RESUMEN
Aluminum (Al) can lead to an exposure of creature in varieties ways for its universality, and it could disturb normal physiological metabolism, with the damage to multisystem including reproduction. Since the oocyte quality is critical for female reproduction, we inspected the toxicity of Al on mouse oocyte maturation. We constructed in vitro exposure mouse model, and we found that 5 mmol/L Al had adverse effects on oocyte maturation by impairing organelle and cytoskeleton. Aberrant spindle and misaligned chromosomes which might be considered to be caused by elevated levels of acetylation, as well as abnormal distribution of actin dynamics could hinder normal meiosis of oocytes. Organelle dysfunction indicated that Al affected proteins synthesis, transport and digestion, which would further damage oocyte maturation. In order to explore the mechanism of Al toxicity, our further investigation demonstrated that Al caused mitochondrial dysfunction and imbalance calcium homeostasis, resulting in limited energy supply. Moreover, high level of reactive oxygen species, DNA damage and apoptosis caused by oxidative stress were also the manifestation of Al toxicity on oocytes. In conclusion, our study provided the evidence that Al exposure affected oocyte quality through its effects on spindle organization, actin dynamics, organelle function and the induction of DNA damage-related apoptosis with mouse model.
Asunto(s)
Actinas , Aluminio , Femenino , Ratones , Animales , Aluminio/toxicidad , Actinas/metabolismo , Oocitos/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Daño del ADN , ApoptosisRESUMEN
Aflatoxin is the most common type of mycotoxins in contaminated corn, peanuts and rice, which affects the livestock and ultimately endangers human health. Aflatoxin is reported to have carcinogenicity, mutation, growth retardation, immunosuppression and reproductive toxicity. In present study we reported the causes for the declined porcine oocyte quality under aflatoxin exposure. We set up an in vitro exposure model and showed that aflatoxin B1 disturbed cumulus cell expansion and oocyte polar body extrusion. We found that aflatoxin B1 exposure disrupted ER distribution and elevated the expression of GRP78, indicating the occurrence of ER stress, and the increased calcium storage also confirmed this. Besides, the structure of cis-Golgi apparatus, another intracellular membrane system was also affected, showing with decreased GM130 expression. The oocytes under aflatoxin B1 exposure showed aberrant lysosome accumulation and higher LAMP2 expression, a marker for lysosome membrane protection, and this might be due to the aberrant mitochondria function with low ATP production and the increase of apoptosis, since we found that BAX expression increased, and ribosomal protein which is also an apoptosis-related factor RPS3 decreased. Taken together, our study revealed that aflatoxin B1 impairs intracellular membrane system ER, Golgi apparatus, lysosome and mitochondria function to affect porcine oocyte maturation quality.
Asunto(s)
Aflatoxina B1 , Oocitos , Humanos , Animales , Porcinos , Aflatoxina B1/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Oocitos/metabolismo , Apoptosis , Membranas Intracelulares , Adenosina Trifosfato/metabolismoRESUMEN
BACKGROUND: Lowered nicotinamide adenine dinucleotide (NAD+) levels in tumor cells drive tumor hyperprogression during immunotherapy, and its restoration activates immune cells. However, the effect of lenvatinib, a first-line treatment for unresectable hepatocellular carcinoma (HCC), on NAD+ metabolism in HCC cells, and the metabolite crosstalk between HCC and immune cells after targeting NAD+ metabolism of HCC cells remain unelucidated. METHODS: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and ultra-high-performance liquid chromatography multiple reaction monitoring-mass spectrometry (UHPLC-MRM-MS) were used to detect and validate differential metabolites. RNA sequencing was used to explore mRNA expression in macrophages and HCC cells. HCC mouse models were used to validate the effects of lenvatinib on immune cells and NAD+ metabolism. The macrophage properties were elucidated using cell proliferation, apoptosis, and co-culture assays. In silico structural analysis and interaction assays were used to determine whether lenvatinib targets tet methylcytosine dioxygenase 2 (TET2). Flow cytometry was performed to assess changes in immune cells. RESULTS: Lenvatinib targeted TET2 to synthesize and increase NAD+ levels, thereby inhibiting decomposition in HCC cells. NAD+ salvage increased lenvatinib-induced apoptosis of HCC cells. Lenvatinib also induced CD8+ T cells and M1 macrophages infiltration in vivo. And lenvatinib suppressed niacinamide, 5-Hydroxy-L-tryptophan and quinoline secretion of HCC cells, and increased hypoxanthine secretion, which contributed to proliferation, migration and polarization function of macrophages. Consequently, lenvatinib targeted NAD+ metabolism and elevated HCC-derived hypoxanthine to enhance the macrophages polarization from M2 to M1. Glycosaminoglycan binding disorder and positive regulation of cytosolic calcium ion concentration were characteristic features of the reverse polarization. CONCLUSIONS: Targeting HCC cells NAD+ metabolism by lenvatinib-TET2 pathway drives metabolite crosstalk, leading to M2 macrophages reverse polarization, thereby suppressing HCC progression. Collectively, these novel insights highlight the role of lenvatinib or its combination therapies as promising therapeutic alternatives for HCC patients with low NAD+ levels or high TET2 levels.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Quinolinas , Animales , Ratones , Humanos , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , NAD/metabolismo , NAD/farmacología , NAD/uso terapéutico , Linfocitos T CD8-positivos , Cromatografía Liquida , Línea Celular Tumoral , Espectrometría de Masas en Tándem , Macrófagos/metabolismo , Quinolinas/farmacología , Quinolinas/uso terapéutico , Hipoxantinas/metabolismo , Hipoxantinas/farmacología , Hipoxantinas/uso terapéuticoRESUMEN
Long noncoding RNA LincIN has been reported to be overexpressed and to be involved in the metastasis of breast cancer. However, the expression and role of LincIN in esophageal squamous cell carcinoma (ESCC) remain unsolved. In the present study, LincIN expression was examined in ESCC by RTqPCR, and the roles of LincIN in ESCC were determined using cell growth, migration and invasion assays. In addition, the effects of LincIN on nuclear factor 90 (NF90) and microRNA/miR (miR)7 were examined by RNA immunoprecipitation assay, RTqPCR, dualluciferase reporter assay and western blot analysis. The results revealed that LincIN expression was significantly increased in ESCC tissues and cell lines. The increased expression of LincIN was positively associated with invasion depth, lymph node metastasis, TNM stage and a poor prognosis. Functional assays revealed that the overexpression of LincIN promoted ESCC cell growth, migration and invasion. Mechanistic analysis revealed that LincIN physically bound to NF90, enhanced the binding between NF90 and primary miR7 (primiR7), and further enhanced the inhibitory effects of NF90 on miR7 biogenesis. Therefore, LincIN downregulated miR7 expression in ESCC. The expression of miR7 inversely correlated with that of LincIN in ESCC tissues. By downregulating miR7, LincIN increased the expression of HOXB13, a target of miR7. The overexpression of miR7 or the depletion of HOXB13 both attenuated the tumorpromoting roles of LincIN in ESCC cell growth, migration and invasion. On the whole, the findings of the present study suggest that LincIN is overexpressed and plays an oncogenic role in ESCC via the regulation of the NF90/miR7/HOXB13 axis. Thus, LincIN may prove to be a promising prognostic biomarker and therapeutic target for ESCC.
Asunto(s)
Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/patología , Proteínas de Homeodominio/genética , Proteínas del Factor Nuclear 90/genética , ARN Largo no Codificante/genética , Anciano , Movimiento Celular/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/mortalidad , Carcinoma de Células Escamosas de Esófago/genética , Carcinoma de Células Escamosas de Esófago/mortalidad , Femenino , Regulación Neoplásica de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Humanos , Masculino , MicroARNs/genética , Persona de Mediana Edad , Proteínas del Factor Nuclear 90/metabolismo , Pronóstico , Regulación hacia ArribaRESUMEN
BACKGROUND: Radioresistance is the major obstacle in radiation therapy (RT) for hepatocellular carcinoma (HCC). Dysregulation of DNA damage response (DDR), which includes DNA repair and cell cycle checkpoints activation, leads to radioresistance and limits radiotherapy efficacy in HCC patients. However, the underlying mechanism have not been clearly understood. METHODS: We obtained 7 pairs of HCC tissues and corresponding non-tumor tissues, and UBE2T was identified as one of the most upregulated genes. The radioresistant role of UBE2T was examined by colony formation assays in vitro and xenograft tumor models in vivo. Comet assay, cell cycle flow cytometry and γH2AX foci measurement were used to investigate the mechanism by which UBE2T mediating DDR. Chromatin fractionation and immunofluorescence staining were used to assess cell cycle checkpoint kinase 1(CHK1) activation. Finally, we analyzed clinical data from HCC patients to verify the function of UBE2T. RESULTS: Here, we found that ubiquitin-conjugating enzyme E2T (UBE2T) was upregulated in HCC tissues, and the HCC patients with higher UBE2T levels exhibited poorer outcomes. Functional studies indicated that UBE2T increased HCC radioresistance in vitro and in vivo. Mechanistically, UBE2T-RNF8, was identified as the E2-E3 pair, physically bonded with and monoubiquitinated histone variant H2AX/γH2AX upon radiation exposure. UBE2T-regulated H2AX/γH2AX monoubiquitination facilitated phosphorylation of CHK1 for activation and CHK1 release from the chromatin to cytosol for degradation. The interruption of UBE2T-mediated monoubiquitination on H2AX/γH2AX, including E2-enzyme-deficient mutation (C86A) of UBE2T and monoubiquitination-site-deficient mutation (K119/120R) of H2AX, cannot effectively activate CHK1. Moreover, genetical and pharmacological inhibition of CHK1 impaired the radioresistant role of UBE2T in HCC. Furthermore, clinical data suggested that the HCC patients with higher UBE2T levels exhibited worse response to radiotherapy. CONCLUSION: Our results revealed a novel role of UBE2T-mediated H2AX/γH2AX monoubiquitination on facilitating cell cycle arrest activation to provide sufficient time for radiation-induced DNA repair, thus conferring HCC radioresistance. This study indicated that disrupting UBE2T-H2AX-CHK1 pathway maybe a promising potential strategy to overcome HCC radioresistance.
Asunto(s)
Carcinoma Hepatocelular/patología , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Histonas/metabolismo , Neoplasias Hepáticas/patología , Tolerancia a Radiación , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación , Animales , Apoptosis , Biomarcadores de Tumor , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/radioterapia , Ciclo Celular , Proliferación Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Daño del ADN , Reparación del ADN , Regulación Neoplásica de la Expresión Génica , Histonas/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/radioterapia , Ratones , Ratones Desnudos , Fosforilación , Pronóstico , Radioterapia , Tasa de Supervivencia , Células Tumorales Cultivadas , Enzimas Ubiquitina-Conjugadoras/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Radioimmunotherapy has a promising antitumor effect in hepatocellular carcinoma (HCC), depending on the regulatory effect of radiotherapy on tumor immune microenvironment. Ionizing radiation (IR)-induced DNA damage repair (DDR) pathway activation leads to the inhibition of immune microenvironment, thus impairing the antitumor effect of radioimmunotherapy. However, it is unclear whether inhibition of the DDR pathway can enhance the effect of radioimmunotherapy. In this study, we aim to explore the role of DDR inhibitor AZD6738 on the combination of radiotherapy and immune checkpoint inhibitors (ICIs) in HCC. METHODS: C57BL/6 mouse subcutaneous tumor model was used to evaluate the ability of different treatment regimens in tumor growth control and tumor recurrence inhibition. Effects of each treatment regimen on the alterations of immunophenotypes including the quantification, activation, proliferating ability, exhaustion marker expression, and memory status were assessed by flow cytometry. RESULTS: AZD6738 further increased radiotherapy-stimulated CD8+ T cell infiltration and activation and reverted the immunosuppressive effect of radiation on the number of Tregs in mice xenografts. Moreover, compared with radioimmunotherapy (radiotherapy plus anti-PD-L1 (Programmed death ligand 1)), the addition of AZD6738 boosted the infiltration, increased cell proliferation, enhanced interferon (IFN)-γ production ability of TIL (tumor-infiltrating lymphocyte) CD8+ T cells, and caused a decreasing trend in the number of TIL Tregs and exhausted T cells in mice xenografts. Thus, the tumor immune microenvironment was significantly improved. Meanwhile, triple therapy (AZD6738 plus radiotherapy plus anti-PD-L1) also induced a better immunophenotype than radioimmunotherapy in mice spleens. As a consequence, triple therapy displayed greater benefit in antitumor efficacy and mice survival than radioimmunotherapy. Mechanism study revealed that the synergistic antitumor effect of AZD6738 with radioimmunotherapy relied on the activation of cyclic GMP-AMP synthase /stimulator of interferon genes (cGAS/STING) signaling pathway. Furthermore, triple therapy led to stronger immunologic memory and lasting antitumor immunity than radioimmunotherapy, thus preventing tumor recurrence in mouse models. CONCLUSIONS: Our findings indicate that AZD6738 might be a potential synergistic treatment for radioimmunotherapy to control the proliferation of HCC cells, prolong survival, and prevent tumor recurrence in patients with HCC by improving the immune microenvironment.
Asunto(s)
Carcinoma Hepatocelular/terapia , Quimioradioterapia/métodos , Neoplasias Hepáticas/terapia , Pirimidinas/farmacología , Radioinmunoterapia/métodos , Sulfóxidos/farmacología , Animales , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/patología , Línea Celular Tumoral/trasplante , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/efectos de los fármacos , Sinergismo Farmacológico , Femenino , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Indoles , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/patología , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Morfolinas , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/uso terapéutico , Sulfonamidas , Sulfóxidos/uso terapéutico , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunologíaRESUMEN
Background: Coronavirus disease (COVID-19) has swept around the globe and led to a worldwide catastrophe. Studies examining the disease progression of patients with non-severe disease on admission are scarce but of profound importance in the early identification of patients at a high risk of deterioration. Objectives: To elucidate the differences in clinical characteristics between patients with progressive and non-progressive COVID-19 and to determine the risk factors for disease progression. Study design: Clinical data of 365 patients with non-severe COVID-19 from 1 January 2020 to 18 March 2020 were retrospectively collected. Patients were stratified into progressive and non-progressive disease groups. Univariate and multivariate logistic regression analyses were performed to determine the independent risk factors for disease progression. Results: Compared with patients with non-progressive disease, those who progressed to severe COVID-19 were older and had significantly decreased lymphocyte and eosinophil counts; increased neutrophil and platelet counts; lower albumin levels; higher levels of lactate dehydrogenase, C-reactive protein (CRP), creatinine, creatinine kinase, and urea nitrogen; and longer prothrombin times. Hypertension, fever, fatigue, anorexia, bacterial coinfection, bilateral patchy shadowing, antibiotic and corticosteroid administration, and oxygen support had a significantly higher incidence among patients with progressive disease. A significantly longer duration of hospital stay was also observed in patients with progressive disease. Bilateral patchy shadowing (OR = 4.82, 95% CI: 1.33-17.50; P = 0.017) and elevated levels of creatinine (OR =6.24, 95% CI: 1.42-27.40; P = 0.015), and CRP (OR = 7.28, 95% CI: 2.56-20.74; P < 0.001) were independent predictors for disease progression. Conclusion: The clinical characteristics of patients with progressive and non-progressive COVID-19 were significantly different. Bilateral patchy shadowing and increased levels of creatinine, and CRP were independent predictors of disease progression.