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1.
Anticancer Res ; 40(9): 5115-5124, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32878800

RESUMO

BACKGROUND/AIM: Pyruvate kinase M2 (PKM2) is an enzyme that is predominantly overexpressed in various types of cancer. The role of PKM2 in liver fluke-associated cholangiocarcinoma (CCA) remains unclear. This study aimed to investigate the antitumor activity of shikonin, a PKM2 inhibitor, in CCA cells. MATERIALS AND METHODS: Immunohistochemistry and immunoblotting were used to determine PKM2 expression in CCA tissues and cells. Antiproliferative effects of shikonin were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony-formation and trypan blue exclusion assays. The anti-metastatic activity of shikonin was determined using the Boyden chamber assay. Mechanisms by which shikonin inhibited CCA progression were determined. RESULTS: PKM2 was overexpressed in CCA compared to normal bile duct epithelial cells. Shikonin significantly inhibited growth, and migration of CCA cells while inducing their death. A mechanistic study revealed that antitumor effects of shikonin in CCA cells depended on increased production of reactive oxygen species. CONCLUSION: Shikonin may be a novel therapeutic agent for patients with CCA.


Assuntos
Antineoplásicos/farmacologia , Neoplasias dos Ductos Biliares/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Colangiocarcinoma/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Naftoquinonas/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células/efeitos dos fármacos , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/patologia , Humanos , Espécies Reativas de Oxigênio/metabolismo , Hormônios Tireóideos
2.
Nat Commun ; 11(1): 4545, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917858

RESUMO

TGF-ß1, ß2 and ß3 bind a common receptor to exert vastly diverse effects in cancer, supporting either tumor progression by favoring metastases and inhibiting anti-tumor immunity, or tumor suppression by inhibiting malignant cell proliferation. Global TGF-ß inhibition thus bears the risk of undesired tumor-promoting effects. We show that selective blockade of TGF-ß1 production by Tregs with antibodies against GARP:TGF-ß1 complexes induces regressions of mouse tumors otherwise resistant to anti-PD-1 immunotherapy. Effects of combined GARP:TGF-ß1/PD-1 blockade are immune-mediated, do not require FcγR-dependent functions and increase effector functions of anti-tumor CD8+ T cells without augmenting immune cell infiltration or depleting Tregs within tumors. We find GARP-expressing Tregs and evidence that they produce TGF-ß1 in one third of human melanoma metastases. Our results suggest that anti-GARP:TGF-ß1 mAbs, by selectively blocking a single TGF-ß isoform emanating from a restricted cellular source exerting tumor-promoting activity, may overcome resistance to PD-1/PD-L1 blockade in patients with cancer.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Membrana/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Animais , Antineoplásicos Imunológicos/uso terapêutico , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral/transplante , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/imunologia , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Neoplasias/imunologia , Neoplasias/patologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta1/metabolismo
3.
Nature ; 585(7823): 96-101, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32814898

RESUMO

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative disorders that overlap in their clinical presentation, pathology and genetic origin. Autoimmune disorders are also overrepresented in both ALS and FTD, but this remains an unexplained epidemiologic observation1-3. Expansions of a hexanucleotide repeat (GGGGCC) in the C9orf72 gene are the most common cause of familial ALS and FTD (C9-ALS/FTD), and lead to both repeat-containing RNA and dipeptide accumulation, coupled with decreased C9orf72 protein expression in brain and peripheral blood cells4-6. Here we show in mice that loss of C9orf72 from myeloid cells alone is sufficient to recapitulate the age-dependent lymphoid hypertrophy and autoinflammation seen in animals with a complete knockout of C9orf72. Dendritic cells isolated from C9orf72-/- mice show marked early activation of the type I interferon response, and C9orf72-/- myeloid cells are selectively hyperresponsive to activators of the stimulator of interferon genes (STING) protein-a key regulator of the innate immune response to cytosolic DNA. Degradation of STING through the autolysosomal pathway is diminished in C9orf72-/- myeloid cells, and blocking STING suppresses hyperactive type I interferon responses in C9orf72-/- immune cells as well as splenomegaly and inflammation in C9orf72-/- mice. Moreover, mice lacking one or both copies of C9orf72 are more susceptible to experimental autoimmune encephalitis, mirroring the susceptibility to autoimmune diseases seen in people with C9-ALS/FTD. Finally, blood-derived macrophages, whole blood and brain tissue from patients with C9-ALS/FTD all show an elevated type I interferon signature compared with samples from people with sporadic ALS/FTD; this increased interferon response can be suppressed with a STING inhibitor. Collectively, our results suggest that patients with C9-ALS/FTD have an altered immunophenotype because their reduced levels of C9orf72 cannot suppress the inflammation mediated by the induction of type I interferons by STING.


Assuntos
Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Inflamação/metabolismo , Inflamação/prevenção & controle , Proteínas de Membrana/metabolismo , Células Mieloides/metabolismo , Envelhecimento/imunologia , Esclerose Amiotrófica Lateral/genética , Animais , Proteína C9orf72/deficiência , Células Dendríticas/citologia , Células Dendríticas/imunologia , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Feminino , Humanos , Inflamação/genética , Inflamação/imunologia , Interferon Tipo I/biossíntese , Interferon Tipo I/imunologia , Proteínas de Membrana/antagonistas & inibidores , Camundongos , Células Mieloides/imunologia , Neoplasias/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia
4.
PLoS Pathog ; 16(8): e1008792, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32813746

RESUMO

Tumor suppressors can exert pro-proliferation functions in specific contexts. In the beta human papillomavirus type 38 (HPV38) experimental model, the viral proteins E6 and E7 promote accumulation of a wild-type (WT) p53 form in human keratinocytes (HKs), promoting cellular proliferation. Inactivation of p53 by different means strongly decreases the proliferation of HPV38 E6/E7 HKs. This p53 form is phosphorylated at S392 by the double-stranded RNA-dependent protein kinase PKR, which is highly activated by HPV38. PKR-mediated S392 p53 phosphorylation promotes the formation of a p53/DNMT1 complex, which inhibits expression of integrin alpha 1 (ITGA1), a repressor of epidermal growth factor receptor (EGFR) signaling. Ectopic expression of ITGA1 in HPV38 E6/E7 HKs promotes EGFR degradation, inhibition of cellular proliferation, and cellular death. Itga1 expression was also inhibited in the skin of HPV38 transgenic mice that have an elevated susceptibility to UV-induced skin carcinogenesis. In summary, these findings reveal the existence of a specific WT p53 form that displays pro-proliferation properties.


Assuntos
Proteínas de Transporte/antagonistas & inibidores , Proliferação de Células , Queratinócitos/patologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/complicações , Proteínas Repressoras/metabolismo , Neoplasias Cutâneas/etiologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Células Cultivadas , Regulação para Baixo , Humanos , Queratinócitos/imunologia , Queratinócitos/virologia , Camundongos , Camundongos Transgênicos , Proteínas Oncogênicas Virais/genética , Papillomaviridae/isolamento & purificação , Proteínas E7 de Papillomavirus/genética , Infecções por Papillomavirus/virologia , Proteínas Repressoras/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Proteína Supressora de Tumor p53/genética
5.
Nat Commun ; 11(1): 4012, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32782388

RESUMO

Transmembrane B cell lymphoma 2-associated X protein inhibitor motif-containing (TMBIM) 6, a Ca2+ channel-like protein, is highly up-regulated in several cancer types. Here, we show that TMBIM6 is closely associated with survival in patients with cervical, breast, lung, and prostate cancer. TMBIM6 deletion or knockdown suppresses primary tumor growth. Further, mTORC2 activation is up-regulated by TMBIM6 and stimulates glycolysis, protein synthesis, and the expression of lipid synthesis genes and glycosylated proteins. Moreover, ER-leaky Ca2+ from TMBIM6, a unique characteristic, is shown to affect mTORC2 assembly and its association with ribosomes. In addition, we identify that the BIA compound, a potentialTMBIM6 antagonist, prevents TMBIM6 binding to mTORC2, decreases mTORC2 activity, and also regulates TMBIM6-leaky Ca2+, further suppressing tumor formation and progression in cancer xenograft models. This previously unknown signaling cascade in which mTORC2 activity is enhanced via the interaction with TMBIM6 provides potential therapeutic targets for various malignancies.


Assuntos
Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Indenos/farmacologia , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Cálcio/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Neoplasias/genética , Neoplasias/patologia , Ligação Proteica , Ribossomos/metabolismo , Transdução de Sinais , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra
6.
PLoS Biol ; 18(7): e3000755, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32644996

RESUMO

Kindlin-1, -2, and -3 directly bind integrin ß cytoplasmic tails to regulate integrin activation and signaling. Despite their functional significance and links to several diseases, structural information on full-length kindlin proteins remains unknown. Here, we report the crystal structure of human full-length kindlin-3, which reveals a novel homotrimer state. Unlike kindlin-3 monomer, which is the major population in insect and mammalian cell expression systems, kindlin-3 trimer does not bind integrin ß cytoplasmic tail as the integrin-binding pocket in the F3 subdomain of 1 protomer is occluded by the pleckstrin homology (PH) domain of another protomer, suggesting that kindlin-3 is auto-inhibited upon trimer formation. This is also supported by functional assays in which kindlin-3 knockout K562 erythroleukemia cells reconstituted with the mutant kindlin-3 containing trimer-disrupting mutations exhibited an increase in integrin-mediated adhesion and spreading on fibronectin compared with those reconstituted with wild-type kindlin-3. Taken together, our findings reveal a novel mechanism of kindlin auto-inhibition that involves its homotrimer formation.


Assuntos
Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/química , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Multimerização Proteica , Movimento Celular , Humanos , Integrinas/metabolismo , Células K562 , Proteínas de Membrana/metabolismo , Modelos Moleculares , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Domínios Proteicos , Homologia Estrutural de Proteína , Relação Estrutura-Atividade
7.
Clin Exp Immunol ; 200(2): 155-162, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32297328

RESUMO

Immune checkpoint blockers improve the overall survival of a limited number of patients among different cancers. Identifying pathways that influence the immunological and clinical response to treatment is critical to improve the therapeutic efficacy and predict clinical responses. Recently, a key role has been assigned to innate immune mechanisms in checkpoint blockade-driven anti-tumor responses. However, inflammatory pathways can both improve and impair anti-tumor immunity. In this review, we discuss how different inflammatory pathways, particularly inflammasome activation, can influence the clinical outcome of immune checkpoint blockers. Inflammasome activation may reinforce anti-tumor immunity by boosting CD8+ T cell priming as well as by enhancing T helper type 17 (Th17) responses. In particular, we focus on the modulation of the cation channel transmembrane protein 176B (TMEM176B) and the ectonucleotidase CD39 as potential targets to unleash inflammasome activation leading to reinforced anti-tumor immunity and improved efficacy of immune checkpoint blockers. Future studies should be aimed at investigating the mechanisms and cell subsets involved in inflammasome-driven anti-tumor responses.


Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Apirase , Inflamassomos/imunologia , Proteínas de Membrana , Proteínas de Neoplasias , Neoplasias , Animais , Apirase/antagonistas & inibidores , Apirase/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/imunologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/imunologia , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/patologia , Células Th17/imunologia , Células Th17/patologia
8.
Gene ; 744: 144608, 2020 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-32234541

RESUMO

Prostate cancer (PCa) is the third most common malignancy worldwide. Novel and effective therapeutic targets are needed for PCa. The purpose of this study was to discover novel therapeutic targets for PCa by performing advanced analysis on PCa RNA sequencing (RNAseq) data from The Cancer Genome Atlas (TCGA). Weighted correlation-network analysis (WGCNA) was performed on the RNAseq data of tumor samples, and the module most relevant to the Gleason score was identified. Combining differential gene-expression analysis and survival analysis, we narrowed down potential therapeutic target genes and found that PKMYT1 might be one. Subsequently, functional studies (i.e., cell-proliferation assays, cell cycle analysis, and colony-formation assays) demonstrated that knockdown of PKMYT1 significantly inhibited the growth of PCa cells. Further investigation illustrated that PKMYT1 promoted the growth of PCa cells through targeting CCNB1 and CCNE1 expression. In addition, fostamatinib, an inhibitor of PKMYT1, effectively inhibited the proliferation of PCa cells. Taken together, our results suggest that PKMYT1 is a gene associated with malignancy of PCa and is a novel therapeutic target.


Assuntos
Neoplasias da Próstata/enzimologia , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Ciclina B1/genética , Ciclina B1/metabolismo , Ciclina E/genética , Ciclina E/metabolismo , Humanos , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Oxazinas/uso terapêutico , Prognóstico , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Piridinas/uso terapêutico
9.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 36(1): 49-55, 2020 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-32314724

RESUMO

Objective To explore the change of the expression of microRNA-124-3p (miR-124-3p) in injured hippocampus of rats and investigate the role of miR-124-3p in neuranagenesis after traumatic brain injury (TBI). Methods The healthy male rats were randomly divided into a sham-operated group, TBI group, miR-124-3p agomir group and miR-124-3p antagomir group. TBI models were constructed by controlled cortical injury (CCI) device for all the groups except for the sham-operated group. The miR-124-3p agomir (1 nmol) was given to the miR-124-3p agomir group and miR-124-3p antagomir (1 nmol) to the miR-124-3p antagomir group via lateral ventricular injection, and equivalent solvent was given to the sham-operated group and TBI group after injury. The injured hippocampus of rats was collected at 12 hours, 1 day, 3, 7 days after injury. The real-time PCR and Western blot analysis were used to examine the expression of miR-124-3p and Delta-like 1 (DLL1) in the injured hippocampus. Immunofluorescence histochemistry was used to examine the expression levels of 5-bromodeoxyuridine (BrdU), neuronal nuclear antigen (NeuN) and nestin in the injured hippocampus. Bioinformatics software was used to predict and dual luciferase reporter assay to validate the regulatory relationship between miR-124-3p and DLL1. Results The miR-124-3p and DLL1 expression in the TBI group were significantly higher than those in the sham-operated group; compared with the TBI group, the miR-124-3p agomir group had significantly increased expression of miR-124-3p and significantly decreased expression of DLL1 in the injured hippocampus, and miR-124-3p antagomir group had significantly decreased expression of miR-124-3p and significantly increased expression of DLL1. Compared with the sham-operated group, the BrdU+NeuN+ cells and BrdU+nestin+ cells in the hippocampus significantly increased in the TBI group at 7 days after injury. The miR-124-3p agomir treatment increased the number of the BrdU+NeuN+ cells and BrdU+nestin+ cells, while the miR-124-3p antagomir treatment decreased the number of the BrdU+NeuN+ cells and BrdU+nestin+ cells. Bioinformatics analysis confirmed that DLL1 was a target of miR-124-3p. Conclusion High expression of miR-124-3p in the trauma region promotes the proliferation and differentiation of neural stem cells by targeting and inhibiting DLL1.


Assuntos
Lesões Encefálicas Traumáticas/genética , Proteínas de Membrana/antagonistas & inibidores , MicroRNAs/genética , Células-Tronco Neurais/citologia , Receptores Notch/genética , Animais , Lesões Encefálicas Traumáticas/patologia , Diferenciação Celular , Proliferação de Células , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Ratos , Ratos Sprague-Dawley , Transdução de Sinais
10.
Biochim Biophys Acta Rev Cancer ; 1873(2): 188364, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32275934

RESUMO

Hyperactivation of the Mitogen Activated Protein Kinase (MAPK) pathway is prevalent in melanoma, principally due to mutations in the BRAF and NRAS genes. MAPK inhibitors are effective only short-term, and recurrence occurs due to functional redundancies or intertwined pathways. The remodeling of Ca2+ signaling is also common in melanoma cells, partly through the increased expression of T-type channels (TTCCs). Here we summarize current knowledge about the prognostic value and molecular targeting of TTCCs. Furthermore, we discuss recent evidence pointing to TTCCs as molecular switches for melanoma chemoresistance, which set the grounds for novel combined therapies against the advanced disease.


Assuntos
Antineoplásicos/uso terapêutico , Canais de Cálcio Tipo T/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Antineoplásicos/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/uso terapêutico , Linhagem Celular Tumoral , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , GTP Fosfo-Hidrolases/antagonistas & inibidores , GTP Fosfo-Hidrolases/genética , Humanos , Estimativa de Kaplan-Meier , Sistema de Sinalização das MAP Quinases/genética , Melanoma/genética , Melanoma/mortalidade , Melanoma/patologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Mutação , Prognóstico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/mortalidade , Neoplasias Cutâneas/patologia , Resultado do Tratamento
11.
Metabolism ; 105: 154182, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32061660

RESUMO

The impairment of podocyte protein filtration function caused by excessive mitochondrial calcium intake is a critical feature of diabetic nephropathy (DN). Ca2+ channel transient receptor potential cation channel subfamily V member 1 (TRPV1) has been reported to protect against ischemia-reperfusion induced acute renal injury, but there is no report about its role in DN. Here, we report that dietary capsaicin potently inhibits and reverses chronic renal structural and functional damages in db/db or streptozotocin (STZ)-induced diabetic mice in a TRPV1-dependent manner. Activation of TRPV1 by capsaicin alleviated hyperglycemia-induced mitochondrial dysfunction in podocytes, accompanied by reduced mitochondria-associated membranes (MAMs) formation and fewer Ca2+ transport from endoplasmic reticulum (ER) to mitochondria. Mechanistically, TRPV1-mediated transient Ca2+ influx activated 5' AMP-activated protein kinase (AMPK) that reduced the transcription of Fundc1, a key molecule participating in MAMs formation. Inhibition of AMPK or overexpression of Fundc1 obviously blocked the inhibitory effect of capsaicin on MAMs formation and functional decline in podocytes. These findings emphasize the critical role of mitochondrial Ca2+ homeostasis in the maintenance of normal renal function and suggest an effective intervention method to counteract DN.


Assuntos
Nefropatias Diabéticas/tratamento farmacológico , Retículo Endoplasmático/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Podócitos/efeitos dos fármacos , Canais de Cátion TRPV/metabolismo , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Canais de Cálcio/metabolismo , Capsaicina/uso terapêutico , Dieta , Inibidores Enzimáticos/farmacologia , Hiperglicemia/tratamento farmacológico , Hiperglicemia/microbiologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Membranas Mitocondriais/efeitos dos fármacos , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/biossíntese
12.
Nat Commun ; 11(1): 484, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980627

RESUMO

ß-Cell dysfunction and reduction in ß-cell mass are hallmark events of diabetes mellitus. Here we show that ß-cells express abundant Kindlin-2 and deleting its expression causes severe diabetes-like phenotypes without markedly causing peripheral insulin resistance. Kindlin-2, through its C-terminal region, binds to and stabilizes MafA, which activates insulin expression. Kindlin-2 loss impairs insulin secretion in primary human and mouse islets in vitro and in mice by reducing, at least in part, Ca2+ release in ß-cells. Kindlin-2 loss activates GSK-3ß and downregulates ß-catenin, leading to reduced ß-cell proliferation and mass. Kindlin-2 loss reduces the percentage of ß-cells and concomitantly increases that of α-cells during early pancreatic development. Genetic activation of ß-catenin in ß-cells restores the diabetes-like phenotypes induced by Kindlin-2 loss. Finally, the inducible deletion of ß-cell Kindlin-2 causes diabetic phenotypes in adult mice. Collectively, our results establish an important function of Kindlin-2 and provide a potential therapeutic target for diabetes.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Fatores de Transcrição Maf Maior/metabolismo , Proteínas Musculares/metabolismo , beta Catenina/metabolismo , Animais , Proliferação de Células , Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/genética , Diabetes Mellitus Experimental/etiologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Feminino , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Insulina/genética , Resistência à Insulina , Ilhotas Pancreáticas/crescimento & desenvolvimento , Ilhotas Pancreáticas/metabolismo , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Musculares/deficiência , Proteínas Musculares/genética , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fenótipo , Estabilidade Proteica , beta Catenina/genética
14.
Biochem Biophys Res Commun ; 524(1): 64-69, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-31980178

RESUMO

PGRMC1 is a protein from the MAPR family with a range of cellular functions. PGRMC1 has been described to play a role in fertility, neuroprotection, steroidogenesis, membrane trafficking and in cancer cell biology. PGRMC1 represents a likely key regulator of cell metabolism and proliferation, as well as a potential target for anti-cancer therapies. To further understand the functions of PGRMC1 and the mechanism of the small molecule inhibitor of PGRMC1, AG-205, proteins differentially bound to PGRMC1 were identified following AG-205 treatment of MIA PaCa-2 cells. Our results suggest that AG-205 influences PGRMC1 interactions with the actin cytoskeleton. The binding of two PGRMC1-associated proteins that support this, RACK1 and alpha-Actinin-1, was reduced following AG-205 treatment. The biology associated with PGRMC1 binding partners identified here merits further investigation.


Assuntos
Actinas/metabolismo , Indóis/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Receptores de Progesterona/antagonistas & inibidores , Citoesqueleto de Actina/metabolismo , Linhagem Celular Tumoral , Humanos , Espectrometria de Massas , Ligação Proteica , Receptores de Quinase C Ativada/metabolismo
15.
EBioMedicine ; 52: 102632, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31981983

RESUMO

BACKGROUND: Small cell lung cancer (SCLC) has a poor prognosis, and its treatment options are limited. Delta-like protein 3 (DLL3) is expressed specifically in SCLC and is considered a promising therapeutic target for patients with this disease. Rovalpituzumab tesirine (Rova-T) was the first antibody-drug conjugate targeting DLL3. Although Rova-T development was unfortunately terminated, DLL3 remains an ideal target for SCLC. Near infrared photoimmunotherapy (NIR-PIT) is a new form of cancer treatment that employs an antibody-photosensitiser conjugate followed by NIR light exposure and damage target cells specifically. In this study, we demonstrate DLL3-targeted NIR-PIT to develop a novel molecularly targeted treatment for SCLC. METHODS: The anti-DLL3 monoclonal antibody rovalpituzumab was conjugated to an IR700 photosensitiser (termed 'rova-IR700'). SCLC cells overexpressing DLL3 as well as non-DLL3-expressing controls were incubated with rova-IR700 and then exposed to NIR-light. Next, mice with SCLC xenografts were injected with rova-IR700 and irradiated with NIR-light. FINDINGS: DLL3-overexpressing cells underwent immediate destruction upon NIR-light exposure, whereas the control cells remained intact. The xenograft in mice treated with rova-IR700 and NIR-light shrank markedly, whereas neither rova-IR700 injection nor NIR-light irradiation alone affected tumour size. INTERPRETATION: Our data suggest that targeting of DLL3 using NIR-PIT could be a novel and promising treatment for SCLC. FUNDING: Research supported by grants from the Program for Developing Next-generation Researchers (Japan Science and Technology Agency), KAKEN (18K15923, JSPS), Medical Research Encouragement Prize of The Japan Medical Association, The Nitto Foundation, Kanae Foundation for the Promotion of Medical Science.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Luz , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/metabolismo , Fármacos Fotossensibilizantes/farmacologia , Fototerapia , Carcinoma de Pequenas Células do Pulmão/metabolismo , Animais , Biomarcadores , Linhagem Celular Tumoral , Modelos Animais de Doenças , Imunofluorescência , Humanos , Imunoconjugados/farmacologia , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Fototerapia/métodos , Ligação Proteica/imunologia , Carcinoma de Pequenas Células do Pulmão/patologia , Carcinoma de Pequenas Células do Pulmão/terapia , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Acta Biochim Biophys Sin (Shanghai) ; 52(1): 9-17, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31867609

RESUMO

Increased glycolysis is involved in the proliferation and migration of vascular smooth muscle cells (VSMCs). Pyruvate kinase isoform M2 (PKM2), a key rate-limiting enzyme in glycolysis, accelerates the proliferation and migration of tumor cells. Although the intracellular mechanisms associated with oxidized low-density lipoprotein (oxLDL)-stimulated VSMC proliferation and migration have been extensively explored, it is still unclear whether oxLDL promotes the proliferation and migration of VSMCs by enhancing PKM2-dependent glycolysis. In the present study, we detected PKM2 expression and pyruvate kinase activity in oxLDL-treated VSMCs and explored the regulation of PKM2 in oxLDL-treated VSMCs and apoE-/- mice. The results showed that PKM2 expression in VSMCs was higher in the intima than in the media in plaques from atherosclerotic rabbits. Moreover, PKM2 level in VSMCs was increased during atherosclerosis progression in apoE-/- mice. Both PKM2 expression and pyruvate kinase activity were found to be upregulated by oxLDL stimulation in VSMCs. Shikonin (SKN), a specific inhibitor of PKM2, was found to inhibit the oxLDL-induced proliferation and migration in VSMCs, in addition to delaying the atherosclerosis progression in apoE-/- mice. More importantly, oxLDL increased glucose uptake, ATP and lactate production, and the extracellular acidification rate in VSMCs, which could be reversed by SKN. Meanwhile, oxygen consumption rate was unchanged after oxLDL stimulation, suggesting that glycolysis is the main contributor to the energy supply in oxLDL-treated VSMCs. Our results suggest that oxLDL induces VSMC proliferation and migration by upregulating PKM2-dependent glycolysis, thereby contributing to the atherosclerosis progression. Thus, targeting PKM2-dependent glycolysis might provide a novel therapeutic approach for the treatment of atherosclerosis.


Assuntos
Aterosclerose/metabolismo , Proteínas de Transporte/metabolismo , Movimento Celular , Proliferação de Células , Glicólise/genética , Proteínas de Membrana/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Piruvato Quinase/metabolismo , Hormônios Tireóideos/metabolismo , Animais , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Células Cultivadas , Técnicas de Silenciamento de Genes , Humanos , Lipoproteínas LDL/metabolismo , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout para ApoE , Naftoquinonas/farmacologia , Piruvato Quinase/antagonistas & inibidores , Piruvato Quinase/genética , Coelhos , Ratos , Ratos Sprague-Dawley , Hormônios Tireóideos/genética
17.
J Med Chem ; 63(2): 847-879, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31860309

RESUMO

Trypanosoma protists are pathogens leading to a spectrum of devastating infectious diseases. The range of available chemotherapeutics against Trypanosoma is limited, and the existing therapies are partially ineffective and cause serious adverse effects. Formation of the PEX14-PEX5 complex is essential for protein import into the parasites' glycosomes. This transport is critical for parasite metabolism and failure leads to mislocalization of glycosomal enzymes, with fatal consequences for the parasite. Hence, inhibiting the PEX14-PEX5 protein-protein interaction (PPI) is an attractive way to affect multiple metabolic pathways. Herein, we have used structure-guided computational screening and optimization to develop the first line of compounds that inhibit PEX14-PEX5 PPI. The optimization was driven by several X-ray structures, NMR binding data, and molecular dynamics simulations. Importantly, the developed compounds show significant cellular activity against Trypanosoma, including the human pathogen Trypanosoma brucei gambiense and Trypanosoma cruzi parasites.


Assuntos
Proteínas de Membrana/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Piridinas/síntese química , Piridinas/farmacologia , Tripanossomicidas/síntese química , Tripanossomicidas/farmacologia , Animais , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/biossíntese , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mioblastos/efeitos dos fármacos , Mioblastos/parasitologia , Proteínas de Protozoários/biossíntese , Ratos , Relação Estrutura-Atividade , Trypanosoma brucei gambiense/efeitos dos fármacos , Trypanosoma brucei gambiense/metabolismo , Trypanosoma brucei rhodesiense/efeitos dos fármacos
18.
EMBO Rep ; 20(12): e47999, 2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31668010

RESUMO

Spatio-temporal regulation of signalling pathways plays a key role in generating diverse responses during the development of multicellular organisms. The role of signal dynamics in transferring signalling information in vivo is incompletely understood. Here, we employ genome engineering in Drosophila melanogaster to generate a functional optogenetic allele of the Notch ligand Delta (opto-Delta), which replaces both copies of the endogenous wild-type locus. Using clonal analysis, we show that optogenetic activation blocks Notch activation through cis-inhibition in signal-receiving cells. Signal perturbation in combination with quantitative analysis of a live transcriptional reporter of Notch pathway activity reveals differential tissue- and cell-scale regulatory modes. While at the tissue-level the duration of Notch signalling determines the probability with which a cellular response will occur, in individual cells Notch activation acts through a switch-like mechanism. Thus, time confers regulatory properties to Notch signalling that exhibit integrative digital behaviours during tissue differentiation.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Proteínas de Membrana/antagonistas & inibidores , Receptores Notch/metabolismo , Animais , Animais Geneticamente Modificados , Diferenciação Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Genes de Insetos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Optogenética , Fenótipo , Receptores Notch/genética , Transdução de Sinais , Análise Espaço-Temporal
19.
BMC Cancer ; 19(1): 958, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31619190

RESUMO

BACKGROUND: We analyzed the changes in permeability of endothelial cell layers after photon irradiation, with a focus on the metalloproteases ADAM10 and ADAM17, and on VE-cadherin, components crucial for the integrity of endothelial intercellular junctions, and their roles in the transmigration of cancer cells through endothelial cell monolayers. METHODS: Primary HUVEC were irradiated with 2 or 4 Gy photons at a dose rate of 5 Gy/min. The permeability of an irradiated endothelial monolayer for macromolecules and tumor cells was analyzed in the presence or absence of the ADAM10/17 inhibitors GI254023X and GW280264X. Expression of ADAM10, ADAM17 and VE-Cadherin in endothelial cells was quantified by immunoblotting and qRT. VE-Cadherin was additionally analyzed by immunofluorescence microscopy and ELISA. RESULTS: Ionizing radiation increased the permeability of endothelial monolayers and the transendothelial migration of tumor cells. This was effectively blocked by a selective inhibition (GI254023X) of ADAM10. Irradiation increased both, the expression and activity of ADAM10, which led to increased degradation of VE-cadherin, but also led to higher rates of VE-cadherin internalization. Increased degradation of VE-cadherin was also observed when endothelial monolayers were exposed to tumor-cell conditioned medium, similar to when exposed to recombinant VEGF. CONCLUSIONS: Our results suggest a mechanism of irradiation-induced increased permeability and transendothelial migration of tumor cells based on the activation of ADAM10 and the subsequent change of endothelial permeability through the degradation and internalization of VE-cadherin.


Assuntos
Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Antígenos CD/metabolismo , Caderinas/metabolismo , Células Endoteliais/efeitos da radiação , Células Endoteliais da Veia Umbilical Humana/efeitos da radiação , Proteínas de Membrana/metabolismo , Proteólise/efeitos da radiação , Radiação Ionizante , Migração Transendotelial e Transepitelial/efeitos da radiação , Proteína ADAM10/antagonistas & inibidores , Proteína ADAM10/genética , Proteína ADAM17/antagonistas & inibidores , Proteína ADAM17/genética , Proteína ADAM17/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/genética , Linhagem Celular Tumoral , Dipeptídeos/farmacologia , Células Endoteliais/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Permeabilidade/efeitos da radiação , Radioterapia/efeitos adversos , Transdução de Sinais/efeitos da radiação , Migração Transendotelial e Transepitelial/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia
20.
BMC Endocr Disord ; 19(1): 115, 2019 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-31664995

RESUMO

BACKGROUND: The prevalence of obesity and its comorbidities, including type 2 diabetes mellitus (T2DM), is dramatically increasing throughout the world; however, the underlying aetiology is incompletely understood. Genome-wide association studies (GWAS) have identified hundreds of genec susceptibility loci for obesity and T2DM, although the causal genes and mechanisms are largely unknown. SPRY2 is a candidate gene identified in GWAS of body fat percentage and T2DM, and has recently been linked to insulin production in pancreatic ß-cells. In the present study, we aimed to further understand SPRY2 via functional characterisation in HepG2 cells, an in vitro model of human hepatocytes widely used to investigate T2DM and insulin resistance. METHODS: CRISPR-Cas9 genome editing was used to target SPRY2 in HepG2 cells, and the functional consequences of SPRY2 knockout (KO) and overexpression subsequently assessed using glucose uptake and lipid droplet assays, measurement of protein kinase phosphorylation and RNA sequencing. RESULTS: The major functional consequence of SPRY2 KO was a significant increase in glucose uptake, along with elevated lipid droplet accumulation. These changes were attenuated, but not reversed, in cells overexpressing SPRY2. Phosphorylation of protein kinases across key signalling pathways (including Akt and mitogen activated protein kinases) was not altered after SPRY2 KO. Transcriptome profiling in SPRY2 KO and mock (control) cells revealed a number of differentially expressed genes related to cholesterol biosynthesis, cell cycle regulation and cellular signalling pathways. Phospholipase A2 group IIA (PLA2G2A) mRNA level was subsequently validated as significantly upregulated following SPRY2 KO, highlighting this as a potential mediator downstream of SPRY2. CONCLUSION: These findings suggest a role for SPRY2 in glucose and lipid metabolism in hepatocytes and contribute to clarifying the function of this gene in the context of metabolic diseases.


Assuntos
Sistemas CRISPR-Cas , Glucose/metabolismo , Hepatócitos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Gotículas Lipídicas/metabolismo , Lipogênese , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Perfilação da Expressão Gênica , Células Hep G2 , Hepatócitos/citologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Fosforilação , Transdução de Sinais
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