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1.
Carcinogenesis ; 37(6): 589-599, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27207648

RESUMO

Tumor microenvironment plays an essential role in prostate carcinogenesis and offers novel opportunities to prevent and treat prostate cancer (PCA). Here, we investigated the ability of cancer-associated fibroblasts (CAFs) to promote PCA progression, and silibinin efficacy to target this response. We collected conditioned media from CAFs treated with vehicle or silibinin, and labeled as control conditioned media (CCM) or silibinin-treatment conditioned media (SBCM), respectively. Next, we characterized the effect of CCM and SBCM treatment in several PCA cell lines (RWPE-1, WPE-1 NA-22, WPE-1 NB-14 and PC3). Result showed that compared with SBCM, CCM significantly reduces E-cadherin expression and increases invasiveness and clonogenicity in PCA cells. Further molecular studies identified monocyte chemotactic protein-1 (MCP-1) as the key component of CCM that promotes PCA invasiveness, whereas silibinin treatment strongly reduced MCP-1 expression in CAFs by inhibiting the DNA-binding activity of MCP-1 transcriptional regulators-nuclear factor-kappaB and AP-1. In vivo, silibinin feeding (200mg/kg body weight) strongly reduced TRAMPC1 allografts growth (by 68%) in syngeneic C57Bl/6 mice. TRAMPC1 tumor analysis showed that silibinin reduced MCP-1 and CAFs' biomarkers (fibroblast activation protein, α-smooth muscle actin, transforming growth factor beta 2, vimentin etc.) and significantly modulated the recruitment of immune cells in the tumor microenvironment. Similar inhibitory effects of silibinin on MCP-1 and immune cells recruitment were also observed in TRAMP PCA tissues with reported silibinin efficacy. Overall, our data suggest that silibinin can target CAF-mediated invasiveness in PCA by inhibiting MCP-1 secretion. This, in turn, was associated with a reduction in immune cell recruitment in vivo along with a marked reduction in tumor growth.


Assuntos
Quimiocina CCL2/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Silimarina/farmacologia , Microambiente Tumoral/efeitos dos fármacos , Animais , Antígenos CD , Antineoplásicos Fitogênicos/farmacologia , Caderinas/metabolismo , Linhagem Celular Tumoral , Quimiocina CCL2/genética , Meios de Cultivo Condicionados/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Neoplasias da Próstata/imunologia , Silibina , Fator de Transcrição AP-1/metabolismo , Microambiente Tumoral/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Oncotarget ; 6(37): 39594-606, 2015 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-26447614

RESUMO

Ultraviolet radiation B (UVB) is the main cause of DNA damage in epidermal cells; and if not repaired, this DNA damage leads to skin cancer. In earlier studies, we have reported that natural flavonolignan silibinin exerts strong chemopreventive efficacy against UVB-induced skin damage and carcinogenesis; however mechanistic studies are still being actively pursued. Here, we investigated the role of nucleotide excision repair (NER) pathway in silibinin's efficacy to repair UVB-induced DNA damage. Normal human dermal fibroblasts (NHDFs) were exposed to UVB (1 mJ/cm2) with pre- or post- silibinin (100 µM) treatment, and cyclobutane pyrimidine dimers (CPDs) formation/repair was measured. Results showed that post-UVB silibinin treatment accelerates DNA repair via activating the NER pathway including the expression of XPA (xeroderma pigmentosum complementation group A), XPB, XPC, and XPG. In UVB exposed fibroblasts, silibinin treatment also increased p53 and GADD45α expression; the key regulators of the NER pathway and DNA repair. Consistently, post-UVB silibinin treatment increased the mRNA transcripts of XPA and GADD45α. Importantly, silibinin showed no effect on UVB-induced DNA damage repair in XPA- and XPB-deficient human dermal fibroblasts suggesting their key role in silibinin-mediated DNA damage repair. Moreover, in the presence of pifithrin-α, an inhibitor of p53, the DNA repair efficacy of silibinin was compromised associated with a reduction in XPA and GADD45α transcripts. Together, these findings suggest that silibinin's efficacy against UVB-induced photodamage is primarily by inhibiting NER and p53; and these findings further support silibinin's usage as a potential inexpensive, effective, and non-toxic agent for skin cancer chemoprevention.


Assuntos
Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Silimarina/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Raios Ultravioleta/efeitos adversos , Antioxidantes/farmacologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Derme/citologia , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Expressão Gênica , Humanos , Microscopia Confocal , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Silibina , Proteína Supressora de Tumor p53/genética , Proteína de Xeroderma Pigmentoso Grupo A/genética , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo
3.
Mol Carcinog ; 54(9): 730-41, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24615813

RESUMO

Tumor microenvironment (TM) is an essential element in prostate cancer (PCA), offering unique opportunities for its prevention. TM includes naïve fibroblasts that are recruited by nascent neoplastic lesion and altered into 'cancer-associated fibroblasts' (CAFs) that promote PCA. A better understanding and targeting of interaction between PCA cells and fibroblasts and inhibiting CAF phenotype through non-toxic agents are novel approaches to prevent PCA progression. One well-studied cancer chemopreventive agent is silibinin, and thus, we examined its efficacy against PCA cells-mediated differentiation of naïve fibroblasts into a myofibroblastic-phenotype similar to that found in CAFs. Silibinin's direct inhibitory effect on the phenotype of CAFs derived directly from PCA patients was also assessed. Human prostate stromal cells (PrSCs) exposed to control conditioned media (CCM) from human PCA PC3 cells showed more invasiveness, with increased alpha-smooth muscle actin (α-SMA) and vimentin expression, and differentiation into a phenotype we identified in CAFs. Importantly, silibinin (at physiologically achievable concentrations) inhibited α-SMA expression and invasiveness in differentiated fibroblasts and prostate CAFs directly, as well as indirectly by targeting PCA cells. The observed increase in α-SMA and CAF-like phenotype was transforming growth factor (TGF) ß2 dependent, which was strongly inhibited by silibinin. Furthermore, induction of α-SMA and CAF phenotype by CCM were also strongly inhibited by a TGFß2-neutralizing antibody. The inhibitory effect of silibinin on TGFß2 expression and CAF-like biomarkers was also observed in PC3 tumors. Together, these findings highlight the potential usefulness of silibinin in PCA prevention through targeting the CAF phenotype in the prostate TM.


Assuntos
Anticarcinógenos/farmacologia , Fibroblastos/efeitos dos fármacos , Próstata/efeitos dos fármacos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/prevenção & controle , Silimarina/farmacologia , Fator de Crescimento Transformador beta2/metabolismo , Anticarcinógenos/química , Antioxidantes/química , Antioxidantes/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Masculino , Silybum marianum/química , Próstata/citologia , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/patologia , Silibina , Silimarina/química
4.
Mol Carcinog ; 54(7): 554-65, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24347249

RESUMO

Hypoxic conditions in prostate cancer (PCA) are associated with poor prognosis; however, precise mechanism/s through which hypoxia promotes malignant phenotype remains unclear. Here, we analyzed the role of exosomes from hypoxic PCA cells in enhancing the invasiveness and stemness of naïve PCA cells, as well as in promoting cancer-associated fibroblast (CAF) phenotype in prostate stromal cells (PrSC). Human PCA LNCaP and PC3 cells were exposed to hypoxic (1% O2 ) or normoxic (21% O2 ) conditions, and exosomes secreted under hypoxic (Exo(Hypoxic) ) and normoxic (Exo(Normoxic) ) conditions were isolated from conditioned media. Nanoparticle tracking analysis revealed that Exo(Hypoxic) have smaller average size as compared to Exo(Normoxic) . Immunoblotting results showed a higher level of tetraspanins (CD63 and CD81), heat shock proteins (HSP90 and HSP70), and Annexin II in Exo(Hypoxic) compared to Exo(Normoxic) . Co-culturing with Exo(Hypoxic) increased the invasiveness and motility of naïve LNCaP and PC3 cells, respectively. Exo(Hypoxic) also promoted prostasphere formation by both LNCaP and PC3 cells, and enhanced α-SMA (a CAF biomarker) expression in PrSC. Compared to Exo(Normoxic) , Exo(Hypoxic) showed higher metalloproteinases activity and increased level of diverse signaling molecules (TGF-ß2, TNF1α, IL6, TSG101, Akt, ILK1, and ß-catenin). Furthermore, proteome analysis revealed a higher number of proteins in Exo(Hypoxic) (160 proteins) compared to Exo(Normoxic) (62 proteins), primarily associated with the remodeling of epithelial adherens junction pathway. Importantly, Exo(Hypoxic) targeted the expression of adherens junction proteins in naïve PC3 cells. These findings suggest that Exo(Hypoxic) are loaded with unique proteins that could enhance invasiveness, stemness, and induce microenvironment changes; thereby, promoting PCA aggressiveness.


Assuntos
Junções Aderentes/patologia , Exossomos/patologia , Hipóxia/complicações , Próstata/patologia , Neoplasias da Próstata/complicações , Neoplasias da Próstata/patologia , Junções Aderentes/metabolismo , Anexina A2/análise , Anexina A2/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Movimento Celular , Técnicas de Cocultura , Exossomos/metabolismo , Proteínas de Choque Térmico/análise , Proteínas de Choque Térmico/metabolismo , Humanos , Hipóxia/metabolismo , Hipóxia/patologia , Masculino , Metaloproteases/análise , Metaloproteases/metabolismo , Invasividade Neoplásica/patologia , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Proteoma/análise , Proteoma/metabolismo , Transdução de Sinais , Tetraspaninas/análise , Tetraspaninas/metabolismo
5.
Mol Cancer ; 13: 37, 2014 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-24565133

RESUMO

BACKGROUND: A better molecular understanding of prostate carcinogenesis is warranted to devise novel targeted preventive and therapeutic strategies against prostate cancer (PCA), a major cause of mortality among men. Here, we examined the role of two epithelial-to-mesenchymal transition (EMT) regulators, the adherens junction protein E-cadherin and its transcriptional repressor SNAI1, in regulating the aggressiveness of PCA cells. METHODS: The growth rate of human prostate carcinoma PC3 cells with stable knock-down of E-cadherin (ShEC-PC3) and respective control cells (Sh-PC3) was compared in MTT and clonogenic assays in cell culture and in nude mouse xenograft model in vivo. Stemness of ShEC-PC3 and Sh-PC3 cells was analyzed in prostasphere assay. Western blotting and immunohistochemistry (IHC) were used to study protein expression changes following E-cadherin and SNAI1 knock-down. Small interfering RNA (siRNA) technique was employed to knock- down SNAI1 protein expression in ShEC-PC3 cells. RESULTS: ShEC-PC3 cells exerted higher proliferation rate both in cell culture and in athymic nude mice compared to Sh-PC3 cells. ShEC-PC3 cells also formed larger and a significantly higher number of prostaspheres suggesting an increase in the stem cell-like population with E-cadherin knock-down. Also, ShEC-PC3 prostaspheres disintegration, in the presence of serum and attachment, generated a bigger mass of proliferating cells as compared to Sh-PC3 prostaspheres. Immunoblotting/IHC analyses showed that E-cadherin knock-down increases the expression of regulators/biomarkers for stemness (CD44, cleaved Notch1 and Egr-1) and EMT (Vimentin, pSrc-tyr416, Integrin ß3, ß-catenin, and NF-κB) in cell culture and xenograft tissues. The expression of several bone metastasis related molecules namely CXCR4, uPA, RANKL and RunX2 was also increased in ShEC-PC3 cells. Importantly, we observed a remarkable increase in SNAI1 expression in cytoplasmic and nuclear fractions, prostaspheres and xenograft tissues of ShEC-PC3 cells. Furthermore, SNAI1 knock-down by specific siRNA strongly inhibited the prostasphere formation, clonogenicity and invasiveness, and decreased the level of pSrc-tyr416, total Src and CD44 in ShEC-PC3 cells. Characterization of RWPE-1, WPE1-NA22, WPE1-NB14 and DU-145 cells further confirmed that low E-cadherin is associated with higher SNAI1 expression and prostasphere formation. CONCLUSIONS: Together, these results suggest that E-cadherin loss promotes SNAI1 expression that controls the aggressiveness of PCA cells.


Assuntos
Caderinas/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Fatores de Transcrição/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Nus , Microscopia Confocal , RNA Interferente Pequeno , Fatores de Transcrição da Família Snail , Transfecção
6.
Mutat Res ; 760: 1-15, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24389535

RESUMO

Prostate cancer (PCA) is the most commonly diagnosed cancer in men in the United States with growing worldwide incidence. Despite intensive investment in improving early detection, PCA often escapes timely detection and mortality remains high; this malignancy being the second highest cancer-associated mortality in American men. Collectively, health care costs of PCA results in an immense financial burden that is only expected to grow. Additionally, even in cases of successful treatment, PCA is associated with long-term and pervasive effects on patients. A proactive alternative to treat PCA is to prevent its occurrence and progression prior to symptomatic malignancy. This may serve to address the issue of burgeoning healthcare costs and increasing number of sufferers. One potential regimen in service of this alternative is PCA chemoprevention. Here, chemical compounds with cancer preventive efficacy are identified on the basis of their potential in a host of categories: their historical medicinal use, correlation with reduced risk in population studies, non-toxicity, their unique chemical properties, or their role in biological systems. PCA chemopreventive agents are drawn from multiple broad classes of chemicals, themselves further subdivided based on source or potential effect, with most derived from natural products. Many such compounds have shown efficacy, varying from inhibiting deregulated PCA cell signaling, proliferation, epithelial to mesenchymal transition (EMT), invasion, metastasis, tumor growth and angiogenesis and inducing apoptosis. Overall, these chemopreventive agents show great promise in PCA pre-clinical models, though additional work remains to be done in effectively translating these findings into clinical use.


Assuntos
Anticarcinógenos/uso terapêutico , Neoplasias da Próstata/prevenção & controle , Animais , Humanos , Masculino
7.
AAPS J ; 15(3): 707-16, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23588585

RESUMO

Despite advances in early detection, prostate cancer remains the second highest cancer mortality in American men, and even successful interventions are associated with enormous health care costs as well as prolonged deleterious effects on quality of patient life. Prostate cancer chemoprevention is one potential avenue to alleviate these burdens. It is a regime whereby long-term treatments are intended to prevent or arrest cancer development, in contrast to more direct intervention upon disease diagnosis. Based on this intention, cancer chemoprevention generally focuses on the use of nontoxic chemical agents which are well-tolerated for prolonged usage that is necessary to address prostate cancer's multistage and lengthy period of progression. One such nontoxic natural agent is the flavonoid silibinin, derived from the milk thistle plant (Silybum marianum), which has ancient medicinal usage and potent antioxidant activity. Based on these properties, silibinin has been investigated in a host of cancer models where it exhibits broad-spectrum efficacy against cancer progression both in vitro and in vivo without noticeable toxicity. Specifically in prostate cancer models, silibinin has shown the ability to modulate cell signaling, proliferation, apoptosis, epithelial to mesenchymal transition, invasion, metastasis, and angiogenesis, which taken together provides strong support for silibinin as a candidate prostate cancer chemopreventive agent.


Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Antioxidantes/uso terapêutico , Neoplasias da Próstata/prevenção & controle , Silybum marianum , Silimarina/uso terapêutico , Animais , Antineoplásicos Fitogênicos/química , Antioxidantes/química , Quimioprevenção/métodos , Humanos , Masculino , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Silibina , Silimarina/química
8.
Biochem Biophys Res Commun ; 421(3): 456-61, 2012 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-22503974

RESUMO

In efforts to define new targets for antithrombotic purposes, there is interest in utilizing antibodies targeting ligand binding domains of platelet receptors. To this end, we have recently shown that an antibody (designated C-EL2Ab), which targets the C-terminus of the 2nd extracellular loop (C-EL2) of the thromboxane A(2) receptor (TPR), selectively blocks TPR-mediated platelet aggregation, under both in vitro and ex vivo experimental conditions. In the current studies we sought to determine whether C-EL2Ab exhibits in vivo antithrombotic activity, by employing a carotid artery injury thrombosis model. It was found that mice treated with C-EL2Ab, exhibited a significant increase in time for occlusion, when compared to controls such as normal rabbit IgG, or an antibody which targets a region separate from the ligand binding site (i.e., EL1). We next examined the effect of C-EL2Ab on hemostasis, and found no increase in tail bleeding times in C-EL2Ab treated mice, compared to the aforementioned controls. Collectively, these results clearly demonstrate that C-EL2Ab has anti-platelet/anti-thrombotic effects, and is devoid of increased bleeding risk. Moreover, the identification of a functionally active TPR sequence should significantly aid molecular modeling study predictions for organic derivatives which possess in vivo activity.


Assuntos
Anticorpos Monoclonais/farmacologia , Fibrinolíticos/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Receptores de Tromboxano A2 e Prostaglandina H2/antagonistas & inibidores , Trombose/tratamento farmacológico , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Modelos Animais de Doenças , Fibrinolíticos/uso terapêutico , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Inibidores da Agregação Plaquetária/uso terapêutico , Estrutura Terciária de Proteína , Receptores de Tromboxano A2 e Prostaglandina H2/química , Receptores de Tromboxano A2 e Prostaglandina H2/imunologia
9.
Biochem Pharmacol ; 83(1): 88-96, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-22008592

RESUMO

There is considerable interest in discovering novel antiplatelet approaches with an enhanced safety profile. To this end, in our efforts to define new targets for antithrombotic activity, we investigated the utility of antibodies which recognize the ligand binding domains of the platelet thromboxane A(2) receptor (TPR). We hypothesized that an antibody (abbreviated as C-EL2Ab), which interacts with the C-terminus of the second extracellular loop (C-EL2; i.e., ligand binding domain) of TPR exhibits antagonistic activity. Our findings demonstrate that C-EL2Ab did indeed inhibit TPR-mediated platelet aggregation. However, it was devoid of any apparent effects on aggregation triggered by ADP or the thrombin receptor activating peptides 1 or 4. Furthermore, results from radiolabeled ligand binding studies indicate that C-EL2Ab competitively displaced the classical TPR antagonist [(3)H]SQ29,548 from its binding sites. On the other hand, control experiments indicated that normal rabbit IgG and an antibody which targets a TPR domain separate from those involved in ligand recognition, failed to inhibit aggregation in response to TPR activation. Collectively, these findings demonstrate that C-EL2 of TPR plays a critical role in platelet activation, and establish C-EL2Ab as a function blocking antibody. Furthermore, our data suggest a potential for the therapeutic application of C-EL2Ab, which may serve either as an alternative to, or a complement for current treatments. Finally, the identification of a functionally active TPR sequence should aid molecular modeling study predictions for organic derivatives which possess in vivo activity.


Assuntos
Fragmentos de Peptídeos/fisiologia , Agregação Plaquetária/fisiologia , Receptores de Tromboxano A2 e Prostaglandina H2/metabolismo , Animais , Sítios de Ligação/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/química , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologia
10.
J Cardiovasc Pharmacol Ther ; 17(3): 308-14, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22123197

RESUMO

OBJECTIVE: The purpose of this study is to investigate the potential in vivo antiplatelet and thromboprotective properties of the antihypertensive drug losartan in mice. METHODS: Aggregometry studies were performed on platelets obtained from mice administered losartan for 5 days, via tail vein to examine the ex vivo effects (dose dependence) of this agent and to select an appropriate dose for the in vivo studies. Next, the tail bleeding time test and the time for occlusion in a carotid artery injury thrombosis model (ferric chloride) were also performed to assess the in vivo effects of losartan treatment. RESULTS: These data indicate that the antihypertensive agent losartan exerts dose-dependent inhibition of the thromboxane receptor-mediated (U46619/agonist)-induced platelet aggregation (ex vivo), whereas it produced no detectable effects on aggregation triggered by adenosine diphosphate or the thrombin receptor activating peptide 4. Findings from the in vivo analysis revealed that tail bleeding time of losartan-treated mice was not different from vehicle-treated mice. On the other hand, in the carotid artery injury thrombosis model, it was found that the losartan-treated mice had significantly longer time for occlusion in comparison with those treated with vehicle control. CONCLUSIONS: These findings provide evidence that administration of the antihypertensive drug losartan into live mice produces thromboxane A(2) receptor-specific antiplatelet effects. Furthermore, interestingly, this antiplatelet activity appears to translate into thromboprotective properties, without resulting in a bleeding phenotype. Consequently, aside from its potential use as an antithrombotic agent, losartan's chemistry may provide a "blueprint" for designing or repurposing novel derivatives which may have the potential to serve as an antiplatelet and thromboprotective agents but are deprived of the usually concomitant bleeding adverse effects.


Assuntos
Anti-Hipertensivos/farmacologia , Losartan/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico , Difosfato de Adenosina/farmacologia , Animais , Ácido Araquidônico , Coagulação Sanguínea/efeitos dos fármacos , Lesões das Artérias Carótidas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Inibidores da Agregação Plaquetária/farmacologia , Receptores de Trombina/metabolismo , Receptores de Tromboxano A2 e Prostaglandina H2/antagonistas & inibidores , Trombose/etiologia , Trombose/prevenção & controle
11.
J Cardiovasc Pharmacol Ther ; 17(3): 248-59, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22064668

RESUMO

While blood platelets express several G-protein-coupled receptors (GPCRs) that play pivotal roles in their activation, several diseases, for example thrombotic disorders, may develop if these receptors are inappropriately activated. Thus, these receptors have been the subject of investigations to design therapeutic interventions for managing multiple thrombosis-based disease states. One such GPCR, the thromboxane A(2) receptor (TPR), remains resistant to such interventions. The present review provides a critical examination of the binding, structural biology, and signaling of TPRs. The review also provides a rationale for using principles of "drug rediscovery" as an alternative/viable approach for the therapeutic targeting of TPRs. To this end, it is noteworthy that many US Food and Drug Administration (FDA)-approved drugs have been found to selectively (and nonselectively) block TPR-mediated functional responses, for example platelet aggregation, as described in this review. Therefore, while none of the antagonists, thus far developed for targeting TPRs, have made it into clinical use, this peculiar receptor can be antagonized by a large number of drugs used for indications unrelated to thrombosis.


Assuntos
Receptores de Tromboxano A2 e Prostaglandina H2/antagonistas & inibidores , Receptores de Tromboxano A2 e Prostaglandina H2/metabolismo , Anticoagulantes/farmacologia , Plaquetas/metabolismo , Desenho de Fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Receptores de Tromboxano A2 e Prostaglandina H2/genética , Trombose/tratamento farmacológico , Trombose/prevenção & controle
12.
Biochem Biophys Res Commun ; 417(2): 853-6, 2012 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-22206677

RESUMO

Although changes in the intracellular levels of calcium (Ca(2+)) are a central step in platelet activation, the underlying mechanism of Ca(2+) entry is still unclear. Previous studies have demonstrated that TRPC6, a member of the canonical transient receptor potential channel (TRPC) family is expressed in platelets in a significant amount, and is predominantly found on the plasma membrane. Based on these considerations, we hypothesized that TRPC6 plays a critical role in platelet function. To characterize the role of TRPC6 in platelet function in vivo, we employed a genetic approach, subjecting TRPC6 knockout mice to the tail bleeding time test and a carotid artery injury thrombosis model. We found that TRPC6-deficient animals displayed a prolonged bleeding time, and an increased time for occlusion of the injured carotid artery, compared to their wild-type littermates. Taken together, our data demonstrate for the first time, that TRPC6 deletion in mice results in defects in hemostasis and protection against thrombogenesis, suggesting a vital role in platelet function. Furthermore, TRPC6 may define a new therapeutic target for managing multiple thrombosis-based disorders.


Assuntos
Plaquetas/fisiologia , Hemostasia/genética , Canais de Cátion TRPC/fisiologia , Trombose/sangue , Trombose/genética , Animais , Tempo de Sangramento , Plaquetas/metabolismo , Deleção de Genes , Camundongos , Camundongos Knockout , Canais de Cátion TRPC/genética , Canal de Cátion TRPC6
13.
Eur J Pharmacol ; 649(1-3): 249-54, 2010 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-20858477

RESUMO

While proper platelet function is a vital component of hemostasis, their inappropriate activation contributes to thrombotic disorders. One pathway for platelet activation involves their synthesis of the lipid mediator thromboxane A2 (TXA2). Although TXA2 acts by binding to a seven-transmembrane receptor (i.e., the prostanoid TP receptor) that participates in the genesis of thrombosis, currently, there are no antagonists available for clinical use. Since the only available drug targeting this pathway (aspirin) is associated with inherent limitations/serious side effects, developing prostanoid TP receptor antagonists is clearly warranted. To this end, we have previously employed the "repurposing old drugs for new uses" approach to identify prostanoid TP receptor antagonists and showed that the antidiabetic agent glybenclamide selectively inhibited human platelet prostanoid TP receptors (in vitro). On this basis, we hypothesized that glybenclamide exhibits in vivo antiplatelet potential, and therefore, may protect against thrombosis development. Using murine platelets, it was found that glybenclamide injections: 1) inhibited platelet aggregation induced by the prostanoid TP receptor agonist U46619 and the TXA2 precursor arachidonic acid, under ex vivo experimental settings, concentration-dependently; 2) lacked any detectable effects on aggregation stimulated by ADP, or the thrombin receptor activating-peptide 4; 3) impaired hemostasis by prolonging tail bleeding time; and 4) delayed the development of occlusive thrombi in a carotid artery injury model. Taken together, these findings indicate that glybenclamide does indeed exert, ex vivo and in vivo, prostanoid TP receptor-dependent inhibitory effects on platelet function. Thus, glybenclamide has the potential to be applied in the management of thromboembolic disorders.


Assuntos
Fibrinolíticos/farmacologia , Glibureto/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Trombose/prevenção & controle , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/antagonistas & inibidores , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Animais , Ácido Araquidônico/antagonistas & inibidores , Ácido Araquidônico/metabolismo , Ácido Araquidônico/farmacologia , Tempo de Sangramento , Trombose das Artérias Carótidas/prevenção & controle , Relação Dose-Resposta a Droga , Reposicionamento de Medicamentos , Fibrinolíticos/administração & dosagem , Fibrinolíticos/uso terapêutico , Glibureto/administração & dosagem , Glibureto/uso terapêutico , Hemostasia/efeitos dos fármacos , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Concentração Osmolar , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/uso terapêutico , Receptores de Tromboxanos/agonistas , Receptores de Tromboxanos/antagonistas & inibidores , Tromboembolia/prevenção & controle , Fatores de Tempo
14.
J Biomed Sci ; 17(1): 24, 2010 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-20370921

RESUMO

While there have been many reports investigating the biological activity and signaling mechanisms of isoprostanes, their role in biology, particularly in platelets, appears to still be underestimated. Moreover, whether these lipids have their own receptors is still debated, despite multiple reports that discrete receptors for isoprostane do exist on platelets, vascular tissues, amongst others. This paper provides a review of the important literature of isoprostanes and provides reasoning that isoprostanes should be classified as orphan ligands until their receptor(s) is/are identified.


Assuntos
Plaquetas/fisiologia , Hemostasia/fisiologia , Isoprostanos/metabolismo , Ativação Plaquetária/fisiologia , Transdução de Sinais/fisiologia , Ácido Araquidônico/química , Plaquetas/metabolismo , Humanos , Isoprostanos/química , Modelos Moleculares , Estrutura Molecular
15.
Acta Pharmacol Sin ; 31(2): 150-9, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20139897

RESUMO

AIM: To investigate the potential antagonistic activity of the antidiabetic agent glybenclamide for the human platelet thromboxane A(2) receptor (abbreviated as TPR). METHODS: Platelets were obtained from healthy donors. Aggregation studies were performed in a model 700 aggregometry system. Radioactivity was counted in a Beckman LS 6000 liquid scintillation counter and calcium imaging was performed using an LS50B PerkinElmer Fluorescence Spectrometer. RESULTS: It was found that glybenclamide: 1) inhibited aggregation induced by the TPR agonist U46619 (IC(50)=2.3+/-0.31 micromol/L) and by the thromboxane A(2) precursor arachidonic acid (IC(50)=2.6+/-0.24 micromol/L); 2) displaced SQ29,548 from its binding sites on platelets; 3) lacked any detectable effects on aggregation stimulated by ADP, or the thrombin receptor activating-peptide 4; 4) blocked calcium mobilization induced by U46619, but not by ADP; and 5) failed to raise cAMP levels. CONCLUSION: The findings indicate that glybenclamide exerts inhibitory effects on platelets by interacting with TPR. Thus, glybenclamide or a rationally designed derivative has the potential to serve as an antithrombotic agent.


Assuntos
Glibureto/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Receptores de Tromboxano A2 e Prostaglandina H2/efeitos dos fármacos , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Difosfato de Adenosina/farmacologia , Sítios de Ligação , Plaquetas/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes , Cálcio/metabolismo , AMP Cíclico/sangue , Ácidos Graxos Insaturados , Humanos , Hidrazinas/farmacologia , Receptores de Tromboxano A2 e Prostaglandina H2/metabolismo , Contagem de Cintilação , Espectrometria de Fluorescência
16.
Microcirculation ; 15(4): 311-23, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18464160

RESUMO

OBJECTIVE: Atherosclerosis is a focal disease that develops at sites of low and oscillatory shear stress in arteries. This study aimed to understand how endothelial cells sense a gradient of fluid shear stress and transduce signals that regulate membrane expression of cell adhesion molecules and monocyte recruitment. METHODS: Human aortic endothelial cells were stimulated with TNF-alpha and simultaneously exposed to a linear gradient of shear stress that increased from 0 to 16 dyne/cm2. Cell adhesion molecule expression and activation of NFkappa B were quantified by immunofluorescence microscopy with resolution at the level of a single endothelial cell. Monocyte recruitment was imaged using custom microfluidic flow chambers. RESULTS: VCAM-1 and E-selectin upregulation was greatest between 2-4 dyne/cm2 (6 and 4-fold, respectively) and above 8 dyne/cm2 expression was suppressed below that of untreated endothelial cells. In contrast, ICAM-1 expression and NFkappa B nuclear translocation increased with shear stress up to a maximum at 9 dyne/cm2. Monocyte recruitment was most efficient in regions where E-selectin and VCAM-1 expression was greatest. CONCLUSIONS: We found that the endothelium can sense a change in shear stress on the order of 0.25 dyne/cm2 over a length of approximately 10 cells, regulating the level of protein transcription, cellular adhesion molecule expression, and leukocyte recruitment during inflammation.


Assuntos
Aorta/metabolismo , Selectina E/metabolismo , Células Endoteliais/metabolismo , Monócitos/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Molécula 1 de Adesão de Célula Vascular/metabolismo , Adolescente , Aorta/patologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/patologia , Feminino , Humanos , Inflamação/metabolismo , Inflamação/patologia , Monócitos/patologia , Resistência ao Cisalhamento , Estresse Mecânico
17.
J Leukoc Biol ; 81(6): 1395-403, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17389580

RESUMO

CD11c, a member of the leukointegrin family, is expressed prominently on tissue macrophages and dendritic cells and binds to complement fragment (iC3b), provisional matrix molecules (fibrinogen), and the Ig superfamily cell adhesion molecule, ICAM-1. CD11c has been proposed to function in phagocytosis, cell migration, and cytokine production by monocytes/macrophages as well as induction of T cell proliferation by Langerhans cells. Using assays to quantify CD11c-mediated cell adhesion, we demonstrate that CD11c recognizes ICAM-2 and VCAM-1. The CD11c-binding site on VCAM-1 appears to be different from that used by the integrin alpha4. CD11c and alpha4beta1 contributed to monocyte capture and transmigration on inflamed human aortic endothelial cells. We discovered that the anti-mouse CD11c mAb N418 blocks CD11c binding to iC3b, ICAM-1, and VCAM-1. Treatment of mice with N418 reduced SRBC-induced delayed-type hypersensitivity significantly. CD11c appeared to contribute predominantly to the sensitization phase and somewhat less to the response to SRBC challenge. This suggests a novel role for CD11c during leukocyte recruitment, antigen uptake, and the survival of APC.


Assuntos
Antígeno CD11c/imunologia , Antígenos CD18/imunologia , Hipersensibilidade Tardia/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Antígenos CD/metabolismo , Aorta/citologia , Sítios de Ligação , Antígeno CD11c/metabolismo , Adesão Celular/imunologia , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Complemento C3b/imunologia , Células Endoteliais/imunologia , Células Endoteliais/fisiologia , Eritrócitos/imunologia , Humanos , Integrina alfa4/metabolismo , Ligantes , Camundongos , Monócitos/imunologia , Monócitos/fisiologia , Ligação Proteica , Ovinos , Molécula 1 de Adesão de Célula Vascular/metabolismo
18.
Circ Res ; 100(3): 381-90, 2007 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-17234968

RESUMO

High levels of triglyceride-rich lipoproteins (TGRLs) in blood are linked to development of atherosclerosis, yet the mechanisms by which these particles initiate inflammation of endothelium are unknown. TGRL isolated from human plasma during the postprandial state was examined for its capacity to bind to cultured human aortic endothelial cells (HAECs) and alter the acute inflammatory response to tumor necrosis factor-alpha. HAECs were repetitively incubated with dietary levels of freshly isolated TGRL for 2 hours per day for 1 to 3 days to mimic postprandial lipidemia. TGRL induced membrane upregulation of the low-density lipoprotein family receptors LRP and LR11, which was inhibited by the low-density lipoprotein receptor-associated protein-1. TGRLs alone did not elicit inflammation in HAECs but enhanced the inflammatory response via a 10-fold increase in sensitivity to cytokine stimulation. This was reflected by increased mitogen-activated protein kinase activation, nuclear translocation of NF-kappaB, amplified expression of endothelial selectin and VCAM-1, and a subsequent increase in monocyte-specific recruitment under shear flow as quantified in a microfabricated vascular mimetic device.


Assuntos
Doenças da Aorta/etiologia , Arteriosclerose/etiologia , Arterite/etiologia , Gorduras na Dieta/efeitos adversos , Células Endoteliais/efeitos dos fármacos , Hipertrigliceridemia/complicações , Proteínas Relacionadas a Receptor de LDL/metabolismo , Lipoproteínas HDL/toxicidade , Lipoproteínas LDL/toxicidade , Lipoproteínas VLDL/toxicidade , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Receptores de LDL/metabolismo , Triglicerídeos/toxicidade , Fator de Necrose Tumoral alfa/farmacologia , Aorta , Apolipoproteína C-III/metabolismo , Apolipoproteína C-III/farmacologia , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Quilomícrons/sangue , Gorduras na Dieta/administração & dosagem , Selectina E/biossíntese , Selectina E/genética , Endocitose , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Emulsões Gordurosas Intravenosas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hipertrigliceridemia/sangue , Hipoglicemia , Molécula 1 de Adesão Intercelular/biossíntese , Molécula 1 de Adesão Intercelular/genética , Proteína Associada a Proteínas Relacionadas a Receptor de LDL/farmacologia , Proteínas Relacionadas a Receptor de LDL/efeitos dos fármacos , Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Lipoproteínas HDL/sangue , Lipoproteínas LDL/sangue , Lipoproteínas VLDL/sangue , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/efeitos dos fármacos , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Modelos Cardiovasculares , Monócitos/citologia , Monócitos/efeitos dos fármacos , NF-kappa B/metabolismo , Estresse Oxidativo , Receptores de LDL/efeitos dos fármacos , Reologia , Transdução de Sinais/efeitos dos fármacos , Triglicerídeos/sangue , Fator de Necrose Tumoral alfa/fisiologia , Molécula 1 de Adesão de Célula Vascular/biossíntese , Molécula 1 de Adesão de Célula Vascular/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
19.
Methods Mol Biol ; 412: 203-10, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-18453113

RESUMO

Neutrophils are among the first cells to respond to acute inflammation through a multistep process initiated by selectin mediated rolling, which transitions to an integrin/intercellular adhesion molecule-dependent arrest and transmigration across endothelium. A conformational shift in the CD11/CD18 adhesion receptor on neutrophils is a critical determinant of the efficiency of recruitment on inflamed endothelium. For instance, beta2-integrin expression level is upregulated up to 10-fold by fusion of cytoplasmic granule pools of CD11b/CD18 (Mac-1). Furthermore, a rapid increase in affinity and membrane clustering of CD11a/CD18 (LFA-1) is necessary for efficient deceleration and arrest in shear flow. We present methods here to quantify the changes in receptor expression and affinity that support neutrophil adhesive phenotypes. Techniques involving real-time fluorescence flow cytometry and parallel plate rheometry coupled with light microscopy are presented.


Assuntos
Fluorescência , Integrinas/metabolismo , Ativação de Neutrófilo/fisiologia , Neutrófilos/metabolismo , Óptica e Fotônica , Antígenos de Superfície/análise , Epitopos , Modelos Biológicos , Neutrófilos/imunologia
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