RESUMO
The signaling network between cancer and stromal cells plays a crucial role in tumor microenvironment. The fate of tumor progression mainly depends on the huge amount of information that these cell populations exchange from the onset of neoplastic transformation. Interfering with such signaling has been producing exciting results in cancer therapy: just think of anti-PD-1/anti-PD-L1/anti-CTLA-4 antibodies that, acting as immune checkpoint inhibitors, interrupt the inhibitory signaling exerted by cancer cells on immune cells or the CAR-T technology that fosters the reactivation of anti-tumoral immunity in a restricted group of leukemias and lymphomas. Nevertheless, many types of cancers, in particular solid tumors, are still refractory to these treatments, so the identification of novel molecular targets in tumor secretome would benefit from implementation of current anti-cancer therapeutical strategies. Neutrophil Gelatinase-Associated Lipocalin (NGAL) is a secreted protein abundantly expressed in the secretome of various human tumors. It represents a promising target for the multiple roles that are played inside cancer and stromal cells, and also overall in their cross-talk. The review focuses on the different roles of NGAL in tumor microenvironment and in cancer senescence-associated secretory phenotype (SASP), highlighting the most crucial functions that could be eventually targetable in cancer therapy.
Assuntos
Lipocalina-2/metabolismo , Neoplasias/metabolismo , Fenótipo Secretor Associado à Senescência , Transdução de Sinais , Microambiente Tumoral , Animais , Anticorpos Monoclonais/uso terapêutico , Sistemas CRISPR-Cas , Edição de Genes/métodos , Humanos , Lipocalina-2/antagonistas & inibidores , Lipocalina-2/genética , Lipocalina-2/imunologia , Neoplasias/terapia , RNA Interferente Pequeno/genética , Terapêutica com RNAi/métodos , Secretoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Células Estromais/metabolismoRESUMO
Although some effective therapies have been available for cancer, it still poses a great threat to human health and life due to its drug resistance and low response in patients. Here, we develop a ferroptosis-based therapy by combining iron nanoparticles and cancer-specific gene interference. The expression of two iron metabolic genes (FPN and LCN2) was selectively knocked down in cancer cells by Cas13a or microRNA controlled by a NF-κB-specific promoter. Cells were simultaneously treated by iron nanoparticles. As a result, a significant ferroptosis was induced in a wide variety of cancer cells. However, the same treatment had little effect on normal cells. By transferring genes with adeno-associated virus and iron nanoparticles, the significant tumor growth inhibition and durable cure were obtained in mice with the therapy. In this work, we thus show a cancer therapy based on gene interference-enhanced ferroptosis.
Assuntos
Proteínas de Transporte de Cátions/antagonistas & inibidores , Ferroptose/genética , Ferro/metabolismo , Lipocalina-2/antagonistas & inibidores , Neoplasias/terapia , Interferência de RNA , Espécies Reativas de Oxigênio/agonistas , Animais , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Linhagem Celular Tumoral , Dependovirus/genética , Dependovirus/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Lipocalina-2/genética , Lipocalina-2/metabolismo , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Nanopartículas/administração & dosagem , Nanopartículas/química , Neoplasias/genética , Neoplasias/mortalidade , Neoplasias/patologia , Regiões Promotoras Genéticas , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Baço/metabolismo , Baço/patologia , Análise de Sobrevida , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Inflammatory Breast Cancer (IBC) is an aggressive form of invasive breast cancer, highly metastatic, representing 2-4% of all breast cancer cases in the United States. Despite its rare nature, IBC is responsible for 7-10% of all breast cancer deaths, with a 5-year survival rate of 40%. Thus, targeted and effective therapies against IBC are needed. Here, we proposed Lipocalin-2 (LCN2)-a secreted glycoprotein aberrantly abundant in different cancers-as a plausible target for IBC. In immunoblotting, we observed higher LCN2 protein levels in IBC cells than non-IBC cells, where the LCN2 levels were almost undetectable. We assessed the biological effects of targeting LCN2 in IBC cells with small interference RNAs (siRNAs) and small molecule inhibitors. siRNA-mediated LCN2 silencing in IBC cells significantly reduced cell proliferation, viability, migration, and invasion. Furthermore, LCN2 silencing promoted apoptosis and arrested the cell cycle progression in the G0/G1 to S phase transition. We used in silico analysis with a library of 25,000 compounds to identify potential LCN2 inhibitors, and four out of sixteen selected compounds significantly decreased cell proliferation, cell viability, and the AKT phosphorylation levels in SUM149 cells. Moreover, ectopically expressing LCN2 MCF7 cells, treated with two potential LCN2 inhibitors (ZINC00784494 and ZINC00640089) showed a significant decrease in cell proliferation. Our findings suggest LCN2 as a promising target for IBC treatment using siRNA and small molecule inhibitors.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Inflamatórias Mamárias/tratamento farmacológico , Lipocalina-2/antagonistas & inibidores , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Inflamatórias Mamárias/genética , Neoplasias Inflamatórias Mamárias/patologia , Lipocalina-2/genética , Células MCF-7 , Terapia de Alvo Molecular/métodos , Invasividade Neoplásica , RNA Interferente Pequeno/farmacologia , RNA Interferente Pequeno/uso terapêuticoRESUMO
Neutrophil gelatinase-associated lipocalin (NGAL) is involved in cardiovascular and renal diseases. Gene inactivation of NGAL blunts the pathophysiological consequences of cardiovascular and renal damage. We aimed to design chemical NGAL inhibitors and investigate its effects in experimental models of myocardial infarction (MI) and chronic kidney disease induced by 5/6 nephrectomy (CKD) on respectively 8-12 weeks old C57Bl6/j and FVB/N male mice. Among the 32 NGAL inhibitors tested, GPZ614741 and GPZ058225 fully blocked NGAL-induced inflammatory and profibrotic markers in human cardiac fibroblasts and primary mouse kidney fibroblasts. The administration of GPZ614741 (100 mg/kg/day) for three months, was able to improve cardiac function in MI mice and reduced myocardial fibrosis and inflammation. The administration of GPZ614741 (100 mg/kg/day) for two months resulting to no renal function improvement but prevented the increase in blood pressure, renal tubulointerstitial fibrosis and profibrotic marker expression in CKD mice. In conclusion, we have identified new compounds with potent inhibitory activity on NGAL-profibrotic and pro-inflammatory effects. GPZ614741 prevented interstitial fibrosis and dysfunction associated with MI, as well as tubulointerstitial fibrosis in a CKD model. These inhibitors could be used for other diseases that involve NGAL, such as cancer or metabolic diseases, creating new therapeutic options.
Assuntos
Lipocalina-2/antagonistas & inibidores , Lipocalina-2/metabolismo , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/metabolismo , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/metabolismo , Animais , Modelos Animais de Doenças , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Masculino , CamundongosRESUMO
Hyperuricemia is an independent risk factor for chronic kidney disease (CKD). Excessive uric acid (UA) level in the blood leads to hyperuricemic nephropathy (HN), which is characterized by glomerular hypertension, arteriolosclerosis and tubulointerstitial fibrosis. Fatty acid binding protein 4 (FABP4) is a potential mediator of inflammatory responses which contributes to renal interstitial fibrosis. However, the roles of FABP4 in HN remains unknown. In the study, a mouse model of HN induced by feeding a mixture of adenine and potassium oxonate, severe kidney injury and interstitial fibrosis, as well as the increased kidney-expressed FABP4 protein level were evident, accompanied by the activation of inflammatory responses. Oral administration of BMS309403, a highly selective FABP4 inhibitor, improved renal dysfunction, inhibited the mRNA level of KIM-1 and NGAL, as well as reduced the expression of proinflammatory cytokines and fibrotic proteins in the injured kidneys. BMS309403 treatment also inhibited the FABP4 activity and further suppressed the activation of JAK2-STAT3 and NF-kB P65 signaling pathways in the hyperuricemia-injured kidneys and UA-stimulated human tubular epithelial (HK-2) cells, respectively. In summary, our study for the first time demonstrated that FABP4 played a crucial role in kidney inflammation and fibrosis via the regulation of JAK2-STAT3 and NF-kB P65 pathways in HN mice. The results suggested that FABP4 inhibition might be a promising therapeutic strategy for HN.
Assuntos
Compostos de Bifenilo/uso terapêutico , Proteínas de Ligação a Ácido Graxo/antagonistas & inibidores , Hiperuricemia/tratamento farmacológico , Nefropatias/tratamento farmacológico , Rim/patologia , Nefrite/tratamento farmacológico , Pirazóis/uso terapêutico , Adenina/farmacologia , Animais , Citocinas/biossíntese , Fibrose , Receptor Celular 1 do Vírus da Hepatite A/antagonistas & inibidores , Humanos , Hiperuricemia/induzido quimicamente , Hiperuricemia/complicações , Janus Quinase 2/antagonistas & inibidores , Nefropatias/etiologia , Lipocalina-2/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ácido Oxônico/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição RelA/efeitos dos fármacosRESUMO
Oxidative stress is a key contributor to the pathogenesis of stroke-reperfusion injury. Neuroinflammatory peptides released after ischemic stroke mediate reperfusion injury. Previous studies, including ours, have shown that lipocalin-2 (LCN2) is secreted in response to cerebral ischemia to promote reperfusion injury. Genetic deletion of LCN2 significantly reduces brain injury after stroke, suggesting that LCN2 is a mediator of reperfusion injury and a potential therapeutic target. Immunotherapy has the potential to harness neuroinflammatory responses and provides neuroprotection against stroke. Here we report that LCN2 was induced on the inner surface of cerebral endothelial cells, neutrophils, and astrocytes that gatekeep the blood-brain barrier (BBB) after stroke. LCN2 monoclonal antibody (mAb) specifically targeted LCN2 in vitro and in vivo, attenuating the induction of LCN2 and pro-inflammatory mediators (iNOS, IL-6, CCL2, and CCL9) after stroke. Administration of LCN2 mAb at 4 h after stroke significantly reduced neurological deficits, cerebral infarction, edema, BBB leakage, and infiltration of neutrophils. The binding epitope of LCN2 mAb was mapped to the ß3 and ß4 strands, which are responsible for maintaining the integrity of LCN2 cup-shaped structure. These data indicate that LCN2 can be pharmacologically targeted using a specific mAb to reduce reperfusion injury after stroke.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Lipocalina-2/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Acidente Vascular Cerebral/tratamento farmacológico , Animais , Anticorpos Monoclonais/farmacologia , Astrócitos/metabolismo , Barreira Hematoencefálica/metabolismo , Cérebro/metabolismo , Modelos Animais de Doenças , Mapeamento de Epitopos , Lipocalina-2/antagonistas & inibidores , Lipocalina-2/química , Masculino , Camundongos , Neutrófilos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/metabolismo , Acidente Vascular Cerebral/complicações , Acidente Vascular Cerebral/metabolismoRESUMO
Lipocalin-2 (LCN2) is a secreted glycoprotein linked to several physiological roles, including transporting hydrophobic ligands across cell membranes, modulating immune responses, maintaining iron homeostasis, and promoting epithelial cell differentiation. Although LNC2 is expressed at low levels in most human tissues, it is abundant in aggressive subtypes of cancer, including breast, pancreas, thyroid, ovarian, colon, and bile duct cancers. High levels of LCN2 have been associated with increased cell proliferation, angiogenesis, cell invasion, and metastasis. Moreover, LCN2 modulates the degradation, allosteric events, and enzymatic activity of matrix metalloprotease-9, a metalloprotease that promotes tumor cell invasion and metastasis. Hence, LCN2 has emerged as a potential therapeutic target against many cancer types. This review summarizes the most relevant findings regarding the expression, biological roles, and regulation of LCN2, as well as the proteins LCN2 interacts with in cancer. We also discuss the approaches to targeting LCN2 for cancer treatment that are currently under investigation, including the use of interference RNAs, antibodies, and gene editing.
Assuntos
Lipocalina-2/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Neoplasias/metabolismo , Regulação para Cima , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Proliferação de Células , Edição de Genes , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lipocalina-2/antagonistas & inibidores , Terapia de Alvo Molecular , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias/tratamento farmacológico , RNA Interferente Pequeno/farmacologia , RNA Interferente Pequeno/uso terapêutico , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: The exact mechanisms underlying neuroinflammation and how they contribute to amyotrophic lateral sclerosis (ALS) pathogenesis remain unclear. One possibility is the secretion of neurotoxic factors, such as lipocalin-2 (LCN2), that lead to neuronal death. METHODS: LCN2 levels were measured in human postmortem tissue using Western blot, quantitative real time polymerase chain reaction, and immunofluorescence, and in plasma by enzyme-linked immunosorbent assay. SH-SY5Y cells were used to test the pro-inflammatory effects of LCN2. RESULTS: LCN2 is increased in ALS postmortem motor cortex, spinal cord, and plasma. Furthermore, we identified several LCN2 variants in ALS patients that may contribute to disease pathogenesis. Lastly, while LCN2 treatment caused cell death and increased pro-inflammatory markers, treatment with an anti-LCN2 antibody prevented these responses in vitro. CONCLUSIONS: LCN2 upregulation in ALS postmortem samples and plasma may be an upstream event for triggering neuroinflammation and neuronal death.
Assuntos
Esclerose Lateral Amiotrófica/genética , Inflamação/metabolismo , Lipocalina-2/genética , Córtex Motor/metabolismo , Medula Espinal/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/fisiopatologia , Western Blotting , Estudos de Casos e Controles , Morte Celular , Linhagem Celular Tumoral , Citocinas/efeitos dos fármacos , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Humanos , Técnicas In Vitro , Lipocalina-2/antagonistas & inibidores , Lipocalina-2/metabolismo , Lipocalina-2/farmacologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Cancer is a leading cause of mortalities worldwide. Over the past few decades, exploration of molecular mechanisms behind cancer initiation and progression has been of great interest in the viewpoint of both basic and clinical scientists. It is generally believed that identification of key molecules implicated in cancer pathology not only improves our understanding of the disease, but also could result in introduction of novel therapeutic strategies. Neutrophil gelatinase-associated lipocalin (NGAL)/lipocalin-2 (LCN2) is a member of lipocalin superfamily with a variety of functions. Although the main function of LCN2 is still unknown, many studies confirmed its significant role in the initiation, progression, and metastasis of various types of cancer. Furthermore, aberrant expression of LCN2 is also concerned with the chemo- and radio-resistant phenotypes of tumors. Here, we will review the contribution of known functions of LCN2 to the pathophysiology of cancer. We also highlight how the deregulated expression of LCN2 is associated with a variety of fatal types of cancer for which there are no effective therapeutic modalities. The unique and multiple functions of LCN2 and its widespread expression in different types of cancer prompted us to suggest LCN2 could be considered either as a valuable diagnostic and prognostic biomarker or as a potential novel therapeutic target.
Assuntos
Suscetibilidade a Doenças , Lipocalina-2/genética , Lipocalina-2/metabolismo , Neoplasias/etiologia , Neoplasias/metabolismo , Animais , Biomarcadores Tumorais , Proliferação de Células , Gerenciamento Clínico , Regulação Neoplásica da Expressão Gênica , Humanos , Lipocalina-2/antagonistas & inibidores , Lipocalina-2/química , Terapia de Alvo Molecular , Neoplasias/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Especificidade de Órgãos , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Lipocalin 2 (Lcn2) has been implicated to play a role in various neurodegenerative diseases, and normalizing its overexpression may be of therapeutic potential. Iron chelators were found to reduce Lcn2 levels in certain animal models of CNS injury. Focusing on Alzheimer's disease (AD), we found that the iron chelators deferoxamine and deferiprone inhibited amyloid-ß (Aß)-induced Lcn2 production in cultured primary astrocytes. Accordingly, Aß-exposure increased astrocytic ferritin production, indicating the possibility that Aß induces iron accumulation in astrocytes. This effect was not significantly modulated by Lcn2. Known neuroprotective effects of iron chelators may rely in part on normalization of Lcn2 levels.
Assuntos
Peptídeos beta-Amiloides/toxicidade , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Quelantes de Ferro/farmacologia , Lipocalina-2/antagonistas & inibidores , Lipocalina-2/biossíntese , Fragmentos de Peptídeos/toxicidade , Animais , Animais Recém-Nascidos , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Camundongos , Camundongos KnockoutRESUMO
Chemoresistance is a major cause of cancer progression and the mortality of cancer patients. Developing a safe strategy for enhancing chemosensitivity is a challenge for biomedical science. Recent studies have suggested that vitamin D supplementation may decrease the risk of many cancers. However, the role of vitamin D in chemotherapy remains unknown. We found that vitamin D sensitised oral cancer cells to cisplatin and partially reversed cisplatin resistance. Using RNA-seq, we discovered that lipocalin 2 (LCN2) is an important mediator. Cisplatin enhanced the expression of LCN2 by decreasing methylation at the promoter, whereas vitamin D enhanced methylation and thereby inhibited the expression of LCN2. Overexpression of LCN2 increased cell survival and cisplatin resistance both in vitro and in vivo. High LCN2 expression was positively associated with differentiation, lymph node metastasis, and T staging and predicted a poor prognosis in oral squamous cell carcinoma (OSCC) patients. LCN2 was also associated with post-chemotherapy recurrence. Moreover, we found that LCN2 promoted the activation of NF-κB by binding to ribosomal protein S3 (RPS3) and enhanced the interaction between RPS3 and p65. Our study reveals that vitamin D can enhance cisplatin chemotherapy and suggests that vitamin D should be supplied during chemotherapy; however, more follow-up clinical studies are needed.
Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/dietoterapia , Cisplatino/farmacologia , Suplementos Nutricionais , Lipocalina-2/metabolismo , Neoplasias Bucais/dietoterapia , NF-kappa B/metabolismo , Proteínas Ribossômicas/metabolismo , Vitamina D/uso terapêutico , Adulto , Animais , Antineoplásicos/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Seguimentos , Humanos , Lipocalina-2/antagonistas & inibidores , Lipocalina-2/genética , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Bucais/tratamento farmacológico , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , Receptores de Calcitriol/genética , Proteínas Ribossômicas/genética , Transdução de Sinais/efeitos dos fármacos , Transfecção , Carga Tumoral/efeitos dos fármacos , Vitamina D/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Astrogliosis can result in astrocytes with hypertrophic morphology after injury, indicated by extended processes and swollen cell bodies. Lipocalin-2 (LCN2), a secreted glycoprotein belonging to the lipocalin superfamily, has been reported to play a detrimental role in ischaemic brains and neurodegenerative diseases. Sailuotong (SLT) capsule is a standardized three-herb preparation composed of ginseng, ginkgo, and saffron for the treatment of vascular dementia. Although recent clinical trials have demonstrated the beneficial effect of SLT on vascular dementia, its potential cellular mechanism has not been fully explored. METHODS: Male adult Sprague-Dawley (SD) rats were subjected to microsphere-embolized cerebral ischaemia. Immunostaining and Western blotting were performed to assess astrocytic reaction. Human astrocytes exposed to oxygen-glucose deprivation (OGD) were used to elucidate the effects of SLT-induced inflammation and astrocytic reaction. RESULTS: A memory recovery effect was found to be associated with the cerebral ischaemia-induced expression of inflammatory proteins and the suppression of LCN2 expression in the brain. Additionally, SLT reduced the astrocytic reaction, LCN2 expression, and the phosphorylation of STAT3 and JAK2. For in vitro experiments, OGD-induced expression of inflammation and LCN2 was also decreased in human astrocyte by the SLT treatment. Moreover, LCN2 overexpression significantly enhanced the above effects. SLT downregulated these effects that were enhanced by LCN2 overexpression. CONCLUSIONS: SLT mediates neuroinflammation, thereby protecting against ischaemic brain injury by inhibiting astrogliosis and suppressing neuroinflammation via the LCN2-JAK2/STAT3 pathway, providing a new idea for the treatment strategy of ischaemic stroke.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Demência Vascular/tratamento farmacológico , Medicamentos de Ervas Chinesas/uso terapêutico , Inflamação/tratamento farmacológico , Lipocalina-2/antagonistas & inibidores , Lipocalina-2/metabolismo , Memória/efeitos dos fármacos , Animais , Medicamentos de Ervas Chinesas/farmacologia , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , TransfecçãoRESUMO
Neuropsychiatric manifestations in lupus (NPSLE) affect â¼20-40% of patients. In the central nervous system, lipocalin-2 (LCN2) can promote injury through mechanisms directly linked to NPSLE, including brain barrier disruption, neurotoxicity, and glial activation. Since LCN2 is elevated in lupus and has been implicated in neuroinflammation, we investigated whether LCN2 is required for the pathogenesis of NPSLE. Here, we investigated the effects of LCN2 deficiency on the development of neurobehavioral deficits in the B6.Sle1.Sle3 (Sle1,3) mouse lupus model. Sle1,3 mice exhibited depression-like behavior and impaired spatial and recognition memory, and these deficits were attenuated in Sle1,3-LCN2KO mice. Whole-brain flow cytometry showed a significant increase in brain infiltrating leukocytes in Sle1,3 mice that was not reduced by LCN2 deficiency. RNA sequencing on sorted microglia revealed that several genes differentially expressed between B6 and Sle1,3 mice were regulated by LCN2, and that these genes are key mediators of the neuroinflammatory cascade. Importantly, LCN2 is upregulated in the cerebrospinal fluid of NPSLE patients across 2 different ethnicities. Our findings establish the Sle1,3 strain as an NPSLE model, demonstrate that LCN2 is a major regulator of the detrimental neuroimmune response in NPSLE, and identify CSF LCN2 as a novel biomarker for NPSLE.
Assuntos
Biomarcadores/metabolismo , Leucócitos/imunologia , Lipocalina-2/metabolismo , Vasculite Associada ao Lúpus do Sistema Nervoso Central/metabolismo , Inflamação Neurogênica/metabolismo , Animais , Barreira Hematoencefálica , Modelos Animais de Doenças , Feminino , Humanos , Lipocalina-2/antagonistas & inibidores , Lipocalina-2/genética , Vasculite Associada ao Lúpus do Sistema Nervoso Central/diagnóstico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Inflamação Neurogênica/diagnóstico , Regulação para CimaRESUMO
Colchicine is a toxic alkaloid prevalent in autumn crocus (Colchicum autumnale) that binds to tubulin and inhibits polymerization of microtubules. Using combinatorial and rational protein design, we have developed an artificial binding protein based on the human lipocalin 2 that binds colchicine with a dissociation constant of 120 pm, i.e. 10000-fold stronger than tubulin. Crystallographic analysis of the engineered lipocalin, dubbed Colchicalin, revealed major structural changes in the flexible loop region that forms the ligand pocket at the open end of the eight-stranded ß-barrel, resulting in a lid-like structure over the deeply buried colchicine. A cis-peptide bond between residues Phe71 and Pro72 in loop #2 constitutes a peculiar feature and allows intimate contact with the tricyclic ligand. Using directed evolution, we achieved an extraordinary dissociation half-life of more than 9 h for the Colchicalin-colchicine complex. Together with the chemical robustness of colchicine and availability of activated derivatives, this also opens applications as a general-purpose affinity reagent, including facile quantification of colchicine in biological samples. Given that engineered lipocalins, also known as Anticalin® proteins, represent a class of clinically validated biopharmaceuticals, Colchicalin may offer a therapeutic antidote to scavenge colchicine and reverse its poisoning effect in situations of acute intoxication.
Assuntos
Antídotos/farmacologia , Colchicina/farmacologia , Lipocalina-2/antagonistas & inibidores , Venenos/farmacologia , Engenharia de Proteínas , Antídotos/química , Sítios de Ligação/efeitos dos fármacos , Colchicina/química , Colchicum/química , Cristalografia por Raios X , Humanos , Lipocalina-2/química , Modelos Moleculares , Estrutura Molecular , Venenos/químicaRESUMO
BACKGROUND/AIMS: Gastric cancer is considered as a common malignancy with a poor prognosis as well as unsatisfactory treatment. Neutrophil gelatinase-associated lipocalin (NGAL) has been reported to affect multiple aspects of human tumor, including gastric cancer. This study aims to explore the effects of NGAL gene silencing on the proliferation as well as apoptosis of human gastric cancer MGC-803 cells. METHODS: This study included 87 patients with gastric cancer. MGC-803 cells were collected and mainly treated with siRNA against NGAL and recombinant NGAL plasmid. The expression of NGAL mRNA and the expressions of NGAL protein and apoptosis-related proteins were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, respectively. Cell cycle and apoptosis were tested by flow cytometry, and cell proliferation was detected by water soluble tetrazolium-1 (WST-1) assay. The effect of NGAL gene silencing on tumorigenicity of MGC-803 cells in vivo was detected through establishment of xenograft in nude mice. RESULTS: NGAL was highly expressed in gastric cancer tissues. The protein and mRNA expressions of NGAL gene in MGC-803 cells treated with NGAL-siRNA were obviously reduced, and the amount of cells in G0/G1 phase was increased. Moreover, MGC-803 cells treated with NGAL-siRNA exhibited inhibited proliferation, enhanced apoptosis, decreased expressions of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) as well as B-cell lymphoma-2 (Bcl-2) and increased expressions of cysteine-aspartic acid specific protease-9 (caspase-9) and Bcl2-associated X (Bax), as well as repressed tumorigenicity in vivo. CONCLUSION: NGAL gene silencing inhibits proliferation and promotes apoptosis of MGC-803 cells, which can provide a novel theory for treatment of gastric cancer.
Assuntos
Apoptose , Proliferação de Células , Lipocalina-2/metabolismo , Neoplasias Gástricas/patologia , Animais , Apoptose/efeitos dos fármacos , Caspase 9/genética , Caspase 9/metabolismo , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Lipocalina-2/antagonistas & inibidores , Lipocalina-2/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , NF-kappa B/genética , NF-kappa B/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/uso terapêutico , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Tea is thought to alleviate neurotoxicity due to the antioxidative effect of ester-type tea catechins (ETC). Neutrophil gelatinase-associated lipocalin (NGAL) can sensitize ß-amyloid (Aß) induced neurotoxicity, and inhibitors of NGAL may relieve associated symptoms. As such, the interactions of ETC with NGAL were investigated by fluorescence spectrometry and molecular simulation. NGAL fluorescence is quenched regularly when being added with six processing types of tea infusion (SPTT) and ETC. Thermodynamic analyses suggest that ETC with more catechol moieties has a stronger binding capacity with NGAL especially in the presence of Fe3+. (-)-Epicatechin 3-O-caffeoate (ECC), a natural product isolated from Zijuan green tea, shows the strongest binding ability with NGAL (Kd = 15.21 ± 8.68 nM in the presence of Fe3+). All ETC are effective in protecting nerve cells against H2O2 or Aß1-42 induced injury. The inhibitory mechanism of ETC against NGAL supports its potential use in attenuation of neurotoxicity.
Assuntos
Catequina/farmacologia , Lipocalina-2/farmacologia , Chá/química , Catequina/metabolismo , Interações Medicamentosas , Ésteres , Peróxido de Hidrogênio/farmacologia , Quelantes de Ferro , Lipocalina-2/antagonistas & inibidores , Lipocalina-2/metabolismo , Modelos Moleculares , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores , Espectrometria de FluorescênciaRESUMO
Cholangiocarcinoma (CCA) is a devastating disease due to resistance to traditional chemotherapies and radiotherapies. New therapeutic strategies against CCA are urgently needed. This study investigated the role of lipocalin-2 (LCN2) in human cholangiocarcinoma as a potential therapeutic target and diagnostic marker. So far, the role of LCN2 in cancer is still controversial and studies regarding the role of LCN2 in CCA are limited. LCN2 knockdown inhibited CCA cell growth in vitro and in vivo through induction of cell cycle arrest at G0/G1 phases and decreased metastatic potential due to repression of epithelial-mesenchymal transition (EMT). Overexpression of LCN2 in CCA cells increased cell metastatic potential. We showed for the first time that the N-myc downstream regulated gene 1 (NDRG1) and NDRG2, known as tumor suppressor genes, are negatively regulated by LCN2 in CCA cells. LCN2 concentration in bile was higher in patients with CCA than that in patients with gallstones, with a cutoff value of 20.08 ng/ml making this a potential diagnostic marker. Higher LCN2 expression was associated with worse survival in patients with CCA. LCN2 is a promising target for CCA treatment and bile LCN2 level is a potential diagnostic marker for CCA.
Assuntos
Neoplasias dos Ductos Biliares/diagnóstico , Bile/metabolismo , Biomarcadores Tumorais/metabolismo , Colangiocarcinoma/diagnóstico , Lipocalina-2/metabolismo , Animais , Neoplasias dos Ductos Biliares/metabolismo , Neoplasias dos Ductos Biliares/mortalidade , Biomarcadores Tumorais/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Colangiocarcinoma/metabolismo , Colangiocarcinoma/mortalidade , Transição Epitelial-Mesenquimal , Pontos de Checagem da Fase G1 do Ciclo Celular , Cálculos Biliares/metabolismo , Cálculos Biliares/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipocalina-2/antagonistas & inibidores , Lipocalina-2/genética , Camundongos , Camundongos Nus , Metástase Neoplásica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
PURPOSE: Lipocalin 2 (LCN2) is a secretory protein that is involved in various physiological processes including iron transport. We previously identified LCN2 as an up-regulated gene in endometrial carcinoma, and found that the overexpression of LCN2 and its receptor, SLC22A17, was associated with a poor prognosis. However, the functions and mechanism of action of LCN2 currently remain unclear. METHODS: The LCN2-overexpressing endometrial carcinoma cell lines, HHUA and RL95-2, and LCN2-low-expressing one, HEC1B, were used. The effects of LCN2 on cell migration, cell viability, and apoptosis under various stresses, including ultraviolet (UV) irradiation and cisplatin treatment, were examined using the scratch wound healing assay, WST-1 assay, and Apostrand assay, respectively. RESULTS: LCN2-silencing using shRNA method significantly reduced the migration ability of cells (p<0.05). Cytotoxic stresses significantly decreased the viability of LCN2-silenced cells more than that of control cells. In contrast, LCN2 overexpression was significantly increased cisplatin resistance. These effects were canceled by the addition of the iron chelator, deferoxamine. After UV irradiation, the expression of phosphorylated Akt (pAkt) was decreased in LCN2-silenced cells, and the PI3K inhibitor canceled the difference induced in UV sensitivity by LCN2. The cisplatin-induced expression of pAkt was not affected by LCN2; however, the expression of p53 and p21 was increased by LCN2-silencing. CONCLUSIONS: These results indicated that LCN2 was involved in the migration and survival of endometrial carcinoma cells under various stresses in an iron-dependent manner. The survival function of LCN2 may be exerted through the PI3K pathway and suppression of the p53-p21 pathway. These functions of LCN2 may increase the malignant potential of endometrial carcinoma cells.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/genética , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Lipocalina-2/genética , Fosfatidilinositol 3-Quinases/genética , Proteína Supressora de Tumor p53/genética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/efeitos da radiação , Cisplatino/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Endométrio/efeitos dos fármacos , Endométrio/metabolismo , Endométrio/patologia , Endométrio/efeitos da radiação , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Células Epiteliais/efeitos da radiação , Feminino , Humanos , Lipocalina-2/antagonistas & inibidores , Lipocalina-2/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Raios UltravioletaRESUMO
AIMS: To study the role of lipocalin-2 (Lcn2) and the effect of Lcn2 blockade via anti-Lcn2 antibody in the development of abdominal aortic aneurysm (AAA). METHODS AND RESULTS: Expression mRNA and protein levels of Lcn2 and its human orthologue neutrophil gelatinase-associated lipocalin (NGAL) in aortic wall samples from experimental mouse and human AAA samples, respectively, were analysed by real-time PCR and immunohistochemistry. Experimental AAA was induced by aortic elastase perfusion in wild-type mice (WT) and Lcn2-deficient mice (Lcn2-/-). NGAL/Lcn2 mRNA and protein levels in human and murine AAA samples were increased compared with healthy aortas. Decreased AAA incidence and reduced aortic expansion were observed in Lcn2-/- mice or mice preoperative treated with a polyclonal anti-Lcn2 antibody compared with WT mice or mice treated with control IgG, respectively, at Day 14 after elastase perfusion. Moreover, immunohistochemical analysis of AAA tissues from Lcn2-/- or anti-Lcn2-treated mice showed diminished elastin damage, reduced microvessels and polymorphonuclear neutrophil (PMN) infiltration, and enhanced preservation of vascular smooth muscle cells compared with WT aortas. Fluorescent molecular tomography revealed decreased MMP activity in AAA of Lcn2-/- mice compared with WT controls. Therapeutic administration of anti-Lcn2 antibody to WT mice 3 days after elastase perfusion decreased aortic dilatation and PMN infiltration compared with WT mice treated with control IgG. CONCLUSION: Either Lcn2 deficiency or anti-Lcn2 antibody blockade limits AAA expansion in mice by decreasing PMN infiltration in the aorta. Lcn2 modulation may therefore be a viable new therapeutic option for the treatment of AAA.
Assuntos
Anticorpos/farmacologia , Aorta Abdominal/efeitos dos fármacos , Aneurisma da Aorta Abdominal/prevenção & controle , Lipocalina-2/antagonistas & inibidores , Lipocalina-2/deficiência , Animais , Aorta Abdominal/metabolismo , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/patologia , Estudos de Casos e Controles , Células Cultivadas , Quimiotaxia de Leucócito/efeitos dos fármacos , Dilatação Patológica , Modelos Animais de Doenças , Elastina/metabolismo , Predisposição Genética para Doença , Humanos , Lipocalina-2/genética , Lipocalina-2/imunologia , Lipocalina-2/metabolismo , Metaloproteinases da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvasos/efeitos dos fármacos , Microvasos/metabolismo , Microvasos/patologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Infiltração de Neutrófilos/efeitos dos fármacos , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
Renal ischemia-reperfusion injury triggers an inflammatory response associated to infiltrating macrophages which determines the further outcome of disease. Brown Norway rats are known to show endogenous resistance to ischemia-induced renal damage. By contrast, Sprague Dawley rats exhibit a higher susceptibility to ischemic injury. In order to ascertain cytoprotective mechanisms, we focused on the implication of lipocalin-2 protein in main resistance mechanisms in renal ischemia/reperfusion injury by using adoptive macrophage administration, genetically modified ex vivo either to overexpress or to knockdown lipocalin-2. In vitro experiments with bone marrow-derived macrophages both from Brown Norway rats and from Sprague Dawley rats under hypoxic conditions showed endogenous differences regarding cytokine and lipocalin-2 expression profile in the two strains. Most interestingly, we observed that macrophages of the resistant strain express significantly more lipocalin-2. In vivo studies showed that tubular epithelial cell apoptosis and renal injury significantly increased and reparative markers decreased in Brown Norway rats after injection of lipocalin-2-knockdown macrophages, while the administration of lipocalin-2-overexpressing cells significantly decreased Sprague Dawley susceptibility. These data point to a crucial role of macrophage-derived lipocalin-2 in endogenous cytoprotective mechanisms. We conclude that expression of lipocalin-2 in tissue-infiltrating macrophages is pivotal for kidney-intrinsic cytoprotective pathways during ischemia reperfusion injury.