RESUMEN
PD-1/PD-L1 monoclonal antibodies exhibit promising therapeutic effectiveness in multiple cancers. However, developing a simple and efficient non-antibody treatment strategy using the PD-1/PD-L1 signaling pathway still remains challenging. In this study, we developed a flow cytometry assay to screen bioactive compounds with PD-L1 inhibitory activity. A total of 409 marine natural products were screened, and sokotrasterol sulfate (SKS) was found to efficiently suppress the IFN-γ-induced PD-L1 expression. SKS sensitizes the tumor cells to antigen-specific T-cell killing in the T cell-tumor cell coculture system. Mechanistically, SKS directly targeted Janus kinase (JAK) to inhibit the downstream activation of signal transducer and activator of transcription (STAT) and the subsequent transcription of PDL1. Our findings highlight the immunological role of SKS that may act as a basis for a potential immunotherapeutic agent.
Asunto(s)
Antígeno B7-H1 , Interferón gamma , Quinasas Janus , Interferón gamma/farmacología , Humanos , Quinasas Janus/metabolismo , Esteroles/farmacología , Transducción de Señal/efectos de los fármacos , Estructura Molecular , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Línea Celular TumoralRESUMEN
Checkpoint inhibitors such as PD-1/PD-L1 antibody therapeutics are a promising option for the treatment of multiple cancers. Due to the inherent limitations of antibodies, great efforts have been devoted to developing small-molecule PD-1/PD-L1 signaling pathway inhibitors. In this study we established a high-throughput AlphaLISA assay to discover small molecules with new skeletons that could block PD-1/PD-L1 interaction. We screened a small-molecule library of 4169 compounds including natural products, FDA approved drugs and other synthetic compounds. Among the 8 potential hits, we found that cisplatin, a first-line chemotherapeutic drug, reduced AlphaLISA signal with an EC50 of 8.3 ± 2.2 µM. Furthermore, we showed that cisplatin-DMSO adduct, but not semplice cisplatin, inhibited PD-1/PD-L1 interaction. Thus, we assessed several commercial platinum (II) compounds, and found that bis(benzonitrile) dichloroplatinum (II) disturbed PD-1/PD-L1 interaction (EC50 = 13.2 ± 3.5 µM). Its inhibitory activity on PD-1/PD-L1 interaction was confirmed in co-immunoprecipitation and PD-1/PD-L1 signaling pathway blockade bioassays. Surface plasmon resonance assay revealed that bis(benzonitrile) dichloroplatinum (II) bound to PD-1 (KD = 2.08 µM) but not PD-L1. In immune-competent wild-type mice but not in immunodeficient nude mice, bis(benzonitrile) dichloroplatinum (II) (7.5 mg/kg, i.p., every 3 days) significantly suppressed the growth of MC38 colorectal cancer xenografts with increasing tumor-infiltrating T cells. These data highlight that platinum compounds are potential immune checkpoint inhibitors for the treatment of cancers.
Asunto(s)
Cisplatino , Inhibidores de Puntos de Control Inmunológico , Neoplasias , Animales , Humanos , Ratones , Anticuerpos , Antígeno B7-H1/metabolismo , Cisplatino/farmacología , Cisplatino/uso terapéutico , Ratones Desnudos , Neoplasias/tratamiento farmacológico , Receptor de Muerte Celular Programada 1 , Inhibidores de Puntos de Control Inmunológico/farmacologíaRESUMEN
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by toxic aggregates of mutant huntingtin protein (mHTT) in the brain. Decreasing mHTT is a potential strategy for therapeutic purpose of HD. Valosin-containing protein (VCP/p97) is a crucial regulator of proteostasis, which regulates the degradation of damaged protein through proteasome and autophagy pathway. Since VCP has been implicated in pathogenesis of HD as well as other neurodegenerative diseases, small molecules that specifically regulate the activity of VCP may be of therapeutic benefits for HD patients. In this study we established a high-throughput screening biochemical assay for VCP ATPase activity measurement and identified gossypol, a clinical approved drug in China, as a novel modulator of VCP. Gossypol acetate dose-dependently inhibited the enzymatic activity of VCP in vitro with IC50 of 6.53±0.6 µM. We further demonstrated that gossypol directly bound to the interface between the N and D1 domains of VCP. Gossypol acetate treatment not only lowered mHTT levels and rescued HD-relevant phenotypes in HD patient iPS-derived Q47 striatal neurons and HD knock-in mouse striatal cells, but also improved motor function deficits in both Drosophila and mouse HD models. Taken together, gossypol acetate acted through a gain-of-function way to induce the formation of VCP-LC3-mHTT ternary complex, triggering autophagic degradation of mHTT. This study reveals a new strategy for treatment of HD and raises the possibility that an existing drug can be repurposed as a new treatment of neurodegenerative diseases.
Asunto(s)
Autofagia/efectos de los fármacos , Gosipol/uso terapéutico , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/tratamiento farmacológico , Fármacos Neuroprotectores/uso terapéutico , Animales , Drosophila , Inhibidores Enzimáticos/uso terapéutico , Femenino , Células HEK293 , Células HeLa , Humanos , Proteína Huntingtina/química , Proteína Huntingtina/genética , Masculino , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación , Multimerización de Proteína/efectos de los fármacos , Proteolisis/efectos de los fármacos , Proteína que Contiene Valosina/antagonistas & inhibidores , Proteína que Contiene Valosina/metabolismoRESUMEN
Grincamycins (GCNs) are a class of angucycline glycosides isolated from actinomycete Streptomyces strains that have potent antitumor activities, but their antitumor mechanisms remain unknown. In this study, we tried to identify the cellular target of grincamycin B (GCN B), one of most dominant and active secondary metabolites, using a combined strategy. We showed that GCN B-selective-induced apoptosis of human acute promyelocytic leukemia (APL) cell line NB4 through increase of ER stress and intracellular reactive oxygen species (ROS) accumulation. Using a strategy of combining phenotype, transcriptomics and protein microarray approaches, we identified that isocitrate dehydrogenase 1(IDH1) was the putative target of GCN B, and confirmed that GCNs were a subset of selective inhibitors targeting both wild-type and mutant IDH1 in vitro. It is well-known that IDH1 converts isocitrate to 2-oxoglutarate (2-OG), maintaining intracellular 2-OG homeostasis. IDH1 and its mutant as the target of GCN B were validated in NB4 cells and zebrafish model. Knockdown of IDH1 in NB4 cells caused the similar phenotype as GCN B treatment, and supplementation of N-acetylcysteine partially rescued the apoptosis caused by IDH1 interference in NB4 cells. In zebrafish model, GCN B effectively restored myeloid abnormality caused by overexpression of mutant IDH1(R132C). Taken together, we demonstrate that IDH1 is one of the antitumor targets of GCNs, suggesting wild-type IDH1 may be a potential target for hematological malignancies intervention in the future.
Asunto(s)
Antraquinonas/farmacología , Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Glicósidos/farmacología , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Animales , Antraquinonas/metabolismo , Antineoplásicos/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Estrés del Retículo Endoplásmico/efectos de los fármacos , Inhibidores Enzimáticos/metabolismo , Glicósidos/metabolismo , Humanos , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Ácidos Cetoglutáricos/metabolismo , Simulación del Acoplamiento Molecular , Mutación , Unión Proteica , Especies Reactivas de Oxígeno/metabolismo , Pez CebraRESUMEN
B-cell lymphomas are heterogeneous blood disorders with limited therapeutic options, largely because of their propensity to relapse and become refractory to treatments. Carabin, a key suppressor of B-cell receptor signaling and proliferation, is inactivated in B-cell lymphoma by unknown mechanisms. Here, we identify prolyl 4-hydroxylase 2 (P4HA2) as a specific proline hydroxylase of Carabin. Carabin hydroxylation leads to its proteasomal degradation, thereby activating the Ras/extracellular signal-regulated kinase pathway and increasing B-cell lymphoma proliferation. P4HA2 is undetectable in normal B cells but upregulated in the diffuse large B-cell lymphoma (DLBCL), driving Carabin inactivation and lymphoma proliferation. Our results indicate that P4HA2 is a potential prognosis marker for DLBCL and a promising pharmacological target for developing treatment of molecularly stratified B-cell lymphomas.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Biomarcadores de Tumor/metabolismo , Proliferación Celular , Linfoma de Células B Grandes Difuso/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas de Neoplasias/metabolismo , Prolil Hidroxilasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Proteínas Activadoras de GTPasa , Humanos , Hidroxilación , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología , Proteínas de Neoplasias/genética , Prolil Hidroxilasas/genética , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , ProteolisisRESUMEN
A series of DLC (delocalized lipophilic cation) modified spinosyn derivatives were synthesized and evaluated for antitumor efficacies both in vitro and in vivo. Cancer cell based antiproliferative assays indicated that the more lipophilic derivatives had stronger inhibitory effects on the tested cancer cell lines. Compound 7b and 8b exhibited strong anti-OXPHOS and apoptosis inducing ability. Notable antitumor efficacies of 7b (5 mg/kg) and 8b (2.5 mg/kg) were observed in the in vivo tumor xenograft experiments, however, lethal toxicities were observed on higher dosages. Our findings indicated that DLC modification is a viable strategy to enhance the anti-OXPHOS and antitumor efficacies of spinosyn derivatives.
Asunto(s)
Macrólidos/síntesis química , Macrólidos/farmacología , Mitocondrias/metabolismo , Fosforilación Oxidativa/efectos de los fármacos , Animales , Antineoplásicos , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Ratones , Mitocondrias/efectos de los fármacos , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Relación Estructura-ActividadRESUMEN
Controlling translation initiation is an efficient way to regulate gene expression at the post-transcriptional level. However, current knowledge regarding regulatory proteins and their modes of controlling translation initiation is still limited. In this study, we employed tandem affinity purification and mass spectrometry to screen for unknown proteins associated with the translation initiation machinery. Ubiquitin specific peptidase 9, X-linked (USP9X), was identified as a novel binding partner, that interacts with the eukaryotic translation initiation factor 4B (eIF4B) in a mRNA-independent manner. USP9X-deficient cells presented significantly impaired nascent protein synthesis, cap-dependent translation initiation and cellular proliferation. USP9X can selectively alter the translation of pro-oncogenic mRNAs, such as c-Myc and XIAP. Moreover, we found that eIF4A1, which is primarily ubiquitinated at Lys-369, is the substrate of USP9X. USP9X dysfunction increases the ubiquitination of eIF4A1 and enhances its degradation. Our results provide evidence that USP9X is a novel regulator of the translation initiation process via deubiquitination of eIF4A1, which offers new insight in understanding the pivotal role of USP9X in human malignancies and neurodevelopmental disorders.
Asunto(s)
Factor 4A Eucariótico de Iniciación/metabolismo , Biosíntesis de Proteínas , Ubiquitina Tiolesterasa/metabolismo , Ubiquitinación , Proliferación Celular/genética , Factor 4A Eucariótico de Iniciación/genética , Factores Eucarióticos de Iniciación/genética , Factores Eucarióticos de Iniciación/metabolismo , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Mutación , Unión Proteica , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especificidad por Sustrato , Ubiquitina Tiolesterasa/genéticaRESUMEN
As a dynamic post-translational modification, O-linked ß- N-acetylglucosamine ( O-GlcNAc) modification (i.e., O-GlcNAcylation) of proteins regulates many biological processes involving cellular metabolism and signaling. However, O-GlcNAc site mapping, a prerequisite for site-specific functional characterization, has been a challenge since its discovery. Herein we present a novel method for O-GlcNAc enrichment and site mapping. In this method, the O-GlcNAc moiety on peptides was labeled with UDP-GalNAz followed by copper-free azide-alkyne cycloaddition with a multifunctional reagent bearing a terminal cyclooctyne, a disulfide bridge, and a biotin handle. The tagged peptides were then released from NeutrAvidin beads upon reductant treatment, alkylated with (3-acrylamidopropyl)trimethylammonium chloride, and subjected to electron-transfer dissociation mass spectrometry analysis. After validation by using standard synthetic peptide gCTD and model protein α-crystallin, such an approach was applied to the site mapping of overexpressed TGF-ß-activated kinase 1/MAP3K7 binding protein 2 (TAB2), with four O-GlcNAc sites unambiguously identified. Our method provides a promising tool for the site-specific characterization of O-GlcNAcylation of important proteins.
Asunto(s)
Acetilglucosamina/análisis , Proteínas Adaptadoras Transductoras de Señales/química , Péptidos/química , Espectrometría de Masas en Tándem/métodos , alfa-Cristalinas/química , Acetilglucosamina/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Alquinos/química , Azidas/química , Química Clic , Reacción de Cicloadición , Glicosilación , Células HEK293 , Humanos , Oxidación-Reducción , Péptidos/metabolismo , Procesamiento Proteico-Postraduccional , Uridina Difosfato N-Acetilgalactosamina/análogos & derivados , Uridina Difosfato N-Acetilgalactosamina/química , alfa-Cristalinas/metabolismoRESUMEN
In order to enhance the mitochondria-targeting ability of spinosad. A series of quartenary ammonium spinosyn derivatives was designed and synthesized. Some of the derivatives displayed greatly enhanced antiproliferative ability towards tested human cancer cell lines. The structure activity relationship study indicated that lipophilicity has a great influence on the antiproliferative effects of these derivatives. The most active compound 11d exhibited remarkably enhanced OXPHS inhibition and apoptosis inducing ability than spinosyn A.
Asunto(s)
Antineoplásicos/farmacología , Macrólidos/farmacología , Compuestos de Amonio Cuaternario/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Macrólidos/síntesis química , Macrólidos/química , Mitocondrias/metabolismo , Estructura Molecular , Fosforilación Oxidativa/efectos de los fármacos , Compuestos de Amonio Cuaternario/síntesis química , Compuestos de Amonio Cuaternario/química , Relación Estructura-ActividadRESUMEN
Eukaryotic translation initiation factor eIF4AI, the founding member of DEAD-box helicases, undergoes ATP hydrolysis-coupled conformational changes to unwind mRNA secondary structures during translation initiation. However, the mechanism of its coupled enzymatic activities remains unclear. Here we report that a gating mechanism for Pi release controlled by the inter-domain linker of eIF4AI regulates the coupling between ATP hydrolysis and RNA unwinding. Molecular dynamic simulations and experimental results revealed that, through forming a hydrophobic core with the conserved SAT motif of the N-terminal domain and I357 from the C-terminal domain, the linker gated the release of Pi from the hydrolysis site, which avoided futile hydrolysis cycles of eIF4AI. Further mutagenesis studies suggested this linker also plays an auto-inhibitory role in the enzymatic activity of eIF4AI, which may be essential for its function during translation initiation. Overall, our results reveal a novel regulatory mechanism that controls eIF4AI-mediated mRNA unwinding and can guide further mechanistic studies on other DEAD-box helicases.
Asunto(s)
Factor 4A Eucariótico de Iniciación/química , Iniciación de la Cadena Peptídica Traduccional , ARN Mensajero/química , Adenosina Difosfato/química , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Secuencias de Aminoácidos , Factor 4A Eucariótico de Iniciación/genética , Factor 4A Eucariótico de Iniciación/metabolismo , Simulación de Dinámica Molecular , Mutación , Fosfatos/química , Fosfatos/metabolismo , Unión Proteica , Conformación Proteica , ARN Mensajero/metabolismoRESUMEN
The change of oligosaccharide structure has been revealed to be crucial for glycoproteins' biological functions and cell biological characteristics. N-acetylglucosaminy transferase V (GnT-V), a key enzyme catalysing the reaction of adding ß1, 6-N-acetylglucosamine (GlcNAc) on asparagine-linked oligosaccharides of cell proteins, has been implicated to a metastastic-promoting oncoprotein in some carcinomas. However, this correlation might not be subjected to all types of cancers, for example, in non-small cell lung cancers, low level of GnT-V expression is associated with relatively short survival time and poor prognosis. To explain the role of GnT-V in lung cancer progression, we studied the association of GnT-V expression with lung cancer EMT behaviour. We found that GnT-V expression was correlated with epithelial marker positively and mesenchymal marker negatively. GnT-V levels, as well as ß1,6-GlcNAc branched N-glycans, were strongly reduced in TGF-ß1-induced EMT of human lung adenocarcinoma A549 cells. Further studies showed that suppression of ß1,6-GlcNAc branched N-glycans by inhibitor or GnT-V silencing in A549 cells could promote TGF-ß1-induced EMT-like changes, cell migration and invasion. Meanwhile, overexpression of GnT-V impaired TGF-ß1-induced EMT, migration and invasion. It suggests that GnT-V suppresses the EMT process of lung cancer cells through inhibiting the TGF-ß/Smad signalling and its downstream transcription factors in a GnT-V catalytic activity-dependent manner. Taken together, the present study reveals a novel mechanism of GnT-V as a suppressor of both EMT and invasion in human lung cancer cells, which may be useful for fully understanding N-glycan's biological roles in lung cancer progression.
Asunto(s)
Acetilglucosamina/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Neoplasias Pulmonares/patología , Polisacáridos/farmacología , Proteínas Smad/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Acetilglucosamina/genética , Apoptosis/efectos de los fármacos , Western Blotting , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Células HEK293 , Humanos , Técnicas para Inmunoenzimas , Luciferasas/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Mesodermo/metabolismo , Mesodermo/patología , N-Acetilglucosaminiltransferasas/genética , N-Acetilglucosaminiltransferasas/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacosRESUMEN
Abnormal DNA methylation is one of the important characteristics in tumor cells. Apoptosis plays an essential role in cell survival and processing. It is not clear whether DNA methyltransferases (DNMTs) change in apoptosis and how DNMTs are regulated in apoptosis. In this study, we found that SMMC-7721 or BEL-7404 cells were induced to apoptosis by STS, meanwhile the DNMT3B protein and mRNA level were decreased. To explore the mechanism of DNMT3B down-regulation, we found that the mRNA decay was not changed and core promoter activity of DNMT3B gene was decreased in STS-induced apoptosis. In order to figure out the signal molecule involved in transcriptional regulation of DNMT3B gene by STS, p-JNK, p-ERK, and p-p38 were examined. In STS-induced apoptosis p-JNK level was increased, and p-ERK and p-p38 were decreased. Furthermore, the inhibitor of p-JNK significantly alleviated the decline of DNMT3B protein. We also found that the siRNA of DNMT3B strengthened the cleavage of PARP and pro-caspase-3 as well as up-regulated the p16 gene expression in STS-treated cells. We concluded here that STS-regulated DNMT3B gene expression via p-JNK and down-regulation of DNMT3B-mediated STS-induced apoptosis through the up-regulation p16 expression.
Asunto(s)
Apoptosis/efectos de los fármacos , Carcinoma Hepatocelular/tratamiento farmacológico , ADN (Citosina-5-)-Metiltransferasas/genética , Neoplasias Hepáticas/tratamiento farmacológico , Estaurosporina/farmacología , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Genes p16 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , MAP Quinasa Quinasa 4/metabolismo , Regiones Promotoras Genéticas , Estabilidad del ARN , ARN Interferente Pequeño , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , ADN Metiltransferasa 3BRESUMEN
Aurora A plays a critical role in G2/M transition and mitosis, making it an attractive target for cancer treatment. Aurora A inhibitors showed remarkable antitumor effects in preclinical studies, but unsatisfactory outcomes in clinical trials have greatly limited their development. In this study, the Aurora A inhibitor alisertib upregulated programmed death ligand 1 (PD-L1) expression in a panel of tumor cells both in vitro and in vivo. Upregulation of the checkpoint protein PD-L1 reduced antitumor immunity in immune-competent mice, paradoxically inhibiting the antitumor effects of alisertib. Mechanistically, Aurora A directly bound to and phosphorylated cyclic GMP-AMP synthase (cGAS), suppressing PD-L1 expression in tumor cells. Aurora A inhibition by alisertib activated the cGAS/stimulator of IFN genes (STING)/NF-κB pathway and promoted PD-L1 expression. Combining alisertib with anti-PD-L1 antibody improved antitumor immunity and enhanced the antitumor effects of alisertib in immune-competent mice. Our results, which reveal the immunomodulatory functions of Aurora A inhibitors and provide a plausible explanation for the poor clinical outcomes with their use, offer a potential approach to improve the antitumor efficacy of these inhibitors.
Asunto(s)
Aurora Quinasa A , Inhibidores de Proteínas Quinasas , Animales , Ratones , Aurora Quinasa A/genética , Aurora Quinasa A/metabolismo , Antígeno B7-H1/genética , Línea Celular Tumoral , Nucleotidiltransferasas , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , HumanosRESUMEN
Bladder cancer (BLCA) remains a difficult malignancy to manage because of its high recurrence, intense follow-up, and invasive diagnostic and treatment techniques. Immune checkpoint inhibitors (ICIs) have forged a new direction for the treatment of BLCA, but it is currently challenging to predict whether an individual patient will be sensitive to ICIs. We collected 43 urine/tumor samples from BLCA patients for primary bladder cancer cells (BCCs) culturing using our previously reported BCC culture platform. We used flow cytometry (FCM) to measure the expression levels of Programmed Death-Ligand 1 (PD-L1) on BCCs before and after interferon-gamma (IFN-γ) treatment and found that PD-L1 expression and the sensitivities to IFN-γ varied among patients. RNA-sequencing, western blotting, and programmed death-1 (PD-1) binding assays confirmed that the BCC FCM-based PD-L1 detection platform (BC-PD-L1) was reliable and was not hindered by the glycosylation of PD-L1. In the subsequent retrospective study, we found that IFN-γ-stimulated PD-L1 (sPD-L1) expression on BCCs detected by BC-PD-L1 could predict the prognosis of BLCA patients. Importantly, the prognostic value was similar or even better in urine-derived BC-PD-L1 (UBC-PD-L1). Transcriptome analysis showed that BCCs with high sPD-L1 tended to enrich genes associated with the collagen-containing extracellular matrix, cell-cell adhesion, and positive regulation of the immune system. In addition, the UBC-PD-L1 also exhibited predictive value for ICI response in BLCA patients. In conclusion, as a novel personalized urine-detection method, UBC-PD-L1 may provide a rapid, accurate, and non-invasive tool for monitoring tumor progression, predicting therapeutic responses, and helping improve BLCA clinical treatment in future.
RESUMEN
Prostate cancer (PCa) is the second most prevalent malignancy in males across the world. A greater knowledge of the relationship between protein abundance and drug responses would benefit precision treatment for PCa. Herein, we establish 35 Chinese PCa primary cell models to capture specific characteristics among PCa patients, including gene mutations, mRNA/protein/surface protein distributions, and pharmaceutical responses. The multi-omics analyses identify Anterior Gradient 2 (AGR2) as a pre-operative prognostic biomarker in PCa. Through the drug library screening, we describe crizotinib as a selective compound for malignant PCa primary cells. We further perform the pharmacoproteome analysis and identify 14,372 significant protein-drug correlations. Surprisingly, the diminished AGR2 enhances the inhibition activity of crizotinib via ALK/c-MET-AKT axis activation which is validated by PC3 and xenograft model. Our integrated multi-omics approach yields a comprehensive understanding of PCa biomarkers and pharmacological responses, allowing for more precise diagnosis and therapies.
Asunto(s)
Multiómica , Neoplasias de la Próstata , Masculino , Humanos , Crizotinib/farmacología , Crizotinib/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Proteínas/metabolismo , Mucoproteínas/uso terapéutico , Proteínas Oncogénicas/uso terapéuticoRESUMEN
Aberrant N-linked glycosylation is a prominent feature of cancers. Perturbance of oligosaccharide structure on cell surfaces directly affects key processes in tumor development and progression. In spite of the critical role played by N-linked glycans in tumor biology, the discovery of small molecules that specifically disturbs the N-linked glycans is still under investigation. To identify more saccharide-structure-perturbing compounds, a repurposed drug screen by using a library consisting of 1530 FDA-approved drugs was performed. Interestingly, an antipsychotic drug, penfluridol, was identified as being able to decrease cell surface Wheat germ agglutinin (WGA) staining. In the presence of penfluridol, cell membrane glycoproteins PD-L1 shifted to a lower molecular weight. Further studies demonstrated that penfluridol treatment caused an accumulation of high-mannose oligosaccharides, especially Man5-7GlcNAc2 glycan structures. Mechanistically, this effect is due to direct targeting of MAN1A1 mannosidase, a Golgi enzyme involved in N-glycan maturation. Moreover, we found that altered glycosylation of PD-L1 caused by penfluridol disrupted interactions between PD-1 and PD-L1, resulting in activation of T-cell tumor immunity. In a mouse xenograft and glioma model, penfluridol enhanced the anti-tumor effect of the anti-PD-L1 antibody in vivo. Overall, these findings revealed an important biological activity of the antipsychotic drug penfluridol as an inhibitor of glycan processing and proposed a repurposed use of penfluridol in anti-tumor therapy through activation of T-cell immunity.
RESUMEN
Induction of hypothermia during hibernation/torpor enables certain mammals to survive under extreme environmental conditions. However, pharmacological induction of hypothermia in most mammals remains a huge challenge. Here we show that a natural product P57 promptly induces hypothermia and decreases energy expenditure in mice. Mechanistically, P57 inhibits the kinase activity of pyridoxal kinase (PDXK), a key metabolic enzyme of vitamin B6 catalyzing phosphorylation of pyridoxal (PL), resulting in the accumulation of PL in hypothalamus to cause hypothermia. The hypothermia induced by P57 is significantly blunted in the mice with knockout of PDXK in the preoptic area (POA) of hypothalamus. We further found that P57 and PL have consistent effects on gene expression regulation in hypothalamus, and they may activate medial preoptic area (MPA) neurons in POA to induce hypothermia. Taken together, our findings demonstrate that P57 has a potential application in therapeutic hypothermia through regulation of vitamin B6 metabolism and PDXK serves as a previously unknown target of P57 in thermoregulation. In addition, P57 may serve as a chemical probe for exploring the neuron circuitry related to hypothermia state in mice.
Asunto(s)
Productos Biológicos , Hipotermia , Animales , Ratones , Regulación de la Temperatura Corporal , Hipotermia/inducido químicamente , Piridoxal Quinasa/genética , Piridoxina , Vitamina B 6 , Productos Biológicos/farmacologíaRESUMEN
Profilin1 (Pfn1) functions as a tumour suppressor against malignant phenotypes of cancer cells. A minimum level of Pfn1 is critical for the differentiation of human epithelial cells, and its lower expression correlates with the tumourigenesis of breast cancer cells and tissues. However, the molecular mechanisms underlying its anti-tumour action remain largely unknown. In this study, we found that stable expression of ectopic Pfn1 sensitized the breast cancer cell line MDA-MB-468 to apoptosis induced by staurosporine, a widely used natural apoptosis-inducing agent. Pfn1 overexpression could also up-regulate the expression of integrin α5ß1, which has been shown to inhibit the transformed phenotype of cancer cells. Furthermore, the Pfn1-facilitated apoptosis induced by staurosporine was blocked in cells attached to a supplementary fibronectin substrate, which serves as a ligand of integrin α5ß1. These results suggest that the insufficient fibronectin caused by the integrin α5ß1 up-regulation might activate a signalling pathway leading to an increase of cellular apoptosis. Moreover, Pfn1 that primarily functions to promote local superstructure formation involving actin filaments and integrin ß1 may contribute to its promotion on apoptosis. Our study indicated a previously uncharacterized role of Pfn1 in mediating staurosporine-inducing apoptosis in breast cancer cells via up-regulating integrin α5ß1, and suggested a new target for breast cancer therapy.
Asunto(s)
Actinas/metabolismo , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/patología , Inhibidores Enzimáticos/farmacología , Integrina beta1/metabolismo , Profilinas/fisiología , Estaurosporina/farmacología , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Femenino , Citometría de Flujo , Humanos , Microscopía Confocal , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
AIM: Glucocorticoid therapy has been used in childhood nephrotic syndrome since the 1950s, where the characteristic change is effacement of the actin-rich foot process of glomerular podocytes. Recent studies have shown that glucocorticoids, in addition to their general immunosuppressive and anti-inflammatory effects, have a direct effect on podocytes, regulate some apoptotic factors, and increase the stability of actin filaments. However, the precise mechanism(s) underlying the protective effects of glucocorticoids on podocytes remain unclear. It is known that adriamycin (ADR) can induce podocyte foot process effacement and trigger massive proteinuria in rodent models. However, few reports have examined the direct role of ADR in podocyte actin rearrangement in vitro. In this study, we investigated how ADR directly induced podocyte actin cytoskeleton rearrangement and further analyzed how dexamethasone prevented such injury. METHODS: We used confocal microscopy to assess podocyte actin rearrangement. Western blot analysis and real-time polymerase chain reaction were performed to measure the protein and mRNA levels of α-actinin-4. RESULTS: We demonstrated that there was a time-dependent ADR-induced podocyte actin rearrangement with less than 12 h of ADR treatment in cultured podocytes. Dexamethasone could protect podocytes from ADR-induced injury and also stabilize the expression of α-actinin-4. CONCLUSION: This study showed that dexamethasone had direct effects on podocytes: α-actinin-4 may be one of the potential target molecules.
Asunto(s)
Citoesqueleto de Actina/efectos de los fármacos , Actinina/metabolismo , Antibióticos Antineoplásicos/toxicidad , Dexametasona/farmacología , Doxorrubicina/toxicidad , Podocitos/efectos de los fármacos , Sustancias Protectoras/farmacología , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/patología , Actinina/genética , Animales , Western Blotting , Células Cultivadas , Citoprotección , Ratones , Microscopía Confocal , Podocitos/metabolismo , Podocitos/patología , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de TiempoRESUMEN
The CRISPR/Cas9 system, originally derived from the prokaryotic adaptive immune system, has been developed as efficient genome editing tools. It enables precise gene manipulation on chromosomal DNA through the specific binding of programmable sgRNA to target DNA, and the Cas9 protein, which has endonuclease activity, will cut a double strand break at specific locus. However, Cas9 is a foreign protein in mammalian cells, and the potential risks associated with its introduction into mammalian cells are not fully understood. In this study, we performed pull-down and mass spectrometry (MS) analysis of Streptococcus pyogenes Cas9 (SpyCas9) interacting proteins in HEK293T cells and showed that the majority of Cas9-associated proteins identified by MS were localized in the nucleolus. Interestingly, we further discovered that the Cas9 protein contains a sequence encoding a nucleolus detention signal (NoDS). Compared with wild-type (WT) Cas9, NoDS-mutated variants of Cas9 (mCas9) are less stable, although their gene editing activity is minimally affected. Overexpression of WT Cas9, but not mCas9, causes general effects on transcription and protein translation in the host cell. Overall, identification of NoDS in Cas9 will improve the understanding of Cas9's biological function in vivo, and the removal of NoDS in Cas9 may enhance its safety for future clinical use.