RESUMO
AIMS/HYPOTHESIS: Finding new treatment alternatives for individuals with diabetes with severe insulin resistance is highly desired. To identify novel mechanisms that improve glucose uptake in skeletal muscle, independently from insulin levels and signalling, we have explored the therapeutic potential of a short peptide sequence, RG54, derived from apolipoprotein A-I (ApoA-I). METHODS: INS-1E rat clonal beta cells, C2C12 rat muscle myotubes and J774 mouse macrophages were used to study the impact of RG54 peptide on glucose-stimulated insulin secretion, glucose uptake and cholesterol efflux, respectively. GTTs were carried out on diet-induced insulin-resistant and Leprdb diabetic mouse models treated with RG54 peptide, and the impact of RG54 peptide on atherosclerosis was evaluated in Apoe-/- mice. Control mice received ApoA-I protein, liraglutide or NaCl. RESULTS: The synthetic RG54 peptide induced glucose uptake in cultured muscle myotubes by a similar amount as insulin, and also primed pancreatic beta cells for improved glucose-stimulated insulin secretion. The findings were verified in diet-induced insulin-resistant and Leprdb diabetic mice, jointly confirming the physiological effect. The RG54 peptide also efficiently catalysed cholesterol efflux from macrophages and prevented the formation of atherosclerotic plaques in Apoe-/- mice. CONCLUSIONS/INTERPRETATION: The RG54 peptide exhibits good prospects for providing glucose control and reducing the risk of cardiovascular disease in individuals with severe insulin resistance.
Assuntos
Apolipoproteína A-I/química , Aterosclerose/prevenção & controle , Glucose/metabolismo , Peptídeos/química , Peptídeos/uso terapêutico , Animais , Aterosclerose/metabolismo , Glicemia/efeitos dos fármacos , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/prevenção & controle , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/prevenção & controle , Resistência à Insulina/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The PDLIM2 protein regulates stability of transcription factors including NF-κB and STATs in epithelial and hemopoietic cells. PDLIM2 is strongly expressed in certain cancer cell lines that exhibit an epithelial-to-mesenchymal phenotype, and its suppression is sufficient to reverse this phenotype. PDLIM2 supports the epithelial polarity of nontransformed breast cells, suggesting distinct roles in tumor suppression and oncogenesis. To better understand its overall function, we investigated PDLIM2 expression and activity in breast cancer. PDLIM2 protein was present in 60% of tumors diagnosed as triple-negative breast cancer (TNBC), and only 20% of other breast cancer subtypes. High PDLIM2 expression in TNBC was positively correlated with adhesion signaling and ß-catenin activity. Interestingly, PDLIM2 was restricted to the cytoplasm/membrane of TNBC cells and excluded from the nucleus. In breast cell lines, PDLIM2 retention in the cytoplasm was controlled by cell adhesion, and translocation to the nucleus was stimulated by insulin-like growth factor-1 or TGFß. Cytoplasmic PDLIM2 was associated with active ß-catenin and ectopic expression of PDLIM2 was sufficient to increase ß-catenin levels and its transcriptional activity in reporter assays. Suppression of PDLIM2 inhibited tumor growth in vivo, whereas overexpression of PDLIM2 disrupted growth in 3D cultures. These results suggest that PDLIM2 may serve as a predictive biomarker for a large subset of TNBC whose phenotype depends on adhesion-regulated ß-catenin activity and which may be amenable to therapies that target these pathways. SIGNIFICANCE: This study shows that PDLIM2 expression defines a subset of triple-negative breast cancer that may benefit from targeting the ß-catenin and adhesion signaling pathways. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/10/2619/F1.large.jpg.
Assuntos
Biomarcadores Tumorais/metabolismo , Adesão Celular , Proteínas com Domínio LIM/metabolismo , Proteínas dos Microfilamentos/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , beta Catenina/metabolismo , Linhagem Celular Tumoral , Citoplasma/metabolismo , Feminino , Células HEK293 , HumanosRESUMO
Over half of colorectal cancers (CRCs) harbor TP53 missense mutations (mutp53). We show that the most common mutp53 allele R248Q (p53Q) exerts gain of function (GOF) and creates tumor dependence in mouse CRC models. mutp53 protein binds Stat3 and enhances activating Stat3 phosphorylation by displacing the phosphatase SHP2. Ablation of the p53Q allele suppressed Jak2/Stat3 signaling, growth, and invasiveness of established, mutp53-driven tumors. Treating tumor-bearing mice with an HSP90 inhibitor suppressed mutp53 levels and tumor growth. Importantly, human CRCs with stabilized mutp53 exhibit enhanced Jak2/Stat3 signaling and are associated with poorer patient survival. Cancers with TP53R248Q/W are associated with a higher patient death risk than are those having nonR248 mutp53. These findings identify GOF mutp53 as a therapeutic target in CRC.
Assuntos
Neoplasias Colorretais/terapia , Mutação , Fator de Transcrição STAT3/fisiologia , Proteína Supressora de Tumor p53/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Humanos , Janus Quinase 2/fisiologia , Perda de Heterozigosidade , Camundongos , Invasividade Neoplásica , Proteína Supressora de Tumor p53/fisiologiaRESUMO
Epithelial cell differentiation and polarized migration associated with epithelial-to-mesenchymal transition (EMT) in cancer requires integration of gene expression with cytoskeletal dynamics. Here we show that the PDZ-LIM domain protein PDLIM2 (Mystique/SLIM), a known cytoskeletal protein and promoter of nuclear nuclear factor κB (NFκB) and signal transducer and activator of transcription (STAT) degradation, regulates transcription factor activity and gene expression through the COP9 signalosome (CSN). Although repressed in certain cancers, PDLIM2 is highly expressed in invasive cancer cells. Here we show that PDLIM2 suppression causes loss of directional migration, inability to polarize the cytoskeleton, and reversal of the EMT phenotype. This is accompanied by altered activity of several transcription factor families, including ß-catenin, Ap-1, NFκB, interferon regulatory factors, STATs, JUN, and p53. We also show that PDLIM2 associates with CSN5, and cells with suppressed PDLIM2 exhibit reduced nuclear accumulation and deneddylation activity of the CSN toward the cullin 1 and cullin 3 subunits of cullin-RING ubiquitin ligases. Thus PDLIM2 integrates cytoskeleton signaling with gene expression in epithelial differentiation by controlling the stability of key transcription factors and CSN activity.
Assuntos
Transição Epitelial-Mesenquimal , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas com Domínio LIM/fisiologia , Proteínas dos Microfilamentos/fisiologia , Complexo do Signalossomo COP9 , Diferenciação Celular , Movimento Celular , Polaridade Celular , Células Epiteliais/fisiologia , Humanos , Células MCF-7 , Complexos Multiproteicos/metabolismo , NF-kappa B/metabolismo , Peptídeo Hidrolases/metabolismo , Transporte Proteico , beta CateninaRESUMO
Inflammatory bowel disease (IBD) is a chronic, inflammatory disorder of the gastrointestinal tract involving an inappropriate immune response to commensal microorganisms in a genetically susceptible host. This study examined the effects of aqueous and ethyl acetate extracts of gold kiwifruit (Actinidia chinensis) or green kiwifruit (Actinidia deliciosa) using in vitro models of IBD. These models comprised primary macrophages and intestinal epithelial cells isolated from C57BL/5J and interleukin-10 gene deficient (Il10(-/-)) mice and RAW 264.7, a murine macrophage-like cell line. All four kiwifruit extracts reduced the activation of these models after lipopolysaccharide stimulation, decreasing nitric oxide and cytokine secretion by both Il10(-/-) and wild-type cells. The ethyl acetate extracts exhibited the highest anti-inflammatory activity, with almost complete suppression of lipopolysaccharide-stimulated macrophage activation. These results suggest that kiwifruit extracts have significant anti-inflammatory activity relevant to IBD. We suggest that the Il10(-/-) mouse is a suitable model for further study of these compounds.
Assuntos
Actinidia , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/imunologia , Interleucina-10/deficiência , Interleucina-10/genética , Ativação de Macrófagos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Linhagem Celular , Citocinas/biossíntese , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Interleucina-10/metabolismo , Mucosa Intestinal/imunologia , Lipopolissacarídeos/farmacologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/biossíntese , FitoterapiaRESUMO
PURPOSE: Hypoxia is a characteristic of solid tumors and a potentially important therapeutic target. Here, we characterize the mechanism of action and preclinical antitumor activity of a novel hypoxia-activated prodrug, the 3,5-dinitrobenzamide nitrogen mustard PR-104, which has recently entered clinical trials. EXPERIMENTAL DESIGN: Cytotoxicity in vitro was evaluated using 10 human tumor cell lines. SiHa cells were used to characterize metabolism under hypoxia, by liquid chromatography-mass spectrometry, and DNA damage by comet assay and gammaH2AX formation. Antitumor activity was evaluated in multiple xenograft models (PR-104 +/- radiation or chemotherapy) by clonogenic assay 18 h after treatment or by tumor growth delay. RESULTS: The phosphate ester "pre-prodrug" PR-104 was well tolerated in mice and converted rapidly to the corresponding prodrug PR-104A. The cytotoxicity of PR-104A was increased 10- to 100-fold by hypoxia in vitro. Reduction to the major intracellular metabolite, hydroxylamine PR-104H, resulted in DNA cross-linking selectively under hypoxia. Reaction of PR-104H with chloride ion gave lipophilic cytotoxic metabolites potentially able to provide bystander effects. In tumor excision assays, PR-104 provided greater killing of hypoxic (radioresistant) and aerobic cells in xenografts (HT29, SiHa, and H460) than tirapazamine or conventional mustards at equivalent host toxicity. PR-104 showed single-agent activity in six of eight xenograft models and greater than additive antitumor activity in combination with drugs likely to spare hypoxic cells (gemcitabine with Panc-01 pancreatic tumors and docetaxel with 22RV1 prostate tumors). CONCLUSIONS: PR-104 is a novel hypoxia-activated DNA cross-linking agent with marked activity against human tumor xenografts, both as monotherapy and combined with radiotherapy and chemotherapy.