleic acid molecules is exploited to develop an innovative LP-MSN-based nanosystem for the topical administration of anti-VEGF siRNA molecules to RPE cells. siRNA is loaded into LP-MSN mesopores, while the external surface of the nanodevices is functionalised with polyethylenimine (PEI) chains that allow the controlled release of siRNA and promote endosomal escape to facilitate cytosolic delivery of the cargo. The successful results obtained for VEGF silencing in ARPE-19 RPE cells demonstrate that the designed nanodevice is suitable as an siRNA transporter.
Assuntos
Nanopartículas , Fator A de Crescimento do Endotélio Vascular , RNA Interferente Pequeno/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Dióxido de Silício/metabolismo , Epitélio Pigmentado da Retina/metabolismoRESUMO
Purpose: In the search for new drug delivery platforms for cardiovascular diseases and coating of medical devices, we synthesized eptifibatide-functionalized silver nanoparticles (AgNPs-EPI) and examined the pharmacological activity of AgNPs-EPI on platelets and endothelial cells in vitro and ex vivo. Methods: Spherical AgNPs linked to eptifibatide were synthesized and characterized. Cytotoxicity was measured in microvascular endothelial cells (HMEC-1), platelets and red blood cells. Platelet mitochondrial respiration was measured using the Oxygraph-2k, a high-resolution modular respirometry system. The effect of AgNPs-EPI on the aggregation of washed platelets was measured by light aggregometry and the ex vivo occlusion time was determined using a reference laboratory method. The surface amount of platelet receptors such as P-selectin and GPIIb/IIIa was measured. The influence of AgNPS-EPI on blood coagulation science was assessed. Finally, the effect of AgNPs-EPI on endothelial cells was measured by the levels of 6-keto-PGF1alpha, tPa, cGMP and vWF. Results: We describe the synthesis of AgNPs using eptifibatide as the stabilizing ligand. The molecules of this drug are directly bonded to the surface of the nanoparticles. The synthesized AgNPs-EPI did not affect the viability of platelets, endothelial cells and erythrocytes. Preincubation of platelets with AgNPs-EPI protected by mitochondrial oxidative phosphorylation capacity. AgNPs-EPI inhibited aggregation-induced P-selectin expression and GPIIb/IIIa conformational changes in platelets. AgNPs-EPI caused prolongation of the occlusion time in the presence of collagen/ADP and collagen/adrenaline. AgNPs-EPI regulated levels of 6-keto-PGF1alpha, tPa, vWf and cGMP produced in thrombin stimulated HMEC-1 cells. Conclusion: AgNPs-EPI show anti-aggregatory activity at concentrations lower than those required by the free drug acting via regulation of platelet aggregation, blood coagulation, and endothelial cell activity. Our results provide proof-of-principle evidence that AgNPs may be used as an effective delivery platform for antiplatelet drugs.
Assuntos
Nanopartículas Metálicas , Selectina-P , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/farmacologia , Plaquetas , Colágeno/metabolismo , Células Endoteliais/metabolismo , Epinefrina/metabolismo , Epinefrina/farmacologia , Eptifibatida/farmacologia , Ligantes , Selectina-P/metabolismo , Agregação Plaquetária , Inibidores da Agregação Plaquetária/farmacologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Prata/metabolismo , Prata/farmacologia , Trombina/metabolismo , Fator de von Willebrand/metabolismoRESUMO
BACKGROUND: Cancer patients have an increased risk of developing venous thromboembolism, with up to 30% dying within a month of their development. Some cancer cells are known to induce platelet aggregation, and this interaction is understood to contribute to thrombosis and haematogenous metastasis. Many researchers have reported on extracellular vesicles (EVs) released from platelets. However, less is known about how cancer cells' EVs may affect platelet function. Here EVs released by triple-negative breast cancer (TNBC) cell line variants were extensively investigated in this regard. METHODS: EVs were separated from conditioned media of TNBC Hs578T and Hs578Ts(i)8 cells using filtration and ultracentrifugation and were characterised by nanoparticle tracking analysis, immunoblots, and transmission electron microscopy. Blood samples from consenting donors were procured, and their platelets collected by differential centrifugation. Light transmission aggregometry and optical microscopy evaluated the potential interaction of TNBC cells and their EVs with platelets. Global proteomic analysis was performed on the EVs, by in-solution digestion and mass spectrometry. Data analysis included the use of Perseus, FunRich, and Vesiclepedia. Immunoblotting was used as a secondary method to investigate some key EV cargo proteins identified by the global proteomics approach. RESULTS: Both TNBC cell variants induced platelet aggregation. Increasing cell numbers significantly reduced the time taken for platelet aggregation to occur. EVs released by the cells also resulted in platelet aggregation. The time to induce platelet aggregation was EV dose-dependent. Proteomics profiling and immunoblotting of the EVs' cargo identified candidate proteins (including uPAR and PDGFRß) that may be involved during this process. CONCLUSIONS: TNBC cells induce platelet aggregation. Furthermore, the cell-free EVs induced this undesirable effect. A number of EV cargo proteins were identified that may be relevant as therapeutic targets.
Assuntos
Vesículas Extracelulares , Neoplasias de Mama Triplo Negativas , Meios de Cultivo Condicionados/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Agregação Plaquetária , Proteômica/métodos , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
Current pathophysiological findings indicate that damage to the alveolar epithelium plays a decisive role in the development of idiopathic pulmonary fibrosis (IPF). The available pharmacological interventions (i.e., oral pirfenidone and nintedanib) only slow down progression of the disease, but do not offer a cure. In order to develop new drug candidates, the pathophysiology of IPF needs to be better understood on a molecular level. It has previously been reported that a loss of caveolin-1 (Cav-1) contributes to profibrotic processes by causing reduced alveolar barrier function and fibrosis-like alterations of the lung-parenchyma. Conversely, overexpression of caveolin-1 appears to counteract the development of fibrosis by inhibiting the inflammasome NLRP3 and the associated expression of interleukin-1ß. In this study, the interaction between Fyn-kinase and caveolin-1 in the alveolar epithelium of various bleomycin (BLM)/TGF-ß damage models using precision-cut lung slices (PCLS), wildtype (WT) and caveolin-1 knockout (KO) mice as well as the human NCI-H441 cell line, were investigated. In WT mouse lung tissues, strong signals for Fyn-kinase were detected in alveolar epithelial type I cells, whereas in caveolin-1 KO animals, expression shifted to alveolar epithelial type II cells. Caveolin-1 and Fyn-kinase were found to be co-localized in isolated lipid rafts of NCI-H441 cell membrane fractions. These findings were corroborated by co-immunoprecipitation studies in which a co-localization of Cav-1 and Fyn-kinase was detected in the cell membrane of the alveolar epithelium. After TGF-ß and BLM-induced damage to the alveolar epithelium both in PCLS and cell culture experiments, a decrease in caveolin-1 and Fyn-kinase was found. Furthermore, TEER (transepithelial electrical resistance) measurements indicated that TGF-ß and BLM have a damaging effect on cell-cell contacts and thus impair the barrier function in NCI-H441 cell monolayers. This effect was attenuated after co-incubation with the Fyn-kinase inhibitor, PP-2. Our data suggest an involvement of Fyn-kinase and caveolin-1 in TGF-ß/bleomycin-induced impairment of alveolar barrier function and thus a possible role in the early stages of pulmonary fibrosis. Fyn-kinase and/or its complex with caveolin-1 might, therefore, be novel therapeutic targets in IPF.
Assuntos
Células Epiteliais Alveolares , Caveolina 1 , Fibrose Pulmonar Idiopática , Proteínas Proto-Oncogênicas c-fyn , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Animais , Bleomicina/farmacologia , Caveolina 1/metabolismo , Fibrose , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
AIMS: We have recently described a novel guanidinium-based compound, VP79s, which induces cytotoxicity in various cancer cell lines. Here, we aim to investigate the activity of VP79s and associated mechanisms of action in multiple myeloma (MM) cells in vitro and ex vivo. MAIN METHODS: The effects of VP79s on cell viability and induction of apoptosis was examined in a panel of drug-sensitive and drug-resistant MM cell lines, as well as ex vivo patient samples and normal donor lymphocytes and platelets. Cell signaling pathways associated with the biological effects of VP79s were analysed by immunoblotting and flow cytometry. Gene expression changes were assessed by quantitative real-time PCR analysis. KEY FINDINGS: VP79s was found to rapidly inhibit both constitutively active and IL-6-induced STAT3 signaling with concurrent downregulation of the IL-6 receptors, CD130 and CD126. VP79s induced a rapid and dose-dependent downregulation of anti-apoptotic Bcl-2 family member, myeloid cell leukaemia-1 (MCL-1). VP79s enhanced bortezomib induced cell death and was also found to overcome bone marrow stromal cell induced drug resistance. VP79s exhibited activity in ex vivo patient samples at concentrations which had no effect on peripheral blood mononuclear cells, lymphocytes and platelets isolated from healthy donors. SIGNIFICANCE: As VP79s resulted in rapid inhibition of the key IL-6/STAT3 signaling pathway and downregulation of MCL-1 expression with subsequent selective anti-myeloma activity, VP79s may be a potential therapeutic agent with a novel mechanism of action in MM cells.
Assuntos
Guanidina/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Fator de Transcrição STAT3/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Expressão Gênica/genética , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/genética , Guanidina/análogos & derivados , Humanos , Interleucina-6/metabolismo , Janus Quinase 1/metabolismo , Janus Quinases/metabolismo , Leucemia/tratamento farmacológico , Leucócitos Mononucleares/metabolismo , Mieloma Múltiplo/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Células Mieloides , Fator de Transcrição STAT3/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Toward improving pharmacokinetics, in vivo efficacy, and selectivity over hERG, structure-activity relationship studies around the central core of antimalarial imidazopyridazines were conducted. This study led to the identification of potent pyrazolopyridines, which showed good in vivo efficacy and pharmacokinetics profiles. The lead compounds also proved to be very potent in the parasite liver and gametocyte stages, which makes them of high interest.
Assuntos
Antimaláricos/química , Antimaláricos/uso terapêutico , Malária/tratamento farmacológico , Plasmodium berghei/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Pirazóis/química , Pirazóis/uso terapêutico , Piridinas/química , Piridinas/uso terapêutico , Animais , Antimaláricos/farmacocinética , Antimaláricos/farmacologia , Canais de Potássio Éter-A-Go-Go/metabolismo , Humanos , Fígado/parasitologia , Malária/parasitologia , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Camundongos , Pirazóis/farmacocinética , Pirazóis/farmacologia , Piridinas/farmacocinética , Piridinas/farmacologia , Ratos , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Engineered nanoparticles (NPs) can induce platelet activation and aggregation, but the mechanisms underlying these interactions are not well understood. This could be due in part to use of devices that study platelet function under quasi-static conditions with low sensitivity to measure platelet microaggregation. Therefore, in this study we investigated the pharmacological pathways and regulators of NP-induced platelet microaggregation under flow conditions at nanoscale using quartz crystal microbalance with dissipation (QCM-D) and compared the data thus obtained with those generated by light aggregometry. METHODS: Blood was collected from healthy volunteers, and platelet-rich plasma was obtained. Thrombin receptor-activating peptide, a potent stimulator of platelet function, and pharmacological inhibitors were used to modulate platelet microaggregation in the presence/absence of silica (10 nm and 50 nm) and polystyrene (23 nm) NPs. Light aggregometry was used to study platelet aggregation in macroscale. Optical, immunofluorescence, and scanning electron microscopy were also used to visualize platelet aggregates. RESULTS: Platelet microaggregation was enhanced by thrombin receptor-activating peptide, whereas prostacyclin, nitric oxide donors, acetylsalicylic acid, and phenanthroline, but not adenosine diphosphate (ADP) blockers, were able to inhibit platelet microaggregation. NPs caused platelet microaggregation, an effect not detectable by light aggregometry. NP-induced microaggregation was attenuated by platelet inhibitors. CONCLUSION: NP-induced platelet microaggregation appears to involve classical proaggregatory pathways (thromboxane A2-mediated and matrix metalloproteinase-2-mediated) and can be regulated by endogenous (prostacyclin) and pharmacological (acetylsalicylic acid, phenanthroline, and nitric oxide donors) inhibitors of platelet function. Quartz crystal microbalance with dissipation, but not light aggregometry, is an appropriate method for studying NP-induced microaggregation.
Assuntos
Plaquetas , Coagulantes , Nanopartículas/química , Agregação Plaquetária/efeitos dos fármacos , Técnicas de Microbalança de Cristal de Quartzo/métodos , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Células Cultivadas , Coagulantes/química , Coagulantes/farmacologia , HumanosRESUMO
In physiological conditions, the interactions between blood platelets and endothelial cells play a major role in vascular reactivity and hemostasis. By contrast, increased platelet activation contributes to the pathogenesis of vascular pathology such as atherosclerosis, thrombosis, diabetes mellitus, hypertension and carcinogenesis. Nanomedicine, including nanodiagnostics and nanotherapeutics is poised to be used in the management of vascular diseases. However, the inherent risk and potential toxicity resultant from the use of nanosized (<100 nm) materials need to be carefully considered. This review, basing on a systematic search of literature provides state-of-the-art and focuses on new discoveries, as well as the potential benefits and threats in the field of nanodiagnostics, nanopharmacology and nanotoxicology of platelet-vessel wall interactions.
Assuntos
Plaquetas/citologia , Adesão Celular , Nanotecnologia , Túnica Íntima/citologia , Plaquetas/ultraestrutura , Humanos , Nanopartículas , Doenças Vasculares/terapiaRESUMO
Several studies have reported that CuO nanoparticles (CuONPs) have the capacity to cross the blood brain barrier and exert a toxic effect. The aims of our study were to investigate mechanisms underlying CuONPs-induced neurotoxicity in vitro and neuroprotective effects of crocetin. We investigated the toxicological effects of exposure of HT22 hippocampal cells to CuONPs (31 nm) in the presence or absence of crocetin. Crocetin is a carotenoid with wide spectrum of pharmacological effects and the ability to cross blood-brain barrier. Exposure of HT22 cells to CuONPs resulted in: (1) increased cell death in a time- and concentration-dependent manner, with a LC50 of 25.9 µg/ml after 24 h; (2) decreased antioxidant/detoxification enzymes activities: glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathione S-tranferase (GST), and reduced glutathione (GSH) levels; (3) decreased gene expression of GPx and SOD; (4) reactive oxygen species (ROS) generation; (5) enhanced apoptosis; and (6) up-regulation of the pro-apoptotic genes Bax, and down-regulation of anti-apoptotic genes Bcl-2. Importantly, all these effects were significantly attenuated by co-incubation of hippocampal cells with 5 µM crocetin.
Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Carotenoides/farmacologia , Cobre/toxicidade , Hipocampo/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Hipocampo/citologia , Luz , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Espalhamento de Radiação , Superóxido Dismutase/metabolismo , Vitamina A/análogos & derivadosRESUMO
Current antimalarials are under continuous threat due to the relentless development of drug resistance by malaria parasites. We previously reported promising in vitro parasite-killing activity with the histone methyltransferase inhibitor BIX-01294 and its analogue TM2-115. Here, we further characterize these diaminoquinazolines for in vitro and in vivo efficacy and pharmacokinetic properties to prioritize and direct compound development. BIX-01294 and TM2-115 displayed potent in vitro activity, with 50% inhibitory concentrations (IC50s) of <50 nM against drug-sensitive laboratory strains and multidrug-resistant field isolates, including artemisinin-refractory Plasmodium falciparum isolates. Activities against ex vivo clinical isolates of both P. falciparum and Plasmodium vivax were similar, with potencies of 300 to 400 nM. Sexual-stage gametocyte inhibition occurs at micromolar levels; however, mature gametocyte progression to gamete formation is inhibited at submicromolar concentrations. Parasite reduction ratio analysis confirms a high asexual-stage rate of killing. Both compounds examined displayed oral efficacy in in vivo mouse models of Plasmodium berghei and P. falciparum infection. The discovery of a rapid and broadly acting antimalarial compound class targeting blood stage infection, including transmission stage parasites, and effective against multiple malaria-causing species reveals the diaminoquinazoline scaffold to be a very promising lead for development into greatly needed novel therapies to control malaria.
Assuntos
Antimaláricos/uso terapêutico , Azepinas/uso terapêutico , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Malária/tratamento farmacológico , Quinazolinas/uso terapêutico , Animais , Antimaláricos/química , Azepinas/química , Feminino , Células Hep G2 , Histona Metiltransferases , Humanos , Malária Falciparum/tratamento farmacológico , Camundongos , Camundongos SCID , Plasmodium berghei/efeitos dos fármacos , Plasmodium berghei/patogenicidade , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/patogenicidade , Quinazolinas/químicaRESUMO
Platelets have been implicated in colon cancer metastasis and prognosis but the underlying molecular mechanisms remain unclear. We evaluated the role of the different mitogen-activated protein kinase (MAPK) pathways in platelet-stimulated matrix metalloproteinase-9 (MMP-9) generation and colon cancer invasion. In addition, proteins released during platelet-tumour cell interactions were studied. For this purpose, interactions of Caco-2 and HT29 cells with platelets were studied using scanning electron microscopy, aggregometry, flow cytometry and cell invasion chambers. Quantitative PCR and zymography were used to study MMP-9 gene expression and activity, respectively, whereas western blot was used to study p38MAPK. Finally, the origin of proteins during platelet-cancer cell interactions was investigated using stable isotope labelling by amino acids in cell culture (SILAC)-based proteomics. We found that platelets promoted p38MAPK phosphorylation and MMP-9 up-regulation in both cell lines, with the subsequent cell-invasion-promoting effects. Pharmacological inhibition of p38MAPK led to a significant down-regulation of MMP-9 and colon cancer cell invasiveness. Also, p38MAPK-small interfering RNA abolished the induction of platelet-stimulated MMP-9. SILAC experiments demonstrated that thrombospondin 1 (TSP1) was released mainly from platelets and clusterin by both platelets and cancer cells. Finally, inhibition of TSP1 and clusterin abolished p38MAPK phosphorylation, MMP-9 activity and platelet-stimulated colon cancer invasion. Our results indicate that platelet-secreted TSP1 and clusterin promote the signal regulation of MMP-9 in platelet-induced colonic cancer invasion via a P38MAPK-regulated pathway. These findings are relevant to the development of therapeutic approaches to preventing and reducing tumour cell metastasis induced by colon adenocarcinoma.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Plaquetas/metabolismo , Clusterina/metabolismo , Neoplasias do Colo/patologia , Regulação Neoplásica da Expressão Gênica , Metaloproteinase 9 da Matriz/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Apoptose , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Western Blotting , Células CACO-2 , Movimento Celular , Proliferação de Células , Cromatografia Líquida , Clusterina/genética , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Citometria de Fluxo , Células HT29 , Humanos , Metaloproteinase 9 da Matriz/genética , Invasividade Neoplásica , Fosforilação , Proteômica , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Espectrometria de Massas em Tandem , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
BACKGROUND AND PURPOSE: Tumour cell-induced platelet aggregation (TCIPA) facilitates cancer cell invasion, angiogenesis and the formation of metastatic foci. TCIPA can be modulated by pharmacological inhibitors of MMP-2 and ADP; however, the COX inhibitor aspirin did not prevent TCIPA. In this study, we have tested the pharmacological effects of a new group of isosorbide-based aspirin prodrugs on TCIPA. EXPERIMENTAL APPROACH: TCIPA was induced in human platelets by mixing with human adenocarcinoma or fibrosarcoma cells under no flow and flow conditions. The release of gelatinases and P-selectin expression during TCIPA were studied by zymography and flow cytometry respectively. KEY RESULTS: Tumour cells caused platelet aggregation. This aggregation resulted in the release of MMP-2 and a significant up-regulation of P-selectin on platelets, indicative of platelet activation. Pharmacological modulation of TCIPA revealed that ST0702, one of the aspirin prodrugs, down-regulated TCIPA while aspirin was ineffective. The deacetylated metabolite of ST0702, 5-nicotinate salicylate (ST0702 salicylate), down-regulated both ADP-stimulated platelet aggregation and TCIPA. CONCLUSIONS AND IMPLICATIONS: Our results show that ST0702 was an effective inhibitor of TCIPA in vitro. Its deacetylated metabolite may contribute to the effects of ST0702 by inhibiting ADP-mediated TCIPA.
Assuntos
Aspirina/análogos & derivados , Plaquetas/efeitos dos fármacos , Comunicação Celular/efeitos dos fármacos , Isossorbida/análogos & derivados , Niacina/análogos & derivados , Agregação Plaquetária/efeitos dos fármacos , Pró-Fármacos/farmacologia , Aspirina/farmacologia , Plaquetas/citologia , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Estabilidade de Medicamentos , Citometria de Fluxo , Humanos , Isossorbida/farmacologia , Microscopia de Contraste de Fase , Niacina/farmacologia , Fisostigmina/farmacologiaRESUMO
Platelets contain and release matrix metalloproteinases (MMPs), their inhibitors (TIMPs) and disintegrin metalloproteinases (ADAMs) including MMP-1, MMP-2, MMP-3, MMP-9, MT1-MMP (MMP-14), ADAM-10, ADAM-17, ADAMTS-13, TIMP-1, TIMP-2 and TIMP-4. These proteins exert several effects regulating platelet functions such as agonist-stimulated platelet adhesion and aggregation, tumour cell-induced platelet aggregation and platelet-leukocyte aggregation. In this review, mechanisms of MMPs, TIMPs and ADAMs on platelets are discussed.
Assuntos
Plaquetas/fisiologia , Metaloproteases/fisiologia , Proteínas ADAM/fisiologia , Proteína ADAM17 , Proteína ADAMTS13 , Animais , Humanos , Inflamação/etiologia , Agregação Plaquetária , Doenças Vasculares/etiologiaRESUMO
Platelets play an important role in carcinogenesis, but the underlying molecular mechanisms remain poorly understood. To investigate the effects of platelets on in vitro invasion of MCF7 human breast cancer cells, human MCF7 cells were used to study their interactions with platelets using aggregometry and cell invasion chambers. Zymography and quantitative polymerase chain reaction (PCR) were used to study matrix metalloproteinases (MMPs), whereas Western blot was used to study protein kinase C (PKC) delta in MCF7 cells. We observed that platelets promoted invasion of MCF7 cells (3-fold increase, p<0.05, n=3) and that this process correlated with a dramatic increase in MMP-9 (8 fold-increase, p<0.001, n=3), which is known to facilitate cancer cell invasion. Because both platelets and MCF7 cells have been shown to release MMP-9, we investigated the cellular source that accounted for this increase. The time course and the use of specific protein synthesis inhibitors demonstrated that most of the increase in MMP-9 levels derived from de novo synthesis of this protease by cancer cells. Furthermore, platelets activated PKCdelta in MCF7 cells after 1 h of incubation (18.45+/-4.75% increase, p<0.05, n=4-7), which, in turn, led to an up-regulation of MMP-9 mRNA (from 60+/-20 to 1040+/-100 pg, p<0.001, n=3) and protein levels (18-fold increase, p<0.001, n=3), with the subsequent cell invasion-promoting effects. PKCdelta plays a crucial role in transducing the invasion-promoting effects of platelets in breast cancer cells, and the specific inhibition of PKCdelta may be a strategy to decrease platelet-mediated cancer cell invasion.
Assuntos
Plaquetas/enzimologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Proteína Quinase C-delta/biossíntese , Células CACO-2 , Humanos , Invasividade Neoplásica , Proteína Quinase C-delta/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Células Tumorais CultivadasRESUMO
Formation of tumor cell-platelet aggregates facilitates hematogenous metastases. However, molecular mechanisms implicated in tumor cell-induced platelet aggregation (TCIPA) in colon cancer are unclear. To investigate mechanisms of TCIPA induced by colon adenocarcinoma cells in vitro, human Caco-2 cells were used to study their interactions with platelets using aggregometry, zymography, phase-contrast microscopy, and flow cytometry. Caco-2-induced platelet aggregation in a concentration-dependent manner. This aggregation resulted in the release of matrix metalloproteinase (MMP)-2, as measured by zymography. In addition, flow cytometry showed a significant up-regulation of activated GpIIb/IIIa, total GpIIb/IIIa, GpIb, and P-selectin receptors on platelets. Inhibition of MMP-2 by phenantroline and degradation of ADP by APT102, respectively, resulted in inhibition of TCIPA. Furthermore, both phenantroline and APT102 significantly down-regulated the surface abundance of platelet receptors. Caco-2 cells aggregate platelets, at least in part, via releasing MMP-2 and ADP. Modulation of MMP-2 and ADP actions could have therapeutic value in colonic cancer.