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1.
Acta Cir Bras ; 39: e391624, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38808816

RESUMO

PURPOSE: To evaluate the chemotherapeutic activity of temozolomide counter to mammary carcinoma. METHODS: In-vitro anticancer activity has been conducted on MCF7 cells, and mammary carcinoma has been induced in Wistar rats by introduction of 7, 12-Dimethylbenz(a)anthracene (DMBA), which was sustained for 24 weeks. Histopathology, immunohistochemistry, cell proliferation study and apoptosis assay via TUNEL method was conducted to evaluate an antineoplastic activity of temozolomide in rat breast tissue. RESULTS: IC50 value of temozolomide in MCF7 cell has been obtained as 103 µM, which demonstrated an initiation of apoptosis. The temozolomide treatment facilitated cell cycle arrest in G2/M and S phase dose dependently. The treatment with temozolomide suggested decrease of the hyperplastic abrasions and renovation of the typical histological features of mammary tissue. Moreover, temozolomide therapy caused the downregulation of epidermal growth factor receptor, extracellular signal-regulated kinase, and metalloproteinase-1 expression and upstream of p53 and caspase-3 proliferation to indicate an initiation of apoptotic events. CONCLUSIONS: The occurrence of mammary carcinoma has been significantly decreased by activation of apoptotic pathway and abrogation of cellular propagation that allowable for developing a suitable mechanistic pathway of temozolomide in order to facilitate chemotherapeutic approach.


Assuntos
Antineoplásicos Alquilantes , Apoptose , Receptores ErbB , Ratos Wistar , Temozolomida , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Feminino , Receptores ErbB/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Antineoplásicos Alquilantes/farmacologia , Antineoplásicos Alquilantes/uso terapêutico , Metaloproteinase 1 da Matriz/efeitos dos fármacos , Metaloproteinase 1 da Matriz/metabolismo , Proliferação de Células/efeitos dos fármacos , Dacarbazina/análogos & derivados , Dacarbazina/farmacologia , Dacarbazina/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Humanos , Células MCF-7 , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , Imuno-Histoquímica , Reprodutibilidade dos Testes , Ratos , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/patologia
2.
Life Sci ; 308: 120917, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36044974

RESUMO

AIM: Endothelial mechanisms underlying the vascular effects of estrogen modulated by the G protein-coupled estrogen receptor (GPER) are not well understood, especially in gonadal sex hormone deprivation. Thus, we investigated vascular function and endothelial signaling pathways involved in the selective activation of GPER in resistance arteries of gonadectomized rats. METHODS: Gonadectomy was performed in Wistar rats of both sexes. After 21 days, the animals were euthanized. Concentration-response curves were obtained by cumulative additions of G-1 in third-order mesenteric arteries. The vasodilatory effects of G-1 were evaluated before and after endothelium removal or incubation with pharmacological inhibitors. Tissue protein expression was measured by western blotting. Assays with 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM) and 2',7' dichlorodihydrofluorescein-diacetate (H2DCF-DA) were performed in the arteries investigated. Immunolocalization was assessed by immunofluorescence. RESULTS: G-1 induced partially endothelium-dependent relaxation in both sexes. The three isoforms of the enzyme nitric oxide synthase contributed to the production and release of nitric oxide in both gonadectomized groups, but the role of inducible nitric oxide synthase is more expressive in males. The mechanistic pathway by which endothelial nitric oxide synthase is phosphorylated appears to differ between sexes, with the rapid signaling pathway phosphatidylinositol-3-kinase/protein kinase B/endothelial nitric oxide synthase (PI3k-Akt-eNOS) being identified for males and mitogen-activated protein kinase/extracellular signal-regulated kinase/endothelial nitric oxide synthase (MEK-ERK-eNOS) for females. The contribution of hydrogen peroxide as an endothelial relaxation mediator seems to be greater in females. CONCLUSION: These results provide new insights into the effects of estrogen-induced responses via GPER on vascular function in gonadal sex hormone deprivation.


Assuntos
Óxido Nítrico Sintase Tipo III , Proteínas Proto-Oncogênicas c-akt , Animais , Endotélio Vascular , Estrogênios/metabolismo , Estrogênios/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Proteínas de Ligação ao GTP/metabolismo , Hormônios Esteroides Gonadais/metabolismo , Peróxido de Hidrogênio/metabolismo , Masculino , Artérias Mesentéricas , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/farmacologia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositóis/metabolismo , Fosfatidilinositóis/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Caracteres Sexuais , Transdução de Sinais , Vasodilatadores/farmacologia
3.
Cell Mol Biol Lett ; 27(1): 23, 2022 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-35260073

RESUMO

BACKGROUND: ROR2 is a tyrosine-kinase receptor whose expression is dysregulated in many human diseases. In cancer, ROR2 stimulates proliferation, survival, migration, and metastasis, and is associated with more aggressive tumor stages. The purpose of this work is to study the role of ROR2 in the chemoresistance of melanoma. METHODS: Gain- and loss-of-function experiments were used to study the biological function of ROR2 in melanoma. Cell death induced by chemotherapeutic drugs and BH-3 mimetics was evaluated using crystal violet cytotoxicity assays and annexin V/propidium iodide staining. Western blots were used to evaluate the expression of proteins implicated in cell death. The differences observed between cells with manipulation of ROR2 levels and control cells were evaluated using both Student's t-test and ANOVA. RESULTS: We describe that ROR2 contributes to tumor progression by enhancing the resistance of melanoma cells to both chemotherapeutic drugs and BH-3 mimetics. We demonstrate that ROR2 reduced cell death upon treatment with cisplatin, dacarbazine, lomustine, camptothecin, paclitaxel, ABT-737, TW-37, and venetoclax. This effect was mediated by the inhibition of apoptosis. In addition, we investigated the molecular mechanisms implicated in this role of ROR2. We identified the MDM2/p53 pathway as a novel target of ROR2 since ROR2 positively regulates MDM2 levels, thus leading to p53 downregulation. We also showed that ROR2 also upregulates Mcl-1 and Bcl2-xL while it negatively regulates Bax and Bid expression. The effect of ROR2 on the expression of these proteins is mediated by the hyperactivation of ERK. CONCLUSIONS: These results demonstrate that ROR2 contributes to melanoma progression by inhibiting apoptosis and increasing chemoresistance. These results not only position ROR2 as a marker of chemoresistance but also support its use as a novel therapeutic target in cancer.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular , Melanoma , Proteínas Proto-Oncogênicas c-bcl-2 , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase , Proteína Supressora de Tumor p53 , Apoptose , Linhagem Celular Tumoral , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Melanoma/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
4.
Mol Neurobiol ; 58(12): 6222-6231, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34476673

RESUMO

Huntington disease (HD) is the most common neurogenetic disorder caused by expansion of the CAG repeat in the HTT gene; nevertheless, the molecular bases of the disease are not fully understood. Non-coding RNAs have demonstrated to be involved in the physiopathology of HD. However, the role of circRNAs has not been investigated. The aim of this study was to identify the circRNAs with differential expression in a murine cell line model of HD and to identify the biological pathways regulated by the differentially expressed circRNAs. CircRNA expression was analyzed through a microarray, which specifically detects circular species of RNA. The expression patterns between a murine cell line expressing mutant Huntingtin and cells expressing wild-type Huntingtin were compared. We predicted the miRNAs with binding sites for the differentially expressed circRNAs and the corresponding target genes for those miRNAs. Using the target genes, we performed a function enrichment analysis. We identified 23 circRNAs differentially expressed, 19 downregulated and four upregulated. Most of the downregulated circRNAs derive from the Rere gene. The dopaminergic synapse, MAPK, and long-term depression pathways were significantly enriched. The three identified pathways have been previously associated with the physiopathology of HD. The understanding of the circRNA-miRNA-mRNA network involved in the molecular mechanisms driving HD can lead us to identify novel biomarkers and potential therapeutic targets. To the best of our knowledge, this is the first study analyzing circRNAs in a model of Huntington disease.


Assuntos
Neurônios Dopaminérgicos/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Doença de Huntington/metabolismo , Depressão Sináptica de Longo Prazo/fisiologia , RNA Circular/metabolismo , Sinapses/metabolismo , Animais , Regulação para Baixo , Perfilação da Expressão Gênica , Doença de Huntington/fisiopatologia , MicroRNAs/metabolismo , Células PC12 , RNA Mensageiro/metabolismo , Ratos
5.
J Biochem Mol Toxicol ; 35(10): e22875, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34350654

RESUMO

Kidney injury molecule-1 (KIM-1) is a membrane receptor upregulated in the proximal tubule cells following various types of kidney injuries. Notably, studies have suggested a correlation between KIM-1 expression and extracellular signal-regulated kinase (ERK) activation. In this study, we aimed to investigate the association between the kidney overexpression pattern of cytoplasmic phosphorylated-ERK (p-ERK) protein and increased urinary KIM-1 levels in rats exposed to gentamicin or lead acetate, both at the end of toxic exposure and after a 4-week recovery period. Although other proteins were evaluated, only kidney overexpression of cytoplasmic p-ERK protein correlated with increased urinary KIM-1 levels. For both toxic substances, the increased urinary KIM-1 levels corresponded with kidney inflammation. Our results suggest that KIM-1 and p-ERK share a common mechanism in kidney injury mediated by both toxic substances that induce proximal tubule damage.


Assuntos
Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/urina , Moléculas de Adesão Celular/urina , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Gentamicinas/toxicidade , Túbulos Renais Proximais/lesões , Túbulos Renais Proximais/metabolismo , Compostos Organometálicos/toxicidade , Transdução de Sinais/efeitos dos fármacos , Animais , Canais de Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Citoplasma/metabolismo , Modelos Animais de Doenças , Histonas/metabolismo , Masculino , Fosforilação , Ratos , Ratos Wistar , Canais de Cátion TRPV/metabolismo
6.
Pharmacol Biochem Behav ; 209: 173244, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34363828

RESUMO

The development of sensitization is one of the hallmarks of addictive drugs such as morphine. We administered morphine (10 mg/kg; MOR) to induce locomotor sensitization and ERK activation in the VTA and NAc. In the first experiment, four groups of rats received five daily 30 min sessions in an open-field, and locomotion was measured. For the first four sessions, one group received MOR pre-test (MOR-P); a second group received vehicle pre-test (MOR-UP) and MOR 30 min post-test; the remaining 2 groups received vehicle (VEH) pre-test. On the fifth session, the MOR-P, MOR-UP, and one VEH group received MOR pre-test and the remaining VEH group received VEH. Sensitization emerged in the first 5 min and progressed over to the second and third 5 min blocks only in the MOR-P group. For the second experiment, 4 groups received MOR and 4 groups VEH, and were then returned to their home cage and after 5, 15, 30 or 60 min post-injection, were euthanized for ERK measurements in VTA and NAc. ERK activation increased and peaked at 5 min post injection in the MOR group and then declined to VEH levels by 30 min. Another two groups received either MOR or VEH immediately before a 5 min arena test and ERK was measured immediately post-test. MOR had no effect on locomotion but increased ERK in the VTA and NAc. The peak ERK activation in VTA reflected activation of reward systems by morphine that reinforced locomotor behavior and with repeated treatments, induced a sensitization effect.


Assuntos
Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Locomoção/efeitos dos fármacos , Morfina/farmacologia , Recompensa , Animais , Encéfalo/metabolismo , Condicionamento Operante/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Masculino , Entorpecentes/farmacologia , Núcleo Accumbens/efeitos dos fármacos , Ratos , Ratos Wistar , Reforço Psicológico , Área Tegmentar Ventral/efeitos dos fármacos
7.
Bioorg Chem ; 113: 104982, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34020277

RESUMO

ERK1/2 inhibitors have attracted special attention concerning the ability of circumventing cases of innate or log-term acquired resistance to RAF and MEK kinase inhibitors. Based on the 4-aminoquinazoline pharmacophore of kinases, herein we describe the synthesis of 4-aminoquinazoline derivatives bearing a 1,2,3-triazole stable core to bridge different aromatic and heterocyclic rings using copper-catalysed azide-alkyne cycloaddition reaction (CuAAC) as a Click Chemistry strategy. The initial screening of twelve derivatives in tumoral cells (CAL-27, HN13, HGC-27, and BT-20) revealed that the most active in BT-20 cells (25a, IC50 24.6 µM and a SI of 3.25) contains a more polar side chain (sulfone). Furthermore, compound 25a promoted a significant release of lactate dehydrogenase (LDH), suggesting the induction of cell death by necrosis. In addition, this compound induced G0/G1 stalling in BT-20 cells, which was accompanied by a decrease in the S phase. Western blot analysis of the levels of p-STAT3, p-ERK, PARP, p53 and cleaved caspase-3 revealed p-ERK1/2 and p-STA3 were drastically decreased in BT-20 cells under 25a incubation, suggesting the involvement of these two kinases in the mechanisms underlying 25a-induced cell cycle arrest, besides loss of proliferation and viability of the breast cancer cell. Molecular docking simulations using the ERK-ulixertinib crystallographic complex showed compound 25a could potentially compete with ATP for binding to ERK in a slightly higher affinity than the reference ERK1/2 inhibitor. Further in silico analyses showed comparable toxicity and pharmacokinetic profiles for compound 25a in relation to ulixertinib.


Assuntos
Antineoplásicos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Triazóis/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Quinazolinas/química , Relação Estrutura-Atividade , Triazóis/química
8.
Tissue Cell ; 72: 101544, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-33892398

RESUMO

During pregnancy, Toxoplasma gondii can triggers serious manifestations and potentially affect the fetal development. In this scenario, differences in susceptibility of trophoblast cells to T. gondii infection might be evaluated in order to establish new therapeutic approaches capable of interfering in the control of fetal infection by T. gondii. This study aimed to evaluate the susceptibility of cytotrophoblast, syncytiotrophoblast and extravillous trophoblast cells to T. gondii infection. Our data demonstrate that HTR-8/SVneo cells (extravillous trophoblast cells) present higher susceptibility to T. gondii infection when compared to syncytiotrophoblast and cytotrophoblast cells, whereas syncytiotrophoblast was the cell type more resistant to the parasite infection. Also, cytotrophoblast and syncytiotrophoblast cells produced significantly more IL-6 than HTR-8/SVneo cells. On the other hand, HTR-8/SVneo cells showed higher ERK1/2 phosphorylation than cytotrophoblast and syncytiotrophoblast cells. ERK1/2 inhibition reduced T. gondii infection and increased IL-6 production in HTR-8/SVneo cells. Thus, it is plausible to conclude that the greater susceptibility of HTR-8/SVneo cells to infection by T. gondii is related to a higher ERK1/2 phosphorylation and lower levels of IL-6 in these cells compared to other cells, suggesting that these mediators may be important to favor the parasite infection in this type of trophoblastic population.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Gigantes/patologia , Interleucina-6/biossíntese , Toxoplasmose/patologia , Trofoblastos/patologia , Trofoblastos/parasitologia , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Suscetibilidade a Doenças , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Humanos , Fosforilação , Regulação para Cima
9.
J Mater Sci Mater Med ; 32(4): 33, 2021 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-33751248

RESUMO

Phytocystatins are endogenous cysteine-protease inhibitors present in plants. They are involved in initial germination rates and in plant defense mechanisms against phytopathogens. Recently, a new phytocystatin derived from sweet orange, CsinCPI-2, has been shown to inhibit the enzymatic activity of human cathepsins, presenting anti-inflammatory potential and pro-osteogenic effect in human dental pulp cells. The osteogenic potential of the CsinCPI-2 protein represents a new insight into plants cysteine proteases inhibitors and this effect needs to be better addressed. The aim of this study was to investigate the performance of pre-osteoblasts in response to CsinCPI-2, mainly focusing on cell adhesion, proliferation and differentiation mechanisms. Together our data show that in the first hours of treatment, protein in CsinCPI-2 promotes an increase in the expression of adhesion markers, which decrease after 24 h, leading to the activation of Kinase-dependent cyclines (CDKs) modulating the transition from G1 to S phases cell cycle. In addition, we saw that the increase in ERK may be associated with activation of the differentiation profile, also observed with an increase in the B-Catenin pathway and an increase in the expression of Runx2 in the group that received the treatment with CsinCPI-2.


Assuntos
Cistatinas/química , Osteoblastos/citologia , beta Catenina/metabolismo , Células 3T3 , Animais , Anti-Inflamatórios/química , Adesão Celular , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Citrus sinensis , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Citoesqueleto/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Camundongos , Osteoblastos/metabolismo , Osteogênese , Compostos Fitoquímicos , Cicatrização
10.
Eur J Pharmacol ; 896: 173913, 2021 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-33508282

RESUMO

Histamine H1 receptor ligands used clinically as antiallergics rank among the most widely prescribed and over-the-counter drugs in the world. They exert the therapeutic actions by blocking the effects of histamine, due to null or negative efficacy towards Gαq-phospholipase C (PLC)-inositol triphosphates (IP3)-Ca2+ and nuclear factor-kappa B cascades. However, there is no information regarding their ability to modulate other receptor responses. The aim of the present study was to investigate whether histamine H1 receptor ligands could display positive efficacy concerning receptor desensitization, internalization, signaling through Gαq independent pathways or even transcriptional regulation of proinflammatory genes. While diphenhydramine, triprolidine and chlorpheniramine activate ERK1/2 (extracellular signal-regulated kinase 1/2) pathway in A549 cells, pre-treatment with chlorpheniramine or triprolidine completely desensitize histamine H1 receptor mediated Ca2+ response, and both diphenhydramine and triprolidine lead to receptor internalization. Unlike histamine, histamine H1 receptor desensitization and internalization induced by antihistamines prove to be independent of G protein-coupled receptor kinase 2 (GRK2) phosphorylation. Also, unlike the reference agonist, the recovery of the number of cell-surface histamine H1 receptors is a consequence of de novo synthesis. On the other hand, all of the ligands lack efficacy regarding cyclooxygenase-2 (COX-2) and interleukin-8 (IL-8) mRNA regulation. However, a prolonged exposure with each of the antihistamines impaires the increase in COX-2 and IL-8 mRNA levels induced by histamine, even after ligand removal. Altogether, these findings demonstrate the biased nature of histamine H1 receptor ligands contributing to a more accurate classification, and providing evidence for a more rational and safe use of them.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Agonistas dos Receptores Histamínicos/farmacologia , Antagonistas dos Receptores Histamínicos H1/farmacologia , Receptores Histamínicos H1/efeitos dos fármacos , Células A549 , Sinalização do Cálcio/efeitos dos fármacos , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Agonismo Inverso de Drogas , Ativação Enzimática , Quinase 2 de Receptor Acoplado a Proteína G/genética , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Ligantes , Fosforilação , Transporte Proteico , Receptores Histamínicos H1/metabolismo , Fosfolipases Tipo C/metabolismo
11.
J Cell Physiol ; 236(6): 4640-4654, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33345322

RESUMO

We have previously reported that angiotensin II receptor type 1 (AT1R) contributes to the hypertrophic effects of thyroid hormones (TH) in cardiac cells. Even though evidence indicates crosstalks between TH and AT1R, the underlying mechanisms are poorly understood. Beta-arrestin (ARRB) signaling has been described as noncanonical signal transduction pathway that exerts important effects in the cardiovascular system through G-protein-coupled receptors, as AT1R. Herein, we investigated the contribution of ARRB signaling in TH-induced cardiomyocyte hypertrophy. Primary cardiomyocyte cultures were treated with Triiodothyronine (T3) to induce cell hypertrophy. T3 rapidly activates extracellular signal-regulated kinase 1/2 (ERK1/2) signaling, which was partially inhibited by AT1R blockade. Also, ERK1/2 inhibition attenuated the hypertrophic effects of T3. ARRB2 was upregulated by T3, and small interfering RNA assays revealed the role of ARRB2-but not ARRB1-on ERK1/2 activation and cardiomyocyte hypertrophy. Corroborating these findings, the ARRB2-overexpressed cells showed increased expression of hypertrophic markers, which were attenuated by ERK1/2 inhibition. Immunocytochemistry and immunoprecipitation assays revealed the increased expression of nuclear AT1R after T3 stimulation and the increased interaction of AT1R/ARRB2. The inhibition of endocytosis also attenuated the T3 effects on cardiac cells. Our results evidence the contribution of ARRB2 on ERK1/2 activation and cardiomyocyte hypertrophy induced by T3 via AT1R.


Assuntos
Cardiomegalia/induzido quimicamente , Miócitos Cardíacos/efeitos dos fármacos , Receptor Tipo 1 de Angiotensina/metabolismo , Tri-Iodotironina/toxicidade , beta-Arrestina 2/metabolismo , Animais , Animais Recém-Nascidos , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Células Cultivadas , Endocitose/efeitos dos fármacos , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fosforilação , Ratos Wistar , Transdução de Sinais , beta-Arrestina 2/genética , Ratos
12.
Am J Physiol Heart Circ Physiol ; 320(1): H352-H363, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33124885

RESUMO

Alamandine is the newest identified peptide of the renin-angiotensin system (RAS) and has protective effects in the cardiovascular system. Although the involvement of classical RAS components in the genesis and progression of cardiac remodeling is well known, less is known about the effects of alamandine. Therefore, in the present study we investigated the effects of alamandine on cardiac remodeling induced by transverse aortic constriction (TAC) in mice. Male mice (C57BL/6), 10-12 wk of age, were divided into three groups: sham operated, TAC, and TAC + ALA (30 µg/kg/day alamandine for 14 days). The TAC surgery was performed under ketamine and xylazine anesthesia. At the end of treatment, the animals were submitted to echocardiographic examination and subsequently euthanized for tissue collection. TAC induced myocyte hypertrophy, collagen deposition, and the expression of matrix metalloproteinase (MMP)-2 and transforming growth factor (TGF)-ß in the left ventricle. These markers of cardiac remodeling were reduced by oral treatment with alamandine. Western blotting analysis showed that alamandine prevents the increase in ERK1/2 phosphorylation and reverts the decrease in 5'-adenosine monophosphate-activated protein kinase (AMPK)α phosphorylation induced by TAC. Although both TAC and TAC + ALA increased SERCA2 expression, the phosphorylation of phospholamban in the Thr17 residue was increased solely in the alamandine-treated group. The echocardiographic data showed that there are no functional or morphological alterations after 2 wk of TAC. Alamandine treatment prevents myocyte hypertrophy and cardiac fibrosis induced by TAC. Our results reinforce the cardioprotective role of alamandine and highlight its therapeutic potential for treating heart diseases related to pressure overload conditions.NEW & NOTEWORTHY Alamandine is the newest identified component of the renin-angiotensin system protective arm. Considering the beneficial effects already described so far, alamandine is a promising target for cardiovascular disease treatment. We demonstrated for the first time that alamandine improves many aspects of cardiac remodeling induced by pressure overload, including cell hypertrophy, fibrosis, and oxidative stress markers.


Assuntos
Fármacos Cardiovasculares/farmacologia , Ventrículos do Coração/efeitos dos fármacos , Hipertrofia Ventricular Esquerda/prevenção & controle , Oligopeptídeos/farmacologia , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Aorta/fisiopatologia , Aorta/cirurgia , Proteínas de Ligação ao Cálcio/metabolismo , Colágeno/metabolismo , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Ventrículos do Coração/metabolismo , Ventrículos do Coração/fisiopatologia , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/fisiopatologia , Ligadura , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Fosforilação , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Camundongos
13.
Int Immunopharmacol ; 86: 106766, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32652504

RESUMO

BACKGROUND: Limonene (LIM) and its main metabolite perillyl alcohol (POH) are ingredients found in food with promising chemical entities due to their pharmacological profile. In this study, we hypothesized that LIM and POH are two molecules capable of accelerating the regenerative process and alleviating neuropathic pain. METHODS: Animals were treated daily (LIM, POH and saline) for 28 days and during this period evaluated for mechanical hyperalgesia, astrocyte participation by immunofluorescence for GFAP, and ELISA was used to quantify IL-1ß and TNF-α in the spinal cord. Western blot analysis of the following proteins was also performed: GFAP, GAP-43, NGF and ERK. For motor deficit analysis, tests were performed to assess hind paw muscle strength and footprints through gait (SFI). RESULTS: Both POH and LIM accelerated the regenerative process and improved motor deficits comparing to positive control; however, POH was more effective, particularly between the 2nd and 3rd week after the nerve injury, increasing GAP-43, NGF and the phosphorylated ERK immunocontent. Moreover, POH and LIM were able to reduce hyperalgesia and astrocytosis. CONCLUSIONS: Both substances, LIM and POH, improved the regeneration process and sensory and motor function recovery in the PNI model in mice by mitigating the inflammatory reactions and up-regulating the neurotrophic process.


Assuntos
Anti-Inflamatórios/uso terapêutico , Aditivos Alimentares/uso terapêutico , Limoneno/uso terapêutico , Monoterpenos/uso terapêutico , Neurônios Motores/fisiologia , Neuralgia/terapia , Traumatismos dos Nervos Periféricos/terapia , Animais , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Interleucina-1beta/metabolismo , Masculino , Camundongos , Fator de Crescimento Neural/metabolismo , Neuralgia/dietoterapia , Regeneração/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo
14.
Front Immunol ; 11: 705, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32425934

RESUMO

Essential thrombocythemia (ET) is comprised among chronic myeloproliferative neoplasms (MPN) and is caused by driver mutations in JAK2, CALR, and MPL, which lead to megakaryocyte proliferation and prominent thrombocytosis. Thrombosis remains the main cause of morbidity in ET and is driven by the interplay between blood cells, the endothelium, the clotting cascade, and host-derived inflammatory mediators. Platelet activation plays a key role in the thrombotic predisposition, although the underlying mechanisms remain poorly defined. In addition to their role in hemostasis, platelets participate in innate immunity and inflammation owing to the expression of toll-like receptors (TLR), which recognize inflammatory signals, triggering platelet functional responses. Considering the impact of inflammation on ET procoagulant state, we assessed the contribution of TLR2 and TLR4 to platelet hemostatic and inflammatory properties in ET patients, by using Pam3CSK4 and lipopolysaccharide (LPS) as specific TLR2 and TLR4 ligands, respectively. TLR2 ligation induced increased surface translocation of α-granule-derived P-selectin and CD40L, which mediate platelet interaction with leukocytes and endothelial cells, respectively, and higher levels of dense granule-derived CD63 in patients, whereas PAC-1 binding was not increased and LPS had no effect on these platelet responses. Platelet-neutrophil aggregate formation was elevated in ET at baseline and after stimulation of both TLR2 and TLR4. In addition, ET patients displayed higher TLR2- and TLR4-triggered platelet secretion of the chemokine RANTES (CCL5), whereas von Willebrand factor release was not enhanced, revealing a differential releasate pattern for α-granule-stored inflammatory molecules. TLR-mediated hyperresponsiveness contrasted with impaired or preserved responses to classic platelet hemostatic agonists, such as TRAP-6 and thrombin. TLR2 and TLR4 expression on the platelet surface was normal, whereas phosphorylation of downstream effector ERK1/2 was higher in patients at baseline and after incubation with Pam3CSK4, which may partly explain the enhanced TLR2 response. In conclusion, exacerbated response to TLR stimulation may promote platelet activation in ET, boosting platelet/leukocyte/endothelial interactions and secretion of inflammatory mediators, overall reinforcing the thromboinflammatory state. These findings highlight the role of platelets as inflammatory sentinels in MPN prothrombotic scenario and provide additional evidence for the close intertwining between thrombosis and inflammation in this setting.


Assuntos
Plaquetas/fisiologia , Inflamação/etiologia , Trombocitemia Essencial/complicações , Trombose/etiologia , Receptores Toll-Like/fisiologia , Adulto , Idoso , Quimiocina CCL5/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neutrófilos/fisiologia , Fosforilação , Ativação Plaquetária , Trombocitemia Essencial/imunologia
15.
Eur J Pharmacol ; 880: 173130, 2020 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-32360975

RESUMO

Pinitol is a natural antidiabetic agent shown to prevent or ameliorate metabolic and overall vascular and neural function. In the present study we have evaluated the potential benefits of pinitol on renal function of streptozotocin (STZ)-induced diabetic rats. Both euglycemic or 8-week or 16-week diabetic rats were treated with either saline (1 ml/kg/12h; p.o) or pinitol (20 mg/kg/12h; p.o). The renal function was evaluated by using metabolic cages, renal hemodynamic and tubular parameters measurements. Histological examination and evaluation of the protein expression of renal markers such as nephrin, TGFß and pERK were also performed. Pinitol decreased by 50% the increased urinary albumin/creatinine ratio in both 8-week and 16 week diabetic rats. In addition, the glomerular volume of 16-week rats increased by 55% and this increase was blunted by pinitol. Remarkably, pressure-natriuresis was completely blunted in both 8 and 16-week diabetic rats but this impairment was prevented by pinitol in both treatment regimens. Pinitol ameliorated renal lesions and also prevented the decrease in nephrin expression and the increase of pERK and TGFß expression in both diabetic groups. Natriuresis due to high renal perfusion pressure increased 7-fold in control animals but was blocked in 16-week diabetic rats and remarkably pinitol partially restored pressure natriuresis (3-fold increase in sodium excretion during pressure natriuresis). Pinitol prevents and ameliorates albuminuria, glomerular expansion, impairment of pressure-natriuresis, renal structural alterations and changes of renal markers and has the potential to be tested for the prevention of diabetic kidney disease.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Hipoglicemiantes/uso terapêutico , Inositol/análogos & derivados , Rim/efeitos dos fármacos , Substâncias Protetoras/uso terapêutico , Albuminúria/tratamento farmacológico , Albuminúria/metabolismo , Albuminúria/patologia , Albuminúria/fisiopatologia , Animais , Creatinina/urina , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/fisiopatologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hemodinâmica , Hipoglicemiantes/farmacologia , Inositol/farmacologia , Inositol/uso terapêutico , Rim/patologia , Rim/fisiologia , Masculino , Proteínas de Membrana/metabolismo , Substâncias Protetoras/farmacologia , Ratos Wistar , Fator de Crescimento Transformador beta/metabolismo
16.
Int J Mol Sci ; 21(7)2020 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-32225112

RESUMO

Inflammatory and wound healing responses take place during liver damage, primarily in the parenchymal tissue. It is known that cellular injury elicits an activation of the purinergic signaling, mainly by the P2X7 receptor; however, the role of P2Y receptors in the onset of liver pathology such as fibrosis has not been explored. Hence, we used mice treated with the hepatotoxin CCl4 to implement a reversible model of liver fibrosis to evaluate the expression and function of the P2Y2 receptor (P2Y2R). Fibrotic livers showed an enhanced expression of P2Y2R that eliminated its zonal distribution. Hepatocytes from CCl4-treated mice showed an exacerbated ERK-phosphorylated response to the P2Y2R-specific agonist, UTP. Cell proliferation was also enhanced in the fibrotic livers. Hepatic transcriptional analysis by microarrays, upon CCl4 administration, showed that P2Y2 activation regulated diverse pathways, revealing complex action mechanisms. In conclusion, our data indicate that P2Y2R activation is involved in the onset of the fibrotic damage associated with the reversible phase of the hepatic damage promoted by CCl4.


Assuntos
Hepatócitos/metabolismo , Cirrose Hepática/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Animais , Tetracloreto de Carbono/toxicidade , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Cirrose Hepática/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL
17.
Cell Biochem Funct ; 38(2): 213-221, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31978253

RESUMO

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that has generated scientific interest because of its prevalence in the population. Studies indicate that physical exercise promotes neuroplasticity and improves cognitive function in animal models and in human beings. The aim of the present study was to investigate the effects of strength exercise on the hippocampal protein contents and memory performance in mice subjected to a model of sporadic AD induced by streptozotocin (STZ). Swiss mice received two injections of STZ (3 mg/kg, intracerebroventricular). After 21 days, they began physical training using a ladde. Mice performed this protocol for 4 weeks. After the last exercise training session, mice performed the Morris Water Maze test. The samples of hippocampus were excised and used to determine protein contents of brain-derived neurotrophic factor (BDNF), extracellular signal-regulated kinase-Ca2+ (ERK), calmodulin-dependent protein kinase (CAMKII) and cAMP-response element-binding protein (CREB) signalling pathway. Strength exercise was effective against the decrease in the time spent and distance travelled in the target quadrant by STZ-injected mice. Strength exercise was also effective against the reduction of mature BDNF, tropomyosin receptor kinase B and neuronal nuclear antigen (NeuN) hippocampal protein levels in STZ mice. The decrease in the hippocampal ratio of pERK/ERK, pCAMKII/CAMKII and pCREB/CREB induced by STZ was reversed by strength exercise. Strength exercise decreased Bax/Bcl2 ratio in the hippocampus of STZ-injected mice. The present study demonstrates that strength exercise modulated the hippocampal BDNF/ERK-CAMKII/CREB signalling pathway and suppressed STZ-induced spatial memory impairment in mice.


Assuntos
Hipocampo/metabolismo , Condicionamento Físico Animal , Transdução de Sinais , Memória Espacial , Animais , Apoptose , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Masculino , Aprendizagem em Labirinto , Memória , Camundongos , Estreptozocina
18.
Cells ; 9(1)2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31906385

RESUMO

Macrophage galactose-C type lectin (MGL)1 receptor is involved in the recognition of Trypanosoma cruzi (T. cruzi) parasites and is important for the modulation of the innate and adaptive immune responses. However, the mechanism by which MGL1 promotes resistance to T. cruzi remains unclear. Here, we show that MGL1 knockout macrophages (MGL1-/- Mφ) infected in vitro with T. cruzi were heavily parasitized and showed decreased levels of reactive oxygen species (ROS), nitric oxide (NO), IL-12 and TNF-α compared to wild-type macrophages (WT Mφ). MGL1-/- Mφ stimulated in vitro with T. cruzi antigen (TcAg) showed low expression of TLR-2, TLR-4 and MHC-II, which resulted in deficient splenic cell activation compared with similar co-cultured WT Mφ. Importantly, the activation of p-ERK1/2, p-c-Jun and p-NF-κB p65 were significantly reduced in MGL1-/- Mφ exposed to TcAg. Similarly, procaspase 1, caspase 1 and NLRP3 inflammasome also displayed a reduced expression that was associated with low IL-ß production. Our data reveal a previously unappreciated role for MGL1 in Mφ activation through the modulation of ERK1/2, c-Jun, NF-κB and NLRP3 signaling pathways, and to the development of protective innate immunity against experimental T. cruzi infection.


Assuntos
Assialoglicoproteínas/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Lectinas Tipo C/metabolismo , Ativação de Macrófagos , Proteínas de Membrana/metabolismo , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Animais , Antígenos de Protozoários/metabolismo , Citocinas/biossíntese , Mediadores da Inflamação/metabolismo , Linfócitos/imunologia , Masculino , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Parasitos/metabolismo , Proteínas de Protozoários/metabolismo , Explosão Respiratória , Trypanosoma cruzi/imunologia
19.
Clin Transl Oncol ; 22(3): 302-310, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31625123

RESUMO

PURPOSE: Long non-coding RNAs (lncRNAs) have been shown to play important roles in tumorigenesis, but their biological functions and the underlying molecular mechanisms remain unclear. Alternative splicing of five exons results in three transcript variants of cancer susceptibility 2 (CASC2): the lncRNAs CASC2a, CASC2b, and CASC2c. CASC2a/b have been found to have crucial regulatory functions in a number of malignancies, but few studies have examined the effects of CASC2c in cancers. The objective of the study was to investigate the role of CASC2c in the proliferation and apoptosis of hepatocellular carcinoma (HCC) cells. METHODS: This study first investigated the expression levels of CASC2c in tumor tissues, corresponding non-tumor tissues and cells using quantitative real-time polymerase chain reaction. The function and underlying molecular mechanism of CASC2c in human HCC were investigated in QGY-7703 cell line, as well as in gastric cancer (GC) cell and colorectal cancer (CRC) cell. RESULTS: In the present work, we observed that CASC2c was significantly down-regulated in HCC tissues and cells. Moreover, its overexpression remarkably inhibited the growth, migration, and invasion of HCC cells in vitro and promoted their apoptosis. Furthermore, we demonstrated that CASC2c overexpression decreased p-ERK1/2 levels in HCC, GC, and CRC cells. Interestingly, while overexpression of CASC2c decreased ß-catenin expression in HCC and GC cells, it increased that in CRC cells. CONCLUSION: The lncRNA-CASC2c has a vital role in tumorigenesis and cancer progression, and may serve as a biomarker or therapeutic target in cancer treatment via down-regulation of the ERK1/2 and Wnt/ß-catenin signaling pathways.


Assuntos
Carcinoma Hepatocelular/patologia , Proliferação de Células , Neoplasias Hepáticas/patologia , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinogênese/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas Supressoras de Tumor/genética , Proteínas Wnt/metabolismo , beta Catenina/metabolismo
20.
Cardiovasc Res ; 116(12): 1995-2008, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31825460

RESUMO

AIMS: Activation of the angiotensin (Ang)-(1-7)/Mas receptor (R) axis protects from sympathetic overactivity. Endocytic trafficking is an essential process that regulates receptor (R) function and its ultimate cellular responses. We investigated whether the blunted responses to Ang-(1-7) in hypertensive rats are associated to an alteration in MasR trafficking. METHODS AND RESULTS: Brainstem neurons from Wistar-Kyoto (WKY) or spontaneously hypertensive rats (SHRs) were investigated for (i) Ang-(1-7) levels and binding and MasR expression, (ii) Ang-(1-7) responses (arachidonic acid and nitric oxide release and Akt and ERK1/2 phosphorylation), and (iii) MasR trafficking. Ang-(1-7) was determined by radioimmunoassay. MasR expression and functionality were evaluated by western blot and binding assays. MasR trafficking was evaluated by immunofluorescence. Ang-(1-7) treatment induced an increase in nitric oxide and arachidonic acid release and ERK1/2 and Akt phosphorylation in WKY neurons but did not have an effect in SHR neurons. Although SHR neurons showed greater MasR expression, Ang-(1-7)-elicited responses were substantially diminished presumably due to decreased Ang-(1-7) endogenous levels concomitant with impaired binding to its receptor. Through immunocolocalization studies, we evidenced that upon Ang-(1-7) stimulation MasRs were internalized through clathrin-coated pits and caveolae into early endosomes and slowly recycled back to the plasma membrane. However, the fraction of internalized MasRs into early endosomes was larger and the fraction of MasRs recycled back to the plasma membrane was smaller in SHR than in WKY neurons. Surprisingly, in SHR neurons but not in WKY neurons, Ang-(1-7) induced MasR translocation to the nucleus. Nuclear MasR expression and Ang-(1-7) levels were significantly greater in the nuclei of Ang-(1-7)-stimulated SHR neurons, indicating that the MasR is translocated with its ligand bound to it. CONCLUSION: MasRs display differential trafficking in brainstem neurons from SHRs, which may contribute to the impaired responses to Ang-(1-7).


Assuntos
Angiotensina I/farmacologia , Tronco Encefálico/efeitos dos fármacos , Hipertensão/metabolismo , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas/agonistas , Receptores Acoplados a Proteínas G/agonistas , Transporte Ativo do Núcleo Celular , Animais , Animais Recém-Nascidos , Ácido Araquidônico/metabolismo , Tronco Encefálico/metabolismo , Tronco Encefálico/fisiopatologia , Células Cultivadas , Modelos Animais de Doenças , Endocitose , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hipertensão/fisiopatologia , Ligantes , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Fosforilação , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Receptores Acoplados a Proteínas G/metabolismo
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