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1.
EMBO J ; 30(18): 3714-28, 2011 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-21804532

RESUMO

C/EBPß is an auto-repressed protein that becomes post-translationally activated by Ras-MEK-ERK signalling. C/EBPß is required for oncogene-induced senescence (OIS) of primary fibroblasts, but also displays pro-oncogenic functions in many tumour cells. Here, we show that C/EBPß activation by H-Ras(V12) is suppressed in immortalized/transformed cells, but not in primary cells, by its 3' untranslated region (3'UTR). 3'UTR sequences inhibited Ras-induced cytostatic activity of C/EBPß, DNA binding, transactivation, phosphorylation, and homodimerization, without significantly affecting protein expression. The 3'UTR suppressed induction of senescence-associated C/EBPß target genes, while promoting expression of genes linked to cancers and TGFß signalling. An AU-rich element (ARE) and its cognate RNA-binding protein, HuR, were required for 3'UTR inhibition. These components also excluded the Cebpb mRNA from a perinuclear cytoplasmic region that contains activated ERK1/2, indicating that the site of C/EBPß translation controls de-repression by Ras signalling. Notably, 3'UTR inhibition and Cebpb mRNA compartmentalization were absent in primary fibroblasts, allowing Ras-induced C/EBPß activation and OIS to proceed. Our findings reveal a novel mechanism whereby non-coding mRNA sequences selectively regulate C/EBPß activity and suppress its anti-oncogenic functions.


Assuntos
Regiões 3' não Traduzidas , Envelhecimento , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Regulação da Expressão Gênica , Proteína Oncogênica p21(ras)/metabolismo , Linhagem Celular , Humanos , Modelos Biológicos , RNA Mensageiro/metabolismo
2.
J Nat Prod ; 77(4): 744-50, 2014 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-24597820

RESUMO

The secondary metabolites illudins C2 (1) and C3 (2), obtained from the culture broth of Coprinus atramentarius, have been shown to possess antimicrobial activity. In the present study, we discovered novel biological activities of 1 and 2 in lipolysis of differentiated 3T3-L1 adipocytes and adipogenesis of 3T3-L1 preadipocytes. Compounds 1 and 2 exhibit a dose-dependent increase in glycerol release and thereby reduce intracellular lipid accumulation. The stimulatory effects of 1 and 2 on lipolysis are prevented by cAMP-dependent protein kinase (PKA) and extracellular signal-regulated kinase (ERK) inhibitors. Compounds 1 and 2 down-regulated perilipin and also affected the mRNA and protein levels of adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL). However, 1 and 2 treatment leads to a significant increase in PKA-mediated phosphorylation of HSL at S563 and S660. In addition, 1 and 2 treatment in 3T3-L1 preadipocytes induces down-regulation of the critical transcription factors, CCAAT/enhancer binding protein α and ß (C/EBPα and C/EBPß), and peroxisome proliferator activated receptor γ (PPARγ), which are required for adipogenesis, and accordingly inhibits adipogenesis. These results suggest that 1 and 2 might be useful for treating obesity due to their modulatory effects on fat by affecting adipocyte differentiation and fat mobilization.


Assuntos
Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Proteína beta Intensificadora de Ligação a CCAAT/efeitos dos fármacos , Coprinus/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Lipase/metabolismo , Lipólise/efeitos dos fármacos , PPAR gama/metabolismo , Sesquiterpenos/isolamento & purificação , Sesquiterpenos/farmacologia , Células 3T3-L1 , Adipócitos/metabolismo , Adipogenia/fisiologia , Animais , Proteína alfa Estimuladora de Ligação a CCAAT , Relação Dose-Resposta a Droga , Glicerol/análise , Glicerol/metabolismo , Lipase/análise , Lipólise/fisiologia , Camundongos , Estrutura Molecular , Obesidade/tratamento farmacológico , Sesquiterpenos Policíclicos , Sesquiterpenos/química
3.
Pharmaceutics ; 15(2)2023 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-36839859

RESUMO

The aim of this study was to encapsulate cannabidiol (CBD) extract in nanostructured lipid carriers (NLCs) to improve the chemical stability and anti-inflammatory activity of CBD for dermal delivery. CBD-loaded NLCs (CBD-NLCs) were prepared using cetyl palmitate (CP) as a solid lipid and stabilized with Tego® Care 450 (TG450) or poloxamer 188 (P188) by high-pressure homogenization (HPH). The CBD extract was loaded at 1% w/w. Three different oils were employed to produce CBD-NLCs, including Transcutol® P, medium-chain triglycerides (MCT), and oleic acid (OA). CBD-NLCs were successfully prepared with an entrapment efficiency (E.E.) of 100%. All formulations showed particle sizes between 160 and 200 nm with PDIs less than 0.10. The type of surfactant and oil used affected the particle sizes, zeta potential, and crystallinity of the CBD-NLCs. CBD-NLCs stabilized with TG450 showed higher crystallinity after production and storage at 30 °C for 30 days as compared to those with P188. Encapsulation of the CBD extract in NLCs enhanced its chemical stability after exposure to simulated sunlight (1000 kJ/m2) compared to that of the CBD extract in ethanolic solution. The CBD-NLCs prepared from MCT and OA showed slower CBD release compared with that from Transcutol® P, and the kinetic data for release of CBD from CBD-NLCs followed Higuchi's release model with a high coefficient of determination (>0.95). The extent of CBD permeation through Strat-M® depended on the oil type. The cytotoxicity of the CBD extract on HaCaT and HDF cells was reduced by encapsulation in the NLCs. The anti-inflammatory activity of the CBD extract in RAW264.7 cell macrophages was enhanced by encapsulation in CBD-NLCs prepared from MCT and OA.

4.
Pharmaceutics ; 15(8)2023 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-37631315

RESUMO

This study aimed to fabricate new variations of glycerol-based polyesters by grafting poly(glycerol adipate) (PGA) with hydrophobic bioactive moieties, tocopherol (TOC), and cholesterol (CHO). Their effects on nanoparticle (NP) formation, drug release, and cellular responses in cancer and normal cells were evaluated. CHO and TOC were successfully grafted onto PGA backbones with 30% and 50% mole grafting. Increasing the percentage of mole grafting in both molecules increased the glass transition temperature and water contact angle of the final polymers but decreased the critical micelle concentration of the formulated particles. PGA-TOC NPs reduced the proliferation of MDA-MB-231 cancer cells. However, they enhanced the proliferation of primary dermal fibroblasts within a specific concentration range. PGA-CHO NPs minimally affected the growth of cancer and normal cells. Both types of NPs did not affect apoptosis or the cell cycle of cancer cells. PGA-CHO and PGA-TOC NPs were able to entrap SN-38, a hydrophobic anticancer drug, with a particle size <200 nm. PGA-CHO NPs had a higher drug loading capacity and a greater drug release than PGA-TOC NPs. However, SN-38-loaded PGA-TOC NPs showed higher toxicity than SN-38 and SN-38-loaded PGA-CHO NPs due to the combined effects of antiproliferation and higher cellular uptake. Compared with SN-38, the drug-loaded NPs more profoundly induced sub-G1 in the cell cycle analysis and apoptosis of cancer cells in a similar pattern. Therefore, PGA-CHO and PGA-TOC polymers have potential applications as delivery systems for anticancer drugs.

5.
Pharmaceutics ; 15(6)2023 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-37376218

RESUMO

This study aimed to improve the anticancer effect of Cordyceps militaris herbal extract (CME) on breast cancer cells with hyaluronic acid (HYA) surface-decorated lipid polymer hybrid nanoparticles (LPNPs) and evaluate the applicability of a synthesized poly(glycerol adipate) (PGA) polymer for LPNP preparation. Firstly, cholesterol- and vitamin E-grafted PGA polymers (PGA-CH and PGA-VE, respectively) were fabricated, with and without maleimide-ended polyethylene glycol. Subsequently, CME, which contained an active cordycepin equaling 9.89% of its weight, was encapsulated in the LPNPs. The results revealed that the synthesized polymers could be used to prepare CME-loaded LPNPs. The LPNP formulations containing Mal-PEG were decorated with cysteine-grafted HYA via thiol-maleimide reactions. The HYA-decorated PGA-based LPNPs substantially enhanced the anticancer effect of CME against MDA-MB-231 and MCF-7 breast cancer cells by enhancing cellular uptake through CD44 receptor-mediated endocytosis. This study demonstrated the successful targeted delivery of CME to the CD44 receptors of tumor cells by HYA-conjugated PGA-based LPNPs and the new application of synthesized PGA-CH- and PGA-VE-based polymers in LPNP preparation. The developed LPNPs showed promising potential for the targeted delivery of herbal extracts for cancer treatment and clear potential for translation in in vivo experiments.

6.
Hum Mol Genet ; 17(19): 2921-33, 2008 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-18621876

RESUMO

The lamin B receptor (LBR) is an integral nuclear envelope protein that interacts with chromatin and has homology to sterol reductases. Mutations in LBR result in Pelger-Huët anomaly and HEM-Greenberg skeletal dysplasia, whereas in mice Lbr mutations result in ichthyosis. To further understand the function of the LBR and its role in disease, we derived a novel mouse model with a gene-trap insertion into the Lbr locus (Lbr(GT/GT)). Phenotypically, the Lbr(GT/GT) mice are similar to ichthyosis mice. The Lbr(GT/GT) granulocytes lack a mature segmented nucleus and have a block in late maturation. Despite these changes in nuclear morphology, the innate granulocyte immune function in the killing of Staphylococcus aureus bacteria appears to be intact. Granulocyte differentiation requires the transcription factor C/EBPepsilon. We identified C/EBPepsilon binding sites within the Lbr promoter and used EMSAs and luciferase assays to show that Lbr is transcriptionally regulated by C/EBPepsilon. Our findings indicate that the Lbr(GT/GT) mice are a model for Pelger-Huët anomaly and that Lbr, under transcriptional regulation of C/EBPepsilon, is necessary for morphological but not necessarily functional granulocyte maturation.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Neutrófilos/citologia , Anomalia de Pelger-Huët/genética , Anomalia de Pelger-Huët/fisiopatologia , Receptores Citoplasmáticos e Nucleares/genética , Transcrição Gênica , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Diferenciação Celular , Forma do Núcleo Celular , Células Cultivadas , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Insercional , Ativação de Neutrófilo , Neutrófilos/fisiologia , Anomalia de Pelger-Huët/embriologia , Anomalia de Pelger-Huët/metabolismo , Regiões Promotoras Genéticas , Receptores Citoplasmáticos e Nucleares/metabolismo , Staphylococcus aureus/fisiologia , Receptor de Lamina B
7.
Colloids Surf B Biointerfaces ; 167: 115-125, 2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-29631222

RESUMO

Polymer-drug conjugates have been actively developed as potential anticancer drug delivery systems. In this study, we report the first polymer-anticancer drug conjugate with poly(glycerol adipate) (PGA) through the successful conjugation of methotrexate (MTX). MTX-PGA conjugates were controllably and simply fabricated by carbodiimide-mediated coupling reaction with various high molar ratios of MTX. The MTX-PGA conjugate self-assembled into nanoparticles with size dependent on the amount of conjugated MTX and the pH of medium. Change in particle size was attributed to steric hindrance and bulkiness inside the nanoparticle core and dissociation of free functional groups of the drug. The MTX-PGA nanoparticles were physically stable in media with pH range of 5-9 and ionic strength of up to 0.15 M NaCl and further chemically stable against hydrolysis in pH 7.4 medium over 30 days but enzymatically degradable to release unchanged free drug. Although 30%MTX-PGA nanoparticles exhibited only slightly less potency than free MTX in 791T cells in contrast to previously reported human serum albumin-MTX conjugates which had >300 times lower potency than free MTX. However, the MTX nanoparticles showed 7 times higher toxicity to Saos-2 cells than MTX. Together with the enzymic degradation experiments, these results suggest that with a suitable biodegradable polymer a linker moiety is not a necessary component. These easily synthesised PGA drug conjugates lacking a linker moiety could therefore be an effective new pathway for development of polymer drug conjugates.


Assuntos
Antineoplásicos/química , Metotrexato/química , Nanopartículas/química , Poliésteres/química , Antimetabólitos Antineoplásicos/química , Antimetabólitos Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Química Farmacêutica , Composição de Medicamentos , Humanos , Espectroscopia de Ressonância Magnética , Metotrexato/farmacologia , Poliésteres/farmacologia
8.
Cancer Res ; 78(4): 891-908, 2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29259016

RESUMO

The precise characteristics that distinguish normal and oncogenic RAS signaling remain obscure. Here, we show that oncogenic RAS and BRAF induce perinuclear relocalization of several RAS pathway proteins, including the kinases CK2 and p-ERK1/2 and the signaling scaffold KSR1. This spatial reorganization requires endocytosis, the kinase activities of MEK-ERK and CK2, and the presence of KSR1. CK2α colocalizes with KSR1 and Rab11, a marker of recycling endosomes, whereas p-ERK associates predominantly with a distinct KSR1-positive endosomal population. Notably, these perinuclear signaling complexes (PSC) are present in tumor cell lines, mouse lung tumors, and mouse embryonic fibroblasts undergoing RAS-induced senescence. PSCs are also transiently induced by growth factors (GF) in nontransformed cells with delayed kinetics (4-6 hours), establishing a novel late phase of GF signaling that appears to be constitutively activated in tumor cells. PSCs provide an essential platform for RAS-induced phosphorylation and activation of the prosenescence transcription factor C/EBPß in primary MEFs undergoing senescence. Conversely, in tumor cells, C/EBPß activation is suppressed by 3'UTR-mediated localization of Cebpb transcripts to a peripheral cytoplasmic domain distinct from the PSC region. Collectively, our findings indicate that sustained PSC formation is a critical feature of oncogenic RAS/BRAF signaling in cancer cells that controls signal transmission to downstream targets by regulating selective access of effector kinases to substrates such as C/EBPß.Significance: In addressing the long-standing question of the difference between normal and oncogenic RAS pathway signaling, this study shows that oncogenic RAS specifically triggers constitutive endocytosis-dependent movement of effector kinases to a perinuclear region, thereby creating connections to unique downstream targets such as the core prosenescence and the inflammatory regulatory transcription factor C/EBPß. Cancer Res; 78(4); 891-908. ©2017 AACR.


Assuntos
Proteínas Quinases/metabolismo , Proteínas ras/metabolismo , Células A549 , Animais , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Células NIH 3T3 , Fosforilação , Transdução de Sinais
9.
Mol Cell Biol ; 24(22): 9873-86, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15509790

RESUMO

Budding yeast polo kinase Cdc5p localizes to the spindle pole body (SPB) and to the bud-neck and plays multiple roles during M-phase progression. To dissect localization-specific mitotic functions of Cdc5p, we tethered a localization-defective N-terminal kinase domain of Cdc5p (Cdc5pDeltaC) to the SPB or to the bud-neck with components specifically localizing to one of these sites and characterized these mutants in a cdc5Delta background. Characterization of a viable, SPB-localizing, CDC5DeltaC-CNM67 mutant revealed that it is defective in timely degradation of Swe1p, a negative regulator of Cdc28p. Loss of BFA1, a negative regulator of mitotic exit, rescued the lethality of a neck-localizing CDC5DeltaC-CDC12 or CDC5DeltaC-CDC3 mutant but yielded severe defects in cytokinesis. These data suggest that the SPB-associated Cdc5p activity is critical for both mitotic exit and cytokinesis, whereas the bud neck-localized Cdc5p is required for proper Swe1p regulation. Interestingly, a cdc5Delta bfa1Delta swe1Delta triple mutant is viable but grows slowly, whereas cdc5Delta cells bearing both CDC5DeltaC-CNM67 and CDC5DeltaC-CDC12 grow well with only a mild cell cycle delay. Thus, SPB- and the bud-neck-localized Cdc5p control most of the critical Cdc5p functions and downregulation of Bfa1p and Swe1p at the respective locations are two critical factors that require Cdc5p.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Divisão Celular , Citocinese , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Genes Fúngicos , Mitose , Mutação , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fuso Acromático/metabolismo , Frações Subcelulares/enzimologia
10.
Mol Biol Cell ; 15(4): 1711-23, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-14767068

RESUMO

The polo-box domain of the budding yeast polo kinase Cdc5p plays an essential role for targeting the catalytic activity of Cdc5p to spindle pole bodies (SPBs) and cytokinetic neck-filaments. Here, we report the isolation of Bbp1p as a polo-box interacting protein by a yeast two-hybrid screen. Bbp1p localizes to the periphery of the central plaque of the SPB and plays an important role in SPB duplication. Similarly, Cdc5p localized to the cytoplasmic periphery of the SPB. In vitro binding studies showed that Cdc5p interacted with the N-terminal domain of Bbp1p (Bbp1pDeltaC), but apparently not with Mps2p, a component shown to form a stable complex with Bbp1p. In addition, Bbp1p, but likely not Mps2p, was required for proper localization of Cdc5p to the SPB. The C-terminal coiled-coil domain of Bbp1p (Bbp1p(243-385)), which is crucial for both the homodimerization and the SPB localization, could target the localization-defective Cdc5pDeltaC to the SPB and induce the release of Cdc14p from the nucleolus. Consistent with this observation, expression of CDC5DeltaC-BBP1(243-385) under CDC5 promoter control partially complemented the cdc5Delta defect. These data suggest that Bbp1pDeltaC interacts with the polo-box domain of Cdc5p, and this interaction is critical for the subcellular localization and mitotic functions of Cdc5p.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas dos Microtúbulos/fisiologia , Mitose , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Fuso Acromático , Catálise , Ciclo Celular , Proteínas de Ciclo Celular/química , Divisão Celular , Nucléolo Celular/metabolismo , Citoplasma/metabolismo , Dimerização , Genótipo , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde , Immunoblotting , Proteínas Luminescentes/metabolismo , Microscopia Imunoeletrônica , Modelos Biológicos , Fosforilação , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Tirosina Fosfatases/química , Proteínas de Ligação a RNA , Proteínas Recombinantes/química , Proteínas de Saccharomyces cerevisiae/química , Temperatura , Fatores de Tempo , Técnicas do Sistema de Duplo-Híbrido
11.
PLoS One ; 12(3): e0173311, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28296906

RESUMO

We have identified the small molecule STK899704 as a structurally novel tubulin inhibitor. STK899704 suppressed the proliferation of cancer cell lines from various origins with IC50 values ranging from 0.2 to 1.0 µM. STK899704 prevented the polymerization of purified tubulin in vitro and also depolymerized microtubule in cultured cells leading to mitotic arrest, associated with increased Cdc25C phosphorylation and the accumulation of both cyclin B1 and polo-like kinase 1 (Plk1), and apoptosis. Unlike many anticancer drugs such as Taxol and doxorubicin, STK899704 effectively displayed antiproliferative activity against multidrug-resistant cancer cell lines. The proposed binding mode of STK899704 is at the interface between αß-tubulin heterodimer overlapping with the colchicine-binding site. Our in vivo carcinogenesis model further showed that STK 899704 is potent in both the prevention and regression of tumors, remarkably reducing the number and volume of skin tumor by STK899704 treatment. Moreover, it was significant to note that the efficacy of STK899704 was surprisingly comparable to 5-fluorouracil, a widely used anticancer therapeutic. Thus, our results demonstrate the potential of STK899704 to be developed as an anticancer chemotherapeutic and an alternative candidate for existing therapies.


Assuntos
Antineoplásicos/farmacologia , Benzofuranos/farmacologia , Moduladores de Tubulina/farmacologia , Animais , Antineoplásicos/química , Benzofuranos/química , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina B1/metabolismo , Humanos , Camundongos , Fosforilação , Polimerização/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fuso Acromático/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Fosfatases cdc25/metabolismo , Quinase 1 Polo-Like
12.
Mol Cell Biol ; 35(5): 866-83, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25535333

RESUMO

Oncogene-induced senescence (OIS) protects normal cells from transformation by Ras, whereas cells lacking p14/p19(Arf) or other tumor suppressors can be transformed. The transcription factor C/EBPß is required for OIS in primary fibroblasts but is downregulated by H-Ras(V12) in immortalized NIH 3T3 cells through a mechanism involving p19(Arf) loss. Here, we report that members of the serum-induced early growth response (Egr) protein family are also downregulated in 3T3(Ras) cells and directly and redundantly control Cebpb gene transcription. Egr1, Egr2, and Egr3 recognize three sites in the Cebpb promoter and associate transiently with this region after serum stimulation, coincident with Cebpb induction. Codepletion of all three Egrs prevented Cebpb expression, and serum induction of Egrs was significantly blunted in 3T3(Ras) cells. Egr2 and Egr3 levels were also reduced in Ras(V12)-expressing p19(Arf) null mouse embryonic fibroblasts (MEFs), and overall Egr DNA-binding activity was suppressed in Arf-deficient but not wild-type (WT) MEFs, leading to Cebpb downregulation. Analysis of human cancers revealed a strong correlation between EGR levels and CEBPB expression, regardless of whether CEBPB was increased or decreased in tumors. Moreover, overexpression of Egrs in tumor cell lines induced CEBPB and inhibited proliferation. Thus, our findings identify the Arf-Egr-C/EBPß axis as an important determinant of cellular responses (senescence or transformation) to oncogenic Ras signaling.


Assuntos
Transformação Celular Neoplásica , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Neoplasias/metabolismo , Proteínas ras/metabolismo , Células 3T3 , Animais , Linhagem Celular Tumoral , Proliferação de Células , Senescência Celular , Fibroblastos/metabolismo , Genótipo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Regiões Promotoras Genéticas
13.
Chem Biol Drug Des ; 85(5): 638-44, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25323822

RESUMO

In this study, we have synthesized novel water soluble derivatives of natural compound aloe emodin 4(a-j) by coupling with various amino acid esters and substituted aromatic amines, in an attempt to improve the anticancer activity and to explore the structure-activity relationships. The structures of the compounds were determined by (1) H NMR and mass spectroscopy. Cell growth inhibition assays revealed that the aloe emodin derivatives 4d, 4f, and 4i effectively decreased the growth of HepG2 (human liver cancer cells) and NCI-H460 (human lung cancer cells) and some of the derivatives exhibited comparable antitumor activity against HeLa (Human epithelial carcinoma cells) and PC3 (prostate cancer cells) cell lines compared to that of the parent aloe emodin at low micromolar concentrations.


Assuntos
Antraquinonas/química , Antraquinonas/farmacologia , Antineoplásicos/síntese química , Antraquinonas/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células HeLa , Células Hep G2 , Humanos , Relação Estrutura-Atividade
14.
Gene ; 513(1): 75-81, 2013 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-23137637

RESUMO

Centrosomal proteins play important roles in cell cycle. Among them, the centrosomal protein of 131kDa (CEP131) has been reported as a critical factor for cilia formation which is related with development, signaling, and various diseases, the malfunction of cilia leading to cancer. Specificity protein 1 (SP1), known as a centrosome regulator, is an essential transcription factor regulating the genes involved in multiple cellular processes such as cell cycle, apoptosis, and DNA damages. In this study, we explored the crucial role of SP1 in the regulation of CEP131 gene transcription. A deletion analysis of the CEP131 promoter region revealed dominant promoter elements within the sequence between -400bp and -200bp, which contained consensus binding sites for SP1. Electrophoretic mobility shift assay (EMSA) and chromatin immuno-precipitation (ChIP) assay further confirmed the direct binding of SP1 to the CEP131 promoter. On the other hand, CEP131 transcription could be inhibited by mithramycin (a GC-rich region inhibitor), but exogenous expression of SP1 could increase CEP131 expression as evidenced by a reporter gene assay. In addition, mutation of several SP1 binding sites revealed four SP1 binding sites at -244/-225, -258/-239, -304/-283 and -323/-304 that strongly affect CEP131 expression. Hence, it is suggested that SP1 is a pivotal transcription factor for the regulation of CEP131 expression, consequently leading the control of centrosome functions.


Assuntos
Proteínas de Ciclo Celular/genética , Regulação da Expressão Gênica , Proteínas dos Microtúbulos/genética , Fator de Transcrição Sp1/metabolismo , Sítios de Ligação/genética , Centrossomo/efeitos dos fármacos , Centrossomo/metabolismo , Imunoprecipitação da Cromatina , Proteínas do Citoesqueleto , Ensaio de Desvio de Mobilidade Eletroforética , Células HEK293 , Células HeLa , Humanos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Plasmídeos , Plicamicina/farmacologia , Regiões Promotoras Genéticas , Ligação Proteica , Deleção de Sequência
15.
PLoS One ; 8(1): e53908, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23349762

RESUMO

STK295900, a small synthetic molecule belonging to a class of symmetric bibenzimidazoles, exhibits antiproliferative activity against various human cancer cell lines from different origins. Examining the effect of STK295900 in HeLa cells indicates that it induces G(2) phase arrest without invoking DNA damage. Further analysis shows that STK295900 inhibits DNA relaxation that is mediated by topoisomerase 1 (Top 1) and topoisomerase 2 (Top 2) in vitro. In addition, STK295900 also exhibits protective effect against DNA damage induced by camptothecin. However, STK295900 does not affect etoposide-induced DNA damage. Moreover, STK295900 preferentially exerts cytotoxic effect on cancer cell lines while camptothecin, etoposide, and Hoechst 33342 affected both cancer and normal cells. Therefore, STK295900 has a potential to be developed as an anticancer chemotherapeutic agent.


Assuntos
Compostos de Anilina/farmacologia , Benzimidazóis/farmacologia , Dano ao DNA , DNA Topoisomerases Tipo II/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Inibidores da Topoisomerase/farmacologia , Compostos de Anilina/química , Antineoplásicos Fitogênicos/farmacologia , Benzimidazóis/química , Proteína Quinase CDC2/metabolismo , Camptotecina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Relação Dose-Resposta a Droga , Etoposídeo/farmacologia , Células HCT116 , Células HT29 , Células HeLa , Células Hep G2 , Humanos , Immunoblotting , Células MCF-7 , Estrutura Molecular , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação/efeitos dos fármacos , Inibidores da Topoisomerase/química
16.
Cell Signal ; 25(2): 552-60, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23178987

RESUMO

Nuclear factor-κB (NF-κB) ligand (RANKL) was shown to induce osteoclast differentiation by increasing the expression of c-Fos, NFATc1 and TRAP. Salubrinal treatment to bone marrow macrophage (BMM) cells, however, significantly blocked NFATc1 expression and osteoclast differentiation by RANKL. Overexpression of NFATc1 further confirmed that NFATc1 is a key factor affected by salubrinal in osteoclast differentiation by RANKL. Unexpectedly, NFATc1 and c-Fos mRNA expressions were not affected by salubrinal, implicating that NFATc1 expression is regulated at a translational stage. In support of this, salubrinal increased the phosphorylation of a translation factor eIF2α, decreasing the global protein synthesis including NFATc1. In contrast, a phosphorylation mutant plasmid pLenti-eIF2α-S51A restored RANKL-induced NFATc1 expression and osteoclast differentiation even in the presence of salubrinal. Furthermore, knockdown of ATF4 significantly reduced salubrinal-induced osteoblast differentiation as evidenced by decreased calcium accumulation and lowered expressions of the osteoblast differentiation markers, alkaline phosphatase and RANKL in MC3T3-E1 osteoblast cells. Salubrinal treatment to co-cultured BMM and MC3T3-E1 cells also showed reduction of osteoclast differentiation. Finally, salubrinal efficiently blocked osteoporosis in mice model treated with RANKL as evidenced by elevated bone mineral density (BMD) and other osteoporosis factors. Collectively, our data indicate that salubrinal could affect the differentiation of both osteoblast and osteoclast, and be developed as an excellent anti-osteoporosis drug. In addition, modulation of ATF4 and NFATc1 expressions through eIF2α phosphorylation could be a valuable target for the treatment of osteoporosis.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Cinamatos/farmacologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Osteoblastos/citologia , Osteoclastos/citologia , Tioureia/análogos & derivados , Fator 4 Ativador da Transcrição/antagonistas & inibidores , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Cálcio/metabolismo , Células Cultivadas , Cinamatos/uso terapêutico , Técnicas de Cocultura , Modelos Animais de Doenças , Fêmur/diagnóstico por imagem , Camundongos , Fatores de Transcrição NFATC/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteoporose/tratamento farmacológico , Osteoporose/metabolismo , Osteoporose/patologia , Fosforilação , Ligante RANK/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Radiografia , Transdução de Sinais , Tioureia/farmacologia , Tioureia/uso terapêutico , Tíbia/diagnóstico por imagem
17.
Mol Cell Biol ; 30(11): 2621-35, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20351173

RESUMO

The bZIP transcription factor C/EBPbeta is a target of Ras signaling that has been implicated in Ras-induced transformation and oncogene-induced senescence (OIS). To gain insights into Ras-C/EBPbeta signaling, we investigated C/EBPbeta activation by oncogenic Ras. We show that C/EBPbeta DNA binding is autorepressed and becomes activated by the Ras-Raf-MEK-ERK-p90(RSK) cascade. Inducible phosphorylation by RSK on Ser273 in the leucine zipper was required for DNA binding. In addition, three other modifications (phosphorylation on Tyr109 [p-Tyr109], p-Ser111, and monomethylation of Arg114 [me-Arg114]) within an N-terminal autoinhibitory domain were important for Ras-induced C/EBPbeta activation and cytostatic activity. Apart from its role in DNA binding, Ser273 phosphorylation also creates an interhelical g<-->e' salt bridge with Lys268 that increases attractive electrostatic interactions between paired leucine zippers and promotes homodimerization. Mutating Ser273 to Ala or Lys268 to Glu decreased C/EBPbeta homodimer formation, whereas heterodimerization with C/EBPgamma was relatively unaffected. The S273A substitution also reduced the antiproliferative activity of C/EBPbeta in Ras(V12)-expressing fibroblasts and decreased binding to target cell cycle genes, while a phosphomimetic substitution (S273D) maintained growth arrest function. Our findings identify four novel C/EBPbeta-activating modifications, including RSK-mediated phosphorylation of a bifunctional residue in the leucine zipper that regulates DNA binding and homodimerization and thereby promotes cell cycle arrest.


Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Ciclo Celular/fisiologia , DNA/metabolismo , Zíper de Leucina , Proteínas Quinases S6 Ribossômicas/metabolismo , Sequência de Aminoácidos , Animais , Proteína beta Intensificadora de Ligação a CCAAT/química , Proteína beta Intensificadora de Ligação a CCAAT/genética , Linhagem Celular , DNA/genética , Substâncias de Crescimento/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Fosforilação , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Ratos , Proteínas Quinases S6 Ribossômicas/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas/genética , Alinhamento de Sequência , Ativação Transcricional , Proteínas ras/genética , Proteínas ras/metabolismo
18.
J Biol Chem ; 281(37): 27090-8, 2006 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-16861226

RESUMO

In budding yeast, Gin4, a Nim1-related kinase, plays an important role in proper organization of the septin ring at the mother-bud neck, a filamentous structure that is critical for diverse cellular processes including mitotic entry and cytokinesis. How Gin4 kinase activity is regulated is not known. Here we showed that a neck-associated Ser/Thr kinase Elm1, which is important for septin assembly, is critical for proper modification of Gin4 and its physiological substrate Shs1. In vitro studies with purified recombinant proteins demonstrated that Elm1 directly phosphorylates and activates Gin4, which in turn phosphorylates Shs1. Consistent with these observations, acute inhibition of Elm1 activity abolished mitotic Gin4 phosphorylation and Gin4-dependent Shs1 modification in vivo. In addition, a gin4 mutant lacking the Elm1-dependent phosphorylation sites exhibited an impaired localization to the bud-neck and, as a result, induced a significant growth defect with an elongated bud morphology. Thus, Elm1 regulates the septin assembly-dependent cellular events by directly phosphorylating and activating the Gin4-dependent pathway(s).


Assuntos
Quinases Ciclina-Dependentes/metabolismo , Proteínas Quinases/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Animais , Quinases Ciclina-Dependentes/química , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Insetos , Proteínas de Membrana/química , Modelos Biológicos , Peptídeos/química , Fosforilação , Plasmídeos/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas de Saccharomyces cerevisiae/química
19.
Cell Cycle ; 4(10): 1346-9, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16123596

RESUMO

In eukaryotes, G(2)/M transition is induced by the activation of cyclin B-bound Cdk1, which is held in check by the protein kinase, Wee1. Recent advances in our understanding of mitotic entry in budding yeast has revealed that these cells utilize the level of Swe1 (Wee1 ortholog) phosphorylation as a means of monitoring cell cycle progression and of coordinating morphogenetic events with mitotic entry. Swe1 is phosphorylated by at least three distinct kinases at different stages of the cell cycle. This cumulative phosphorylation leads to the hyperphosphorylation and degradation of Swe1 through ubiquitin-mediated proteolysis. Thus, Swe1 functions as an important cell cycle modulator that integrates multiple upstream signals from prior cell cycle events before its ultimate degradation permits passage into mitosis.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Fosfotransferases/metabolismo , Saccharomycetales/citologia , Saccharomycetales/metabolismo , Regulação Fúngica da Expressão Gênica , Humanos , Fosforilação , Saccharomycetales/enzimologia
20.
EMBO J ; 24(12): 2194-204, 2005 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-15920482

RESUMO

In eukaryotes, entry into mitosis is induced by cyclin B-bound Cdk1, which is held in check by the protein kinase, Wee1. In budding yeast, Swe1 (Wee1 ortholog) is targeted to the bud neck through Hsl1 (Nim1-related kinase) and its adaptor Hsl7, and is hyperphosphorylated prior to ubiquitin-mediated degradation. Here, we show that Hsl1 and Hsl7 are required for proper localization of Cdc5 (Polo-like kinase homolog) to the bud neck and Cdc5-dependent Swe1 phosphorylation. Mitotic cyclin (Clb2)-bound Cdc28 (Cdk1 homolog) directly phosphorylated Swe1 and this modification served as a priming step to promote subsequent Cdc5-dependent Swe1 hyperphosphorylation and degradation. Clb2-Cdc28 also facilitated Cdc5 localization to the bud neck through the enhanced interaction between the Clb2-Cdc28-phosphorylated Swe1 and the polo-box domain of Cdc5. We propose that the concerted action of Cdc28/Cdk1 and Cdc5/Polo on their common substrates is an evolutionarily conserved mechanism that is crucial for effectively triggering mitotic entry and other critical mitotic events.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Fosfotransferases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/fisiologia , Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Mitose/fisiologia , Fosforilação , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Fosfatases/fisiologia , Proteína-Arginina N-Metiltransferases , Proteínas de Ligação a RNA , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomycetales/enzimologia , ras-GRF1
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