RESUMO
UFMylation is an emerging ubiquitin-like post-translational modification that regulates various biological processes. Dysregulation of the UFMylation pathway leads to human diseases, including cancers. However, the physiological role of UFMylation in T cells remains unclear. Here, we report that mice with conditional knockout (cKO) Ufl1, a UFMylation E3 ligase, in T cells exhibit effective tumor control. Single-cell RNA sequencing analysis shows that tumor-infiltrating cytotoxic CD8+ T cells are increased in Ufl1 cKO mice. Mechanistically, UFL1 promotes PD-1 UFMylation to antagonize PD-1 ubiquitination and degradation. Furthermore, AMPK phosphorylates UFL1 at Thr536, disrupting PD-1 UFMylation to trigger its degradation. Of note, UFL1 ablation in T cells reduces PD-1 UFMylation, subsequently destabilizing PD-1 and enhancing CD8+ T cell activation. Thus, Ufl1 cKO mice bearing tumors have a better response to anti-CTLA-4 immunotherapy. Collectively, our findings uncover a crucial role of UFMylation in T cells and highlight UFL1 as a potential target for cancer treatment.
Assuntos
Neoplasias , Receptor de Morte Celular Programada 1 , Animais , Humanos , Camundongos , Linfócitos T CD8-Positivos/metabolismo , Neoplasias/metabolismo , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
This study aimed to investigate the impact of epidemic prevention and isolation policies on residents' health and well-being and assess the effectiveness of implementing intervention measures to maintain their quality of life. This mixed-methods research study involved a retrospective record review of residents' daily life diaries and descriptive statistical analysis. Data were collected between March 2021 and June 2022, and epidemic-prevention measures were implemented using Taiwan's Centers for Disease Control guidelines. Three interventions were developed to address residents' health, social, and rehabilitation needs. Despite an overall infection rate of 10% at various times between 2021 and 2022, there were no reported outbreaks of nosocomial infections. The concept of reablement proved effective in helping residents maintain their independence and physical function, with a maintenance rate of 66.6%, thereby improving their quality of life. By implementing epidemic-prevention measures, we found that proper hand washing and the use of surgical masks were effective in controlling infections. Furthermore, the decline in physical function is a continuous and gradual process for older adults. Even under the restriction of social interaction, it is essential to incorporate rehabilitation plans into residents' daily activities and encourage their active participation, as this promotes improved physical function and enhances their overall quality of life.
RESUMO
The prolyl hydroxylation of hypoxia-inducible factor 1α (HIF-1α) mediated by the EGLN-pVHL pathway represents a classic signalling mechanism that mediates cellular adaptation under hypoxia. Here we identify RIPK1, a known regulator of cell death mediated by tumour necrosis factor receptor 1 (TNFR1), as a target of EGLN1-pVHL. Prolyl hydroxylation of RIPK1 mediated by EGLN1 promotes the binding of RIPK1 with pVHL to suppress its activation under normoxic conditions. Prolonged hypoxia promotes the activation of RIPK1 kinase by modulating its proline hydroxylation, independent of the TNFα-TNFR1 pathway. As such, inhibiting proline hydroxylation of RIPK1 promotes RIPK1 activation to trigger cell death and inflammation. Hepatocyte-specific Vhl deficiency promoted RIPK1-dependent apoptosis to mediate liver pathology. Our findings illustrate a key role of the EGLN-pVHL pathway in suppressing RIPK1 activation under normoxic conditions to promote cell survival and a model by which hypoxia promotes RIPK1 activation through modulating its proline hydroxylation to mediate cell death and inflammation in human diseases, independent of TNFR1.
Assuntos
Necroptose , Receptores Tipo I de Fatores de Necrose Tumoral , Humanos , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Hidroxilação , Hipóxia , Prolina/metabolismo , Inflamação , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismoRESUMO
Adenosine monophosphate-activated protein kinase (AMPK) activity is stimulated to promote metabolic adaptation upon energy stress. However, sustained metabolic stress may cause cell death. The mechanisms by which AMPK dictates cell death are not fully understood. We report that metabolic stress promoted receptor-interacting protein kinase 1 (RIPK1) activation mediated by TRAIL receptors, whereas AMPK inhibited RIPK1 by phosphorylation at Ser415 to suppress energy stress-induced cell death. Inhibiting pS415-RIPK1 by Ampk deficiency or RIPK1 S415A mutation promoted RIPK1 activation. Furthermore, genetic inactivation of RIPK1 protected against ischemic injury in myeloid Ampkα1-deficient mice. Our studies reveal that AMPK phosphorylation of RIPK1 represents a crucial metabolic checkpoint, which dictates cell fate response to metabolic stress, and highlight a previously unappreciated role for the AMPK-RIPK1 axis in integrating metabolism, cell death, and inflammation.
Assuntos
Proteínas Quinases Ativadas por AMP , Metabolismo Energético , Necroptose , Proteína Serina-Treonina Quinases de Interação com Receptores , Estresse Fisiológico , Animais , Camundongos , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Fosforilação , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Inflamação/metabolismo , Isquemia/metabolismoRESUMO
The programmed cell death protein 1 (PD-1) is an inhibitory receptor on T cells and plays an important role in promoting cancer immune evasion. While ubiquitin E3 ligases regulating PD-1 stability have been reported, deubiquitinases governing PD-1 homeostasis to modulate tumor immunotherapy remain unknown. Here, we identify the ubiquitin-specific protease 5 (USP5) as a bona fide deubiquitinase for PD-1. Mechanistically, USP5 interacts with PD-1, leading to deubiquitination and stabilization of PD-1. Moreover, extracellular signal-regulated kinase (ERK) phosphorylates PD-1 at Thr234 and promotes PD-1 interaction with USP5. Conditional knockout of Usp5 in T cells increases the production of effector cytokines and retards tumor growth in mice. USP5 inhibition in combination with Trametinib or anti-CTLA-4 has an additive effect on suppressing tumor growth in mice. Together, this study describes a molecular mechanism of ERK/USP5-mediated regulation of PD-1 and identifies potential combinatorial therapeutic strategies for enhancing anti-tumor efficacy.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular , Receptor de Morte Celular Programada 1 , Animais , Camundongos , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo , Homeostase , ImunoterapiaRESUMO
Banana (Musa acuminata) blossom contains high nutritional value and bioactive compounds. In this study, Macrobrachium rosenbergii were fed with diets containing banana blossom powder (BBP) at 10 and 20 g kg-1, hot-banana blossom (BBH) extract at 10 and 20 g kg-1, and the basal diet for 56 days. The growth performance, physiological response and immune parameters were evaluated. The results showed that a significantly higher percentage weight gain (PWG) and percentage length gain (PLG) in prawns fed with BBH diet. The feed efficiency (FE) significantly increased in prawns fed BBP. The prawn fed both BBH and BBP diet showed higher survival rate than control group. The prawn fed with BBH showed a significant increase in total haemocyte count (THC) and different haemocyte count (DHC), whereas phenoloxidase (PO) activity and respiratory bursts (RBs) significant increase in prawns fed both BBP and BBH diet. Furthermore, M. rosenbergii fed with both BBP and BBH diets showed significantly higher phagocytic activity and clearance efficiency against Lactococcus garvieae infection. At the end of the 56 days of feeding trial, the susceptibility of prawns to L. garvieae infection and hypothermal (18 °C) stress were evaluated. The results showed that prawns fed BBH diets had a significantly higher survival rate against L. garvieae than those of fed with the basal diet. Anti-hypothermal stress was observed in prawns fed both BBP and BBH diets showing no significant difference in haemolymph glucose in prawns subjected to 18 °C and 28 °C, whereas the norepinephrine level in haemolymph of prawns fed with BBH diets subjected to 18 °C was significantly lower than in prawns subjected to 28 °C. In summary, we recommend addition of hot-banana blossom extract to the diet of M. rosenbergii at 20 g kg-1 to promote growth performance, improve physiological function, enhance immunity, increase anti-hypothermal stress, and to increase resistance against L. gavieae.
Assuntos
Musa , Palaemonidae , Extratos Vegetais , Animais , Resistência à Doença , Flores/química , Musa/química , Palaemonidae/efeitos dos fármacos , Palaemonidae/imunologia , Extratos Vegetais/farmacologiaRESUMO
Activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway is a pervasive event in tumorigenesis due to PI3K mutation and dysfunction of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Pharmacological inhibition of PI3K has resulted in variable clinical outcomes, however, raising questions regarding the possible mechanisms of unresponsiveness and resistance to treatment. WWP1 is an oncogenic HECT-type ubiquitin E3 ligase frequently amplified and mutated in multiple cancers, as well as in the germ lines of patients predisposed to cancer, and was recently found to activate PI3K signaling through PTEN inactivation. Here, we demonstrate that PTEN dissociated from the plasma membrane upon treatment with PI3K inhibitors through WWP1 activation, whereas WWP1 genetic or pharmacological inhibition restored PTEN membrane localization, synergizing with PI3K inhibitors to suppress tumor growth both in vitro and in vivo. Furthermore, we demonstrate that WWP1 inhibition attenuated hyperglycemia and the consequent insulin feedback, which is a major tumor-promoting side effect of PI3K inhibitors. Mechanistically, we found that AMPKα2 was ubiquitinated and, in turn, inhibited in its activatory phosphorylation by WWP1, whereas WWP1 inhibition facilitated AMPKα2 activity in the muscle to compensate for the reduction in glucose uptake observed upon PI3K inhibition. Thus, our identification of the cell-autonomous and systemic roles of WWP1 inhibition expands the therapeutic potential of PI3K inhibitors and reveals new avenues of combination cancer therapy.
Assuntos
Neoplasias da Mama/enzimologia , Neoplasias Mamárias Experimentais/enzimologia , Proteínas de Neoplasias/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Feminino , Humanos , Células MCF-7 , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/genética , Camundongos , Camundongos Knockout , Camundongos Nus , Camundongos SCID , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/efeitos adversos , Ubiquitina-Proteína Ligases/genéticaRESUMO
The architecture of chromatin regulates eukaryotic cell states by controlling transcription factor access to sites of gene regulation. Here we describe a dual transposase-peroxidase approach, integrative DNA and protein tagging (iDAPT), which detects both DNA (iDAPT-seq) and protein (iDAPT-MS) associated with accessible regions of chromatin. In addition to direct identification of bound transcription factors, iDAPT enables the inference of their gene regulatory networks, protein interactors and regulation of chromatin accessibility. We applied iDAPT to profile the epigenomic consequences of granulocytic differentiation of acute promyelocytic leukemia, yielding previously undescribed mechanistic insights. Our findings demonstrate the power of iDAPT as a platform for studying the dynamic epigenomic landscapes and their transcription factor components associated with biological phenomena and disease.
Assuntos
Cromatina/metabolismo , DNA/genética , Regulação da Expressão Gênica/genética , Histonas/metabolismo , Leucemia Promielocítica Aguda/genética , Redes Reguladoras de Genes , Humanos , Leucemia Promielocítica Aguda/patologia , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Patients with PTEN hamartoma tumor syndrome (PHTS) have germline mutations in the tumor-suppressor gene encoding phosphatase and tensin homologue (PTEN). Such mutations have been associated with a hereditary predisposition to multiple types of cancer, including the Cowden syndrome. However, a majority of patients who have PHTS-related phenotypes have tested negative for PTEN mutations. In a previous study, we found that the E3 ubiquitin ligase WWP1 negatively regulates the function of PTEN. METHODS: In a prospective cohort study conducted from 2005 through 2015, we enrolled 431 patients with wild-type PTEN who met at least the relaxed diagnostic criteria of the International Cowden Consortium. Patients were scanned for WWP1 germline variants. We used the Cancer Genome Atlas (TCGA) data set as representative of apparently sporadic cancers and the Exome Aggregation Consortium data set excluding TCGA (non-TCGA ExAC) and the noncancer Genome Aggregation Database (gnomAD) as representative of population controls without a reported cancer diagnosis. We established both in vitro and murine in vivo models to functionally characterize representative WWP1 variants. RESULTS: The existence of germline WWP1 variants was first established in a family with wild-type PTEN who had oligopolyposis and early-onset colon cancers. A validation series indicated that WWP1 germline variants occurred in 5 of 126 unrelated patients (4%) with oligopolyposis as a predominant phenotype. Germline WWP1 variants, particularly the WWP1 K740N and N745S alleles, were enriched in patients who did not have PHTS but had prevalent sporadic cancers, including PTEN-related cancer types in TCGA (odds ratio, 1.5; 95% confidence interval, 1.1 to 2.1; P = 0.01). The prioritized WWP1 variants resulted in gain-of-function effects, which led to aberrant enzymatic activation with consequent PTEN inactivation, thereby triggering hyperactive growth-promoting PI3K signaling in cellular and murine models. CONCLUSIONS: In this study involving patients with disorders resulting in a predisposition to the development of multiple malignant neoplasms without PTEN germline mutations, we confirmed the function of WWP1 as a cancer-susceptibility gene through direct aberrant regulation of the PTEN-PI3K signaling axis. (Funded by the National Institutes of Health and others.).
Assuntos
Mutação com Ganho de Função , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Síndrome do Hamartoma Múltiplo/genética , PTEN Fosfo-Hidrolase/genética , Ubiquitina-Proteína Ligases/genética , Adulto , Animais , Modelos Animais de Doenças , Feminino , Variação Genética , Humanos , Masculino , Camundongos , Camundongos Mutantes , Linhagem , Estudos ProspectivosRESUMO
Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is one of the most frequently mutated, deleted, and functionally inactivated tumor suppressor genes in human cancer. PTEN is found mutated both somatically and in the germline of patients with PTEN hamartoma tumor syndrome (PHTS). PTEN encodes a dual lipid and protein phosphatase that dephosphorylates the lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3), in turn negatively regulating the oncogenic PI3K-AKT pathway, a key proto-oncogenic player in cancer development and progression. Because of importance of PTEN in tumorigenesis, a large number of sophisticated genetically engineered mouse models (GEMMs) has been designed to elucidate the underlying mechanisms by which the "PTEN pathway" promotes tumorigenesis, while simultaneously providing a well-tailored system for the identification of novel therapies and offering platforms for new drug discoveries. This review summarizes the major cancer mouse models through which the PTEN pathway has been genetically deconstructed, and outlines the rapid development of GEMMs toward more detailed functional and tissue-specific analysis.
Assuntos
Modelos Animais de Doenças , Neoplasias/metabolismo , PTEN Fosfo-Hidrolase/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Carcinogênese/genética , Síndrome do Hamartoma Múltiplo/metabolismo , Síndrome do Hamartoma Múltiplo/patologia , Humanos , Camundongos , Mutação , Neoplasias/patologia , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de SinaisRESUMO
The function of PTEN in the cytoplasm largely depends on its lipid-phosphatase activity, though which it antagonizes the PI3K-AKT oncogenic pathway. However, molecular mechanisms underlying the role of PTEN in the nucleus remain largely elusive. Here, we report that DNA double-strand breaks (DSB) promote PTEN interaction with MDC1 upon ATM-dependent phosphorylation of T/S398-PTEN. Importantly, DNA DSBs enhance NSD2 (MMSET/WHSC1)-mediated dimethylation of PTEN at K349, which is recognized by the tudor domain of 53BP1 to recruit PTEN to DNA-damage sites, governing efficient repair of DSBs partly through dephosphorylation of γH2AX. Of note, inhibiting NSD2-mediated methylation of PTEN, either through expressing methylation-deficient PTEN mutants or through inhibiting NSD2, sensitizes cancer cells to combinatorial treatment with a PI3K inhibitor and DNA-damaging agents in both cell culture and in vivo xenograft models. Therefore, our study provides a novel molecular mechanism for PTEN regulation of DSB repair in a methylation- and protein phosphatase-dependent manner. SIGNIFICANCE: NSD2-mediated dimethylation of PTEN is recognized by the 53BP1 tudor domain to facilitate PTEN recruitment into DNA-damage sites, governing efficient repair of DNA DSBs. Importantly, inhibiting PTEN methylation sensitizes cancer cells to combinatorial treatment with a PI3K inhibitor combined with DNA-damaging agents in both cell culture and in vivo xenograft models.This article is highlighted in the In This Issue feature, p. 1143.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Neoplasias/genética , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Repressoras/metabolismo , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Feminino , Células HCT116 , Humanos , Metilação , Camundongos , Células NIH 3T3 , Neoplasias/metabolismo , PTEN Fosfo-Hidrolase/química , Fosforilação , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismoRESUMO
Activation of tumor suppressors for the treatment of human cancer has been a long sought, yet elusive, strategy. PTEN is a critical tumor suppressive phosphatase that is active in its dimer configuration at the plasma membrane. Polyubiquitination by the ubiquitin E3 ligase WWP1 (WW domain-containing ubiquitin E3 ligase 1) suppressed the dimerization, membrane recruitment, and function of PTEN. Either genetic ablation or pharmacological inhibition of WWP1 triggered PTEN reactivation and unleashed tumor suppressive activity. WWP1 appears to be a direct MYC (MYC proto-oncogene) target gene and was critical for MYC-driven tumorigenesis. We identified indole-3-carbinol, a compound found in cruciferous vegetables, as a natural and potent WWP1 inhibitor. Thus, our findings unravel a potential therapeutic strategy for cancer prevention and treatment through PTEN reactivation.
Assuntos
Anticarcinógenos/farmacologia , Indóis/farmacologia , Neoplasias/tratamento farmacológico , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Anticarcinógenos/uso terapêutico , Carcinogênese/efeitos dos fármacos , Células HEK293 , Humanos , Indóis/uso terapêutico , Masculino , Neoplasias/metabolismo , PTEN Fosfo-Hidrolase/genética , Multimerização Proteica , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/efeitos dos fármacosRESUMO
PTEN is a potent tumour suppressor, and its loss of function is frequently observed in both heritable and sporadic cancers. PTEN has phosphatase-dependent and phosphatase-independent (scaffold) activities in the cell and governs a variety of biological processes, including maintenance of genomic stability, cell survival, migration, proliferation and metabolism. Even a subtle decrease in PTEN levels and activity results in cancer susceptibility and favours tumour progression. Regulation of PTEN has therefore emerged as a subject of intense research in tumour biology. Recent discoveries, including the existence of distinct PTEN isoforms and the ability of PTEN to form dimers, have brought to light new modes of PTEN function and regulation. These milestone findings have in turn opened new therapeutic avenues for cancer prevention and treatment through restoration of PTEN tumour suppressor activity.
Assuntos
Neoplasias/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Progressão da Doença , Humanos , Neoplasias/patologiaRESUMO
Resistance to chemotherapy plays a significant role in cancer mortality. To identify genetic units affecting sensitivity to cytarabine, the mainstay of treatment for acute myeloid leukemia (AML), we developed a comprehensive and integrated genome-wide platform based on a dual protein-coding and non-coding integrated CRISPRa screening (DICaS). Putative resistance genes were initially identified using pharmacogenetic data from 760 human pan-cancer cell lines. Subsequently, genome scale functional characterization of both coding and long non-coding RNA (lncRNA) genes by CRISPR activation was performed. For lncRNA functional assessment, we developed a CRISPR activation of lncRNA (CaLR) strategy, targeting 14,701 lncRNA genes. Computational and functional analysis identified novel cell-cycle, survival/apoptosis, and cancer signaling genes. Furthermore, transcriptional activation of the GAS6-AS2 lncRNA, identified in our analysis, leads to hyperactivation of the GAS6/TAM pathway, a resistance mechanism in multiple cancers including AML. Thus, DICaS represents a novel and powerful approach to identify integrated coding and non-coding pathways of therapeutic relevance.
Assuntos
Sistemas CRISPR-Cas , Resistencia a Medicamentos Antineoplásicos , Genoma Humano , RNA Longo não Codificante/genética , Animais , Citarabina/farmacologia , Feminino , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Células HEK293 , Células HL-60 , Humanos , Células K562 , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Masculino , Camundongos , Farmacogenética , Proteínas/genética , RNA/análise , RNA Mensageiro/genética , Transdução de SinaisRESUMO
Lipids, either endogenously synthesized or exogenous, have been linked to human cancer. Here we found that PML is frequently co-deleted with PTEN in metastatic human prostate cancer (CaP). We demonstrated that conditional inactivation of Pml in the mouse prostate morphs indolent Pten-null tumors into lethal metastatic disease. We identified MAPK reactivation, subsequent hyperactivation of an aberrant SREBP prometastatic lipogenic program, and a distinctive lipidomic profile as key characteristic features of metastatic Pml and Pten double-null CaP. Furthermore, targeting SREBP in vivo by fatostatin blocked both tumor growth and distant metastasis. Importantly, a high-fat diet (HFD) induced lipid accumulation in prostate tumors and was sufficient to drive metastasis in a nonmetastatic Pten-null mouse model of CaP, and an SREBP signature was highly enriched in metastatic human CaP. Thus, our findings uncover a prometastatic lipogenic program and lend direct genetic and experimental support to the notion that a Western HFD can promote metastasis.
Assuntos
Lipogênese/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas de Ligação a Elemento Regulador de Esterol/fisiologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Humanos , Masculino , Redes e Vias Metabólicas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Metástase Neoplásica , Células PC-3 , PTEN Fosfo-Hidrolase/genética , Neoplasias da Próstata/metabolismo , Proteínas de Ligação a Elemento Regulador de Esterol/genéticaRESUMO
The novel compounds NSC745885 and NSC757963 developed at our laboratory were tested against a panel of 60 cancer cell lines at the National Cancer Institute, USA, and a panel of 39 cancer cell lines at the Japanese Foundation of Cancer Research. Both compounds demonstrated selective unique multi-log differential patterns of activity, with GI50 values in the sub-micro molar range against cancer cells rather than normal cardiac cells. NSC757963 showed high selectivity towards the leukemia subpanel. Activities of both compounds strongly correlated to expression of NFKB1 and CSNK2B genes, implying that they may inhibit the NF-κB pathway. Immunocytochemical microscopy of OVCAR-3 cells showed clear cytosolic accumulation of the NF-κB p65 subunit following treatment. Western blotting showed dose dependent inhibition of the nuclear expression of the NF-κB p65 subunit with subsequent accumulation in the cytosol following treatment. Docking experiments showed binding of both compounds to the NF-κB activator IKKß subunit preventing its translocation to the nucleus. Collectively, these results confirm the ability of our compounds to inhibit the constitutively active NF-κB pathway of OVCAR-3 cells. Furthermore, COMPARE analysis indicated that the activity of NSC757963 is similar to the antituberculosis agent rifamycin SV, this was confirmed by testing the antimycobacterial activity of NSC757963 against Mycobacterium tuberculosis, results revealed potent activity suitable for use in clinical practice. Molecular properties and Lipinski's parameters predicted acceptable bioavailability properties with no indication of mutagenicity, tumorigenicity, irritability and reproductive effects. Oral absorption experiments using the human Caco-2 model showed high intestinal absorption of NSC745885 by passive transport mechanism with no intestinal efflux or active transport mechanisms. The unique molecular characterization as well as the illustrated anticancer spectra of activity and bioavailability properties warrant further development of our compounds and present a foundation brick in the pre-clinical investigations to implement such compounds in clinical practice.
Assuntos
Antineoplásicos/farmacologia , Antituberculosos/farmacologia , Regulação Neoplásica da Expressão Gênica , Tiadiazóis/farmacologia , Fator de Transcrição RelA/antagonistas & inibidores , Antineoplásicos/síntese química , Antituberculosos/síntese química , Disponibilidade Biológica , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Perfilação da Expressão Gênica , Humanos , Quinase I-kappa B/antagonistas & inibidores , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Absorção Intestinal/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Modelos Biológicos , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Transdução de Sinais , Tiadiazóis/síntese química , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismoRESUMO
The bioprospecting of potentially mixotrophic microalgae in a constructed wetland was conducted. A locally isolated microalga, Chlorella sp., was grown to determine the effect of temperature, aeration rate, and cultivation time on simultaneous biomass production and nutrient removal from piggery wastewater using central composite design (CCD). The most important variable for the biomass productivity of Chlorella sp. was aeration rate, while that for lipid content and nutrient removal efficiency was cultivation time. Total nitrogen (TN) and total phosphorus (TP) removal efficiencies were higher than that of chemical oxygen demand (COD) from piggery wastewater. The CCD results indicate that the highest biomass productivity (79.2 mg L(-1) d(-1)) and simultaneous nutrient removal efficiency (TN 80.9%, TP 99.2%, COD 74.5%) were obtained with a cultivation temperature of 25 °C, a cultivation time of 5 days, and an air aeration rate of 1.6 L L(-1) min(-1). Palmitic acid (C16:0) and linoleic acid (C18:2) were both abundant in Chlorella sp. cells under mixotrophic cultivation with piggery wastewater.
Assuntos
Biomassa , Chlorella , Eliminação de Resíduos Líquidos/métodos , Águas Residuárias/química , Criação de Animais Domésticos , Animais , Análise da Demanda Biológica de Oxigênio , Resíduos Industriais , Lipídeos/biossíntese , Microalgas , Nitrogênio/farmacologia , Fósforo , Suínos , Áreas AlagadasRESUMO
A series of sulfur-substituted anthra[1,2-c][1,2,5]thiadiazole-6,11-diones were synthesized and evaluated using the cell proliferations, apoptosis and NCI-60 cell panel assays. Also, the signaling pathways that account for their activities were investigated. Compounds 2, 3, 4a, 4d, 4f, 4i, 4k, 5b, 5c, 5d, 5f, 5g, 6b, 6c, 6d, 6e, 6g, 7a and 7g were selected by NCI. Among the tested compounds, 6g appeared to be the most active compound of this series that not only induced apoptosis in DU-145 cancer cells but also attenuated the ERK1/2 and p38 signaling pathways. All test compounds exhibited diverse cytostatic and cytotoxic activities that warrant further development as potential anticancer agents.
Assuntos
Antraquinonas/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Enxofre/farmacologia , Tiadiazóis/farmacologia , Antraquinonas/síntese química , Antraquinonas/química , Antineoplásicos/síntese química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Enxofre/química , Tiadiazóis/síntese química , Tiadiazóis/químicaRESUMO
Ubiquitin chains are formed as structurally distinct polymers via different linkages, and several chain types including K33-linkage remain uncharacterized. Here, we describe a role for K33-polyubiquitination in protein trafficking. We show that the Cullin 3 (Cul3) substrate adaptor KLHL20 is localized to the trans-Golgi network (TGN) and is important for post-Golgi trafficking by promoting the biogenesis of TGN-derived transport carriers. The Cul3-KLHL20 ubiquitin E3 ligase catalyzes a nondegradable, K33-linked polyubiquitination on coronin 7 (Crn7), which facilitates Crn7 targeting to TGN through a ubiquitin-dependent interaction with Eps15. Blockage of K33-chain formation, Crn7 ubiquitination, or disruption of Crn7-Eps15 interaction impairs TGN-pool F-actin assembly, a process essential for generating transport carriers. Enforced targeting of Crn7 to TGN bypasses the requirement of K33-ubiquitination for TGN-pool F-actin assembly and post-Golgi trafficking. Our study reveals a role of KLHL20-mediated K33-ubiquitination of Crn7 in post-Golgi transport and identifies a cellular recognition mechanism for this ubiquitin chain type.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Culina/metabolismo , Proteínas dos Microfilamentos/metabolismo , Transporte Proteico , Ubiquitina-Proteína Ligases/metabolismo , Actinas/genética , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Células COS , Proteínas de Transporte/genética , Linhagem Celular , Chlorocebus aethiops , Proteínas Culina/genética , Complexo de Golgi/metabolismo , Células HEK293 , Células HeLa , Humanos , Lisina/metabolismo , Proteínas dos Microfilamentos/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Rede trans-Golgi/metabolismoRESUMO
Metastasis is responsible for most cancer deaths but it remains a poorly understood process. Recent evidence has emerged that death-associated protein kinase (DAPK) is a candidate of metastasis suppressor. DAPK downregulation or inactivation has been observed in a number of metastatic cancers through epigenetic, transcriptional, post-transcriptional, or post-translational mechanism. In certain cases, DAPK downregulation correlates with metastatic recurrence. Animal studies further show that DAPK impedes both early-stage and late-stage metastatic process, which suggests that DAPK possesses multiple mechanisms to suppress metastasis. Cell-based studies revealed that DAPK mediates several types of cell death, including apoptosis, autophagic death and necrosis, depending on death stimuli and cell context. DAPK also regulates cytoskeleton proteins to mediate death-associated cell morphological alterations and to inhibit cell motility. Besides tumor cells, DAPK can influence on stromal cells to regulate their survival and functions. These effects likely all contribute to the metastasis suppressive role of DAPK. The detail molecular mechanisms of these anti-metastatic effects of DAPK are reviewed in this article.