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1.
Nat Commun ; 12(1): 1052, 2021 02 16.
Artículo en Inglés | MEDLINE | ID: mdl-33594070

RESUMEN

The parasitic protist Trypanosoma brucei is the causative agent of Human African Trypanosomiasis, also known as sleeping sickness. The parasite enters the blood via the bite of the tsetse fly where it is wholly reliant on glycolysis for the production of ATP. Glycolytic enzymes have been regarded as challenging drug targets because of their highly conserved active sites and phosphorylated substrates. We describe the development of novel small molecule allosteric inhibitors of trypanosome phosphofructokinase (PFK) that block the glycolytic pathway resulting in very fast parasite kill times with no inhibition of human PFKs. The compounds cross the blood brain barrier and single day oral dosing cures parasitaemia in a stage 1 animal model of human African trypanosomiasis. This study demonstrates that it is possible to target glycolysis and additionally shows how differences in allosteric mechanisms may allow the development of species-specific inhibitors to tackle a range of proliferative or infectious diseases.


Asunto(s)
Glucólisis/efectos de los fármacos , Fosfofructoquinasas/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Trypanosoma/enzimología , Tripanosomiasis Africana/metabolismo , Tripanosomiasis Africana/parasitología , Enfermedad Aguda , Regulación Alostérica/efectos de los fármacos , Animales , Células Hep G2 , Humanos , Concentración 50 Inhibidora , Estimación de Kaplan-Meier , Ratones , Parásitos/efectos de los fármacos , Fosfofructoquinasas/química , Fosfofructoquinasas/metabolismo , Unión Proteica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/uso terapéutico , Multimerización de Proteína , Relación Estructura-Actividad , Trypanosoma/efectos de los fármacos , Tripanosomiasis Africana/tratamiento farmacológico
2.
Theranostics ; 11(2): 649-664, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33391497

RESUMEN

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide epidemic of the lethal respiratory coronavirus disease (COVID-19), necessitating urgent development of specific and effective therapeutic tools. Among several therapeutic targets of coronaviruses, the spike protein is of great significance due to its key role in host invasion. Here, we report a potential anti-SARS-CoV-2 strategy based on the CRISPR-Cas13a system. Methods: A comprehensive set of bioinformatics methods, including sequence alignment, structural comparison, and molecular docking, was utilized to identify a SARS-CoV-2-spike(S)-specific segment. A tiling crRNA library targeting this specific RNA segment was designed, and optimal crRNA candidates were selected using in-silico methods. The efficiencies of the crRNA candidates were tested in human HepG2 and AT2 cells. Results: The most effective crRNA sequence inducing a robust cleavage effect on S and a potent collateral cleavage effect were identified. Conclusions: This study provides a rapid design pipeline for a CRISPR-Cas13a-based antiviral tool against SARS-CoV-2. Moreover, it offers a novel approach for anti-virus study even if the precise structures of viral proteins are indeterminate.


Asunto(s)
Antivirales/administración & dosificación , ARN Guia/genética , Glicoproteína de la Espiga del Coronavirus/genética , /virología , Sistemas CRISPR-Cas/genética , Biología Computacional , Evaluación Preclínica de Medicamentos , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Células Hep G2 , Humanos , Simulación del Acoplamiento Molecular , Homología de Secuencia de Aminoácido
3.
Nat Commun ; 12(1): 240, 2021 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-33431832

RESUMEN

CAR-T cell therapy targeting CD19 demonstrated strong activity against advanced B cell leukemia, however shows less efficacy against lymphoma with nodal dissemination. To target both B cell Non-Hodgkin's lymphoma (B-NHLs) and follicular T helper (Tfh) cells in the tumor microenvironment (TME), we apply here a chimeric antigen receptor (CAR) that recognizes human CXCR5 with high avidity. CXCR5, physiologically expressed on mature B and Tfh cells, is also highly expressed on nodal B-NHLs. Anti-CXCR5 CAR-T cells eradicate B-NHL cells and lymphoma-supportive Tfh cells more potently than CD19 CAR-T cells in vitro, and they efficiently inhibit lymphoma growth in a murine xenograft model. Administration of anti-murine CXCR5 CAR-T cells in syngeneic mice specifically depletes endogenous and malignant B and Tfh cells without unexpected on-target/off-tumor effects. Collectively, anti-CXCR5 CAR-T cells provide a promising treatment strategy for nodal B-NHLs through the simultaneous elimination of lymphoma B cells and Tfh cells of the tumor-supporting TME.


Asunto(s)
Linfocitos B/inmunología , Linfoma no Hodgkin/inmunología , Neoplasias/inmunología , Receptores CXCR5/metabolismo , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T/inmunología , Animales , Antígenos de Neoplasias/inmunología , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Células HEK293 , Células Hep G2 , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Ratones , Ensayos Antitumor por Modelo de Xenoinjerto
4.
Nat Commun ; 12(1): 98, 2021 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-33397935

RESUMEN

Glucose metabolism and innate immunity evolved side-by-side. It is unclear if and how the two systems interact with each other during hepatitis B virus (HBV) infections and, if so, which mechanisms are involved. Here, we report that HBV activates glycolysis to impede retinoic acid-inducible gene I (RIG-I)-induced interferon production. We demonstrate that HBV sequesters MAVS from RIG-I by forming a ternary complex including hexokinase (HK). Using a series of pharmacological and genetic approaches, we provide in vitro and in vivo evidence indicating that HBV suppresses RLR signaling via lactate dehydrogenase-A-dependent lactate production. Lactate directly binds MAVS preventing its aggregation and mitochondrial localization during HBV infection. Therefore, we show that HK2 and glycolysis-derived lactate have important functions in the immune escape of HBV and that energy metabolism regulates innate immunity during HBV infection.


Asunto(s)
Virus de la Hepatitis B/fisiología , Inmunidad Innata , Metaboloma , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Anaerobiosis , Animales , Células Cultivadas , Proteína 58 DEAD Box/metabolismo , Glucosa/metabolismo , Glucólisis , Células Hep G2 , Hepatocitos/metabolismo , Hepatocitos/patología , Hepatocitos/virología , Humanos , Evasión Inmune , Interferones/metabolismo , Ácido Láctico/metabolismo , Ratones Endogámicos C57BL , Modelos Biológicos , Transducción de Señal , Virión/metabolismo
5.
Nat Commun ; 12(1): 150, 2021 01 08.
Artículo en Inglés | MEDLINE | ID: mdl-33420011

RESUMEN

Novel bacterial type II topoisomerase inhibitors (NBTIs) stabilize single-strand DNA cleavage breaks by DNA gyrase but their exact mechanism of action has remained hypothetical until now. We have designed a small library of NBTIs with an improved DNA gyrase-binding moiety resulting in low nanomolar inhibition and very potent antibacterial activity. They stabilize single-stranded cleavage complexes and, importantly, we have obtained the crystal structure where an NBTI binds gyrase-DNA in a single conformation lacking apparent static disorder. This directly proves the previously postulated NBTI mechanism of action and shows that they stabilize single-strand cleavage through asymmetric intercalation with a shift of the scissile phosphate. This crystal stucture shows that the chlorine forms a halogen bond with the backbone carbonyls of the two symmetry-related Ala68 residues. To the best of our knowledge, such a so-called symmetrical bifurcated halogen bond has not been identified in a biological system until now.


Asunto(s)
Antibacterianos/farmacología , Cloro/metabolismo , Girasa de ADN/metabolismo , Inhibidores de Topoisomerasa II/farmacología , Alanina/química , Alanina/metabolismo , Antibacterianos/química , Cristalografía por Rayos X , Girasa de ADN/química , ADN-Topoisomerasas de Tipo II , ADN de Cadena Simple/metabolismo , Diseño de Fármacos , Canal de Potasio ERG1/metabolismo , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Células Hep G2 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Concentración 50 Inhibidora , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Proteínas de Unión a Poli-ADP-Ribosa/antagonistas & inhibidores , Quinolinas/química , Quinolinas/farmacología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/enzimología , Inhibidores de Topoisomerasa II/química
6.
Anticancer Res ; 41(2): 773-782, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33517282

RESUMEN

BACKGROUND/AIM: Oncolytic adenoviruses (OAds) have attracted much attention as novel anticancer therapeutics. The proper design of an expression cassette containing the E1A gene, which is indispensable for self-replication of the Ad genome, is crucial for efficient tumor cell-specific infection of an OAd. Various types of oncolytic adenoviruses (OAds) possessing different types of the E1A gene expression cassettes have been developed, but their oncolytic activities and safety profiles have not been systematically evaluated. Herein we examined the oncolytic activities and safety profiles of five types of OAds possessing different types of the E1A gene expression cassette in order to optimize the E1A gene expression cassette for development of an efficient and safe OAd. MATERIALS AND METHODS: We prepared five types of OAds containing different types of E1 gene expression cassettes, and examined the oncolytic activities and safety profiles of the OAds. RESULTS: Among the OAds examined, OAd-Δ24, which had a 24-bp deletion in the E1A gene, mediated the most efficient oncolytic activities against the human tumor cell lines, although OAd-Δ24 showed slightly higher cytotoxicity to normal human cells than the other OAds. CONCLUSION: These results provide important clues for the development of safe and efficient OAds.


Asunto(s)
Adenoviridae/fisiología , Proteínas E1A de Adenovirus/genética , Eliminación de Secuencia , Survivin/genética , Telomerasa/genética , Adenoviridae/genética , Apoptosis , Línea Celular Tumoral , Expresión Génica , Células HCT116 , Células HEK293 , Células HeLa , Células Hep G2 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Células MCF-7 , Viroterapia Oncolítica , Virus Oncolíticos/genética , Virus Oncolíticos/fisiología , Regiones Promotoras Genéticas , Replicación Viral
7.
Cell Prolif ; 54(2): e12978, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33393114

RESUMEN

OBJECTIVES: The final stage of liver development is the production of hepatocytes and cholangiocytes (biliary epithelial cells) from bipotent hepatic progenitor cells. We used HepaRG cells, which are bipotent and able to differentiate into both hepatocytes and cholangiocytes, as a model to study the action of a novel lncRNA (lnc-RHL) and its role in the regulation of bipotency leading to hepatocytes and cholangiocytes. MATERIALS AND METHODS: Differentiation of HepaRG cells was assessed by marker expression and morphology which revealed their ability to differentiate into hepatocytes and cholangiocytes (modelling the behaviour of hepatoblasts in vivo). Using a qRT-PCR and RACE, we cloned a novel lncRNA (lnc-RHL; regulator of hepatic lineages) that is upregulated upon HepaRG differentiation. Using inducible knockdown of lnc-RHL concurrently with differentiation, we show that lnc-RHL is required for proper HepaRG cell differentiation resulting in diminution of the hepatocyte lineage. RESULTS: Here, we report the discovery of lnc-RHL, a spliced and polyadenylated 670 base lncRNA expressed from the 11q23.3 apolipoprotein gene cluster. lnc-RHL expression is confined to hepatic lineages and is upregulated when bipotent HepaRG cells are caused to differentiate. HepaRG cells made deficient for lnc-RHL have reduced ability to differentiate into hepatocytes, but retain their ability to differentiate into cholangiocytes. CONCLUSIONS: Deficiency for lnc-RHL in HepaRG cells converts them from bipotent progenitor cells to unipotent progenitor cells with impaired ability to yield hepatocytes. We conclude that lnc-RHL is a key regulator of bipotency in HepaRG cells.


Asunto(s)
Diferenciación Celular/genética , Hepatocitos/metabolismo , ARN Largo no Codificante/metabolismo , Apolipoproteínas/genética , Apolipoproteínas/metabolismo , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula , Niño , Cromosomas Humanos Par 11 , Doxorrubicina/farmacología , Femenino , Células Hep G2 , Factor Nuclear 4 del Hepatocito/genética , Factor Nuclear 4 del Hepatocito/metabolismo , Hepatocitos/citología , Humanos , Hígado/metabolismo , Masculino , Familia de Multigenes , Interferencia de ARN , ARN Largo no Codificante/antagonistas & inhibidores , ARN Largo no Codificante/genética , ARN Interferente Pequeño/metabolismo , Células Madre/citología , Células Madre/metabolismo , Adulto Joven
8.
Molecules ; 26(2)2021 Jan 10.
Artículo en Inglés | MEDLINE | ID: mdl-33435264

RESUMEN

Diabetes mellitus (DM) is a complex disease which currently affects more than 460 million people and is one of the leading cause of death worldwide. Its development implies numerous metabolic dysfunctions and the onset of hyperglycaemia-induced chronic complications. Multiple ligands can be rationally designed for the treatment of multifactorial diseases, such as DM, with the precise aim of simultaneously controlling multiple pathogenic mechanisms related to the disease and providing a more effective and safer therapeutic treatment compared to combinations of selective drugs. Starting from our previous findings that highlighted the possibility to target both aldose reductase (AR) and protein tyrosine phosphatase 1B (PTP1B), two enzymes strictly implicated in the development of DM and its complications, we synthesised 3-(5-arylidene-4-oxothiazolidin-3-yl)propanoic acids and analogous 2-butenoic acid derivatives, with the aim of balancing the effectiveness of dual AR/PTP1B inhibitors which we had identified as designed multiple ligands (DMLs). Out of the tested compounds, 4f exhibited well-balanced AR/PTP1B inhibitory effects at low micromolar concentrations, along with interesting insulin-sensitizing activity in murine C2C12 cell cultures. The SARs here highlighted along with their rationalization by in silico docking experiments into both target enzymes provide further insights into this class of inhibitors for their development as potential DML antidiabetic candidates.


Asunto(s)
Aldehído Reductasa/antagonistas & inhibidores , Diabetes Mellitus/tratamiento farmacológico , Inhibidores Enzimáticos , Hipoglucemiantes , Proteína Tirosina Fosfatasa no Receptora Tipo 1/antagonistas & inhibidores , Aldehído Reductasa/metabolismo , Animales , Diabetes Mellitus/enzimología , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Células Hep G2 , Humanos , Hipoglucemiantes/química , Hipoglucemiantes/farmacología , Ligandos , Ratones , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , Relación Estructura-Actividad
9.
Nat Commun ; 12(1): 476, 2021 01 20.
Artículo en Inglés | MEDLINE | ID: mdl-33473107

RESUMEN

Endonuclease G (ENDOG), a mitochondrial nuclease, is known to participate in many cellular processes, including apoptosis and paternal mitochondrial elimination, while its role in autophagy remains unclear. Here, we report that ENDOG released from mitochondria promotes autophagy during starvation, which we find to be evolutionally conserved across species by performing experiments in human cell lines, mice, Drosophila and C. elegans. Under starvation, Glycogen synthase kinase 3 beta-mediated phosphorylation of ENDOG at Thr-128 and Ser-288 enhances its interaction with 14-3-3γ, which leads to the release of Tuberin (TSC2) and Phosphatidylinositol 3-kinase catalytic subunit type 3 (Vps34) from 14-3-3γ, followed by mTOR pathway suppression and autophagy initiation. Alternatively, ENDOG activates DNA damage response and triggers autophagy through its endonuclease activity. Our results demonstrate that ENDOG is a crucial regulator of autophagy, manifested by phosphorylation-mediated interaction with 14-3-3γ, and its endonuclease activity-mediated DNA damage response.


Asunto(s)
Autofagia/fisiología , Daño del ADN/fisiología , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Transducción de Señal/fisiología , Serina-Treonina Quinasas TOR/metabolismo , Proteínas 14-3-3/metabolismo , Animales , Apoptosis , Caenorhabditis elegans , Línea Celular , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Drosophila , Técnicas de Inactivación de Genes , Células Hep G2 , Humanos , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Fosforilación , Transcriptoma , Proteína 2 del Complejo de la Esclerosis Tuberosa/metabolismo
10.
Life Sci ; 269: 119028, 2021 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-33444618

RESUMEN

BACKGROUND: Hepatocellular carcinoma (HCC) is the second most common cancer-related death in the world. No effective curative option exists for the treatment of HCC. The available drugs exhibit severe toxic effects and low therapeutic index. AIM: This work aimed to examine different monocationic arylthiophene derivatives for possible use as chemotherapeutic agents against HCC. METHODS: The IC50 values for the compounds were determined. The mechanism of cytotoxicity was further investigated using different methods. RESULTS: Compound 2j proved to retain the highest cytotoxicity in comparison to as a positive control. The selectivity index of compound 2j revealed the safety to normal cells. Moreover, compound 2j was able to inhibit HepG2 cells´ migration and division. The anticancer effect of compound 2j was found to be partially via cell cycle arrest, activation of the tumour suppressor p53 protein, and induction of apoptosis via both intrinsic and extrinsic pathways. Compound 2j has a potential sensitization activity and significantly reduced the IC50 values for the anticancer drugs doxorubicin, cisplatin, and taxol. CONCLUSION: The tested arylthiophenes showed a potent cytotoxicity against HepG2 cells and were safe to normal cells. The most active compound 2j was found to be able to inhibit cell division and migration and also to induce apoptosis. Compound 2j also proved to have a sensitization effect on standard anticancer drugs.


Asunto(s)
Antineoplásicos/uso terapéutico , Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Tiofenos/uso terapéutico , Antineoplásicos/química , Antineoplásicos/farmacología , Carcinoma Hepatocelular/patología , Caspasas/metabolismo , Cationes , Puntos de Control del Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cisplatino/farmacología , Doxorrubicina/farmacología , Activación Enzimática/efectos de los fármacos , Fase G2/efectos de los fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/patología , Mitosis/efectos de los fármacos , Paclitaxel/farmacología , Relación Estructura-Actividad , Tiofenos/química , Tiofenos/farmacología , Ensayo de Tumor de Célula Madre , Cicatrización de Heridas/efectos de los fármacos
11.
BMC Infect Dis ; 21(1): 56, 2021 Jan 12.
Artículo en Inglés | MEDLINE | ID: mdl-33435880

RESUMEN

BACKGROUND: Hepatitis B virus (HBV) infection is a high-risk factor of hepatocellular carcinoma (HCC). Cellular immune responses are essential for HCC development, and the CD4+ and CD8+ T subtypes are identified as the primary anti-tumor immune cells. In the study, we investigated the effect and mechanism of amygdalin in the cellular immune response in HBV-related HCC and HCC progression. METHODS: The cell proliferation was examined by MTT analysis. Cells metastasis ability was detected by Invasion and migration assays. Quantification of apoptotic cells was performed with Flow cytometer assay. The protein levels of p-STAT3, STAT3, p-JAK2, JAK2, caspase-3, cleaved caspase-3 were detected by performing immunoblotting assays. RESULTS: We demonstrate that amygdalin treatment could rescue the HBV-T cell viability and IFN-γ and TNF-αproduction. In HBV-T cells, the MFI levels of CD8+ are lower than that in NC-T cells. Moreover, the phosphorylation levels of STAT3 and JAK2 are higher in HBV-T cells, compared to those in NC-T cells, and then reduced by amygdalin treatment. Co-culture with HBV-T cells could reduce IFN-γ and TNF-α, production while increase IL-6 and IL-10 production in HepG2.2.15 cells; these alterations could be partially reversed by amygdalin pretreatment. Finally, co-culture with HBV-T cells significantly promoted the cell viability, inhibited the apoptosis, and promoted the migration of HepG2.2.15 cells, and these alterations could be partially reversed by amygdalin treatment. CONCLUSION: Our findings provide a rationale for further studies on the functions and mechanism of amygdalin inhibiting HBV-related HCC cell proliferation, invasion, and migration via T cell-mediated tumor immunity.


Asunto(s)
Amigdalina/farmacología , Carcinoma Hepatocelular/sangre , Carcinoma Hepatocelular/virología , Progresión de la Enfermedad , Hepatitis B/complicaciones , Janus Quinasa 2/metabolismo , Neoplasias Hepáticas/sangre , Neoplasias Hepáticas/virología , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Anciano , Amigdalina/metabolismo , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Técnicas de Cocultivo , Citocinas/metabolismo , Femenino , Células Hep G2 , Hepatitis B/virología , Virus de la Hepatitis B/fisiología , Humanos , Masculino , Persona de Mediana Edad , Linfocitos T/metabolismo , Linfocitos T/virología
12.
Anticancer Res ; 41(2): 645-660, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33517269

RESUMEN

BACKGROUND/AIM: Sorafenib is a multikinase inhibitor approved as a first-line therapy for hepatocellular carcinoma. This study examined the sorafenib resistance mechanism. MATERIALS AND METHODS: Hepatoma HepG2 cells were exposed to sorafenib, and the biological activity of the conditioned media was analyzed using cell proliferation/apoptosis assays, multiplex immunoassays, ELISA, and western blot analyses. The effect of urokinase-type plasminogen activator (uPA) inhibitors or siRNA-mediated gene silencing was examined in culture experiments and a mouse xenograft tumor model. RESULTS: Sorafenib increased uPA secretion, which was abrogated by an Akt inhibitor. The growth-inhibitory effect of sorafenib was significantly enhanced by the uPA inhibitors UK122 and amiloride. Sorafenib-induced apoptosis was increased 2.4-fold in uPA siRNA-transduced cells (p<0.05). Combined therapy with sorafenib and amiloride significantly decreased tumor volumes [mean volume: 759 mm3 (sorafenib) vs. 283 mm3 (sorafenib plus amiloride), p<0.05]. CONCLUSION: uPA may play a critical role in sorafenib resistance.


Asunto(s)
Antineoplásicos/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Resistencia a Antineoplásicos , Neoplasias Hepáticas/tratamiento farmacológico , Proteínas de la Membrana/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Sorafenib/farmacología , Amilorida/farmacología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
13.
Anticancer Res ; 41(2): 699-706, 2021 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-33517274

RESUMEN

BACKGROUND/AIM: Benzimidazoles are considered potential anticancer candidates. We herein studied the anticancer activity of CCL299, 4-(1H-1,3-benzodiazol-1-yl) benzonitrile. MATERIALS AND METHODS: In this in vitro study, we used ATP assays, flow cytometry, western blotting, and caspase-3/7 assays to evaluate the effects of CCL299 on cell proliferation, cell-cycle progression and apoptosis. RESULTS: ATP assays showed that CCL299 inhibited cell growth in the hepatoblastoma cell line HepG2 and the cervical cancer cell line HEp-2, without exhibiting cytotoxic effects on non-cancer cells and TIG-1-20 fibroblasts. Flow cytometry, western blotting, and caspase-3/7 assays revealed that CCL299 induced G1-phase cell-cycle arrest followed by apoptosis that was associated with up-regulation of p-p53 (Ser15) and p21 expression and the down-regulation of p-CDK2 (Thr160) expression. CONCLUSION: CCL299 exhibits cytotoxic activity via apoptosis in a subset of cancer cells, and should be considered as a promising anticancer candidate agent.


Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Bencimidazoles/farmacología , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Células A549 , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Femenino , Células HeLa , Células Hep G2 , Humanos , Neoplasias/metabolismo , Neoplasias/patología , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo
14.
Nat Commun ; 12(1): 269, 2021 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-33431834

RESUMEN

Chemical matter is needed to target the divergent biology associated with the different life cycle stages of Plasmodium. Here, we report the parallel de novo screening of the Medicines for Malaria Venture (MMV) Pandemic Response Box against Plasmodium asexual and liver stage parasites, stage IV/V gametocytes, gametes, oocysts and as endectocides. Unique chemotypes were identified with both multistage activity or stage-specific activity, including structurally diverse gametocyte-targeted compounds with potent transmission-blocking activity, such as the JmjC inhibitor ML324 and the antitubercular clinical candidate SQ109. Mechanistic investigations prove that ML324 prevents histone demethylation, resulting in aberrant gene expression and death in gametocytes. Moreover, the selection of parasites resistant to SQ109 implicates the druggable V-type H+-ATPase for the reduced sensitivity. Our data therefore provides an expansive dataset of compounds that could be redirected for antimalarial development and also point towards proteins that can be targeted in multiple parasite life cycle stages.


Asunto(s)
Antimaláricos/uso terapéutico , Descubrimiento de Drogas , Malaria/tratamiento farmacológico , Malaria/transmisión , Pandemias , Aedes/parasitología , Animales , Antimaláricos/química , Antimaláricos/farmacología , Análisis por Conglomerados , Relación Dosis-Respuesta a Droga , Células Hep G2 , Humanos , Concentración 50 Inhibidora , Estadios del Ciclo de Vida/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/parasitología , Malaria/epidemiología , Masculino , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/crecimiento & desarrollo
15.
Toxicon ; 190: 58-64, 2021 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-33338448

RESUMEN

The metabolic toxicity of Fumonisin B1 (FB1) converges at the accumulation of sphingoid bases and reduced ceramide levels. Several studies have alluded to a hypercholesterolemic endpoint after FB1 exposure, yet the molecular mechanisms remain elusive. Cell surface receptors are important regulators of cholesterol metabolism by regulating influx of lipids and efflux of cholesterol. Western blot analysis showed that FB1 elevates the expression of ABCA1 (a cholesterol efflux promoter) in an LXR dependent mechanism. We further highlight the potential role of PCSK9 in the degradation of LDL receptor. These data provide important evidence for the mechanism underlying hypercholesterolemia in FB1 treated models. The disruption of lipid homeostasis by FB1 is beginning to shift away from canonical ceramide synthase inhibition, and this changed perspective may shed light on diseases caused by dysregulated cholesterol metabolism such as cancer initiation and promotion.


Asunto(s)
Fumonisinas/toxicidad , Receptores de LDL/metabolismo , Transportador 1 de Casete de Unión a ATP , Animales , Células Hep G2 , Humanos , Proproteína Convertasa 9
16.
Metabolism ; 114: 154349, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-32888949

RESUMEN

BACKGROUND: The functions of Acly in regulating nonalcoholic fatty liver disease (NAFLD) have been identified; however, the dynamic control of Acly expression under the pathological state of metabolic disorders has not been fully elucidated. Previous studies reported an ubiquitin-proteasome-mediated degradation of Acly, but the mechanism is still largely unknown. METHODS: Co-IP-based mass spectrum (MS/MS) assays were performed in HepG2 and Hepa1-6 hepatocytes and mouse liver tissue. The protein-protein interaction and ubiquitin modification of Hrd1 on Acly were confirmed by co-IP based immuno-blotting. Acetyl-CoA levels and lipogenesis rates were determined. The roles of Hrd1 on NAFLD and insulin resistance were tested by adenovirus-mediated overexpression in db/db mice or in separated primary hepatocytes. RESULTS: Hrd1, a subunit of the endoplasmic reticulum-associated degradation (ERAD) complex, interacted with and ubiquitinated Acly, thereby reducing its protein level. Hrd1 suppressed the acetyl-CoA level and inhibited lipogenesis through an Acly-dependent pathway. The expression of hepatic Hrd1 was negatively associated with NAFLD, whereas overexpression of Hrd1 ameliorated hepatic steatosis and enhanced insulin sensitivity, both in db/db mice and in separated mouse primary hepatocytes. CONCLUSIONS: Our results suggest that Acly, a master enzyme that regulates lipogenesis, is degraded by Hrd1 through ubiquitin modification. The activation of Hrd1 in hepatocytes might therefore represent a strategic approach for NAFLD therapy.


Asunto(s)
ATP Citrato (pro-S)-Liasa/metabolismo , Hígado/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Obesidad/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/fisiología , Animales , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Resistencia a la Insulina/fisiología , Lipogénesis/fisiología , Ratones , Espectrometría de Masas en Tándem
17.
Phytomedicine ; 81: 153416, 2021 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-33321412

RESUMEN

BACKGROUND: Bulbine natalensis is an African-folk medicinal plant used as a dietary supplement for enhancing sexual function and muscle strength in males by presumably boosting testosterone levels, but no scientific information is available about the possible herb-drug interaction (HDI) risk when bulbine-containing supplements are concomitantly taken with prescription drugs. PURPOSE: This study was aimed to investigate the HDI potential of B. natalensis in terms of the pregnane X receptor (PXR)-mediated induction of major drug-metabolizing cytochrome P450 enzyme isoforms (i.e., CYP3A4 and CYP2C9) as well as inhibition of their catalytic activity. RESULTS: We found that a methanolic extract of B. natalensis activated PXR (EC50 6.2 ± 0.6 µg/ml) in HepG2 cells resulting in increased mRNA expression of CYP3A4 (2.40 ± 0.01 fold) and CYP2C9 (3.37 ± 0.3 fold) at 30 µg/ml which was reflected in increased activites of the two enzymes. Among the constituents of B. natalensis, knipholone was the most potent PXR activator (EC50 0.3 ± 0.1 µM) followed by bulbine-knipholone (EC50 2.0 ± 0.5 µM), and 6'-methylknipholone (EC50 4.0 ± 0.5 µM). Knipholone was also the most effective in increasing the expression of CYP3A4 (8.47 ± 2.5 fold) and CYP2C9 (2.64 ± 0.3 fold) at 10 µM. Docking studies further confirmed the unique structural features associated with knipholones for their superior inductive potentials in the activation of PXR compared to other anthraquinones. In a CYP inhibition assay, the methanolic extract as well as the anthraquinones strongly inhibited the catalytic activity of CYP2C9 while, inhibition of CYP3A4 was weak. CONCLUSIONS: These results suggest that consumption of B. natalensis may pose a potential risk for HDI if taken with conventional medications that are substrates of CYP3A4 and CYP2C9 and may contribute to unanticipated adverse reactions or therapeutic failures. Further studies are warranted to validate these findings and establish their clinical relevancy.


Asunto(s)
Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP3A/metabolismo , Suplementos Dietéticos , Interacciones de Hierba-Droga , Xanthorrhoeaceae/química , Inhibidores del Citocromo P-450 CYP2C9/química , Inhibidores del Citocromo P-450 CYP2C9/farmacología , Inhibidores del Citocromo P-450 CYP3A/química , Inhibidores del Citocromo P-450 CYP3A/farmacología , Suplementos Dietéticos/efectos adversos , Células Hep G2 , Humanos , Masculino , Simulación del Acoplamiento Molecular , Extractos Vegetales/química , Extractos Vegetales/farmacología , Plantas Medicinales/química , Receptor X de Pregnano/química , Receptor X de Pregnano/genética , Receptor X de Pregnano/metabolismo
18.
Am J Hum Genet ; 108(1): 115-133, 2021 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-33308444

RESUMEN

Signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) is a member of a small family of multifunctional cell surface-anchored glycoproteins functioning as co-receptors for a variety of growth factors. Here we report that bi-allelic inactivating variants in SCUBE3 have pleiotropic consequences on development and cause a previously unrecognized syndromic disorder. Eighteen affected individuals from nine unrelated families showed a consistent phenotype characterized by reduced growth, skeletal features, distinctive craniofacial appearance, and dental anomalies. In vitro functional validation studies demonstrated a variable impact of disease-causing variants on transcript processing, protein secretion and function, and their dysregulating effect on bone morphogenetic protein (BMP) signaling. We show that SCUBE3 acts as a BMP2/BMP4 co-receptor, recruits the BMP receptor complexes into raft microdomains, and positively modulates signaling possibly by augmenting the specific interactions between BMPs and BMP type I receptors. Scube3-/- mice showed craniofacial and dental defects, reduced body size, and defective endochondral bone growth due to impaired BMP-mediated chondrogenesis and osteogenesis, recapitulating the human disorder. Our findings identify a human disease caused by defective function of a member of the SCUBE family, and link SCUBE3 to processes controlling growth, morphogenesis, and bone and teeth development through modulation of BMP signaling.


Asunto(s)
Huesos/metabolismo , Proteínas de Unión al Calcio/metabolismo , Discapacidades del Desarrollo/metabolismo , Osteogénesis/fisiología , Transducción de Señal/fisiología , Animales , Proteína Morfogenética Ósea 2/metabolismo , Proteína Morfogenética Ósea 4/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Línea Celular , Línea Celular Tumoral , Femenino , Regulación del Desarrollo de la Expresión Génica/fisiología , Células HEK293 , Células Hep G2 , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Células MCF-7 , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL
19.
Mol Pharmacol ; 99(1): 1-16, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33130557

RESUMEN

Aberrant activation of Wnt/ß-catenin axis occurs in several gastrointestinal malignancies due to inactivating mutations of adenomatous polyposis coli (in colorectal cancer) or activating mutations of ß-catenin itself [in hepatocellular carcinoma (HCC)]. These lead to ß-catenin stabilization, increase in ß-catenin/T-cell factor (TCF)-mediated transcriptional activation, and target gene expression, many of which are involved in tumor progression. While studying pharmaceutical agents that can target ß-catenin in cancer cells, we observed that the plant compound berberine (BBR), a potent activator of AMP-activated protein kinase (AMPK), can reduce ß-catenin expression and downstream signaling in HCC cells in a dose-dependent manner. More in-depth analyses to understand the mechanism revealed that BBR-induced reduction of ß-catenin occurs independently of AMPK activation and does not involve transcriptional or post-translational mechanisms. Pretreatment with protein synthesis inhibitor cycloheximide antagonized BBR-induced ß-catenin reduction, suggesting that BBR affects ß-catenin translation. BBR treatment also antagonized mammalian target of rapamycin (mTOR) activity and was associated with increased recruitment of eukaryotic translation initiation factor 4E-binding protein (4E-BP) 1 in the translational complex, which was revealed by 7-methyl-cap-binding assays, suggesting inhibition of cap-dependent translation. Interestingly, knocking down 4E-BP1 and 4E-BP2 significantly attenuated BBR-induced reduction of ß-catenin levels and expression of its downstream target genes. Moreover, cells with 4E-BP knockdown were resistant to BBR-induced cell death and were resensitized to BBR after pharmacological inhibition of ß-catenin. Our findings indicate that BBR antagonizes ß-catenin pathway by inhibiting ß-catenin translation and mTOR activity and thereby reduces HCC cell survival. These also suggest that BBR could be used for targeting HCCs that express mutated/activated ß-catenin variants that are currently undruggable. SIGNIFICANCE STATEMENT: ß-catenin signaling is aberrantly activated in different gastrointestinal cancers, including hepatocellular carcinoma, which is currently undruggable. In this study we describe a novel mechanism of targeting ß-catenin translation via utilizing a plant compound, berberine. Our findings provide a new avenue of targeting ß-catenin axis in cancer, which can be utilized toward the designing of effective therapeutic strategies to combat ß-catenin-dependent cancers.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Berberina/farmacología , Carcinoma Hepatocelular/metabolismo , Proteínas de Ciclo Celular/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Neoplasias Hepáticas/metabolismo , beta Catenina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/genética , Carcinoma Hepatocelular/genética , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Factores Eucarióticos de Iniciación/antagonistas & inhibidores , Factores Eucarióticos de Iniciación/genética , Células HEK293 , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/fisiología , beta Catenina/antagonistas & inhibidores , beta Catenina/genética
20.
Anal Chem ; 93(3): 1604-1611, 2021 01 26.
Artículo en Inglés | MEDLINE | ID: mdl-33356171

RESUMEN

Carboxylic metabolites are an important class of metabolites, which widely exist in mammals with various types. Chemical isotope labeling liquid chromatography-mass spectrometry (CIL-LC-MS) has been widely used for the detection of carboxylated metabolites. However, high coverage analysis of carboxylated metabolites in biological samples is still challenging due to improper reactivity and selectivity of labeling reagents to carboxylated metabolites. In this study, we used N-methylphenylethylamine (MPEA) to label various types of carboxylated metabolites including short-chain fatty acids (SCFAs), medium-chain fatty acids (MCFAs), long-chain fatty acids (LCFAs), polycarboxylic acids (polyCAs), amino acids (AAs), and aromatic acids. Additionally, metabolites containing other functional groups, such as phenol, sulfhydryl, and phosphate groups, could not be labeled under the conditions of MPEA labeling. After MPEA labeling, the detection sensitivity of carboxylic acids was increased by 1-2 orders of magnitude, and their chromatographic retention on a reversed-phase (RP) column was enhanced (RT > 3 min). Under optimized labeling conditions, we used MPEA and d3-N-methylphenylethylamine (d3-MPEA) for high coverage screening of carboxylated metabolites in HepG2 cells by ultrahigh-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). As a result, a total of 403 potential carboxylated metabolites were obtained of which 68 were confirmed based on our established in-house chemically labeled metabolite database (CLMD). SCFAs, MCFAs, LCFAs, polyCAs, AAs, and aromatic acids were all detected in HepG2 cell extracts. Due to the successful identification of AAs, the current method increased the coverage of carboxylated metabolites compared with our previous work. Moreover, 133 and 109 carboxylated metabolites with changed contents were obtained in HepG2 cells incubated with curcumin and R-3-hydroxybutyric acid, respectively. In general, our established method realized high coverage analysis of carboxylated metabolites in HepG2 cells.


Asunto(s)
Aminoácidos/análisis , Ácidos Carboxílicos/análisis , Ácidos Grasos/análisis , Metanfetamina/análogos & derivados , Aminoácidos/metabolismo , Ácidos Carboxílicos/metabolismo , Cromatografía Líquida de Alta Presión , Ácidos Grasos/metabolismo , Células Hep G2 , Humanos , Espectrometría de Masas , Metanfetamina/química , Metanfetamina/metabolismo , Estructura Molecular
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