RESUMO
The molecular mechanisms of skeletal muscle atrophy under extended periods of either disuse or microgravity are not yet fully understood. The transition of Homer isoforms may play a key role during neuromuscular junction (NMJ) imbalance/plasticity in space. Here, we investigated the expression pattern of Homer short and long isoforms by gene array, qPCR, biochemistry, and laser confocal microscopy in skeletal muscles from male C57Bl/N6 mice (n = 5) housed for 30 days in space (Bion-flight = BF) compared to muscles from Bion biosatellite on the ground-housed animals (Bion ground = BG) and from standard cage housed animals (Flight control = FC). A comparison study was carried out with muscles of rats subjected to hindlimb unloading (HU). Gene array and qPCR results showed an increase in Homer1a transcripts, the short dominant negative isoform, in soleus (SOL) muscle after 30 days in microgravity, whereas it was only transiently increased after four days of HU. Conversely, Homer2 long-form was downregulated in SOL muscle in both models. Homer immunofluorescence intensity analysis at the NMJ of BF and HU animals showed comparable outcomes in SOL but not in the extensor digitorum longus (EDL) muscle. Reduced Homer crosslinking at the NMJ consequent to increased Homer1a and/or reduced Homer2 may contribute to muscle-type specific atrophy resulting from microgravity and HU disuse suggesting mutual mechanisms.
Assuntos
Proteínas de Arcabouço Homer/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Isoformas de Proteínas/metabolismo , Animais , Elevação dos Membros Posteriores/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Junção Neuromuscular/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Voo Espacial/métodos , Ausência de PesoRESUMO
Accurate modeling of intratumor heterogeneity presents a bottleneck against drug testing. Flexibility in a preclinical platform is also desirable to support assessment of different endpoints. We established the model system, OHC-NB1, from a bone marrow metastasis from a patient diagnosed with MYCN-amplified neuroblastoma and performed whole-exome sequencing on the source metastasis and the different models and passages during model development (monolayer cell line, 3D spheroid culture and subcutaneous xenograft tumors propagated in mice). OHC-NB1 harbors a MYCN amplification in double minutes, 1p deletion, 17q gain and diploid karyotype, which persisted in all models. A total of 80-540 single-nucleotide variants (SNVs) was detected in each sample, and comparisons between the source metastasis and models identified 34 of 80 somatic SNVs to be propagated in the models. Clonal reconstruction using the combined copy number and SNV data revealed marked clonal heterogeneity in the originating metastasis, with four clones being reflected in the model systems. The set of OHC-NB1 models represents 43% of somatic SNVs and 23% of the cellularity in the originating metastasis with varying clonal compositions, indicating that heterogeneity is partially preserved in our model system.
Assuntos
Modelos Animais de Doenças , Neuroblastoma/genética , Neuroblastoma/patologia , Neoplasias Abdominais/genética , Neoplasias Abdominais/patologia , Animais , Feminino , Heterogeneidade Genética , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos SCID , Neoplasias Torácicas/genética , Neoplasias Torácicas/patologia , Células Tumorais CultivadasRESUMO
BACKGROUND: The biological mechanisms underlying early- and advanced-stage epithelial ovarian cancers (EOCs) are still poorly understood. This study explored kinase-driven metabolic signalling in early and advanced EOCs, and its role in tumour progression and response to carboplatin-paclitaxel treatment. METHODS: Tumour epithelia were isolated from two independent sets of primary EOC (n=72 and 30 for the discovery and the validation sets, respectively) via laser capture microdissection. Reverse phase protein microarrays were used to broadly profile the kinase-driven metabolic signalling of EOC with particular emphasis on the LBK1-AMPK and AKT-mTOR axes. Signalling activation was compared between early and advanced lesions, and carboplatin-paclitaxel-sensitive and -resistant tumours. RESULTS: Advanced EOCs were characterised by a heterogeneous kinase-driven metabolic signature and decreased phosphorylation of the AMPK-AKT-mTOR axis compared to early EOC (P<0.05 for AMPKα T172, AMPKα1 S485, AMPKß1 S108, AKT S473 and T308, mTOR S2448, p70S6 S371, 4EBP1 S65, GSK-3 α/ß S21/9, FOXO1 T24/FOXO3 T32, and FOXO1 S256). Advanced tumours with low relative activation of the metabolic signature and increased FOXO1 T24/FOXO3 T32 phosphorylation (P=0.041) were associated with carboplatin-paclitaxel resistance. CONCLUSIONS: If validated in a larger cohort of patients, the decreased AMPK-AKT-mTOR activation and phosphorylation of FOXO1 T24/FOXO3 T32 may help identify carboplatin-paclitaxel-resistant EOC patients.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Epiteliais e Glandulares/tratamento farmacológico , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Carboplatina/administração & dosagem , Carcinoma Epitelial do Ovário , Quimioterapia Adjuvante , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Epitélio/metabolismo , Feminino , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O3/metabolismo , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Epiteliais e Glandulares/cirurgia , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/cirurgia , Paclitaxel/administração & dosagem , Fosforilação , Análise Serial de Proteínas , Adulto JovemRESUMO
BACKGROUND: Although malaria is a preventable and curable human disease, millions of people risk to be infected by the Plasmodium parasites and to develop this illness. Therefore, there is an urgent need to identify new anti-malarial drugs. Ca2+ signalling regulates different processes in the life cycle of Plasmodium falciparum, representing a suitable target for the development of new drugs. RESULTS: This study investigated for the first time the effect of a highly specific inhibitor of nicotinic acid adenine dinucleotide phosphate (NAADP)-induced Ca2+ release (Ned-19) on P. falciparum, revealing the inhibitory effect of this compound on the blood stage development of this parasite. Ned-19 inhibits both the transition of the parasite from the early to the late trophozoite stage and the ability of the late trophozoite to develop to the multinucleated schizont stage. In addition, Ned-19 affects spontaneous intracellular Ca2+ oscillations in ring and trophozoite stage parasites, suggesting that the observed inhibitory effects may be associated to regulation of intracellular Ca2+ levels. CONCLUSIONS: This study highlights the inhibitory effect of Ned-19 on progression of the asexual life cycle of P. falciparum. The observation that Ned-19 inhibits spontaneous Ca2+ oscillations suggests a potential role of NAADP in regulating Ca2+ signalling of P. falciparum.
Assuntos
Antimaláricos/farmacologia , Carbolinas/farmacologia , NADP/análogos & derivados , Piperazinas/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Transdução de Sinais , Eritrócitos/parasitologia , Humanos , NADP/fisiologia , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/fisiologia , Esquizontes/efeitos dos fármacos , Esquizontes/crescimento & desenvolvimento , Esquizontes/fisiologiaRESUMO
Vascular endothelial growth factor (VEGF) and its receptors VEGFR1/VEGFR2 play major roles in controlling angiogenesis, including vascularization of solid tumors. Here we describe a specific Ca(2+) signaling pathway linked to the VEGFR2 receptor subtype, controlling the critical angiogenic responses of endothelial cells (ECs) to VEGF. Key steps of this pathway are the involvement of the potent Ca(2+) mobilizing messenger, nicotinic acid adenine-dinucleotide phosphate (NAADP), and the specific engagement of the two-pore channel TPC2 subtype on acidic intracellular Ca(2+) stores, resulting in Ca(2+) release and angiogenic responses. Targeting this intracellular pathway pharmacologically using the NAADP antagonist Ned-19 or genetically using Tpcn2(-/-) mice was found to inhibit angiogenic responses to VEGF in vitro and in vivo. In human umbilical vein endothelial cells (HUVECs) Ned-19 abolished VEGF-induced Ca(2+) release, impairing phosphorylation of ERK1/2, Akt, eNOS, JNK, cell proliferation, cell migration, and capillary-like tube formation. Interestingly, Tpcn2 shRNA treatment abolished VEGF-induced Ca(2+) release and capillary-like tube formation. Importantly, in vivo VEGF-induced vessel formation in matrigel plugs in mice was abolished by Ned-19 and, most notably, failed to occur in Tpcn2(-/-) mice, but was unaffected in Tpcn1(-/-) animals. These results demonstrate that a VEGFR2/NAADP/TPC2/Ca(2+) signaling pathway is critical for VEGF-induced angiogenesis in vitro and in vivo. Given that VEGF can elicit both pro- and antiangiogenic responses depending upon the balance of signal transduction pathways activated, targeting specific VEGFR2 downstream signaling pathways could modify this balance, potentially leading to more finely tailored therapeutic strategies.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Neovascularização Fisiológica/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Canais de Cálcio/genética , Sinalização do Cálcio/efeitos dos fármacos , Carbolinas/farmacologia , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Camundongos , Camundongos Knockout , NADP/análogos & derivados , NADP/antagonistas & inibidores , NADP/genética , NADP/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Piperazinas/farmacologia , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Adherent/invasive Escherichia coli (AIEC) strains have recently been receiving increased attention because they are more prevalent and persistent in the intestine of Crohn's disease (CD) patients than in healthy subjects. Since AIEC strains show a high percentage of similarity to extraintestinal pathogenic E. coli (ExPEC), neonatal meningitis-associated E. coli (NMEC), and uropathogenic E. coli (UPEC) strains, here we compared AIEC strain LF82 with a UPEC isolate (strain EC73) to assess whether LF82 would be able to infect prostate cells as an extraintestinal target. The virulence phenotypes of both strains were determined by using the RWPE-1 prostate cell line. The results obtained indicated that LF82 and EC73 are able to adhere to, invade, and survive within prostate epithelial cells. Invasion was confirmed by immunofluorescence and electron microscopy. Moreover, cytochalasin D and colchicine strongly inhibited bacterial uptake of both strains, indicating the involvement of actin microfilaments and microtubules in host cell invasion. Moreover, both strains belong to phylogenetic group B2 and are strong biofilm producers. In silico analysis reveals that LF82 shares with UPEC strains several virulence factors: namely, type 1 pili, the group II capsule, the vacuolating autotransporter toxin, four iron uptake systems, and the pathogenic island (PAI). Furthermore, compared to EC73, LF82 induces in RWPE-1 cells a marked increase of phosphorylation of mitogen-activated protein kinases (MAPKs) and of NF-κB already by 5 min postinfection, thus inducing a strong inflammatory response. Our in vitro data support the hypothesis that AIEC strains might play a role in prostatitis, and, by exploiting host-cell signaling pathways controlling the innate immune response, likely facilitate bacterial multiplication and dissemination within the male genitourinary tract.
Assuntos
Aderência Bacteriana/fisiologia , Células Epiteliais/microbiologia , Escherichia coli/patogenicidade , Próstata/citologia , Biofilmes/crescimento & desenvolvimento , Linhagem Celular , Doença de Crohn/microbiologia , Células Epiteliais/metabolismo , Escherichia coli/fisiologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Humanos , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Fenótipo , Filogenia , Virulência , Fatores de Virulência/metabolismoAssuntos
Biomarcadores Tumorais , Neoplasias Colorretais/patologia , Neoplasias Peritoneais/diagnóstico , Neoplasias Peritoneais/secundário , Neoplasias Peritoneais/terapia , Medicina de Precisão/métodos , Animais , Neoplasias Colorretais/etiologia , Modelos Animais de Doenças , Genômica/métodos , Genômica/normas , Humanos , Técnicas In Vitro , Medicina de Precisão/normas , Prognóstico , Pesquisa Translacional BiomédicaRESUMO
Epithelial ovarian carcinoma (EOC) is a deadly disease, with a 5-year survival of 30%. The aim of the study was to perform broad-scale protein signaling activation mapping to evaluate if EOC can be redefined based on activated protein signaling network architecture rather than histology. Tumor cells were isolated using laser capture microdissection (LCM) from 72 EOCs. Tumors were classified as serous (n = 38), endometrioid (n = 13), mixed (n = 8), clear cell (CCC; n = 7), and others (n = 6). LCM tumor cells were lysed and subjected to reverse-phase protein microarray to measure the expression/activation level of 117 protein drug targets. Unsupervised hierarchical clustering analysis was utilized to explore the overall signaling network. ANOVA was used to detect significant differences among the groups (p < 0.05). Regardless of histology, unsupervised analysis revealed five pathway-driven clusters. When the EOC histotypes were compared by ANOVA, only CCC showed a distinct signaling network, with activation of EGFR, Syk, HER2/ErbB2, and SHP2 (p = 0.0007, p = 0.0021, p < 0.0001, and p = 0.0410, respectively). The histological classification of EOC fails to adequately describe the underpinning protein signaling network. Nevertheless, CCC presents unique signaling characteristics compared to the other histotypes. EOC may need to be characterized by functional signaling activation mapping rather than pure histology.
Assuntos
Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/patologia , Ovário/patologia , Mapas de Interação de Proteínas , Transdução de Sinais , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Epitelial do Ovário , Receptores ErbB/análise , Receptores ErbB/metabolismo , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/análise , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Ovarianas/metabolismo , Ovário/metabolismo , Medicina de Precisão , Análise Serial de Proteínas , Proteína Tirosina Fosfatase não Receptora Tipo 11/análise , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteínas Tirosina Quinases/análise , Proteínas Tirosina Quinases/metabolismo , Receptor ErbB-2/análise , Receptor ErbB-2/metabolismo , Quinase Syk , Adulto JovemRESUMO
Toll-like receptors (TLRs) are a family of highly conserved transmembrane proteins expressed in epithelial and immune cells that recognize pathogen associated molecular patterns. Besides their role in immune response against infections, numerous studies have shown an important role of different TLRs in cancer, indicating these receptors as potential targets for cancer therapy. We previously demonstrated that the activation of TLR3 by the synthetic double-stranded RNA analogue poly I:C induces apoptosis of androgen-sensitive prostate cancer (PCa) LNCaP cells and, much less efficiently, of the more aggressive PC3 cell line. Therefore, in this study we selected LNCaP cells to investigate the mechanism of TLR3-mediated apoptosis and the in vivo efficacy of poly I:C-based therapy. We show that interferon regulatory factor-3 (IRF-3) signalling plays an essential role in TLR3-mediated apoptosis in LNCaP cells through the activation of the intrinsic and extrinsic apoptotic pathways. Interestingly, hardly any apoptosis was induced by poly I:C in normal prostate epithelial cells RWPE-1. We also demonstrate for the first time the direct anticancer effect of poly I:C as a single therapeutic agent in a well-established human androgen-sensitive PCa xenograft model, by showing that tumour growth is highly impaired in poly I:C-treated immunodeficient mice. Immunohistochemical analysis of PCa xenografts highlights the antitumour role of poly I:C in vivo both on cancer cells and, indirectly, on endothelial cells. Notably, we show the presence of TLR3 and IRF-3 in both human normal and PCa clinical samples, potentially envisaging poly I:C-based therapy for PCa.
Assuntos
Androgênios/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fator Regulador 3 de Interferon/metabolismo , Próstata/efeitos dos fármacos , Próstata/metabolismo , Animais , Apoptose/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NODRESUMO
Toll-Like receptors (TLRs) are a family of evolutionary conserved transmembrane proteins that recognize highly conserved molecules in pathogens. TLR-expressing cells represent the first line of defence sensing pathogen invasion, triggering innate immune responses and subsequently priming antigen-specific adaptive immunity. In vitro and in vivo studies on experimental cancer models have shown both anti- and pro-tumoural activity of different TLRs in prostate cancer, indicating these receptors as potential targets for cancer therapy. In this review, we highlight the intriguing duplicity of TLR stimulation by pathogens: their protective role in cases of acute infections, and conversely their negative role in favouring hyperplasia and/or cancer onset, in cases of chronic infections. This review focuses on the role of TLRs in the pathophysiology of prostate infection and cancer by exploring the biological bases of the strict relation between TLRs and prostate cancer. In particular, we highlight the debated question of how reliable mutations or deregulated expression of TLRs are as novel diagnostic or prognostic tools for prostate cancer. So far, the anticancer activity of numerous TLR ligands has been evaluated in clinical trials only in organs other than the prostate. Here we review recent clinical trials based on the most promising TLR agonists in oncology, envisaging a potential application also in prostate cancer therapy.
Assuntos
Infecções Bacterianas/imunologia , Próstata/imunologia , Hiperplasia Prostática/imunologia , Neoplasias da Próstata/imunologia , Receptores Toll-Like/genética , Imunidade Adaptativa , Infecções Bacterianas/complicações , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/patologia , Biomarcadores/metabolismo , Ensaios Clínicos como Assunto , Expressão Gênica , Humanos , Imunidade Inata , Fatores Imunológicos/farmacologia , Masculino , Próstata/efeitos dos fármacos , Próstata/patologia , Hiperplasia Prostática/complicações , Hiperplasia Prostática/tratamento farmacológico , Hiperplasia Prostática/patologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Transdução de Sinais , Receptores Toll-Like/imunologiaRESUMO
A variety of endothelial agonist-induced responses are mediated by rises in intracellular Ca(2+), suggesting that different Ca(2+) signatures could fine-tune specific inflammatory and thrombotic activities. In search of new intracellular mechanisms modulating endothelial effector functions, we identified nicotinic acid adenine dinucleotide phosphate (NAADP) as a crucial second messenger in histamine-induced Ca(2+) release via H1 receptors (H1R). NAADP is a potent intracellular messenger mobilizing Ca(2+) from lysosome-like acidic compartments, functionally coupled to the endoplasmic reticulum. Using the human EA.hy926 endothelial cell line and primary human umbilical vein endothelial cells, we show that selective H1R activation increases intracellular NAADP levels and that H1R-induced calcium release involves both acidic organelles and the endoplasmic reticulum. To assess that NAADP links H1R to Ca(2+)-signaling we used both microinjection of self-inactivating concentrations of NAADP and the specific NAADP receptor antagonist, Ned-19, both of which completely abolished H1R-induced but not thrombin-induced Ca(2+) mobilization. Interestingly, H1R-mediated von Willebrand factor (VWF) secretion was completely inhibited by treatment with Ned-19 and by siRNA knockdown of 2-pore channel NAADP receptors, whereas thrombin-induced VWF secretion failed to be affected. These findings demonstrate a novel and specific Ca(2+)-signaling mechanism activated through H1R in human endothelial cells, which reveals an obligatory role of NAADP in the control of VWF secretion.
Assuntos
Células Endoteliais/metabolismo , NADP/análogos & derivados , Receptores Histamínicos H1/metabolismo , Fator de von Willebrand/metabolismo , Sequência de Bases , Canais de Cálcio/genética , Sinalização do Cálcio/efeitos dos fármacos , Carbolinas/farmacologia , Linhagem Celular , Células Endoteliais/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Ácidos Heptanoicos/farmacologia , Histamina/farmacologia , Humanos , NADP/metabolismo , Antagonistas Nicotínicos/farmacologia , Piperazinas/farmacologia , Piperidinas/farmacologia , RNA Interferente Pequeno/genética , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Sistemas do Segundo Mensageiro/fisiologiaRESUMO
BACKGROUND: Melanoma represents only 4% of all skin cancers, but nearly 80% of skin cancer deaths. This manuscript applies several new measurement technologies with the purpose of elucidating molecular signatures of melanoma aggressiveness. PURPOSE: We sought to determine whether low-abundant serum proteins related to apoptotic pathways could be measured and correlated with defined melanoma subtypes. Hydrogel core shell nanoparticles, a new technology capable of selectively entrapping low molecular weight proteins and protecting them from enzymatic degradation, were used to capture candidate serum biomarkers. Biomarker levels were correlated with confocal microscopy, thereby representing a combination of new technologies for in vivo histologic documentation. RESULTS: Among a panel of analyzed serum proteins, Bak was differentially expressed between nevi and melanomas. Melanomas with higher Bak serum levels exhibited more pronounced junctional activity on confocal imaging, whereas lesions with 'sparse' dermal nests had weak Bak expression. CONCLUSIONS: Our study links serum proteome analysis with confocal microscopic clinical in vivo histologic classification of melanomas. Bak has not been previously measured in serum. Bak differential expression among melanoma subtypes confirms the importance of the apoptotic pathway as a contributor to melanoma aggressiveness.
Assuntos
Biomarcadores Tumorais/sangue , Melanoma/sangue , Neoplasias Cutâneas/sangue , Proteína Killer-Antagonista Homóloga a bcl-2/sangue , Adulto , Becaplermina , Humanos , Melanoma/diagnóstico , Melanoma/patologia , Melanoma/secundário , Microscopia Confocal , Pessoa de Meia-Idade , Nanopartículas , Nanotecnologia , Nevo Pigmentado/sangue , Nevo Pigmentado/diagnóstico , Fator de Crescimento Derivado de Plaquetas/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas c-bcl-2/sangue , Proteínas Proto-Oncogênicas c-sis , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/patologiaRESUMO
Increased oxidative stress by reactive oxygen species (ROS) and reactive nitrogen species (RNS) is a major determinant of disuse-induced muscle atrophy. Muscle biopsies (thigh vastus lateralis, VL) obtained from healthy male subjects enrolled in the Toulouse Cocktail bedrest (BR) study were used to assess efficacy of an antioxidant cocktail (polyphenols, omega-3, vitamin E, and selenium) to counteract the increased redox homeostasis and enhance the antioxidant defense response by using label-free LC-MS/MS and NITRO-DIGE (nitrosated proteins), qPCR, and laser confocal microscopy. Label-free LC-MS/MS indicated that treatment prevented the redox homeostasis dysregulation and promoted structural remodeling (TPM3, MYH7, MYBPC, MYH1, MYL1, HRC, and LUM), increment of RyR1, myogenesis (CSRP3), and skeletal muscle development (MUSTN1, LMNA, AHNAK). These changes were absent in the Placebo group. Glycolysis, tricarboxylic acid cycle (TCA), oxidative phosphorylation, fatty acid beta-oxidation, and mitochondrial transmembrane transport were normalized in treated subjects. Proteins involved in protein folding were also normalized, whereas protein entailed in ion homeostasis decreased. NITRO-DIGE analysis showed significant protein nitrosylation changes for CAT, CA3, SDHA, and VDAC2 in Treatment vs. Placebo. Similarly, the nuclear factor erythroid 2-related factor 2 (Nrf-2) antioxidant response element (Nrf-2 ARE) signaling pathway showed an enhanced response in the Treatment group. Increased nitrosative redox homeostasis and decreased antioxidant defense response were found in post-BR control (Placebo, n = 10) vs. the antioxidant cocktail treated group (Treatment, n = 10). Taken together, increased nitrosative redox homeostasis and muscle deterioration during BR-driven physical inactivity were prevented, whereas decreased antioxidant nitrosative stress defense response was attenuated by Treatment suggesting positive effects of the nutritional intervention protocol in bedrest.
RESUMO
BACKGROUND: Anti-programmed cell death protein 1 and programmed cell death ligand 1 (PD-L1) agents are broadly used in first-line and second-line treatment across different tumor types. While immunohistochemistry-based assays are routinely used to assess PD-L1 expression, their clinical utility remains controversial due to the partial predictive value and lack of standardized cut-offs across antibody clones. Using a high throughput immunoassay, the reverse phase protein microarray (RPPA), coupled with a fluorescence-based detection system, this study compared the performance of six anti-PD-L1 antibody clones on 666 tumor samples. METHODS: PD-L1 expression was measured using five antibody clones (22C3, 28-8, CAL10, E1L3N and SP142) and the therapeutic antibody atezolizumab on 222 lung, 71 ovarian, 52 prostate and 267 breast cancers, and 54 metastatic lesions. To capture clinically relevant variables, our cohort included frozen and formalin-fixed paraffin-embedded samples, surgical specimens and core needle biopsies. Pure tumor epithelia were isolated using laser capture microdissection from 602 samples. Correlation coefficients were calculated to assess concordance between antibody clones. For two independent cohorts of patients with lung cancer treated with nivolumab, RPPA-based PD-L1 measurements were examined along with response to treatment. RESULTS: Median-center PD-L1 dynamic ranged from 0.01 to 39.37 across antibody clones. Correlation coefficients between the six antibody clones were heterogeneous (range: -0.48 to 0.95) and below 0.50 in 61% of the comparisons. In nivolumab-treated patients, RPPA-based measurement identified a subgroup of tumors, where low PD-L1 expression equated to lack of response. CONCLUSIONS: Continuous RPPA-based measurements capture a broad dynamic range of PD-L1 expression in human specimens and heterogeneous concordance levels between antibody clones. This high throughput immunoassay can potentially identify subgroups of tumors in which low expression of PD-L1 equates to lack of response to treatment.
Assuntos
Neoplasias/genética , Medicina de Precisão/métodos , Receptor de Morte Celular Programada 1/uso terapêutico , Análise Serial de Proteínas/métodos , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Identification of countermeasures able to prevent disuse-induced muscle wasting is crucial to increase performance of crew members during space flight as well as ameliorate patient's clinical outcome after long immobilization periods. We report on the outcome of short but high-impact reactive jumps (JUMP) as countermeasure during 60 days of 6° head-down tilt (HDT) bed rest on myofiber size, type composition, capillarization, and oxidative capacity in tissue biopsies (pre/post/recovery) from the knee extensor vastus lateralis (VL) and deep calf soleus (SOL) muscle of 22 healthy male participants (Reactive jumps in a sledge, RSL-study 2015-2016, DLR:envihab, Cologne). Bed rest induced a slow-to-fast myofiber shift (type I ->II) with an increased prevalence of hybrid fibers in SOL after bed rest without jumps (control, CTRL, p = 0.016). In SOL, JUMP countermeasure in bed rest prevented both fast and slow myofiber cross-sectional area (CSA) decrements (p = 0.005) in CTRL group. In VL, bed rest only induced capillary rarefaction, as reflected by the decrease in local capillary-to-fiber ratio (LCFR) for both type II (pre vs. post/R + 10, p = 0.028/0.028) and type I myofibers (pre vs. R + 10, p = 0.012), which was not seen in the JUMP group. VO2 max Fiber (pL × mm-1 × min-1) calculated from succinate dehydrogenase (SDH)-stained cryosections (OD660 nm) showed no significant differences between groups. High-impact jump training in bed rest did not prevent disuse-induced myofiber atrophy in VL, mitigated phenotype transition (type I - >II) in SOL, and attenuated capillary rarefaction in the prime knee extensor VL however with little impact on oxidative capacity changes.
RESUMO
We have investigated the role of NAADP-mediated Ca(2+) mobilization in endothelin (ET) signaling via endothelin receptor subtype A (ETA) and endothelin receptor subtype B (ETB) in rat peritubular smooth muscle cells. Microinjection and extracellular application of NAADP were both able to elicit Ca(2+) release which was blocked by inhibitory concentrations of NAADP, by impairing Ca(2+) uptake in acidic stores with bafilomycin, and by thapsigargin. Ca(2+) release in response to selective ETB stimulation was abolished by inhibition of NAADP signaling through the same strategies, while these treatments only partially impaired ETA-dependent Ca(2+) signaling, showing that transduction of the ETB signal is dependent on NAADP. In addition, we show that lipid rafts/caveolae contain ETA, ETB, and NAADP/cADPR generating enzyme CD38 and that stimulation of ETB receptors results in increased CD38 activity; interestingly, ETB- (but not ETA-) mediated Ca(2+) responses were antagonized by disruption of lipid rafts/caveolae with methyl-beta-cyclodextrin. These data demonstrate a primary role of NAADP in ETB-mediated Ca(2+) signaling and strongly suggest a novel role of lipid rafts/caveolae in triggering ET-induced NAADP signaling.
Assuntos
Sinalização do Cálcio/fisiologia , Cavéolas/metabolismo , Endotelina-1/metabolismo , Microdomínios da Membrana/metabolismo , NADP/análogos & derivados , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/metabolismo , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1/metabolismo , Animais , Cálcio/metabolismo , Caveolina 1/metabolismo , Células Cultivadas , Endotelina-1/genética , Endotelinas/metabolismo , Inibidores Enzimáticos/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/fisiologia , NADP/metabolismo , Fragmentos de Peptídeos/metabolismo , Ratos , Ratos Wistar , Receptor de Endotelina A/genética , Receptor de Endotelina B/genética , Túbulos Seminíferos/citologia , Transdução de Sinais/fisiologia , beta-Ciclodextrinas/metabolismoRESUMO
The dystrophin-related and -associated protein dystrobrevin is a component of the dystrophin-associated protein complex, which directly links the cytoskeleton to the extracellular matrix. It is now thought that this complex also serves as a dynamic scaffold for signaling proteins, and dystrobrevin may play a role in this context. Since dystrobrevin involvement in signaling pathways seems to be dependent on its interaction with other proteins, we sought new insights and performed a two-hybrid screen of a mouse brain cDNA library using beta-dystrobrevin, the isoform expressed in non-muscle tissues, as bait. Among the positive clones characterized after the screen, one encodes the regulatory subunit RIalpha of the cAMP-dependent protein kinase A (PKA). We confirmed the interaction by in vitro and in vivo association assays, and mapped the binding site of beta-dystrobrevin on RIalpha to the amino-terminal region encompassing the dimerization/docking domain of PKA regulatory subunit. We also found that the domain of interaction for RIalpha is contained in the amino-terminal region of beta-dystrobrevin. We obtained evidence that beta-dystrobrevin also interacts directly with RIIbeta, and that not only beta-dystrobrevin but also alpha-dystrobrevin interacts with PKA regulatory subunits. We show that both alpha and beta-dystrobrevin are specific phosphorylation substrates for PKA and that protein phosphatase 2A (PP2A) is associated with dystrobrevins. Our results suggest a new role for dystrobrevin as a scaffold protein that may play a role in different cellular processes involving PKA signaling.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Associadas à Distrofina/química , Proteínas Associadas à Distrofina/fisiologia , Animais , Sítios de Ligação , Encéfalo/metabolismo , Células COS , Chlorocebus aethiops , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico , Matriz Extracelular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Transdução de Sinais , Técnicas do Sistema de Duplo-HíbridoRESUMO
Recent advances in next-generation sequencing and other omics technologies capable to map cell fate provide increasing evidence on the crucial role of intra-tumor heterogeneity (ITH) for cancer progression. The different facets of ITH, from genomic to microenvironmental heterogeneity and the hierarchical cellular architecture originating from the cancer stem cell compartment, contribute to the range of tumor phenotypes. Decoding these complex data resulting from the analysis of tumor tissue complexity poses a challenge for developing novel therapeutic strategies that can counteract tumor evolution and cellular plasticity. To achieve this aim, the development of in vitro and in vivo cancer models that resemble the complexity of ITH is crucial in understanding the interplay of cells and their (micro)environment and, consequently, in testing the efficacy of new targeted treatments and novel strategies of tailoring combinations of treatments to the individual composition of the tumor. This challenging approach may be an important cornerstone in overcoming the development of pharmaco-resistances during multiple lines of treatment. In this paper, we report the latest advances in patient-derived 3D (PD3D) cell cultures and patient-derived tumor xenografts (PDX) as in vitro and in vivo models that can retain the genetic and phenotypic heterogeneity of the tumor tissue.
RESUMO
Microgravity exposure as well as chronic disuse are two main causes of skeletal muscle atrophy in animals and humans. The antigravity calf soleus is a reference postural muscle to investigate the mechanism of disuse-induced maladaptation and plasticity of human and rodent (rats or mice) skeletal musculature. Here, we report microgravity-induced global gene expression changes in space-flown mouse skeletal muscle and the identification of yet unknown disuse susceptible transcripts found in soleus (a mainly slow phenotype) but not in extensor digitorum longus (a mainly fast phenotype dorsiflexor as functional counterpart to soleus). Adult C57Bl/N6 male mice (n = 5) flew aboard a biosatellite for 30 days on orbit (BION-M1 mission, 2013), a sex and age-matched cohort were housed in standard vivarium cages (n = 5), or in a replicate flight habitat as ground control (n = 5). Next to disuse atrophy signs (reduced size and myofiber phenotype I to II type shift) as much as 680 differentially expressed genes were found in the space-flown soleus, and only 72 in extensor digitorum longus (only 24 genes in common) compared to ground controls. Altered expression of gene transcripts matched key biological processes (contractile machinery, calcium homeostasis, muscle development, cell metabolism, inflammatory and oxidative stress response). Some transcripts (Fzd9, Casq2, Kcnma1, Ppara, Myf6) were further validated by quantitative real-time PCR (qRT-PCR). Besides previous reports on other leg muscle types we put forth for the first time a complete set of microgravity susceptible gene transcripts in soleus of mice as promising new biomarkers or targets for optimization of physical countermeasures and rehabilitation protocols to overcome disuse atrophy conditions in different clinical settings, rehabilitation and spaceflight.
Assuntos
Fibras Musculares de Contração Lenta/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/genética , Voo Espacial , Ausência de Peso , Animais , Perfilação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise em Microsséries , Atrofia Muscular/etiologia , Atrofia Muscular/metabolismo , Fatores de Tempo , Ausência de Peso/efeitos adversosRESUMO
Microgravity as well as chronic muscle disuse are two causes of low back pain originated at least in part from paraspinal muscle deconditioning. At present no study investigated the complexity of the molecular changes in human or mouse paraspinal muscles exposed to microgravity. The aim of this study was to evaluate longissimus dorsi adaptation to microgravity at both morphological and global gene expression level. C57BL/N6 male mice were flown aboard the BION-M1 biosatellite for 30 days (BF) or housed in a replicate flight habitat on ground (BG). Myofiber cross sectional area and myosin heavy chain subtype patterns were respectively not or slightly altered in longissimus dorsi of BF mice. Global gene expression analysis identified 89 transcripts differentially regulated in longissimus dorsi of BF vs. BG mice. Microgravity-induced gene expression changes of lipocalin 2 (Lcn2), sestrin 1(Sesn1), phosphatidylinositol 3-kinase, regulatory subunit polypeptide 1 (p85 alpha) (Pik3r1), v-maf musculoaponeurotic fibrosarcoma oncogene family protein B (Mafb), protein kinase C delta (Prkcd), Muscle Atrophy F-box (MAFbx/Atrogin-1/Fbxo32), and Muscle RING Finger 1 (MuRF-1) were further validated by real time qPCR analysis. In conclusion, our study highlighted the regulation of transcripts mainly linked to insulin sensitivity and metabolism in longissimus dorsi following 30 days of microgravity exposure. The apparent absence of robust signs of back muscle atrophy in space-flown mice, despite the overexpression of Atrogin-1 and MuRF-1, opens new questions on the possible role of microgravity-sensitive genes in the regulation of peripheral insulin resistance following unloading and its consequences on paraspinal skeletal muscle physiology.