RESUMO
Previous studies have shown that the occurrence of atherosclerosis is closely related to changes of α2, 6-sialic acid transferase I (ST6Gal-I). Bace1 has been identified as a protease responsible for the cleavage and secretion of Golgi-resident ST6Gal-I. There have been only a few attempts to clarify the direct connection between Bace1 and atherosclerosis. The purpose of this study was to investigate the relationship between Bace1 gene and atherosclerosis. Expressions of Bace1 protein and mRNA in ApoE-/- mice fed on high-fat diet were evaluated and the development of atherosclerosis was assessed in Bace1-/- mice fed on high-fat diet. In vitro, the expression of Bace1 gene was detected in foam cell model and the formation of foam cells was examined after knocking down Bace1 by siRNA. We observed a significant increase in Bace1 expression in the aortic root in the model of atherosclerosis in ApoE-/-mice. The expression of Bace1 protein and mRNA levels had a remarkable increase in high-fat group. After knocking out the Bace1 gene, serum lipid levels were significantly lower and intimal thickness was obvious thinner than those in wild-type mice with high-fat diet. Expression of Bace1 protein and mRNA levels were significantly elevated in foam cell. The formation of foam cells was blocked when Bace1 was knocked down by siRNA interferes. Our results suggested that elevated Bace1 gene had a positive role in the progression of atherosclerosis. Affecting the glycosyltransferase may be one of its mechanisms.
Assuntos
Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Aterosclerose/etiologia , Células Espumosas/metabolismo , Secretases da Proteína Precursora do Amiloide/deficiência , Animais , Aorta/metabolismo , Aorta/patologia , Ácido Aspártico Endopeptidases/deficiência , Aterosclerose/genética , Aterosclerose/metabolismo , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Células Espumosas/patologia , Técnicas de Silenciamento de Genes , Técnicas In Vitro , Masculino , Camundongos , Camundongos Knockout , Camundongos Knockout para ApoE , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Regulação para CimaRESUMO
BACKGROUND: Human immunodeficiency virus infection and acquired immunodeficiency syndrome (HIV/AIDS) are infectious diseases with high mortality. Early diagnosis is crucial. Combining HIV antibody and p24 antigen, the Elecsys HIV combi PT assay is a fourth generation HIV screening assay. The sensitivity and specificity of this assay was examined. METHODS: A total of 111,556 samples was conducted from January 1, 2016, to June 30, 2018 in Zhongshan Hospital of Yat-Sen University. We conducted a fourth-generation HIV test of retrospective HIV screening samples and assessed the reliability of using signal-to-cutoff (S/CO) ratios to distinguish false positive HIV antibody reactions and analyzed false positives. RESULTS: A total of 122 specimens were confirmed as HIV-1 infected by western blot (WB) and HIV nucleic acid assays. The median S/CO ratio for HIV false positive specimens was 3.27, while for the HIV-infected specimen it was 391.7. Receiver operating characteristic (ROC) analysis showed that the best diagnostic point for HIV was 22.85 S/CO. The sensitivity, specificity, and Youden index were 100%, 97.8%, and 0.978, respectively. The highest false positive rate of 26.4% was found in patients with malignant tumors and blood diseases. CONCLUSIONS: The results of this study show that the fourth-generation Elecsys HIV combination PT test can identify early HIV infected and can be a useful adjunct to help clinicians to manage the disease by viral load testing and starting an appropriate therapy. Our research data provides a reference for subsequent research and HIV testing in the region.
Assuntos
Diagnóstico Precoce , Técnicas Eletroquímicas/métodos , Infecções por HIV/diagnóstico , Imunoensaio/métodos , Medições Luminescentes/métodos , Programas de Rastreamento/métodos , Reações Antígeno-Anticorpo/imunologia , Reações Falso-Positivas , Anticorpos Anti-HIV/sangue , Anticorpos Anti-HIV/imunologia , Anticorpos Anti-HIV/metabolismo , Proteína do Núcleo p24 do HIV/sangue , Proteína do Núcleo p24 do HIV/imunologia , Proteína do Núcleo p24 do HIV/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/metabolismo , HIV-1/fisiologia , Humanos , Kit de Reagentes para Diagnóstico/normas , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
Tamoxifen has been reported to be associated with antagonism of estrogen-mediated cell growth signaling and activation of estrogen receptor-independent apoptosis events. It has been demonstrated that mammalian sterile 20-like kinase 1 is a direct target of Caspases to amplify the apoptotic signaling pathway. Here, we presented that breast cancer MCF-7 and SKBR3 cells under treatment with 4-hydroxytamoxifen displayed decreased level of pyruvate kinase M2. Western blot results also showed that 4-hydroxytamoxifen induced the activity of pro-apoptotic protein Caspase-3 in MCF-7 and SKBR3 cells, as evidenced by the cleavage of mammalian sterile 20-like kinase 1 substrate in a dose-dependent manner. Co-immunoprecipitation and immunofluorescence experiments were performed to clarify the relationship between pyruvate kinase M2 and mammalian sterile 20-like kinase 1. The results indicated that mammalian sterile 20-like kinase 1 was associated with pyruvate kinase M2 in cultured mammalian cells, and the interaction between mammalian sterile 20-like kinase 1 and pyruvate kinase M2 was decreased in response to 4-hydroxytamoxifen treatment. In addition, knockdown of pyruvate kinase M2 upregulated the level of cleaved Caspase-3 and subsequently facilitated the nuclear translocation of mammalian sterile 20-like kinase 1. Our data further supplemented the extensive functions of pyruvate kinase M2 in mediating breast cancer cell viability by substantially abating the mammalian sterile 20-like kinase 1-mediated apoptosis. In summary, our results identified that mammalian sterile 20-like kinase 1 is a novel downstream target of pyruvate kinase M2, and knockdown of pyruvate kinase M2 contributes apoptosis via promoting nuclear translocation of mammalian sterile 20-like kinase 1 by enhancing Caspase-3-dependent cleavage.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Caspase 3/genética , Proteínas Quinases/metabolismo , Piruvato Quinase/metabolismo , Tamoxifeno/administração & dosagem , Apoptose/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Caspase 3/metabolismo , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Estrogênios/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Células MCF-7 , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Quinases/genética , Piruvato Quinase/genética , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Transdução de Sinais/genéticaRESUMO
An isothermal colorimetric method is described for amplified detection of the CaMV 35S promoter sequence in genetically modified organism (GMO). It is based on (a) target DNA-triggered unlabeled molecular beacon (UMB) termini binding, and (b) exonuclease III (Exo III)-assisted target recycling, and (c) hemin/G-quadruplex (DNAzyme) based signal amplification. The specific binding of target to the G-quadruplex sequence-locked UMB triggers the digestion of Exo III. This, in turn, releases an active G-quadruplex segment and target DNA for successive hybridization and cleavage. The Exo III impellent recycling of targets produces numerous G-quadruplex sequences. These further associate with hemin to form DNAzymes and hence will catalyze H2O2-mediated oxidation of the chromogenic enzyme substrate ABTS2- causing the formation of a green colored product. This finding enables a sensitive colorimetric determination of GMO DNA (at an analytical wavelength of 420 nm) at concentrations as low as 0.23 nM. By taking advantage of isothermal incubation, this method does not require sophisticated equipment or complicated syntheses. Analyses can be performed within 90 min. The method also discriminates single base mismatches. In our perception, it has a wide scope in that it may be applied to the detection of many other GMOs. Graphical abstract An isothermal and sensitive colorimetric method is described for amplified detection of CaMV 35S promoter sequence in genetically modified organism (GMO). It is based on target DNA-triggered molecular beacon (UMB) termini-binding and exonuclease III assisted target recycling, and on hemin/G-quadruplex (DNAzyme) signal amplification.
Assuntos
Colorimetria/métodos , DNA Catalítico/genética , Exodesoxirribonucleases/metabolismo , Técnicas de Amplificação de Ácido Nucleico/métodos , Organismos Geneticamente Modificados/genética , Técnicas Biossensoriais/métodos , DNA/metabolismo , Quadruplex G , Hemina/químicaRESUMO
OBJECTIVE: To establish a laboratory method for detection of Mycobacterium tuberculosis in sputum based on variable number tandem repeat (VNTR). METHODS: Mycobacterium tuberculosis was tested by VNTR and fluorescent quantitative reverse transcription polymerase chain reaction (FQ-PCR) in 130 sputum samples from patients with pulmonary tuberculosis and 200 specimens from patients with other lung diseases. According to the amplification conditions and clinical detection needs, MTUB21, MUTB04, QUB18, QUB26, QUB11b, MIRU31, MIRU10 and MIRU26 were selected as test targets. The results of VNTR and FQ-PCR were compared with Lowenstein-Jensen culture and clinical diagnosis, and analyzed by chi-square test. RESULTS: With the results of L-J culture as the standard, the sensitivity and specificity of VNTR were 93.1% (108/116) and 97.7% (209/214), and those of FQ-PCR were 94.0% (109/116) and 96.7% (207/214), respectively; no significant difference was observed between two groups (χ2=0.352, P=0.569). Using the clinical diagnosis as the standard, the sensitivity and specificity of VNTR were 86.9% (113/130) and 100% (200/200), and those of FQ-PCR were 87.7% (114/130) and 99.0% (198/200), respectively; the difference was not statistically significant (χ2=0.030, P=0.862). In 113 VNTR positive samples, the molecular codes differed from each other in 98.2% samples (111/113); only 2 samples had identical code (5-4-6-8-5-5-3-8). CONCLUSION: The study suggests that VNTR provides a promising method for diagnosis of clinical tuberculosis.
Assuntos
Repetições Minissatélites , Mycobacterium tuberculosis/isolamento & purificação , Escarro/microbiologia , Tuberculose Pulmonar/diagnóstico , Humanos , Reação em Cadeia da Polimerase , Sensibilidade e EspecificidadeRESUMO
With adequate serum concentration of antiepileptic drugs, the epilepsy symptoms in many patients still cannot be controlled well. The alteration of glycosyltransferase has obvious influence on the pathogenesis of epilepsy. In this study, we focus on the diagnostic and prognostic value of fucosyltransferase 8 (Fut8) on epilepsy and refractory epilepsy. Serum samples of 199 patients with epilepsy, 59 patients with refractory epilepsy and 22 healthy controls who were diagnosed in Shenzhen Children's hospital from August 2018 to August 2019 were collected. The level of lectins was further analyzed by lectin chip and enzyme linked immunosorbent assay (ELISA). The diagnostic value of serum Fut8 for epilepsy and refractory epilepsy was evaluated by receiver operating characteristic curve. Finally, the difference in the recurrence rate of convulsion in patients with epilepsy or refractory epilepsy within 2 years were observed in different Fut8 expression patients. The concentration of valproic acid (VPA) were significant different between epilepsy and refractory epilepsy group. The expression of α1, 6-fucosylation and Fut8 was significantly increased in the refractory epilepsy group compared with healthy controls. The area under the curve of Fut8 as a biomarker for predicting epilepsy or refractory epilepsy was 0.620 and 0.856, respectively. There was a significant difference in the recurrence rate of convulsion within 2 years in the children with refractory epilepsy (p = 0.0493) not epilepsy (p = 0.1865) between the high and low Fut8 expression groups. Fut8 was one of the effective indicators for the diagnosis and prognosis of refractory epilepsy.
Assuntos
Epilepsia Resistente a Medicamentos , Fucosiltransferases , Criança , Humanos , Epilepsia Resistente a Medicamentos/diagnóstico , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Glicosilação , Prognóstico , ConvulsõesRESUMO
Atherosclerosis is the primary origin of acute coronary syndrome (ACS) diseases. Previous studies have shown that lncRNA plaque-enriched long noncoding RNA in atherosclerotic macrophage regulation (lncRNA PELATON) is a specific lncRNA in macrophage nuclei. This study aims to identify serum lncRNA PELATON as a biomarker for assessing the incidence and prognosis of ACS. Levels of serum lncRNA PELATON were detected by real-time polymerase chain reaction (RT-PCR) in patients with ACS and healthy individuals. The clinical significance of lncRNA PELATON in patients with ACS was assessed by analyzing receiver operating characteristic and survival curves. The serum levels of lncRNA PELATON in patients with ACS were significantly higher than those in healthy individuals. LncRNA PELATON expression was positively correlated with the expression levels of high sensitivity C-reactive protein (hs-CRP), cardiac troponin T (cTnT) and creatine kinase MB (CK-MB) (p < 0.05). LncRNA PELATON can be used as a potential diagnostic index with an AUC of 0.706 for unstable angina pectoris (UA), 0.782 for acute non-ST-segment elevation myocardial infarction (NSTEMI) and 0.900 for acute ST-segment elevation myocardial infarction (STEMI). The incidence of major cardiovascular events in patients with ACS with high lncRNA PELATON expression was higher than that in those with low lncRNA PELATON expression. However, the mortality between patients in the high and low lncRNA PELATON groups was not significantly different. This study showed that higher levels of lncRNA PELATON were negatively correlated with the prognosis of ACS, revealing the potential of this measurement to serve as an index to assess the incidence and prognosis of ACS.
Assuntos
Síndrome Coronariana Aguda , Infarto do Miocárdio , RNA Longo não Codificante , Síndrome Coronariana Aguda/diagnóstico , Síndrome Coronariana Aguda/epidemiologia , Síndrome Coronariana Aguda/genética , Aterosclerose/genética , Aterosclerose/metabolismo , Biomarcadores , Humanos , Incidência , Infarto do Miocárdio/diagnóstico , Infarto do Miocárdio/genética , Prognóstico , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Fatores de RiscoRESUMO
Inhibitor of beta-catenin and TCF (ICAT) is a key protein in the Wnt-ß-catenin signaling pathway. However, its role in acute myeloid leukemia (AML) remains unknown. In this study, we evaluated its expression level as well as its prognostic value in AML patients. A total of 72 patients with AML and 30 control subjects were enrolled in this study during the period of January 2017 and December 2019 at Zhongshan Hospital of SunYat-sen University. ICAT and ß-catenin expression levels in peripheral blood were determined via enzyme-linked immunosorbent assays. ICAT levels in AML patients were significantly lower and ß-catenin levels were higher than those of the control group. After the first course of standard chemotherapy, the concentration of ICAT in the partial remission group (93.79 ng/mL) was significantly higher than that in the initial diagnosis group (49.38 ng/mL) and the no response group (39.94 ng/mL). AML subtypes had lower ICAT expression levels than controls, and ICAT levels were significantly correlated with body mass index, bone marrow/peripheral blood blast cell proportions, and white blood cell and red blood cell counts at initial diagnosis. Furthermore, low ICAT expression was found to be associated with poor disease-free survival and overall survival in AML. ICAT is closely associated with AML progression and can be used as an indicator to monitor AML treatment efficacy.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Leucemia Mieloide Aguda , beta Catenina , Proteínas Adaptadoras de Transdução de Sinal/genética , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Prognóstico , beta Catenina/metabolismoRESUMO
The objective of the study was to investigate the value of anti-α-enolase antibody (Ab) combined with RDW in evaluating the activity of systemic lupus erythematosus (SLE). Levels of serum anti-α-enolase Ab and RDW were detected in 193 SLE patients and 98 healthy controls by ELISA and automatic blood cell counter (XN9000), respectively. Furthermore, the correlation between anti-α-enolase Ab and RDW in evaluating the activity of SLE was evaluated by correlation analysis. The level of anti-α-enolase Ab (9.16 ± 0.44 ng/mL in stable group and 10.26 ± 0.36 ng/mL in activity group) was significantly higher than that in the healthy control (7.05 ± 0.27 ng/mL). The level of RDW (12.92% ± 1.23% in stable group and 13.57% ± 2.12% in activity group) was significantly higher than that in the healthy control (12.46% ± 0.61%). The levels of anti-α-enolase Ab or RDW in SLE patients were positively correlated with SLEDAI-2 K score (r= 0.75, r = 0.73), respectively. Compared with the anti-α-enolase Ab (AUC: 78.0%) or RDW (AUC:80.0%) alone, anti-α-enolase Ab combined with RDW (AUC: 81.0%) had the best of the effectiveness of evaluating activity of SLE. These data suggested that combined anti-α-enolase Ab with RDW might be good biomarker to predict the activity of SLE in clinical.
Assuntos
Anticorpos Antinucleares/imunologia , Biomarcadores/sangue , Índices de Eritrócitos , Eritrócitos/química , Lúpus Eritematoso Sistêmico/patologia , Fosfopiruvato Hidratase/imunologia , Adulto , Estudos de Casos e Controles , Feminino , Seguimentos , Humanos , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Prognóstico , Estudos RetrospectivosRESUMO
PURPOSE: To explore the clinical significance of plasma pyruvate kinase M2 (PKM2) in assessing the incidence and prognosis of acute leukemia. METHODS: Plasma samples from 56 acute myeloid leukemia (AML) patients, 40 acute lymphoblastic leukemia (ALL) patients, and 66 plasma samples from healthy individuals were collected. The level of plasma PKM2 was detected by enzyme-linked immunosorbent assay. The clinical significance of PKM2 in acute leukemia was assessed by analyzing receiver operating characteristic and survival curves. RESULTS: The plasma levels of PKM2 in AML or ALL patients were significantly higher than those in healthy individuals, respectively. PKM2 can be used as a potential diagnostic index with the AUC of 0.827 for AML and 0.837 for ALL. The level of plasma PKM2 in ALL patients with a BCR/ABL-positive genotype was significantly higher than that in patients with a BCR/ABL-negative genotype (p<0.05). The event-free survival and the overall survival of acute leukemia patients with higher PKM2 expression was worse than those with lower PKM2 expression. CONCLUSION: This study showed that higher levels of PKM2 was negatively correlated with the prognosis of acute leukemia. Therefore, PKM2 can be used as a potential index to assess the incidence and prognosis of acute leukemia.
RESUMO
BACKGROUND: Previously, it has been shown that obesity may be considered as a risk factor for breast cancer in postmenopausal women. Leptin, a hormone whose level is elevated in obesity, has been suggested to be involved in the development of breast cancer, and univariate survival analyses have shown that over-expression of ACAT2, an enzyme that is involved in the production of cholesteryl esters, may be associated with a poor prognosis. Here, we aimed to investigate the effect of leptin on the proliferation, migration and invasion of breast cancer cells, as well as to elucidate its underlying mode of action. METHODS: Gene expression changes in leptin treated breast cancer-derived MCF-7, T47D and BT474 cells were assessed using PCR array, qRT-PCR and Western blot analyses. The expression patterns of Ob-R (leptin receptor) and ACAT2 in breast cancer cells and primary breast cancer tissue samples were analyzed using immunofluorescence and immunohistochemistry, respectively. Leptin-induced proliferation of breast cancer cells was assessed using a CCK8 assay, and scratch wound and Transwell assays were used to assess breast cancer cell invasion and migration. RESULTS: We found that, among the genes tested, ACAT2 expression exhibited the most significant changes in the leptin treated cells. In addition, we found that inhibition of ACAT2 expression using pyripyropene A (PPPA) or siRNA-mediated gene silencing significantly decreased leptin-induced proliferation, migration and invasion of MCF-7 and T47D cells. Subsequent Western blot analyses strongly indicated that the PI3K/AKT/SREBP2 signaling pathway was involved in leptin-induced ACAT2 upregulation in both MCF-7 and T47D cells. Finally, through the analysis of primary breast cancer tissue samples we found that ACAT2 may affect cancer progression through activation of the Ob-R. CONCLUSIONS: Our data indicate that leptin may enhance the proliferation, migration and invasion of breast cancer cells via ACAT2 up-regulation through the PI3K/AKT/SREBP2 signaling pathway. Therefore, the leptin/ACAT2 axis may represent an attractive therapeutic target for breast cancer, particularly in postmenopausal and/or obese women.
Assuntos
Acetil-CoA C-Acetiltransferase/metabolismo , Neoplasias da Mama/metabolismo , Leptina/farmacologia , Receptores para Leptina/metabolismo , Western Blotting , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Técnicas In Vitro , Células MCF-7 , Transdução de Sinais/efeitos dos fármacosRESUMO
Breast cancer is one of the most malignant diseases world-wide and it ranks the first among female cancers. Masses of intrinsic and extrinsic factors, especially the inflammatory factors can lead to breast cancer. Aberrant activation and accumulation of key molecules can lead to inflammation associated carcinogenesis. The signal transducers and activators of transcription 3 (STAT3) is one of them. Therefore, to evaluate the novel molecular mechanisms, STAT3 has become our focus for breast cancer targeted therapy. At present, many tumor suppressing microRNAs have been validated, and are the highlights in research on microRNAs. Thus, we predicted microRNAs which could putatively regulate STAT3 through databases and selected six to screen with Dual-luciferase assay. The result hinted that miR520c could bind with STAT3 3'UTR. We mutated the seed sequence of miR520c on STAT3 3'UTR, which illustrated a reverse effect compared with wild-type of STAT3 3'UTR. Subsequently, STAT3, p-STAT3 and miR520c were assessed in three different grades of breast cancer cells, with the degree of malignancy, we found an escalating trend of STAT3 and p-STAT3, on the contrary, a downward trend of miR520c. We observed STAT3 was deactivated by miR520c. Epithelial to mesenchymal transition (EMT) is a fatal transfer of cancer progression. To find out whether the downregulation of STAT3 can repress breast cancer motility and invasion ability, we detected EMT markers. The result implied a suppression effect on EMT. We overexpressed STAT3 to conduct rescue experiments, the result showed a recovery of STAT3 and EMT characteristics. Cell motility and invasion property were regained as well. In the study, we elucidated miR520c could inhibit breast cancer EMT by targeting STAT3. It can enrich the mechanism of breast cancer and may lay the foundation for breast cancer targeted treatment.
Assuntos
Neoplasias da Mama/prevenção & controle , Transição Epitelial-Mesenquimal/genética , MicroRNAs/genética , Fator de Transcrição STAT3/antagonistas & inibidores , Apoptose , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Movimento Celular , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Células Tumorais CultivadasRESUMO
In recent years, crosstalk between tumor microenvironment and cancer cells have received increasing attention. Accumulating research data suggests that leptin, a key adipokine secreted from adipocytes, plays important roles in breast cancer development. In our study, the effects of leptin on polarization of tumor-associated macrophages (TAMs) and promotion of the invasiveness of tumor cells were investigated. THP1 cells were used to differentiate M2 polarization macrophages. After stimulated by leptin, we established a co-culture system of tumor cells and macrophages to evaluate the function of leptin-induced macrophages in the migration and invasion of breast cancer cells. The gene and protein expressions were analyzed and the underlying mechanisms were evaluated. Moreover, pathological human specimens, and xenografts in nude mice, were detected to strengthen the in vitro results. Leptin elevated the expression of an array of cytokines in TAMs, IL-18 was the most increased, with an activation of the NF-κB/NF-κB1 signalling pathway. Additionally, after treated with leptin, TAMs significantly promoted the migration and invasion of breast cancer cells. However, these effects of leptin were abolished by the co-incubation of Bay117082, a pharmacological NF-κB inhibitor. Leptin also directly stimulated IL-18 expression in breast cancer cells, which, differently, was via the PI3K/AKT-ATF-2 signaling pathway. In vivo studies showed that malignant breast carcinoma exhibited strong higher expression of Leptin, IL-8, and TAMs markers. Xenograft tumor-bearing mouse models showed that leptin significantly increased tumor volume, enhanced lung metastases, and increased expression of IL-8 and TAM markers, which were abolished by depletion of macrophages by clophosome-clodronate liposomes (CCL). Leptin could induce IL-18 expression both in TAMs and breast cancer cells. Leptin-induced IL-18 expression was regulated via NF-κB/NF-κB1 signaling in TAMs, while via PI3K-AKT/ATF-2 signaling in breast cancer cells, which, eventually, lead to invasion and metastasis of breast cancer cells.
Assuntos
Neoplasias da Mama/patologia , Movimento Celular/efeitos dos fármacos , Interleucina-18/genética , Leptina/metabolismo , Macrófagos/efeitos dos fármacos , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Técnicas de Cocultura , Feminino , Humanos , Interleucina-18/metabolismo , Células MCF-7 , Macrófagos/citologia , Macrófagos/patologia , Camundongos , Invasividade Neoplásica , Transplante de Neoplasias , Transdução de Sinais/efeitos dos fármacos , Microambiente TumoralRESUMO
This study aims to investigate the mechanisms underlying leptin-mediated crosstalk between tumor-associated macrophages (M2 macrophages) and breast cancer cells. THP1 human leukemic monocytes were induced to differentiate into M2 macrophages by PMA (100 nM) and IL-4 (20 ng/mL). Quantitative RT-PCR and Western blot revealed that leptin (100 nM) significantly increased the expression of leptin receptor (ObR) in the M2 macrophages (P < 0.01) and stimulated interleukin (IL)-8 expression in the M2 macrophages, mouse macrophage cells RAW264.7, and primary mouse peritoneal macrophages in a dose- and time-dependent manner. Leptin-induced IL-8 production was sensitive to the ERK inhibitor PD980590 (10 µmol/L), p38 MAPK inhibitor SB203580 (20 µmol/L), and anti-ObR neutralizing antibody (4 µg/mL). Leptin (100 ng/mL) substantially increased the phosphorylation of p38 and ERK1/2. Thus, leptin may induce IL-8 production in M2 macrophages by interacting with ObR to activate the p38 and ERK signaling pathways. Scratch and transwell chamber assay showed that both recombinant IL-8 and leptin-induced M2 macrophage-derived IL-8 promoted the migration and invasion of human breast cancer cells MCF7 and MDA-MB-231 (All P < 0.01). In a nude mice xenograft model of breast cancer (n = 5 per group), injection of leptin (0.1 µg/g) dramatically increased tumor volume and mass, reduced survival, exacerbated pulmonary metastasis, and elevated IL-8 and Ki67 expression in the tumor tissue (All P < 0.05) compared with PBS injection. Depletion of mouse macrophage by Clophosome®-clodronate liposome and injection of anti-mouse IL-8 neutralizing antibodies in the xenograft tumor significantly attenuated those leptin-mediated stimulations (All P < 0.05). These findings indicate that leptin may promote tumor growth and metastasis by stimulating IL-8 production in tumor-associated macrophage.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Movimento Celular , Interleucina-8/metabolismo , Leptina/metabolismo , Macrófagos/imunologia , Animais , Anticorpos Bloqueadores/farmacologia , Diferenciação Celular , Feminino , Humanos , Interleucina-8/imunologia , Sistema de Sinalização das MAP Quinases , Células MCF-7 , Camundongos , Camundongos Nus , Fosforilação , Células RAW 264.7 , Receptores para Leptina/metabolismo , Acetato de Tetradecanoilforbol/imunologia , Células Th1/imunologia , Carga Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND: Accumulating researches have shown that epithelial-mesenchymal transition (EMT) contributes to tumor metastasis. Leptin, a key adipokine secreted from adipocytes, shapes the tumor microenvironment, potentiates the migration of breast cancer cells and angiogenesis, and is also involved in EMT. However, the potential mechanism remains unknown. This study aims to explore the effect of leptin on EMT in breast cancer cells and the underlying mechanism. METHODS: With the assessment of EMT-associated marker expression in MCF-7, SK-BR-3, and MDA-MB-468 cells, the effect of leptin on breast cancer cells was analyzed. Besides, an array of pathway inhibitors as well as RNA interference targeting pyruvate kinase M2 (PKM2) were used to clarify the underlying mechanism of leptin-mediated EMT in vitro and in vivo. RESULTS: The results demonstrated that leptin promoted breast cancer cells EMT, visibly activated the PI3K/AKT signaling pathway, and upregulated PKM2 expression. An antibody against the leptin receptor (anti-ObR) and the PI3K/AKT signaling pathway inhibitor LY294002 significantly abolished leptin-induced PKM2 expression and EMT-associated marker expression. SiRNA targeting PKM2 partially abolished leptin-induced migration, invasion, and EMT-associated marker expression. In vivo xenograft experiments indicated that RNA interference against PKM2 suppressed breast cancer growth and metastasis. CONCLUSIONS: Our data suggest that leptin promotes EMT in breast cancer cells via the upregulation of PKM2 expression as well as activation of PI3K/AKT signaling pathway, and PKM2 might be one of the key points and potential targets for breast cancer therapy.
Assuntos
Neoplasias da Mama/patologia , Proteínas de Transporte/metabolismo , Transição Epitelial-Mesenquimal , Leptina/metabolismo , Proteínas de Membrana/metabolismo , Hormônios Tireóideos/metabolismo , Regulação para Cima , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Cromonas/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Camundongos , Morfolinas/farmacologia , Invasividade Neoplásica , Transplante de Neoplasias , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas de Ligação a Hormônio da TireoideRESUMO
OBJECTIVE: To investigate the effect of interleukin-8 (IL-8) on the apoptosis of MCF-7 human breast cancer cells and the molecular mechanism. METHODS: The expressions of IL-8 receptors (CXCR1, CXCR2) in MCF-7 cells were detected by Western blotting. The effects of 0, 20, 40, 80, 160 ng/mL IL-8 on the expressions of apoptosis-related genes Bcl-2 and caspase-3 in MCF-7 cells were observed by reverse transcription PCR(RT-PCR) and Western blotting. Cell proliferation was determined by CCK-8 assay after 0, 40, 80 ng/mL IL-8 treatment. Phase contrast microscope was used to examine cell morphology of MCF-7 cells after 80 ng/mL IL-8 treatment. The effects of 80 ng/mL IL-8 combined with PD980590 (10 µmol/L), LY294002 (10 µmol/L) or AG490 (50 µmol/L) [mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), Janus kinase/signal transducer and activator of transcription (JAK/STAT) signal pathway inhibitors, respectively], on the expression of Bcl-2 were detected by Western blotting. The effects of 0, 20, 40, 80, 160 ng/mL IL-8 on the expression of p-AKT in MCF-7 cells were observed by Western blotting. The effects of 80 ng/mL IL-8 combined with 10 µmol/L LY294002 on the apoptosis of MCF-7 cells, the expressions of Bcl-2 and caspase-3 were determined by flow cytometry, RT-PCR and Western blotting, respectively. RESULTS: Both CXCR1 and CXCR2 were expressed in MCF-7 cells. IL-8 markedly up-regulated the anti-apoptotic gene Bcl-2, down-regulated the pro-apoptotic gene caspase-3, and significantly inhibited the apoptosis of MCF-7 cells. However, these effects were blocked by phosphoinositide 3-kinase/protein kinase B (PI3K/AKT), signal pathway inhibitor LY294002. As predicted, IL-8 markedly increased the expression of p-AKT in MCF-7 cells. CONCLUSION: IL-8 might significantly inhibit the apoptosis of MCF-7 cells. This effect may be achieved by up-regulating Bcl-2 and down-regulating caspase-3 via PI3K/AKT signal pathway.
Assuntos
Apoptose , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Caspase 3/genética , Interleucina-8/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Neoplasias da Mama/fisiopatologia , Caspase 3/metabolismo , Proliferação de Células , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , Regulação para CimaRESUMO
OBJECTIVE: To investigate the effect of NF-κB inhibitor BAY11-7082 on proliferation and apoptosis of breast carcinoma MCF-7 cells and the underlying mechanism. METHODS: MCF-7 cells in the logarithmic growth phase were divided into control group, 5 µmol/L BAY11-7082 group and 10 µmol/L LY294002 group. After the treatment of BAY11-7082 and LY294002, the protein levels of ATP citrate lyase (ACL), phosphated-ACL (p-ACL), phosphated-Akt (p-Akt) and phosphated nuclear factor κB (p-NF-κB) were determined by Western blotting. The proliferation of MCF-7 cells treated with BAY11-7082 or siACL were detected by CCK-8 assay. The apoptosis of MCF-7 cells treated with BAY11-7082 or siACL were observed by flow cytometry combined with annexin V-FITC/PI staining. RESULTS: The proliferation of MCF-7 cells was inhibited by BAY11-7082 in a dose-dependent manner. Compared with the control group, the expressions of p-ACL and p-NF-κB protein in MCF-7 cells treated with BAY11-7082 were lowered. The expressions of p-ACL and p-NF-κB protein in MCF-7 cells treated with 10 µmol/L LY294002 were also reduced significantly. The proliferation of MCF-7 cells treated with BAY11-7082 or siACL for 48 hours was inhibited and the apoptosis was promoted significantly, as shown by CCK-8 assay and flow cytometry. CONCLUSION: BAY11-7082 could inhibit the proliferation of MCF-7 breast carcinoma cells and promote the apoptosis by inhibiting the phosphorylation of ACL.
Assuntos
ATP Citrato (pro-S)-Liase/antagonistas & inibidores , Apoptose/efeitos dos fármacos , NF-kappa B/antagonistas & inibidores , Nitrilas/farmacologia , Sulfonas/farmacologia , ATP Citrato (pro-S)-Liase/metabolismo , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Humanos , Células MCF-7 , FosforilaçãoRESUMO
BACKGROUND INFORMATION: Previous studies have revealed that leptin may be involved in epithelial-mesenchymal transition (EMT), a crucial initiator of cancer progression to facilitate metastatic cascade, increase tumor recurrence, and ultimately cause poor prognosis. However, the underlying mechanism remains unclear. The aim of our present study was to investigate the effect of leptin on EMT of breast cancer cells and the underlying mechanism. RESULTS: Our data demonstrated that leptin significantly increased the phosphorylation of STAT3, Akt, and ERK1/2, elevated the expression of IL-8, and induced breast cancer cells to undergo EMT. The effect of leptin on IL-8 could visibly abolished by the inhibitor of PI3K LY294002. In addition, leptin-induced EMT of breast cancer cells was blocked by anti-IL-8 antibodies. Examination of the expression of ObR, leptin, IL-8 and EMT-related biomarkers in patient specimens demonstrated that malignant breast carcinoma with lymph node metastases (LNM), which represents poor prognosis, expressed higher levels of ObR, leptin, IL-8 than other types of breast cancer, and displayed more obvious EMT transversion. In vivo xenograft experiment revealed that leptin signally promoted tumor growth and metastasis and increased the expressions of IL-8 and EMT-related biomarkers. CONCLUSIONS: Our results support that leptin-induced EMT in breast cancer cells requires IL-8 activation via the PI3K/Akt signal pathway.