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1.
Oncol Rep ; 42(4): 1475-1486, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31364740

RESUMO

Gallbladder cancer (GBC) is a lethal aggressive malignant neoplasm of the biliary tract. Potential prognostic markers and therapeutic targets for this disease are urgently required. Cancer­associated fibroblasts (CAFs) play a key role in tumorigenesis and the development of cancer. Nicotinamide adenine dinucleotide phosphate oxidase 1 (NOX1) expression has been reported to be involved in tumorigenesis and useful for tumor prognosis. However, NOX1 expression in the stroma of GBCs, particularly gallbladder cancer­associated fibroblasts (GCAFs), and its prognostic significance in GBC patients remains unclear. In the present study, NOX1 expression in the stroma of human gallbladder lesions in vivo was investigated, as well as in GCAFs and co­cultures of GBC­SD+GCAFs in vitro, and their correlation with clinicopathological parameters and the prognosis of GBC patients were evaluated. The results revealed that NOX1 expression was significantly upregulated in the stroma of GBCs compared with precancerous and benign lesions of the gallbladder; NOX1 expression was localized to gallbladder stromal fibroblasts expressing α­smooth muscle actin and fibroblast secreted protein­1. Furthermore, these observations were confirmed by the fact that NOX1 expression was upregulated in GCAFs as determined by Affymetrix gene profile chip analysis and reverse transcription­quantitative PCR. In addition, overexpression was observed in formed spheroids of GBC­SD+GCAF co­cultures by immunohistochemistry and western blotting in vitro. Thus, it was verified that NOX1 expression was upregulated in GCAFs. Furthermore, upregulated stromal NOX1 expression was correlated with aggressive characteristics such as differentiation degree (P=0.042), venous invasion (P=0.041), resection methods (P=0.002), and a lower survival rate (P=0.025, log­rank test) of patients with GBC. Stromal NOX1 expression (P=0.047) was an independent prognostic factor for the overall survival rate of patients with GBC. GBC patients with upregulated NOX1 expression in GCAFs had a poorer prognosis. These results revealed that stromal NOX1 may be a novel biomarker and/or target, and may contribute to the discovery of new tumor markers and potential targeted therapeutics for human GBCs.


Assuntos
Fibroblastos Associados a Câncer/enzimologia , Neoplasias da Vesícula Biliar/enzimologia , NADPH Oxidase 1/biossíntese , Actinas/biossíntese , Idoso , Proteínas de Ligação ao Cálcio/biossíntese , Fibroblastos Associados a Câncer/patologia , Feminino , Neoplasias da Vesícula Biliar/patologia , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Prognóstico , Regulação para Cima
2.
Oncol Rep ; 42(4): 1319-1328, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31364748

RESUMO

Oral squamous cell carcinoma (OSCC), with high potential for metastasis, is the most common malignant tumor of the head and neck. Cancer­associated fibroblasts (CAFs) are the main stromal cells in the microenvironment and aggravate tumor progression. However, whether CAFs are associated with the progression of OSCC remains unknown and the underlying mechanism remains unclear. In the present study, the role of CAFs in mediating OSCC cell migration and invasion was investigated, and the participation of exosomal miR­382­5p in this process was elucidated. In this study, according to the α­SMA staining with immunohistochemistry, 47 OSCC patients were divided into CAFs­rich and CAFs poor groups, and association of CAF density and clinicopathologic features of the OSCC patients were analyzed with Pearson χ2 test. Transwell assay was used for evaluating cell migration and invasion ability of OSCC cells after being co­cultured with NFs or CAFs, or after added exosomes. qPCR was used to detect the expression of miR­382­5p. Western blot analysis was used to measure the expression of migration and invasion­associated proteins. In the present study, the CAF density in tumor tissues was found to be relevant to OSCC lymph node metastasis and TNM stage. Furthermore, we revealed that miR­382­5p was overexpressed in CAFs compared with that in fibroblasts of adjacent normal tissue and miR­382­5p overexpression was responsible for OSCC cell migration and invasion. Finally, we demonstrated that CAF­derived exosomes transported miR­382­5p to OSCC cells. The present study confirmed a new mechanism of CAF­facilitated OSCC progression and may be beneficial for identifying new cancer therapeutic targets.


Assuntos
Fibroblastos Associados a Câncer/patologia , Exossomos/genética , MicroRNAs/biossíntese , Neoplasias Bucais/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Actinas/biossíntese , Adulto , Idoso , Fibroblastos Associados a Câncer/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Exossomos/metabolismo , Exossomos/patologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , Metástase Neoplásica , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo
3.
Mol Carcinog ; 58(8): 1362-1375, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30997718

RESUMO

The main focus of this study is exploring the effect and mechanism of two HIV-protease inhibitors: Ritonavir and Ritonavir-nitric oxide (Ritonavir-NO) on in vitro growth of melanoma cell lines. NO modification significantly improved the antitumor potential of Ritonavir, as the IC50 values of Ritonavir-NO were approximately two times lower than IC50 values of the parental compound. Our results showed for the first time, that both compounds induced senescence in primary and metastatic melanoma cell lines. This transformation was manifested as a change in cell morphology, enlargement of nuclei, increased cellular granulation, upregulation of ß-galactosidase activity, lipofuscin granules appearance, higher production of reactive oxygen species and persistent inhibition of proliferation. The expression of p53, as one of the key regulators of senescence, was upregulated after 48 hours of Ritonavir-NO treatment only in metastatic B16F10 cells, ranking it as a late-response event. The development of senescent phenotype was consistent with the alteration of the cytoskeleton-as we observed diminished expression of vinculin, α-actin, and ß-tubulin. Permanent inhibition of S6 protein by Ritonavir-NO, but not Ritonavir, could be responsible for a stronger antiproliferative potential of the NO-modified compound. Taken together, induction of senescent phenotype may provide an excellent platform for developing therapeutic approaches based on selective killing of senescent cells.


Assuntos
Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Inibidores da Protease de HIV/farmacologia , Melanoma/tratamento farmacológico , Ritonavir/farmacologia , Actinas/biossíntese , Linhagem Celular Tumoral , Humanos , Lipofuscina/metabolismo , Melanoma/patologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases S6 Ribossômicas/antagonistas & inibidores , Tubulina (Proteína)/biossíntese , Proteína Supressora de Tumor p53/biossíntese , Vinculina/biossíntese , beta-Galactosidase/metabolismo
4.
Mol Med Rep ; 19(2): 877-884, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30535476

RESUMO

Actin­like protein 8 (ACTL8) is a member of the cancer­testis antigens (CTA) family, which is mainly localized in the cytoplasm and generally expressed in the testis. The association between ACTL8 and various types of cancer, including glioblastoma and breast cancer, has previously been demonstrated. However, whether ACTL8 is involved in the development of head and neck squamous cell carcinoma (HNSCC) remains unknown. In the present study, the expression of ACTL8 in patients with HNSCC was analyzed in The Cancer Genome Atlas (TCGA) dataset, clinical tissues and cell lines. Correlations between the expression levels of ACTL8 and HNSCC clinical outcomes were analyzed with the Kaplan­Meier method and the Cox proportional hazards model. Cell Counting Kit­8, plate colony formation and Transwell assays were used to assess the effects of ACTL8 interference on the proliferation, migration and invasion of HNSCC PCI­13 cells. Reverse transcription­quantitative polymerase chain reaction and western blotting were used to evaluate the expression levels of ACTL8 in PCI­13 cells. Furthermore, alterations in the expression levels of key proteins in the phosphatidylinositol­4,5­bisphosphate 3­kinase (PI3K)/RAC­α protein kinase B (AKT) signaling pathway were determined by western blotting. Increased expression of ACTL8 in HNSCC was observed in TCGA dataset, cancerous tissue samples and HNSCC cell line. Cox regression analysis indicated that ACTL8 expression could be regarded as an independent prognostic factor for HNSCC, since increased expression of ACTL8 was associated with a poor prognosis. Knocking down ACTL8 markedly inhibited the proliferation, invasion and migration of PCI­13 cells. Additionally, activation of the PI3K/AKT signaling pathway was suppressed by reduced expression levels of certain key proteins in this pathway. The present data indicate that ACTL8 serves a role in the progression and clinical prognosis of HNSCC. Therefore, ACTL8 may be a potential prognostic marker and novel therapeutic target for HNSCC.


Assuntos
Actinas/genética , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Actinas/biossíntese , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Fosfatidilinositol 3-Quinases/genética , Prognóstico , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo
5.
PLoS One ; 13(12): e0202829, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30557388

RESUMO

Bactrocera cucurbitae (melon flies) are prominent invasive pests in southern China. To screen for a stable reference gene in melon flies suitable for comparing tissue samples subjected to different conditions in four categories (temperature, insect stage, days of age and gender), the expression of 12 candidate reference genes under different treatment conditions was analyzed by real-time fluorescent quantitative PCR. The results obtained from a comprehensive analysis with geNorm, NormFinder, BestKeeper and RefFinder software showed that the most stable reference gene was RPL60, and the least stable reference gene was actin-5. We used a heat shock protein gene (HSP-90) to verify the results, and the conclusion was consistent. When the reference gene RPL60 was used as the reference gene, the relative expression of HSP-90 was essentially constant with the prolongation of treatment time. When actin-5 was used, HSP-90 expression changed markedly with treatment time. The results of this study can be used for further research on gene expression inBactrocera cucurbitae.


Assuntos
Actinas/genética , Regulação da Expressão Gênica , Genes de Insetos , Proteínas de Choque Térmico HSP90/genética , Proteínas de Insetos/genética , Reação em Cadeia da Polimerase em Tempo Real , Tephritidae/genética , Actinas/biossíntese , Animais , Proteínas de Choque Térmico HSP90/biossíntese , Proteínas de Insetos/biossíntese , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência , Tephritidae/metabolismo
6.
Proc Natl Acad Sci U S A ; 115(28): E6487-E6496, 2018 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-29941587

RESUMO

Many organisms possess multiple and often divergent actins whose regulation and roles are not understood in detail. For example, Chlamydomonas reinhardtii has both a conventional actin (IDA5) and a highly divergent one (NAP1); only IDA5 is expressed in normal proliferating cells. We showed previously that the drug latrunculin B (LatB) causes loss of filamentous (F-) IDA5 and strong up-regulation of NAP1, which then provides essential actin function(s) by forming LatB-resistant F-NAP1. RNA-sequencing analyses now show that this up-regulation of NAP1 reflects a broad transcriptional response, much of which depends on three proteins (LAT1, LAT2, and LAT3) identified previously as essential for NAP1 transcription. Many of the LAT-regulated genes contain a putative cis-acting regulatory site, the "LRE motif." The LatB transcriptional program appears to be activated by loss of F-IDA5 and deactivated by formation of F-NAP1, thus forming an F-actin-dependent negative-feedback loop. Multiple genes encoding proteins of the ubiquitin-proteasome system are among those induced by LatB, resulting in rapid degradation of IDA5 (but not NAP1). Our results suggest that IDA5 degradation is functionally important because nonpolymerizable LatB-bound IDA5 interferes with the formation of F-NAP1. The genes for the actin-interacting proteins cofilin and profilin are also induced. Cofilin induction may further the clearance of IDA5 by promoting the scission of F-IDA5, whereas profilin appears to function in protecting monomeric IDA5 from degradation. This multifaceted regulatory system allows rapid and quantitative turnover of F-actin in response to cytoskeletal perturbations and probably also maintains F-actin homeostasis under normal growth conditions.


Assuntos
Actinas/biossíntese , Chlamydomonas reinhardtii/metabolismo , Proteínas de Plantas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Transcrição Genética , Actinas/genética , Chlamydomonas reinhardtii/genética , Proteínas de Plantas/genética , Complexo de Endopeptidases do Proteassoma/genética
7.
Life Sci ; 205: 176-183, 2018 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-29752961

RESUMO

AIMS: Dihydroartemisinin has been shown to inhibit the development of pulmonary fibrosis in rats, but its mechanism has yet to be elucidated. This study aimed to determine the mechanisms of dihydroartemisinin in bleomycin-induced pulmonary fibrosis in a rat model. MAIN METHODS: Morphological changes and collagen deposition were analyzed via hematoxylin-eosin staining and Masson staining and the expression of biotic-factor-related oxidative stress in lung tissues was assayed with standard assay kits. The expressions of α-SMA, E-cadherin, and Nrf2/HO-1 were detected by Western blot and RT-PCR, and the cell morphology and proliferation of cultured type II alveolar epithelial cells (AECs) were assessed via microscopy and immunocytochemical assay. KEY FINDINGS: Dihydroartemisinin treatment significantly decreased the level of oxidative stress and collagen synthesis and inhibited AECs differentiation in bleomycin-induced pulmonary fibrosis compared to the control group (P < 0.001). SIGNIFICANCE: Our results indicated that dihydroartemisinin might decrease oxidative damage to attenuate lung injury and fibrosis.


Assuntos
Antimetabólitos Antineoplásicos , Antioxidantes/farmacologia , Artemisininas/farmacologia , Bleomicina , Estresse Oxidativo/efeitos dos fármacos , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/tratamento farmacológico , Actinas/biossíntese , Células Epiteliais Alveolares/efeitos dos fármacos , Animais , Antioxidantes/metabolismo , Caderinas/biossíntese , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Miofibroblastos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/biossíntese , Ratos , Ratos Sprague-Dawley
8.
Pharmacology ; 102(1-2): 1-8, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29669350

RESUMO

BACKGROUND: Periplaneta americana is one of the ancient insect groups with the strongest vitality. Periplaneta americana extract (PAE) has been explored as an alternative remedy for many diseases. Although much progress has been made in the study about PAE, the role of the drug in renal disease is rarely reported, especially in renal fibrosis. This study was designed to evaluate the renoprotective effect of PAE treatment to renal fibrosis. METHOD: An in vivo, unilateral ureteral obstruction (UUO) mouse model was built. Then the mice were treated with PAE (100 mg/kg body weight) once daily by oral gavage, again starting on the day of UUO and continued for 1 week. At the end of 1 week, the mice were sacrificed; kidney samples were collected for further analysis. In vitro, Boston University mouse proximal tubular cells were plated in 35-mm dishes at a density of 0.3 * 106 cells/dish. Then the cells were treated with 5-ng/mL TGF-ß1 in serum-free DMEM medium for an indicated length of time. The experimental groups were pretreated with the indicated concentrations of PAE (0.3125 mg/mL). The cells were further cultured for 24 h, and then cells were monitored morphologically or collected for biochemical analyses. RESULTS: Both in vivo and vitro PAE inhibits the expression of FN and alpha-smooth muscle actin and suppresses renal fibrosis. Importantly, PAE protects against renal fibrosis by inhibiting Janus tyrosine kinase 2 (JAK)/signal transducer and activator of transcription 3 (STAT) tyrosine phosphorylation. CONCLUSION: PAE attenuates renal fibrosis through the suppression of the JAK2/STAT3 pathway.


Assuntos
Fibrose/prevenção & controle , Janus Quinase 2/antagonistas & inibidores , Rim/patologia , Periplaneta , Extratos Vegetais/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Actinas/biossíntese , Animais , Células Cultivadas , Fibronectinas/biossíntese , Masculino , Camundongos , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
9.
PLoS One ; 13(3): e0194053, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29518138

RESUMO

Sphingosine Kinase-2 (Sphk2) is responsible for the production of the bioactive lipid Sphingosine-1 Phosphate, a key regulator of tissue repair. Here we address the in vivo significance of Sphingosine Kinase -2 in renal inflammation/fibrosis in response to unilateral ureteral obstruction using both genetic and pharmacological strategies. Obstructed kidneys of Sphk2-/- mice showed reduced renal damage and diminished levels of the renal injury markers TGFß1 and αSMA when compared to wild type controls. We found a consistently significant increase in anti-inflammatory (M2) macrophages in obstructed Sphk2-/- kidneys by flow cytometry and a decrease in mRNA levels of the inflammatory cytokines, MCP1, TNFα, CXCL1 and ILß1, suggesting an anti-inflammatory bias in the absence of Sphk2. Indeed, metabolic profiling showed that the pro-inflammatory glycolytic pathway is largely inactive in Sphk2-/- bone marrow-derived macrophages. Furthermore, treatment with the M2-promoting cytokines IL-4 or IL-13 demonstrated that macrophages lacking Sphk2 polarized more efficiently to the M2 phenotype than wild type cells. Bone marrow transplant studies indicated that expression of Sphk2-/- on either the hematopoietic or parenchymal cells did not fully rescue the pro-healing phenotype, confirming that both infiltrating M2-macrophages and the kidney microenvironment contribute to the damaging Sphk2 effects. Importantly, obstructed kidneys from mice treated with an Sphk2 inhibitor recapitulated findings in the genetic model. These results demonstrate that reducing Sphk2 activity by genetic or pharmacological manipulation markedly decreases inflammatory and fibrotic responses to obstruction, resulting in diminished renal injury and supporting Sphk2 as a novel driver of the pro-inflammatory macrophage phenotype.


Assuntos
Macrófagos/fisiologia , Nefrite Intersticial/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Actinas/biossíntese , Actinas/genética , Animais , Microambiente Celular , Citocinas/biossíntese , Citocinas/genética , Fibrose , Regulação da Expressão Gênica/imunologia , Glicólise , Rim/enzimologia , Rim/patologia , Lisofosfolipídeos/sangue , Lisofosfolipídeos/fisiologia , Ativação de Macrófagos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nefrite Intersticial/etiologia , Nefrite Intersticial/imunologia , Nefrite Intersticial/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/deficiência , Isoformas de Proteínas/fisiologia , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Esfingosina/análogos & derivados , Esfingosina/sangue , Esfingosina/fisiologia , Fator de Crescimento Transformador beta1/biossíntese , Fator de Crescimento Transformador beta1/genética , Obstrução Ureteral/complicações
10.
Ann Hematol ; 97(7): 1251-1258, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29589107

RESUMO

We have previously demonstrated that recombinant human erythropoietin (rHuEpo) is involved in the regulation of the angiogenic response in multiple myeloma (MM) through a direct effect on macrophages and endothelial cells isolated from the bone marrow of patients with MM. The aim of the present study was designed to determine the effects of rHuEpo on cancer-associated fibroblasts (CAFs) from monoclonal gammopathy of undetermined significance (MGUS) and MM patients by means of in vitro and in vivo assays. rHuEpo treatment reduces the expression of mRNA levels of fibroblast activation markers, namely alpha smooth actin (αSMA) and fibroblast activation protein (FAP) in MGUS and MM CAFs, and of pro-inflammatory and pro-angiogenic cytokines, including interleukin (IL)-6 and IL-8, vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor-2 (FGF-2), and hepatocyte growth factor (HGF) in MM CAFs. Moreover, rHuEpo inhibits the proliferative activity of MM CAFs and increased the apoptosis of MGUS and MM CAFs. Overall, these data suggest that rHu-Epo down-regulates CAFs pro-tumorigenic activity. Moreover, these results are not suggestive for a pro-angiogenic activity of rHuEpo on CAFs. In fact, rHuEpo pre-treatment induces a low angiogenic response in vivo in the chorioallantoic membrane (CAM) assay of MGUS and MM CAFs conditioned medium, not comparable to that of a well-known angiogenic cytokine, VEGF-A, tested in the same assay.


Assuntos
Fibroblastos/efeitos dos fármacos , Gamopatia Monoclonal de Significância Indeterminada/patologia , Mieloma Múltiplo/patologia , Actinas/biossíntese , Actinas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/efeitos dos fármacos , Citocinas/biossíntese , Citocinas/genética , Método Duplo-Cego , Epoetina alfa , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Gelatinases/biossíntese , Gelatinases/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Neovascularização Fisiológica/efeitos dos fármacos , Receptores da Eritropoetina/biossíntese , Receptores da Eritropoetina/genética , Serina Endopeptidases/biossíntese , Serina Endopeptidases/genética
11.
PLoS One ; 13(2): e0192146, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29444136

RESUMO

Fibroblast growth factor 21 (FGF21) is an important metabolic regulator expressed predominantly in the liver. In this study, we evaluated the role of LY2405319, an analogue of FGF21, in hepatic stellate cell (HSC) activation and in a methionine and choline-deficient (MCD)-diet induced mouse model of liver fibrosis. During liver injury, HSCs trans-differentiate into activated myofibroblasts which produce alpha-smooth muscle actin (α-SMA) and become a major cell type in hepatic fibrogenesis. Succinate and succinate receptor (GPR91) signaling has emerged as a regulator to promote α-SMA production in MCD diet- induced mice. Treatment with palmitate or MCD medium on LX-2 cells (HSCs) increased succinate concentration in the conditioned medium and cell lysate of LX-2 cells and increased production of GPR91 and α-SMA. However, LY2405319 administration ameliorates palmitate or MCD media-induced succinate production and decreases over-expression of GPR91 and α-SMA in LX2-cells. In an in vivo study, the MCD diet treatment caused increased steatohepatitis and liver fibrosis compared with the control diet in mice. Administration of LY2405319 improved steatohepatitis ameliorated GPR91 and α -SMA production in the liver, decreased succinate concentration in both liver and serum of MCD diet -induced mice. These results suggest that FGF21 reduces production of α-SMA by inhibiting the succinate-GPR91 pathway. We conclude that FGF21 acts as an inhibitor of the succinate-GPR91 pathway to control liver fibrosis. This suggests that FGF21 has therapeutic potential for treating liver fibrogenesis.


Assuntos
Actinas/antagonistas & inibidores , Fatores de Crescimento de Fibroblastos/farmacologia , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Succinatos/antagonistas & inibidores , Actinas/biossíntese , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I , Cirrose Hepática/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL
12.
Biochim Biophys Acta Mol Basis Dis ; 1864(4 Pt A): 1129-1137, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29366776

RESUMO

Mature crosslinked-poly-elastin deposition has been found to be associated with liver fibrosis. However, the regulation of crosslinked/insoluble elastin in liver fibrosis remains largely unknown. Here, we investigated the contribution of lysyl oxidases (LOXs) family, mediated elastin crosslinking, to liver fibrogenesis. We established carbon tetrachloride (CCl4)-induced liver fibrotic and cirrhotic models and found that crosslinked/insoluble elastin levels spiked only in cirrhosis stage during disease progression, in comparison to collagen Ι levels which increased continuously though all stages. Among the LOXs family members, only LOX-like 1 (LOXL1) levels were coincident with the appearance of crosslinked/insoluble elastin. These coincidences included that LOXL1 expression increased (34 fold) in cirrhosis, localized with α-smooth muscle actin (SMA) and was absent in normal and fibrotic livers. In LX-2 cells, LOXL1 silencing arrested expression of α-SMA, elastin and collagen Ι. Our previously characterized adeno-associated vector (AAV) 2/8 shRNA was shown to effectively downregulate LOXL1 expression in CCl4 induced fibrosis mice models. These resulted in delicate and thinner septa and less crosslinked elastin, with a 58% loss of elastin area and 51% decrease of collagen area. Our findings strongly suggested that elastin crosslinking and LOXL1 were co-associated with liver cirrhosis, while selective inhibition of LOXL1 arrested disease progression by reducing crosslinking of elastin.


Assuntos
Aminoácido Oxirredutases/biossíntese , Elastina/metabolismo , Regulação Enzimológica da Expressão Gênica , Cirrose Hepática/metabolismo , Actinas/biossíntese , Actinas/genética , Aminoácido Oxirredutases/genética , Animais , Intoxicação por Tetracloreto de Carbono/genética , Intoxicação por Tetracloreto de Carbono/metabolismo , Intoxicação por Tetracloreto de Carbono/patologia , Colágeno Tipo I/biossíntese , Colágeno Tipo I/genética , Elastina/genética , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/genética , Cirrose Hepática/patologia , Masculino , Camundongos
13.
Am J Dermatopathol ; 40(6): 449-451, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29329129

RESUMO

Alpha smooth muscle actin (SMA) belongs to the actin proteins. It is a known immunohistochemical marker for tumors of mesenchymal origin. There have been reports of expression of SMA in certain epithelial malignancies in the head and neck and genital regions. In this study, the authors report a primary cutaneous spindle cell squamous carcinoma expressing SMA. Both high- and low-molecular-weight keratins and p63 were positive, and S100 protein, SOX10, MART-1/Melan-A, and muscle-specific actin stains were negative. This case highlights that an epithelial tumor could express a mesenchymal marker, thereby making the diagnosis problematic.


Assuntos
Actinas/biossíntese , Carcinoma de Células Escamosas/patologia , Neoplasias Cutâneas/patologia , Actinas/análise , Biomarcadores Tumorais/análise , Feminino , Humanos , Pessoa de Meia-Idade , Músculo Liso/patologia
14.
J Pharm Pharmacol ; 70(3): 393-403, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29341132

RESUMO

OBJECTIVES: In alcoholic liver disease, alcohol and lipopolysaccharide (LPS) are major stimulation factors of hepatic lipogenesis. Our objective was to determine the protective mechanism of acanthoic acid (AA) in EtOH- and LPS-induced hepatic lipogenesis. METHODS: HSC-T6 cells were treated with ethanol (200 mm) plus LPS (1 µg/ml) for 1 h, followed by AA (10 or 20 µm) for another 6 h. C57BL/6 mice were pretreated with of AA (20 and 40 mg/kg) or equal volume of saline and then exposed to three doses of ethanol (5 g/kg body weight) within 24 h. The mice were sacrificed at 6 h after the last ethanol dosing. KEY FINDINGS: Acanthoic acid significantly decreased the expressions of α-SMA, collagen-I, SREBP-1, and lipin1/2 induced, also decreased fat droplets caused by EtOH/LPS. AA treatment decreased the protein expressions of TLR4, CD14, IRAK4, TRAF3, p-TAK1 and NF-κB increased by EtOH/LPS on HSC cells. Results in vivo were consistent with results in vitro. CONCLUSIONS: Our data demonstrated that AA might modulate hepatic fibrosis and lipid deposition in HSC-T6 cell stimulated with ethanol combined with LPS by decreasing lipin1/2 via TLR4 and IRAK4 signalling pathways, and AA might be considered as a potential therapeutic candidate for alcoholic liver disease.


Assuntos
Diterpenos/farmacologia , Lipogênese/efeitos dos fármacos , Hepatopatias Alcoólicas/prevenção & controle , Fosfatidato Fosfatase/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Actinas/biossíntese , Animais , Células Cultivadas , Colágeno/biossíntese , Diterpenos/isolamento & purificação , Etanol , Receptores de Lipopolissacarídeos/biossíntese , Lipopolissacarídeos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Hepatopatias Alcoólicas/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Camundongos , NF-kappa B/metabolismo , Fosfatidato Fosfatase/biossíntese , Proteínas Serina-Treonina Quinases/biossíntese , Ratos , Proteínas de Ligação a Elemento Regulador de Esterol/biossíntese , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Fator 3 Associado a Receptor de TNF/biossíntese , Receptor 4 Toll-Like/biossíntese
15.
Life Sci ; 192: 183-189, 2018 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-29197497

RESUMO

AIMS: Peroxisome-proliferator activated receptor γ (PPARγ) plays a pivotal role in inhibition of hepatic stellate cell (HSC) activation, a key step for liver fibrogenesis. Adipocyte-derived hormone leptin has been shown to promote liver fibrosis in murine and human. PPARγ includes two subtypes, PPARγ1 and PPARγ2. Our previous study indicated that leptin down-regulated PPARγ1 expression in HSCs. The aim of this study was to investigate the effect of leptin on PPARγ2 expression and the underlying mechanisms in HSCs. MAIN METHODS: Real-time PCR and western blot analyses were used to examine gene expression. The promoter activities were detected by luciferase assay. KEY FINDINGS: Leptin reduced PPARγ2 expressions at promoter level, mRNA level, and protein level in HSCs, which required ß-catenin, p38 mitogen-activated protein kinase, and delta-like homolog1 (DLK1) signaling pathways. Leptin induced GATA binding protein 2 (GATA2) expression through DLK1 pathway and GATA2 reduced PPARγ2 expression. Ectopic expression of PPARγ2 reduced the protein levels of α-smooth muscle actin and α1(I)collagen in HSCs. SIGNIFICANCE: Since obese patients, often accompanied by hyperleptinemia, are more prone to liver fibrosis, the data from this study might have potential implications for clarifying the mechanisms for liver fibrogenesis in obese patients with hyperleptinemia.


Assuntos
Fator de Transcrição GATA2/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Leptina/farmacologia , PPAR gama/antagonistas & inibidores , PPAR gama/biossíntese , Actinas/biossíntese , Animais , Colágeno Tipo I/biossíntese , Fator de Transcrição GATA2/biossíntese , Fator de Transcrição GATA2/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos Endogâmicos C57BL , Cultura Primária de Células , Regiões Promotoras Genéticas/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
16.
Int J Oncol ; 52(1): 89-102, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29115590

RESUMO

Lamins are the major components of the nuclear lamina and play important roles in many cellular processes. The role of lamins in cancer development and progression is still unclear but it is known that reduced expression of lamin B1 has been observed in colon cancer. Thus, the aim of the present study was to elucidate the influence of LMNB1 upregulation on colon cancer cell line after treatment with 5-FU. The results indicate, that overexpression of LMNB1 induced dose-dependent cell death mainly by mitotic catastrophe pathway. Furthermore, after upregulation of this intermediate protein, lower expression of lamin A/C was observed. Moreover, we observed an increase in fluorescence intensity of nuclear ß-catenin and decrease in cell-cell interaction area, that was connected with inhibition of colon cancer cells migration. We present the reorganization of actin filament and ß-tubulin, because these cytoskeletal proteins are directly or indirectly linked with lamins, and analyzing publicly available mRNA data we show that patients with overexpression of LMNB1 are characterized by lower survival rates within the first 30 months from diagnosis. Summarizing our results, upregulation of LMNB1 induce mitotic catastrophe and only small percentage of apoptosis. Moreover, we showed inhibition of cell migration and promotion of cell-cell contact as a results of direct and indirect regulation of ß-catenin, lamin A/C, actin and tubulin. However, it is possible that mitotic catastrophe cells in patients with colorectal cancer may be a reservoir of the cells responsible for faster disease progression, and further investigations are necessary to confirm this hypothesis.


Assuntos
Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Lamina Tipo B/biossíntese , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/patologia , Actinas/biossíntese , Antimetabólitos Antineoplásicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Fluoruracila/farmacologia , Humanos , Lamina Tipo B/genética , Mitose/fisiologia , Transfecção , Tubulina (Proteína)/biossíntese , Tubulina (Proteína)/metabolismo , Regulação para Cima , beta Catenina/biossíntese
17.
Drug Res (Stuttg) ; 68(3): 153-158, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28992660

RESUMO

BACKGROUND: Alpha mangostin has been reported to have activity for the treatment of liver fibrosis in the rats. However, the mechanisms of action are poorly understood. This study was aimed to investigate the effect of alpha mangostin on hepatic stellate cells (HSC) activation and proliferation through TGF-ß/Smad and Akt signaling pathways. METHODS: Immortalized HSC, LX2 cells, were incubated with TGF-ß with or without alpha mangostin (5 or 10 µM). Sorafenib 10 µM was used as positive control. LX2 viability was counted using trypan blue exclusion method. The effect of alpha mangostin on TGF-ß concentrations, and the expressions of proliferation and fibrogenic markers were evaluated. RESULTS: Alpha mangostin treatment resulted in a reduced proliferation of HSC, decreased Ki-67 and p-Akt expressions. These findings were followed with decreased concentrations of TGF-ß in the medium of cells treated with alpha mangostin, decreased expressions of COL1A1, TIMP1, PAI1, α-SMA, and p-Smad3 as fibrogenic markers. These effects were shown to be dose-dependent. CONCLUSIONS: Alpha mangostin inhibits hepatic stellate cells proliferation and activation through TGF-ß/Smad and Akt signaling pathways in dose dependent manner.


Assuntos
Actinas/biossíntese , Colágeno Tipo I/biossíntese , Células Estreladas do Fígado/efeitos dos fármacos , Inibidor 1 de Ativador de Plasminogênio/biossíntese , Proteínas Proto-Oncogênicas c-akt/biossíntese , Proteína Smad3/biossíntese , Inibidor Tecidual de Metaloproteinase-1/biossíntese , Fator de Crescimento Transformador beta/metabolismo , Xantonas/farmacologia , Animais , Biomarcadores/metabolismo , Proliferação de Células , Células Cultivadas , Relação Dose-Resposta a Droga , Antígeno Ki-67/biossíntese , Masculino , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Compostos de Fenilureia/farmacologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Proteína Smad3/metabolismo , Sorafenibe
18.
Am J Respir Cell Mol Biol ; 58(3): 378-390, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29019707

RESUMO

Glutaminolysis is the metabolic process of glutamine, aberration of which has been implicated in several pathogeneses. Although we and others recently found a diversity of metabolic dysregulation in organ fibrosis, it is unknown if glutaminolysis regulates the profibrotic activities of myofibroblasts, the primary effector in this pathology. In this study, we found that lung myofibroblasts demonstrated significantly augmented glutaminolysis that was mediated by elevated glutaminase 1 (Gls1). Inhibition of glutaminolysis by specific Gls1 inhibitors CB-839 and BPTES as well as Gls1 siRNA blunted the expression of collagens but not that of fibronectin, elastin, or myofibroblastic marker smooth muscle actin-α. We found that glutaminolysis enhanced collagen translation and stability, which were mediated by glutaminolysis-dependent mTOR complex 1 activation and collagen proline hydroxylation, respectively. Furthermore, we found that the amount of the glutaminolytic end product α-ketoglutarate (α-KG) was increased in myofibroblasts. Similar to glutaminolysis, α-KG activated mTOR complex 1 and promoted the expression of collagens but not of fibronectin, elastin, or smooth muscle actin-α. α-KG also remarkably inhibited collagen degradation in fibroblasts. Taken together, our studies identified a previously unrecognized mechanism by which a major metabolic program regulates the exuberant production of collagens in myofibroblasts and suggest that glutaminolysis is a novel therapeutic target for treating organ fibrosis, including idiopathic pulmonary fibrosis.


Assuntos
Glutamina/metabolismo , Ácidos Cetoglutáricos/metabolismo , Miofibroblastos/metabolismo , Prolina/química , Fibrose Pulmonar/patologia , Serina-Treonina Quinases TOR/metabolismo , Actinas/biossíntese , Animais , Benzenoacetamidas/farmacologia , Células Cultivadas , Colágeno/biossíntese , Modelos Animais de Doenças , Elastina/biossíntese , Ativação Enzimática/fisiologia , Fibronectinas/biossíntese , Glutaminase/antagonistas & inibidores , Glutaminase/genética , Glutaminase/metabolismo , Humanos , Hidroxilação , Camundongos , Camundongos Endogâmicos C57BL , Interferência de RNA , RNA Interferente Pequeno/genética , Sulfetos/farmacologia , Tiadiazóis/farmacologia
19.
PLoS One ; 13(12): e0210087, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30596787

RESUMO

The imbalance in homocysteine (Hcy) metabolism has been implicated in the pathogenesis of human diseases, including cardiovascular and neurodegenerative disorders. When attempting to identify gene expression profiles using quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR), the selection of suitable reference genes is important. Here, the expression levels of 10 commonly used reference genes were assessed for normalization of RT-qPCR in Hcy-treated human umbilical vein endothelial cells (HUVECs) and control cells. The suitability of eight selected candidate genes was comparatively analyzed across the tested samples and separately ranked by four programs, geNorm, NormFinder, BestKeeper, and the ΔCt method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was the most stable gene in the final ranking using the RankAggreg package. Surprisingly, the ß-actin (ACTB) levels decreased significantly in Hcy-treated HUVECs compared with control HUVECs (P<0.05), and further study indicated that Hcy suppressed the expression of ACTB by upregulating the miR-145-5p level in Hcy-treated HUVECs. Our data suggest that GAPDH can be used as a reliable reference gene, while ACTB cannot; normalization of gene expression in RT-qPCR experiments in Hcy-treated HUVECs. The data, which identifies a suitable reference gene in Hcy-treated HUVECs, will contribute to the design of an effective and accurate method for quantitation of gene expression.


Assuntos
Actinas/biossíntese , Regulação da Expressão Gênica/efeitos dos fármacos , Gliceraldeído-3-Fosfato Desidrogenases/biossíntese , Homocisteína/farmacologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/normas , Actinas/genética , Gliceraldeído-3-Fosfato Desidrogenases/genética , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Padrões de Referência
20.
Am J Hum Genet ; 101(6): 1021-1033, 2017 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-29220674

RESUMO

ACTB encodes ß-actin, an abundant cytoskeletal housekeeping protein. In humans, postulated gain-of-function missense mutations cause Baraitser-Winter syndrome (BRWS), characterized by intellectual disability, cortical malformations, coloboma, sensorineural deafness, and typical facial features. To date, the consequences of loss-of-function ACTB mutations have not been proven conclusively. We describe heterozygous ACTB deletions and nonsense and frameshift mutations in 33 individuals with developmental delay, apparent intellectual disability, increased frequency of internal organ malformations (including those of the heart and the renal tract), growth retardation, and a recognizable facial gestalt (interrupted wavy eyebrows, dense eyelashes, wide nose, wide mouth, and a prominent chin) that is distinct from characteristics of individuals with BRWS. Strikingly, this spectrum overlaps with that of several chromatin-remodeling developmental disorders. In wild-type mouse embryos, ß-actin expression was prominent in the kidney, heart, and brain. ACTB mRNA expression levels in lymphoblastic lines and fibroblasts derived from affected individuals were decreased in comparison to those in control cells. Fibroblasts derived from an affected individual and ACTB siRNA knockdown in wild-type fibroblasts showed altered cell shape and migration, consistent with known roles of cytoplasmic ß-actin. We also demonstrate that ACTB haploinsufficiency leads to reduced cell proliferation, altered expression of cell-cycle genes, and decreased amounts of nuclear, but not cytoplasmic, ß-actin. In conclusion, we show that heterozygous loss-of-function ACTB mutations cause a distinct pleiotropic malformation syndrome with intellectual disability. Our biological studies suggest that a critically reduced amount of this protein alters cell shape, migration, proliferation, and gene expression to the detriment of brain, heart, and kidney development.


Assuntos
Anormalidades Múltiplas/genética , Actinas/genética , Deficiências do Desenvolvimento/genética , Haploinsuficiência/genética , Actinas/biossíntese , Adolescente , Adulto , Idoso , Animais , Ciclo Celular/genética , Proliferação de Células/genética , Criança , Pré-Escolar , Códon sem Sentido/genética , Coloboma/genética , Facies , Feminino , Mutação da Fase de Leitura/genética , Deleção de Genes , Humanos , Lactente , Recém-Nascido , Deficiência Intelectual/genética , Masculino , Malformações do Desenvolvimento Cortical/genética , Camundongos , Interferência de RNA , RNA Interferente Pequeno/genética , Adulto Jovem
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