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1.
Toxicol Lett ; 313: 130-136, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31276767

RESUMO

We previously demonstrated that based on their potency, contact allergens differently modulate Blimp-1/NLRP12 expression in human keratinocytes, with the extreme allergen 2,4-dinitrochlorobenzene (DNCB) more rapidly upregulating Blimp-1, leading to downregulation of NLRP12, and to the production of interleukin-18 (IL-18). The purpose of this study was to further investigate the effects of DNCB and para-phenylenediamine (PPD) on the expression of the proteins of the inflammasome, namely NLRP3, ASC and caspase 1 by western blot analysis; to define the intracellular localization and co-localization of NLRP3 and NLPR12 by immunoprecipitation and immunohistochemistry; and to define the role of NF-κB in Blimp-1 induction by pharmacological inhibition. The human keratinocyte cell line NCTC2544 was used for all experiments. Dose and time course experiments were performed to evaluate the effect of the selected contact allergens on the parameters investigated. Results indicate, that consistent with previous finding, DNCB more rapidly (3 h) induces NLRP3, ASC protein expression and caspase-1 activation compared to PPD. Immunoprecipitation studies show the recruitment of ASC to the inflammasome following exposure to both allergens, while high level of NLRP12 and less ASC protein were found associated in control cells. By immunohistochemistry, we found increased NLRP3 expression following exposure to contact allergens, and observed a nuclear co-localization of the two proteins, indicating the NLRP12 likely acts preventing the cytosolic localization of NLRP3 and inflammasome assembly. Finally, contact allergen-induced Blimp-1 mRNA and protein expression can be completely blocked by inhibiting NF-κB activation, confirming the central role of NF-κB in contact allergen-induced keratinocyte activation.


Assuntos
Alérgenos/toxicidade , Dermatite Alérgica de Contato/etiologia , Dinitroclorobenzeno/toxicidade , Inflamassomos/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Queratinócitos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fenilenodiaminas/toxicidade , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Caspase 1/metabolismo , Linhagem Celular , Dermatite Alérgica de Contato/genética , Dermatite Alérgica de Contato/metabolismo , Relação Dose-Resposta a Droga , Humanos , Inflamassomos/genética , Inflamassomos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Queratinócitos/metabolismo , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Fatores de Tempo
2.
Anticancer Res ; 39(6): 2689-2697, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31177103

RESUMO

Colorectal cancer (CRC) is the second most prevalent type of cancer among males and the third among females. CRC recurrence and poor prognosis may be related to the prevalence of chemotherapy-resistant cancer stem cells (CSCs). Recent studies have indicated the role of doublecortin-like kinase 1 (DCLK1) protein as a marker of CSC in CRC. This review focuses on the role of DCLK1 in CRC. Long-lived DCLK1-positive tuft cells can function as cancer-initiating cells. Numerous studies have shown DCLK1 overexpression to be significantly correlated with the stage of disease, the presence of metastasis and poor survival rate. DCLK1 may also be used to identify patients at high risk and those with chemotherapy-resistant tumors. DCLK1-specific drugs are examined as potential cancer treatments.


Assuntos
Neoplasias Colorretais/patologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Regulação para Cima , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Metástase Neoplásica , Estadiamento de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Prognóstico , Análise de Sobrevida
3.
Nat Commun ; 10(1): 2532, 2019 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-31182717

RESUMO

Targeted inhibition of the ERK-MAPK pathway, upregulated in a majority of human cancers, has been hindered in the clinic by drug resistance and toxicity. The MRAS-SHOC2-PP1 (SHOC2 phosphatase) complex plays a key role in RAF-ERK pathway activation by dephosphorylating a critical inhibitory site on RAF kinases. Here we show that genetic inhibition of SHOC2 suppresses tumorigenic growth in a subset of KRAS-mutant NSCLC cell lines and prominently inhibits tumour development in autochthonous murine KRAS-driven lung cancer models. On the other hand, systemic SHOC2 ablation in adult mice is relatively well tolerated. Furthermore, we show that SHOC2 deletion selectively sensitizes KRAS- and EGFR-mutant NSCLC cells to MEK inhibitors. Mechanistically, SHOC2 deletion prevents MEKi-induced RAF dimerization, leading to more potent and durable ERK pathway suppression that promotes BIM-dependent apoptosis. These results present a rationale for the generation of SHOC2 phosphatase targeted therapies, both as a monotherapy and to widen the therapeutic index of MEK inhibitors.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/genética , Inibidores de Proteínas Quinases/farmacologia , Quinases raf/metabolismo , Animais , Apoptose , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos Knockout , Camundongos Nus , Mutação , Transplante de Neoplasias , Multimerização Proteica , Quinases raf/antagonistas & inibidores , Quinases raf/genética , Proteínas ras/metabolismo
4.
Nat Commun ; 10(1): 2761, 2019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31235698

RESUMO

Cerebral cavernous malformation (CCM) is a neurovascular familial or sporadic disease that is characterised by capillary-venous cavernomas, and is due to loss-of-function mutations to any one of three CCM genes. Familial CCM follows a two-hit mechanism similar to that of tumour suppressor genes, while in sporadic cavernomas only a small fraction of endothelial cells shows mutated CCM genes. We reported that in mouse models and in human patients, endothelial cells lining the lesions have different features from the surrounding endothelium, as they express mesenchymal/stem-cell markers. Here we show that cavernomas originate from clonal expansion of few Ccm3-null endothelial cells that express mesenchymal/stem-cell markers. These cells then attract surrounding wild-type endothelial cells, inducing them to express mesenchymal/stem-cell markers and to contribute to cavernoma growth. These characteristics of Ccm3-null cells are reminiscent of the tumour-initiating cells that are responsible for tumour growth. Our data support the concept that CCM has benign tumour characteristics.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Neoplasias do Sistema Nervoso Central/patologia , Células Endoteliais/patologia , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas/genética , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Biomarcadores/metabolismo , Encéfalo/irrigação sanguínea , Encéfalo/citologia , Encéfalo/patologia , Diferenciação Celular/genética , Linhagem Celular , Neoplasias do Sistema Nervoso Central/genética , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/patologia , Feminino , Técnicas de Inativação de Genes , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutação com Perda de Função , Proteínas de Membrana/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas/metabolismo
5.
Nat Commun ; 10(1): 2882, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253783

RESUMO

NLR Family CARD Domain Containing 5 (NLRC5), an important immune regulator in innate immunity, is involved in regulating inflammation and antigen presentation. However, the role of NLRC5 in vascular remodeling remains unknown. Here we report the role of NLRC5 on vascular remodeling and provide a better understanding of its underlying mechanism. Nlrc5 knockout (Nlrc5-/-) mice exhibit more severe intimal hyperplasia compared with wild-type mice after carotid ligation. Ex vivo data shows that NLRC5 deficiency leads to increased proliferation and migration of human aortic smooth muscle cells (HASMCs). NLRC5 binds to PPARγ and inhibits HASMC dedifferentiation. NACHT domain of NLRC5 is essential for the interaction with PPARγ and stimulation of PPARγ activity. Pioglitazone significantly rescues excessive intimal hyperplasia in Nlrc5-/- mice and attenuates the increased proliferation and dedifferentiation in NLRC5-deficient HASMCs. Our study demonstrates that NLRC5 regulates vascular remodeling by directly inhibiting SMC dysfunction via its interaction with PPARγ.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Túnica Íntima/metabolismo , Animais , Aorta , Apoptose , Pressão Sanguínea , Transplante de Medula Óssea , Proliferação de Células , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Frequência Cardíaca , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Knockout , Miócitos de Músculo Liso/metabolismo , Plasmídeos , Transcriptoma , Remodelação Vascular
6.
Food Chem Toxicol ; 131: 110533, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31150783

RESUMO

Hepatocellular carcinoma is the fifth most common and the third most lethal cancer worldwide. In recent years, natural flavonoids have drawn great attention as repository for the exploitation of novel antineoplastic agents. 2',4'-Dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC), a functional chalcone isolated from the buds of Cleistocalyx operculatus, has been reported to exert potent cytotoxicity against multi-drug resistant BEL-7402/5-FU cells. In this study, the precise mechanisms of DMC-mediated growth inhibition in BEL-7402/5-FU cells were further investigated. DMC was found to trigger apoptosis predominantly via the mitochondria-dependent pathway and the enhancement of reactive oxygen species (ROS) generation. Meanwhile, DMC induced G1 cell cycle arrest through downregulation of cyclin D1 and CDK4. Furthermore, DMC increased p53 level and inhibited NF-κB nuclear-localization via suppression of PI3K/AKT signaling axis, which might be the underlying mechanism of DMC-induced apoptosis and cell cycle arrest in BEL-7402/5-FU cells. Collectively, the study elucidated the mechanisms by which DMC may inhibit the growth of BEL-7402/5-FU cells and suggested the possibility that DMC might be a promising candidate therapeutic agent for hepatoma treatment in the future.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Chalconas/farmacologia , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Flores/química , Humanos , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Syzygium/química , Proteína Supressora de Tumor p53/metabolismo
7.
Nat Commun ; 10(1): 2850, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253801

RESUMO

Cancer cells secrete matrix metalloproteinases to remodel the extracellular matrix, which enables them to overcome tissue barriers and form metastases. The membrane-bound matrix metalloproteinase MT1-MMP (MMP14) is internalized by endocytosis and recycled in endosomal compartments. It is largely unknown how endosomal sorting and recycling of MT1-MMP are controlled. Here, we show that the endosomal protein WDFY2 controls the recycling of MT1-MMP. WDFY2 localizes to endosomal tubules by binding to membranes enriched in phosphatidylinositol 3-phosphate (PtdIns3P). We identify the v-SNARE VAMP3 as an interaction partner of WDFY2. WDFY2 knockout causes a strong redistribution of VAMP3 into small vesicles near the plasma membrane. This is accompanied by increased, VAMP3-dependent secretion of MT1-MMP, enhanced degradation of extracellular matrix, and increased cell invasion. WDFY2 is frequently lost in metastatic cancers, most predominantly in ovarian and prostate cancer. We propose that WDFY2 acts as a tumor suppressor by serving as a gatekeeper for VAMP3 recycling.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Metaloproteinases da Matriz/metabolismo , Invasividade Neoplásica , Proteína 3 Associada à Membrana da Vesícula/metabolismo , Actinas/fisiologia , Linhagem Celular Tumoral , Membrana Celular , Exocitose/fisiologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Metaloproteinases da Matriz/genética , Microtúbulos , Fosfatos de Fosfatidilinositol/fisiologia , Transporte Proteico , Proteína 3 Associada à Membrana da Vesícula/genética , Proteínas rab4 de Ligação ao GTP/genética , Proteínas rab4 de Ligação ao GTP/metabolismo
8.
Braz J Med Biol Res ; 52(6): e8399, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31166382

RESUMO

Imatinib is the first line of therapy for patients with metastatic or gastrointestinal stromal tumors (GIST). However, drug resistance limits the long-term effect of imatinib. Long non-coding RNAs (lncRNAs) are emerging as key players in regulating drug resistance in cancer. In this study, we investigated the association between lncRNA CCDC26 and IGF-1R in GIST and their involvement in drug resistance. Considering the key role of lncRNAs in drug resistance in cancer, we hypothesized that IGF-1R is regulated by lncRNAs. The expression of a series of reported drug resistance-related lncRNAs, including CCDC26, ARF, H19, NBR2, NEAT1, and HOTAIR, in GIST cells treated with imatinib H19 was examined at various time-points by qRT-PCR. Based on our results and published literature, CCDC26, a strongly down-regulated lncRNA following imatinib treatment, was chosen as our research target. GIST cells with high expression of CCDC26 were sensitive to imatinib treatment while knockdown of CCDC26 significantly increased the resistance to imatinib. Furthermore, we found that CCDC26 interacted with c-KIT by RNA pull down, and that CCDC26 knockdown up-regulated the expression of IGF-1R. Moreover, IGF-1R inhibition reversed CCDC26 knockdown-mediated imatinib resistance in GIST. These results indicated that treatments targeting CCDC26-IGF-1R axis would be useful in increasing sensitivity to imatinib in GIST.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Mesilato de Imatinib/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , RNA Longo não Codificante/genética , Receptores de Somatomedina/genética , Apoptose , Linhagem Celular Tumoral , Regulação para Baixo/genética , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , RNA Longo não Codificante/metabolismo , Receptores de Somatomedina/metabolismo , Transdução de Sinais
9.
Nat Commun ; 10(1): 2230, 2019 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-31110180

RESUMO

LNK (SH2B3) is a key negative regulator of JAK-STAT signaling which has been extensively studied in malignant hematopoietic diseases. We found that LNK is significantly elevated in cutaneous melanoma; this elevation is correlated with hyperactive signaling of the RAS-RAF-MEK pathway. Elevated LNK enhances cell growth and survival in adverse conditions. Forced expression of LNK inhibits signaling by interferon-STAT1 and suppresses interferon (IFN) induced cell cycle arrest and cell apoptosis. In contrast, silencing LNK expression by either shRNA or CRISPR-Cas9 potentiates the killing effect of IFN. The IFN-LNK signaling is tightly regulated by a negative feedback mechanism; melanoma cells exposed to IFN upregulate expression of LNK to prevent overactivation of this signaling pathway. Our study reveals an unappreciated function of LNK in melanoma and highlights the critical role of the IFN-STAT1-LNK signaling axis in this potentially devastating disease. LNK may be further explored as a potential therapeutic target for melanoma immunotherapy.


Assuntos
Interferons/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Melanoma/patologia , Proteínas/metabolismo , Neoplasias Cutâneas/patologia , Animais , Apoptose , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Células HEK293 , Humanos , Interferons/imunologia , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Fator de Transcrição STAT1/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Medicine (Baltimore) ; 98(19): e15629, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31083261

RESUMO

OBJECTIVE: To determine the effects and mechanism of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1, CC1)-mediated regulation of the Coxsackie and Adenovirus Receptor (CAR) after Coxsackievirus B3 (CVB3) infection. METHODS: A mouse CC1 overexpression recombinant virus was constructed, followed by insertion of a pLVX-CEACAM 1-zsgreen-puro (rLV-CEACAM 1) plasmid into the recombinant retrovirus. Cardiac myocytes were assigned into different groups according to various treatments. The apoptosis rate and cell activity in each group were observed. Further, CAR expression and SYK, IL-1ß, and p-SYK levels were measured. RESULTS: The recombinant retrovirus titer was measured as 1.5 × 10 TUs/ml. The apoptosis rate of cardiac myocytes in the CC1 overexpression plus CVB3 group was significantly elevated, and the relative expression of the CAR gene was the highest in the CC1 overexpression plus CVB3 group. TNF-α and IL-1ß levels increased due to CC1 overexpression and further increased after CVB3 infection. CAR protein expression also changed along with the levels of CC1, SYK, and TNF-α after infection. CONCLUSION: CC1 may promote CAR expression after CVB3 infection and regulate CAR protein expression by activating the CC1-SYK-TNF-α signaling axis during the infection process.


Assuntos
Antígeno Carcinoembrionário/metabolismo , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/metabolismo , Infecções por Coxsackievirus/metabolismo , Cardiopatias/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Animais , Apoptose/fisiologia , Antígeno Carcinoembrionário/genética , Infecções por Coxsackievirus/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Cardiopatias/etiologia , Cardiopatias/patologia , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Células Musculares/metabolismo , Células Musculares/patologia , Organismos Livres de Patógenos Específicos , Quinase Syk/antagonistas & inibidores , Quinase Syk/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
11.
Nat Commun ; 10(1): 2055, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-31053714

RESUMO

Autophagy is an essential recycling and quality control pathway. Mammalian ATG8 proteins drive autophagosome formation and selective removal of protein aggregates and organelles by recruiting autophagy receptors and adaptors that contain a LC3-interacting region (LIR) motif. LIR motifs can be highly selective for ATG8 subfamily proteins (LC3s/GABARAPs), however the molecular determinants regulating these selective interactions remain elusive. Here we show that residues within the core LIR motif and adjacent C-terminal region as well as ATG8 subfamily-specific residues in the LIR docking site are critical for binding of receptors and adaptors to GABARAPs. Moreover, rendering GABARAP more LC3B-like impairs autophagy receptor degradation. Modulating LIR binding specificity of the centriolar satellite protein PCM1, implicated in autophagy and centrosomal function, alters its dynamics in cells. Our data provides new mechanistic insight into how selective binding of LIR motifs to GABARAPs is achieved, and elucidate the overlapping and distinct functions of ATG8 subfamily proteins.


Assuntos
Motivos de Aminoácidos/fisiologia , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Autofagia , Ligação Proteica/fisiologia , Autoantígenos/isolamento & purificação , Autoantígenos/metabolismo , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/isolamento & purificação , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/isolamento & purificação , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteínas de Ciclo Celular/isolamento & purificação , Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/isolamento & purificação , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Proteólise , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
12.
Nat Commun ; 10(1): 2208, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101817

RESUMO

Cortical force generators connect epithelial polarity sites with astral microtubules, allowing dynein movement to orient the mitotic spindle as astral microtubules depolymerize. Complexes of the LGN and NuMA proteins, fundamental components of force generators, are recruited to the cortex by Gαi-subunits of heterotrimeric G-proteins. They associate with dynein/dynactin and activate the motor activity pulling on astral microtubules. The architecture of cortical force generators is unknown. Here we report the crystal structure of NuMA:LGN hetero-hexamers, and unveil their role in promoting the assembly of active cortical dynein/dynactin motors that are required in orchestrating oriented divisions in polarized cells. Our work elucidates the basis for the structural organization of essential spindle orientation motors.


Assuntos
Antígenos Nucleares/metabolismo , Polaridade Celular , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Fuso Acromático/metabolismo , Antígenos Nucleares/química , Antígenos Nucleares/genética , Antígenos Nucleares/isolamento & purificação , Células CACO-2 , Cristalografia por Raios X , Complexo Dinactina/metabolismo , Dineínas/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/isolamento & purificação , Microtúbulos/metabolismo , Proteínas Associadas à Matriz Nuclear/química , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/isolamento & purificação , Ligação Proteica/fisiologia , Multimerização Proteica/fisiologia , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
13.
Zhong Nan Da Xue Xue Bao Yi Xue Ban ; 44(4): 399-405, 2019 Apr 28.
Artigo em Chinês | MEDLINE | ID: mdl-31113915

RESUMO

OBJECTIVE: To explore the clinical significance of the altered expression of polycomb group (PcG)-associated protein RYBP in hepatocellular carcinoma (HCC) specimens.
 Methods: The expression levels of RYBP in tumor tissues and adjacent normal tissues in 77 HCC cases were detected by immunohistochemistry (IHC), and the relationships between RYBP expression levels and HCC clinicopathological characteristics, five-year survival rates or prognosis of HCC patients were analyzed.
 Results: RYBP expression level was significantly decreased in HCC tumor tissues than that in the adjacent normal tissues (P<0.05). The expression levels of RYBP in HCC specimens were highly correlated with HBsAg, ALT, GGT, Type III procollagen, tumor size, distant metastasis, and tumor differentiation (P<0.05). The RFS and OS for patients with RYBP-low expression were markedly lower than those with RYPB-high expression (P<0.05). Both age and RYBP expression level were protective factors for RFS, while GGT, lymph node metastasis, TNM stage, tumor differentiation and tumor size were risk factors for RFS (P<0.05). As to OS, RYBP expression level was a protective factor, while tumor number, ALT, GGT, AFP, pCEA, lymph node metastasis, TNM stage, tumor differentiation and tumor size were risk factors (P<0.05). The age, GGT, lymph node metastasis and TNM stage were independent prognostic factors for RFS (P<0.05), and both lymph node metastasis and TNM stage were independent risk factors for OS (P<0.05). Comparing to serum alpha fetoprotein (AFP) level, RYBP expression level in tumor tissues was applied to predict the prognosis of HCC patients more accurately.
 Conclusion: PcG associated protein RYBP displays a reduced expression in HCC tissues, which is related to poor prognosis of HCC patients. It might be a promising therapeutic target for HCC treatment.


Assuntos
Carcinoma Hepatocelular , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Hepáticas , Biomarcadores Tumorais , Humanos , Imuno-Histoquímica , Metástase Linfática , Prognóstico
14.
Nat Commun ; 10(1): 2356, 2019 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-31142743

RESUMO

Centrosomes control cell motility, polarity and migration that is thought to be mediated by their microtubule-organizing capacity. Here we demonstrate that WNT signalling drives a distinct form of non-directional cell motility that requires a key centrosome module, but not microtubules or centrosomes. Upon exosome mobilization of PCP-proteins, we show that DVL2 orchestrates recruitment of a CEP192-PLK4/AURKB complex to the cell cortex where PLK4/AURKB act redundantly to drive protrusive activity and cell motility. This is mediated by coordination of formin-dependent actin remodelling through displacement of cortically localized DAAM1 for DAAM2. Furthermore, abnormal expression of PLK4, AURKB and DAAM1 is associated with poor outcomes in breast and bladder cancers. Thus, a centrosomal module plays an atypical function in WNT signalling and actin nucleation that is critical for cancer cell motility and is associated with more aggressive cancers. These studies have broad implications in how contextual signalling controls distinct modes of cell migration.


Assuntos
Aurora Quinase B/metabolismo , Movimento Celular , Centrossomo/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Desgrenhadas/metabolismo , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Via de Sinalização Wnt , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/metabolismo , Carcinoma de Células de Transição/metabolismo , Linhagem Celular Tumoral , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Prognóstico , Mapas de Interação de Proteínas , Reação em Cadeia da Polimerase em Tempo Real , Neoplasias da Bexiga Urinária/metabolismo
15.
Dis Markers ; 2019: 6171782, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31061682

RESUMO

Renal cancer is one of the most common malignant urological tumors; however, its diagnosis and treatment are not well established. In the present study, we identified that CDK5 regulatory subunit-associated protein 3 (CDK5RAP3), a putative tumor suppressor in many cancers, was downregulated in renal cancer tissues. Through loss- and gain-of-function experiments, we observed that the action of CDK5RAP3 in renal cancer cells was different in Caki-1 and 769-P cell lines. Knockdown of endogenous CDK5RAP3 in Caki-1 slightly increased cell viability, whereas overexpression of CDK5RAP3 in 769-P cells inhibited cell viability. In addition, we observed that CDK5RAP3 participated in the regulation of autophagy in renal cancer. Knockdown of CDK5RAP3 induced significant inhibition of autophagy in Caki-1 cells but not in 769-P cells. In contrast, overexpression of CDK5RAP3 significantly activated autophagy in 769-P cells, as evidenced by increased LC3-II levels. However, the LC3-II could not be altered by CDK5RAP3 overexpression in Caki-1 cells. These findings demonstrated that CDK5RAP3 is downregulated in renal cancer and may be associated with autophagy.


Assuntos
Autofagia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Renais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Idoso , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Renais/genética , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética
16.
Int J Mol Sci ; 20(9)2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31052191

RESUMO

Tubulins and microtubules (MTs) represent targets for taxane-based chemotherapy. To date, several lines of evidence suggest that effectiveness of compounds binding tubulin often relies on different post-translational modifications on tubulins. Among them, methylation was recently associated to drug resistance mechanisms impairing taxanes binding. The sea urchin is recognized as a research model in several fields including fertilization, embryo development and toxicology. To date, some α- and ß-tubulin genes have been identified in P. lividus, while no data are available in echinoderms for arginine methyl transferases (PRMT). To evaluate the exploiting of the sea urchin embryo in the field of antiproliferative drug development, we carried out a survey of the expressed α- and ß-tubulin gene sets, together with a comprehensive analysis of the PRMT gene family and of the methylable arginine residues in P. lividus tubulins. Because of their specificities, the sea urchin embryo may represent an interesting tool for dissecting mechanisms of tubulin targeting drug action. Therefore, results herein reported provide evidences supporting the P. lividus embryo as animal system for testing antiproliferative drugs.


Assuntos
Citostáticos/toxicidade , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Ouriços-do-Mar/efeitos dos fármacos , Testes de Toxicidade/métodos , Moduladores de Tubulina/toxicidade , Tubulina (Proteína)/metabolismo , Animais , Embrião não Mamífero/efeitos dos fármacos , Metilação , Processamento de Proteína Pós-Traducional , Ouriços-do-Mar/embriologia
17.
Emerg Microbes Infect ; 8(1): 773-786, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31132962

RESUMO

Enterovirus 71 (EV71) is typically transmitted by the oral-faecal route and initiates infection upon crossing the intestinal mucosa. Our limited understanding of the mechanisms by which it crosses the intestinal mucosa has hampered the development of effective therapeutic options. Here, using an RNA interference screen combined with chemical inhibitors or the overexpression of dominant negative proteins, we found that EV71 entry into Caco-2 cells, a polarized human intestinal epithelial cell line, does not involve clathrin- and caveolae-dependent endocytic pathways or macropinocytosis but requires GTP-binding protein dynamin 2 and cytoskeleton remodelling. The use of siRNAs targeting endophilin family members revealed that endophlin-A2 is essential for the uptake of EV71 particles by Caco-2 cells. Subcellular analysis revealed that internalized EV71 virions largely colocalized with endophilin-A2 at cytomembrane ruffles and in the perinuclear area. Combined with viral entry kinetics, these data suggest that EV71 enters Caco-2 cells mainly via an endophilin-A2-mediated endocytic (EME) pathway. Finally, we showed that internalized EV71 virions were transported to endosomal sorting complex required for transport (ESCRT)-related multivesicular bodies (MVBs). These data provide attractive therapeutic targets to block EV71 infection.


Assuntos
Endocitose , Enterovirus Humano A/fisiologia , Infecções por Enterovirus/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Internalização do Vírus , Células CACO-2 , Enterovirus Humano A/genética , Infecções por Enterovirus/virologia , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Humanos , Mucosa Intestinal/virologia , Peptídeos e Proteínas de Sinalização Intracelular/genética
18.
Int J Oncol ; 54(6): 1995-2004, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31081045

RESUMO

Harmine (HM) is a ß­carboline alkaloid found in multiple medicinal plants. It has been used in folk medicine for anticancer therapy; however, the molecular mechanism of HM on human breast cancer remains unclear. Transcriptional co­activator with PDZ­binding motif (TAZ), also known as WW domain­containing transcription regulator 1, serves an important role in the carcinogenesis and progression of breast cancer. The aim of the present study was to elucidate the potential anticancer activity and mechanism of HM in breast cancer, in vitro and in vivo. Cell proliferation was measured using a CCK­8 assay, apoptotic activity was detected by flow cytometry and DAPI staining, and cell migration was examined using a wound healing assay. The expression of proteins, including extracellular signal­regulate kinase (Erk), phosphorylated (p­) Erk, protein kinase B (Akt), p­Akt, B­cell lymphoma 2 (Bcl­2) and Bcl­2­associated X protein (Bax), were determined by western blotting. The mRNA expression of TAZ was detected using reverse transcription­quantitative polymerase chain reaction analysis. The expression of proteins in mouse tumor tissues were examined by immunohistochemistry. HM significantly suppressed cellular proliferation and migration, promoted apoptosis in vitro and inhibited tumor growth in vivo. In addition, HM significantly decreased the expression of TAZ, p­Erk, p­Akt and Bcl­2, but increased that of Bax. The overexpression of TAZ in breast cancer cells inhibited the antitumor effect of HM. In conclusion, HM was found to induce apoptosis and prevent the proliferation and migration of human breast cancer cell lines, possibly via the downregulation of TAZ.


Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Neoplasias da Mama/tratamento farmacológico , Harmina/administração & dosagem , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Harmina/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células MCF-7 , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Int J Oncol ; 54(6): 2117-2126, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31081052

RESUMO

Transforming growth factor-ß1 (TGF-ß1) is a multifunctional cytokine that functions as a growth suppressor in normal epithelial cells and early stage tumors, but acts as a tumor promoter during malignant progression. However, the molecular basis underlying the conversion of TGF­ß1 function remains largely undefined. X­linked inhibitor of apoptosis­associated factor 1 (XAF1) is a pro­apoptotic tumor suppressor that frequently displays epigenetic inactivation in various types of human malignancies, including colorectal cancer. The present study explored whether the anti­apoptotic effect of TGF­ß1 is linked to its regulatory effect on XAF1 induction in human colon cancer cells under stressful conditions. The results revealed that TGF­ß1 treatment protected tumor cells from various apoptotic stresses, including 5­fluorouracil, etoposide and γ­irradiation. XAF1 expression was activated at the transcriptional level by these apoptotic stresses and TGF­ß1 blocked the stress­mediated activation of the XAF1 promoter. The study also demonstrated that mitogen­activated protein kinase kinase inhibition or extracellular signal­activated kinase (Erk)1/2 depletion induced XAF1 induction, while the activation of K­Ras (G12C) led to its reduction. In addition, TGF­ß1 blocked the stress­mediated XAF1 promoter activation and induction of apoptosis. This effect was abrogated if Erk1/2 was depleted, indicating that TGF­ß1 represses XAF1 transcription through Erk activation, thereby protecting tumor cells from apoptotic stresses. These findings point to a novel molecular mechanism underlying the tumor­promoting function of TGF­ß1, which may be utilized in the development of a novel therapeutic strategy for the treatment of colorectal cancer.


Assuntos
Neoplasias do Colo/metabolismo , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Neoplasias/genética , Fator de Crescimento Transformador beta1/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Neoplasias do Colo/genética , Progressão da Doença , Etoposídeo/farmacologia , Fluoruracila/farmacologia , Raios gama/efeitos adversos , Regulação Neoplásica da Expressão Gênica , Células HT29 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas de Neoplasias/metabolismo , Regiões Promotoras Genéticas , Proteínas ras/metabolismo
20.
Int J Oncol ; 54(6): 1907-1920, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31081062

RESUMO

The p53 protein is a tumour suppressor and transcription factor that regulates the expression of target genes involved in numerous stress responses systems. In this study, we designed a screening strategy using DNA damage­induced mouse and human transcriptome data to identify novel downstream targets of p53. Our method selected genes with an induced expression in multiple organs of X­ray­irradiated p53 wild­type mice. The expression of inka box actin regulator 2 gene, known as Inka2, was upregulated in 12 organs when p53 expression was induced. Similarly, INKA2 was induced in a p53­dependent manner at both the mRNA and protein level in human cells treated with adriamycin. Reporter assays confirmed that p53 directly regulated INKA2 through an intronic binding site. The overexpression of INKA2 produced a slight decrease in cancer cell growth in the colony formation assay. Moreover, the analysis of The Cancer Genome Atlas (TCGA) data revealed a decreased INKA2 expression in tumour samples carrying p53 mutations compared with p53 wild­type samples. In addition, significantly higher levels of DNA methylation were observed in the INKA2 promoter in tumour samples, concordant with the reduced INKA2 expression in tumour tissues. These results demonstrate the potential of INKA2 as a cancer cell growth inhibitor. Furthermore, INKA2 protein interacts with the serine/threonine­protein kinase, p21 (RAC1) activated kinase (PAK)4, which phosphorylates ß­catenin to prevent ubiquitin­proteasomal degradation. As ß­catenin was downregulated in a stable INKA2­expressing cell line, the findings of this study suggest that INKA2 is a novel, direct downstream target of p53 that potentially decreases cell growth by inhibiting the PAK4­ß­catenin pathway.


Assuntos
Perfilação da Expressão Gênica/métodos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias/genética , Proteína Supressora de Tumor p53/metabolismo , Quinases Ativadas por p21/metabolismo , Animais , Linhagem Celular Tumoral , Metilação de DNA , Doxorrubicina/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Células HCT116 , Humanos , Camundongos , Mutação , Neoplasias/metabolismo , Fosforilação , Regiões Promotoras Genéticas , Análise de Sequência de RNA/métodos , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , beta Catenina/metabolismo
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