RESUMEN
Asthma has reached epidemic levels, yet progress in developing specific therapies is slow. One of the main reasons for this is the fact that asthma is an umbrella term for various distinct subsets. Due to its high heterogeneity, it is difficult to establish biomarkers for each subset of asthma and to propose endotype-specific treatments. This review focuses on protein glycosylation as a process activated in asthma and ways to utilize it to develop novel biomarkers and treatments. We discuss known and relevant glycoproteins whose functions control disease development. The key role of glycoproteins in processes integral to asthma, such as inflammation, tissue remodeling, and repair, justifies our interest and research in the field of glycobiology. Altering the glycosylation states of proteins contributing to asthma can change the pathological processes that we previously failed to inhibit. Special emphasis is placed on chitotriosidase 1 (CHIT1), an enzyme capable of modifying LacNAc- and LacdiNAc-containing glycans. The expression and activity of CHIT1 are induced in human diseased lungs, and its pathological role has been demonstrated by both genetic and pharmacological approaches. We propose that studying the glycosylation pattern and enzymes involved in glycosylation in asthma can help in patient stratification and in developing personalized treatment.
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Asma , Glicoproteínas , Humanos , Asma/metabolismo , Asma/genética , Glicosilación , Glicoproteínas/metabolismo , Glicoproteínas/genética , Hexosaminidasas/metabolismo , Hexosaminidasas/genética , Biomarcadores/metabolismo , Animales , Polisacáridos/metabolismoRESUMEN
BACKGROUND: Impaired neutrophil activity is an important issue in chronic lymphocytic leukemia (CLL), as it contributes to a dysfunctional immune response leading to life-threatening infections in patients. Some features typical of CLL neutrophils, e.g., the B-cell-supportive secretion profile, have already been described. However, most of these studies were performed on cells isolated from peripheral blood. It is still unclear which molecular factors and cell types are involved in shaping neutrophil function and phenotype in the CLL microenvironment. Since regulatory T cells (Treg) play an important role in CLL progression and influence the activity of neutrophils, we investigated the crosstalk between Treg and neutrophils in the spleen using a murine model of CLL. METHODS: In this work, we used an Eµ-TCL1 mouse model of human CLL. For our in vivo and ex vivo experiments, we inoculated wild-type mice with TCL1 leukemic cells isolated from Eµ-TCL1 transgenic mice and then monitored disease progression by detecting leukemic cells in peripheral blood. We analyzed both the phenotype and activity of neutrophils isolated from the spleens of TCL1 leukemia-bearing mice. To investigate the interrelation between Treg and neutrophils in the leukemia microenvironment, we performed experiments using TCL1-injected DEREG mice with Treg depletion or RAG2KO mice with adoptively transferred TCL1 cells alone or together with Treg. RESULTS: The obtained results underline the plasticity of the neutrophil phenotype, observed under the influence of leukemic cells alone and depending on the presence of Treg. In particular, Treg affect the expression of CD62L and IL-4 receptor in neutrophils, both of which are crucial for the function of these cells. Additionally, we show that Treg depletion and IL-10 neutralization induce changes in the leukemia microenvironment, partially restoring the "healthy" phenotype of neutrophils. CONCLUSIONS: Altogether, the results indicate that the crosstalk between Treg and neutrophils in CLL may play an important role in CLL progression by interfering with the immune response.
RESUMEN
Neutrophils are specialized immune cells that are essential constituents of the innate immune response. They defend the organism against pathogens through various mechanisms. It was reported that phosphatidylinositols are key players in neutrophil functions, especially in the activity of class-I phosphoinositide 3-kinases (PI3Ks). P110δ, one of the PI3K subunits, is mostly expressed in immune cells, and its activity plays an important role in inflammatory responses. The aim of this study was to investigate the role of p110δ in neutrophil antimicrobial functions, activation status and cytokine production. To this end, we used bone marrow and splenic neutrophils isolated from a murine model expressing catalytically inactive p110δD910A/D910A. The level of phagocytosis and degranulation, the expressions of activation markers and cytokine production were determined by flow cytometry. ROS generation and NET release were assessed by fluorometry and fluorescent microscopy. We observed a significantly higher percentage of CD80-positive cells among the splenic granulocytes and found granulocytes subpopulations of differing phenotypes between WT and p110δD910A/D910A mice by multiparametric tSNE analysis. Moreover, we detected some differences in the expressions of activation markers, intracellular production of cytokines and bacterial killing. However, we did not observe any alterations in the selected neutrophil functions in p110δ mutant mice. Altogether, our data suggest that the catalytic p110 subunit(s), other than p110δ, is a key player in most neutrophil functions in mice. A follow-up study to correlate these in vitro results with in vivo observations is highly recommended.
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Fosfatidilinositol 3-Quinasa Clase I/farmacocinética , Neutrófilos , Fosfatidilinositol 3-Quinasas , Animales , Antígeno B7-1 , Citocinas , Estudios de Seguimiento , Ratones , Neutrófilos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Bazo/metabolismoRESUMEN
Photofrin-based photodynamic therapy (PDT) is approved for clinical use by the US Food and Drug Administration and the European Medicines Agency and is among the most widely used photosensitizer for the treatment of cancer. It was broadly reported that both the innate and the adaptive arms of immune response can be activated by PDT and play a critical role in the anticancer outcome of this treatment. PDT leads to the induction of acute local inflammation that includes leukocyte infiltration as well as increased activation and production of pro-inflammatory factors and cytokines. These events can lead to the development of systemic and specific antitumor immune response. Combining Photofrin-PDT with the epigenetic agent 5-aza-2'-deoxycytidine results in potentiated antitumor effects in vivo. Understanding the molecular mechanisms underlying this phenomenon would be invaluable for clinical development of this therapeutic approach. This chapter describes a detailed protocol allowing evaluation of specific antitumor immune response induced by PDT.
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Éter de Dihematoporfirina , Fotoquimioterapia , Decitabina/farmacología , Decitabina/uso terapéutico , Éter de Dihematoporfirina/farmacología , Éter de Dihematoporfirina/uso terapéutico , Epigénesis Genética , Inmunidad , Estados UnidosRESUMEN
Regulatory T cells (Tregs) are capable of inhibiting the proliferation, activation and function of T cells and play an important role in impeding the immune response to cancer. In chronic lymphocytic leukemia (CLL) a dysfunctional immune response and elevated percentage of effector-like phenotype Tregs have been described. In this study, using the Eµ-TCL1 mouse model of CLL, we evaluated the changes in the Tregs phenotype and their expansion at different stages of leukemia progression. Importantly, we show that Tregs depletion in DEREG mice triggered the expansion of new anti-leukemic cytotoxic T cell clones leading to leukemia eradication. In TCL1 leukemia-bearing mice we identified and characterized a specific Tregs subpopulation, the phenotype of which suggests its role in the formation of an immunosuppressive microenvironment, supportive for leukemia survival and proliferation. This observation was also confirmed by the gene expression profile analysis of these TCL1-specific Tregs. The obtained data on Tregs are consistent with those described so far, however, above all show that the changes in the Tregs phenotype described in CLL result from the formation of a specific, described in this study Tregs subpopulation. In addition, functional tests revealed the ability of Tregs to inhibit T cells that recognize model antigens expressed by leukemic cells. Moreover, inhibition of Tregs with a MALT1 inhibitor provided a therapeutic benefit, both as monotherapy and also when combined with an immune checkpoint inhibitor. Altogether, activation of Tregs appears to be crucial for CLL progression.
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Leucemia Linfocítica Crónica de Células B , Animales , Modelos Animales de Enfermedad , Inmunidad , Inmunosupresores/uso terapéutico , Ratones , Linfocitos T Reguladores , Microambiente TumoralRESUMEN
The prognosis for B-cell precursor acute lymphoblastic leukemia patients with Mixed-Lineage Leukemia (MLL) gene rearrangements (MLLr BCP-ALL) is still extremely poor. Inhibition of anti-apoptotic protein BCL-2 with venetoclax emerged as a promising strategy for this subtype of BCP-ALL, however, lack of sufficient responses in preclinical models and the possibility of developing resistance exclude using venetoclax as monotherapy. Herein, we aimed to uncover potential mechanisms responsible for limited venetoclax activity in MLLr BCP-ALL and to identify drugs that could be used in combination therapy. Using RNA-seq, we observed that long-term exposure to venetoclax in vivo in a patient-derived xenograft model leads to downregulation of several tumor protein 53 (TP53)-related genes. Interestingly, auranofin, a thioredoxin reductase inhibitor, sensitized MLLr BCP-ALL to venetoclax in various in vitro and in vivo models, independently of the p53 pathway functionality. Synergistic activity of these drugs resulted from auranofin-mediated upregulation of NOXA pro-apoptotic protein and potent induction of apoptotic cell death. More specifically, we observed that auranofin orchestrates upregulation of the NOXA-encoding gene Phorbol-12-Myristate-13-Acetate-Induced Protein 1 (PMAIP1) associated with chromatin remodeling and increased transcriptional accessibility. Altogether, these results present an efficacious drug combination that could be considered for the treatment of MLLr BCP-ALL patients, including those with TP53 mutations.
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Linfoma de Burkitt , Leucemia-Linfoma Linfoblástico de Células Precursoras , Apoptosis , Proteínas Reguladoras de la Apoptosis/metabolismo , Auranofina/farmacología , Auranofina/uso terapéutico , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Línea Celular Tumoral , Humanos , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Sulfonamidas , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND: Immune checkpoint inhibitors and chimeric antigen receptor (CAR)-based therapies have transformed cancer treatment. Recently, combining these approaches into a strategy of PD-L1-targeted CAR has been proposed to target PD-L1high tumors. Our study provides new information on the efficacy of such an approach against PD-L1low targets. METHODS: New atezolizumab-based PD-L1-targeted CAR was generated and introduced into T, NK, or NK-92 cells. Breast cancer MDA-MB-231 and MCF-7 cell lines or non-malignant cells (HEK293T, HMEC, MCF-10A, or BM-MSC) were used as targets to assess the reactivity or cytotoxic activity of the PD-L1-CAR-bearing immune effector cells. Stimulation with IFNγ or with supernatants from activated CAR T cells were used to induce upregulation of PD-L1 molecule expression on the target cells. HER2-CAR T cells were used for combination with PD-L1-CAR T cells against MCF-7 cells. RESULTS: PD-L1-CAR effector cells responded vigorously with degranulation and cytokine production to PD-L1high MDA-MB-231 cells, but not to PD-L1low MCF-7 cells. However, in long-term killing assays, both MDA-MB-231 and MCF-7 cells were eliminated by the PD-L1-CAR cells, although with a delay in the case of PD-L1low MCF-7 cells. Notably, the coculture of MCF-7 cells with activated PD-L1-CAR cells led to bystander induction of PD-L1 expression on MCF-7 cells and to the unique self-amplifying effect of the PD-L1-CAR cells. Accordingly, PD-L1-CAR T cells were active not only against MDA-MD-231 and MCF-7-PD-L1 but also against MCF-7-pLVX cells in tumor xenograft models. Importantly, we have also observed potent cytotoxic effects of PD-L1-CAR cells against non-malignant MCF-10A, HMEC, and BM-MSC cells, but not against HEK293T cells that initially did not express PD-L1 and were unresponsive to the stimulation . Finally, we have observed that HER-2-CAR T cells stimulate PD-L1 expression on MCF-7 cells and therefore accelerate the functionality of PD-L1-CAR T cells when used in combination. CONCLUSIONS: In summary, our studies show that CAR-effector cells trigger the expression of PD-L1 on target cells, which in case of PD-L1-CAR results in the unique self-amplification phenomenon. This self-amplifying effect could be responsible for the enhanced cytotoxicity of PD-L1-CAR T cells against both malignant and non-malignant cells and implies extensive caution in introducing PD-L1-CAR strategy into clinical studies.
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Neoplasias de la Mama/terapia , Citotoxicidad Inmunológica , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunoterapia Adoptiva/métodos , Receptores Quiméricos de Antígenos/inmunología , Animales , Antígeno B7-H1/análisis , Antígeno B7-H1/antagonistas & inhibidores , Línea Celular Tumoral , Femenino , Células HEK293 , Humanos , Ratones , Receptor ErbB-2/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tumor-associated neutrophils appear to be a crucial element of the tumor microenvironment that actively participates in the development and progression of cancerous diseases. The increased lifespan, plasticity in changing of phenotype, and functions of neutrophils influence the course of the disease and may significantly affect survival. In patients with chronic lymphocytic leukemia (CLL), disturbances in neutrophils functions impede the effective immune defense against pathogens. Therefore, understanding the mechanism underlying such a phenomenon in CLL seems to be of great importance. Here we discuss the recent reports analyzing the phenotype and functions of neutrophils in CLL, the most common leukemia in adults. We summarize the data concerning both the phenotype and the mechanisms by which neutrophils directly support the proliferation and survival of malignant B cells.
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Leucemia Linfocítica Crónica de Células B/etiología , Leucemia Linfocítica Crónica de Células B/patología , Neutrófilos/patología , Linfocitos B/inmunología , Humanos , Inmunidad , Leucemia Linfocítica Crónica de Células B/inmunología , Infiltración Neutrófila/inmunología , Fenotipo , Microambiente Tumoral/inmunologíaRESUMEN
Neutrophils represent the first line of defense against pathogens using various strategies, such as phagocytosis, production of reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) formation. Recently, an autophagy-independent role of autophagy related (ATG) gene 5 in immune cells, including neutrophils, was emphasized. Our aim was to investigate the role of ATG5 protein in neutrophils' antimicrobial functions, proliferation and apoptosis. To this end, we used genetically modified human promyelocytic leukemia (HL-60) cells overexpressing ATG5, differentiated toward granulocyte-like cells with all-trans retinoic acid (ATRA) and dimethylformamide. The level of differentiation, phagocytosis, proliferation and apoptosis were determined by flow cytometry. ROS production and NETs release was assessed by fluorometry and fluorescent microscopy. ATG5 gene expression was evaluated by real-time PCR, whereas the protein level of ATG5 and LC3-II was determined by Western blot. We did not observe the induction of autophagy in differentiated HL-60 cells overexpressing ATG5. The increased expression of ATG5 affects the differentiation of HL-60 cells with ATRA, ROS production and phagocytosis. However, we did not detect changes in NETs release. Moreover, ATG5 protects differentiated HL-60 cells from apoptosis but does not cause changes in proliferation rate.
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Apoptosis/efectos de los fármacos , Proteína 5 Relacionada con la Autofagia/metabolismo , Autofagia/genética , Diferenciación Celular/efectos de los fármacos , Granulocitos/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Fagocitosis/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Proteína 5 Relacionada con la Autofagia/genética , Proliferación Celular/efectos de los fármacos , Dimetilformamida/farmacología , Citometría de Flujo , Células HL-60 , Humanos , Leucemia Promielocítica Aguda/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Neutrófilos/metabolismo , Tretinoina/farmacología , Regulación hacia ArribaRESUMEN
It was previously reported that the activation of antitumor immune response by photodynamic therapy (PDT) is crucial for its therapeutic outcome. Excessive PDT-mediated inflammation is accompanied by immunosuppressive mechanisms that protect tissues from destruction. Thus, the final effect of PDT strongly depends on the balance between the activation of an adoptive arm of immune response and a range of activated immunosuppressive mechanisms. Here, with flow cytometry and functional tests, we evaluate the immunosuppressive activity of tumor-associated myeloid cells after PDT. We investigate the antitumor potential of PDT combined with indoleamine 2,3-dioxygenase 1 (IDO) inhibitor in the murine 4T1 and E0771 orthotopic breast cancer models. We found that the expression of IDO, elevated after PDT, affects the polarization of T regulatory cells and influences the innate immune response. Our results indicate that, depending on a therapeutic scheme, overcoming IDO-induced immunosuppressive mechanisms after PDT can be beneficial or can lead to a systemic toxic reaction. The inhibition of IDO, shortly after PDT, activates IL-6-dependent toxic reactions that can be diminished by the use of anti-IL-6 antibodies. Our results emphasize that deeper investigation of the physiological role of IDO, an attractive target for immunotherapies of cancer, is of great importance.
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Indolamina-Pirrol 2,3,-Dioxigenasa/efectos adversos , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Inflamación/complicaciones , Interleucina-6/efectos adversos , Fotoquimioterapia/métodos , Animales , Femenino , Humanos , RatonesRESUMEN
Despite great interest, the mechanism of neutrophil extracellular traps (NETs) release is not fully understood and some aspects of this process, e.g. the role of reactive nitrogen species (RNS), still remain unclear. Therefore, our aim was to investigate the mechanisms underlying RNS-induced formation of NETs and contribution of RNS to NETs release triggered by various physiological and synthetic stimuli. The involvement of RNS in NETs formation was studied in primary human neutrophils and differentiated human promyelocytic leukemia cells (HL-60 cells). RNS (peroxynitrite and nitric oxide) efficiently induced NETs release and potentiated NETs-inducing properties of platelet activating factor and lipopolysaccharide. RNS-induced NETs formation was independent of autophagy and histone citrullination, but dependent on the activity of phosphoinositide 3-kinases (PI3K) and myeloperoxidase, as well as selective degradation of histones H2A and H2B by neutrophil elastase. Additionally, NADPH oxidase activity was required to release NETs upon stimulation with NO, as shown in NADPH-deficient neutrophils isolated from patients with chronic granulomatous disease. The role of RNS was further supported by increased RNS synthesis upon stimulation of NETs release with phorbol 12-myristate 13-acetate and calcium ionophore A23187. Scavenging or inhibition of RNS formation diminished NETs release triggered by these stimuli while scavenging of peroxynitrite inhibited NO-induced NETs formation. Our data suggest that RNS may act as mediators and inducers of NETs release. These processes are PI3K-dependent and ROS-dependent. Since inflammatory reactions are often accompanied by nitrosative stress and NETs formation, our studies shed a new light on possible mechanisms engaged in various immune-mediated conditions.
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Trampas Extracelulares/efectos de los fármacos , Neutrófilos/metabolismo , Ácido Peroxinitroso/farmacología , Adenina/análogos & derivados , Adenina/farmacología , ADN/metabolismo , Trampas Extracelulares/metabolismo , Enfermedad Granulomatosa Crónica/metabolismo , Enfermedad Granulomatosa Crónica/patología , Humanos , Elastasa de Leucocito/metabolismo , Neutrófilos/citología , Neutrófilos/inmunología , Óxido Nítrico , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/metabolismo , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , S-Nitroso-N-Acetilpenicilamina/farmacología , Transducción de Señal/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is a genetically heterogeneous blood cancer characterized by abnormal expansion of immature B cells. Although intensive chemotherapy provides high cure rates in a majority of patients, subtypes harboring certain genetic lesions, such as MLL rearrangements or BCR-ABL1 fusion, remain clinically challenging, necessitating a search for other therapeutic approaches. Herein, we aimed to validate antioxidant enzymes of the thioredoxin system as potential therapeutic targets in BCP-ALL. We observed oxidative stress along with aberrant expression of the enzymes associated with the activity of thioredoxin antioxidant system in BCP-ALL cells. Moreover, we found that auranofin and adenanthin, inhibitors of the thioredoxin system antioxidant enzymes, effectively kill BCP-ALL cell lines and pediatric and adult BCP-ALL primary cells, including primary cells cocultured with bone marrow-derived stem cells. Furthermore, auranofin delayed the progression of leukemia in MLL-rearranged patient-derived xenograft model and prolonged the survival of leukemic NSG mice. Our results unveil the thioredoxin system as a novel target for BCP-ALL therapy, and indicate that further studies assessing the anticancer efficacy of combinations of thioredoxin system inhibitors with conventional anti-BCP-ALL drugs should be continued.
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Auranofina/farmacología , Diterpenos de Tipo Kaurano/farmacología , Sistemas de Liberación de Medicamentos , Proteínas de Neoplasias/antagonistas & inhibidores , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamiento farmacológico , Tiorredoxinas/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Femenino , Proteínas de Fusión bcr-abl/metabolismo , Reordenamiento Génico , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
L-ascorbate (L-ASC) is a widely-known dietary nutrient which holds promising potential in cancer therapy when given parenterally at high doses. The anticancer effects of L-ASC involve its autoxidation and generation of H2O2, which is selectively toxic to malignant cells. Here we present that thioredoxin antioxidant system plays a key role in the scavenging of extracellularly-generated H2O2 in malignant B-cells. We show that inhibition of peroxiredoxin 1, the enzyme that removes H2O2 in a thioredoxin system-dependent manner, increases the sensitivity of malignant B-cells to L-ASC. Moreover, we demonstrate that auranofin (AUR), the inhibitor of the thioredoxin system that is used as an antirheumatic drug, diminishes the H2O2-scavenging capacity of malignant B-cells and potentiates pharmacological ascorbate anticancer activity in vitro and in vivo. The addition of AUR to L-ASC-treated cells triggers the accumulation of H2O2 in the cells, which results in iron-dependent cytotoxicity. Importantly, the synergistic effects are observed at as low as 200⯵Mâ¯L-ASC concentrations. In conclusion, we observed strong, synergistic, cancer-selective interaction between L-ASC and auranofin. Since both of these agents are available in clinical practice, our findings support further investigations of the efficacy of pharmacological ascorbate in combination with auranofin in preclinical and clinical settings.
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Ácido Ascórbico/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Peróxido de Hidrógeno/metabolismo , Leucemia de Células B/metabolismo , Linfoma de Células B/metabolismo , Tiorredoxinas/metabolismo , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Linfocitos B/patología , Línea Celular , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Hierro/metabolismo , Leucemia de Células B/tratamiento farmacológico , Leucemia de Células B/patología , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/patología , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Our previous work has shown peroxiredoxin-1 (PRDX1), one of major antioxidant enzymes, to be a biomarker in human breast cancer. Hereby, we further investigate the role of PRDX1, compared to its close homolog PRDX2, in mammary malignant cells. METHODS: CRISPR/Cas9- or RNAi-based methods were used for genetic targeting PRDX1/2. Cell growth was assessed by crystal violet, EdU incorporation or colony formation assays. In vivo growth was assessed by a xenotransplantation model. Adenanthin was used to inhibit the thioredoxin-dependent antioxidant defense system. The prooxidant agents used were hydrogen peroxide, glucose oxidase and sodium L-ascorbate. A PY1 probe or HyPer-3 biosensor were used to detect hydrogen peroxide content in samples. RESULTS: PRDX1 downregulation significantly impaired the growth rate of MCF-7 and ZR-75-1 breast cancer cells. Likewise, xenotransplanted PRDX1-deficient MCF-7 cells presented a retarded tumour growth. Furthermore, genetic targeting of PRDX1 or adenanthin, but not PRDX2, potently sensitised all six cancer cell lines studied, but not the non-cancerous cells, to glucose oxidase and ascorbate. CONCLUSIONS: Our study pinpoints the dominant role for PRDX1 in management of exogeneous oxidative stress by breast cancer cells and substantiates further exploration of PRDX1 as a target in this disease, especially when combined with prooxidant agents.
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Antioxidantes/administración & dosificación , Neoplasias de la Mama/terapia , Diterpenos de Tipo Kaurano/administración & dosificación , Técnicas de Silenciamiento del Gen/métodos , Peroxirredoxinas/genética , Animales , Antioxidantes/farmacología , Ácido Ascórbico/administración & dosificación , Ácido Ascórbico/farmacología , Neoplasias de la Mama/genética , Sistemas CRISPR-Cas , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Diterpenos de Tipo Kaurano/farmacología , Femenino , Glucosa Oxidasa/administración & dosificación , Glucosa Oxidasa/farmacología , Humanos , Células MCF-7 , Ratones , Estrés Oxidativo/efectos de los fármacos , Interferencia de ARN , Regulación hacia Arriba/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A cute myeloid leukemia is a malignant disease of immature myeloid cells. Despite significant therapeutic effects of differentiation-inducing agents in some acute myeloid leukemia subtypes, the disease remains incurable in a large fraction of patients. Here we show that SK053, a thioredoxin inhibitor, induces differentiation and cell death of acute myeloid leukemia cells. Considering that thioredoxin knock-down with short hairpin RNA failed to exert antiproliferative effects in one of the acute myeloid leukemia cell lines, we used a biotin affinity probe-labeling approach to identify potential molecular targets for the effects of SK053. Mass spectrometry of proteins precipitated from acute myeloid leukemia cells incubated with biotinylated SK053 used as a bait revealed protein disulfide isomerase as a potential binding partner for the compound. Biochemical, enzymatic and functional assays using fluorescence lifetime imaging confirmed that SK053 binds to and inhibits the activity of protein disulfide isomerase. Protein disulfide isomerase knockdown with short hairpin RNA was associated with inhibition of cell growth, increased CCAAT enhancer-binding protein α levels, and induction of differentiation of HL-60 cells. Molecular dynamics simulation followed by the covalent docking indicated that SK053 binds to the fourth thioredoxin-like domain of protein disulfide isomerase. Differentiation of myeloid precursor cells requires the activity of CCAAT enhancer-binding protein α, the function of which is impaired in acute myeloid leukemia cells through various mechanisms, including translational block by protein disulfide isomerase. SK053 increased the levels of CCAAT enhancer-binding protein α and upregulated mRNA levels for differentiation-associated genes. Finally, SK053 decreased the survival of blasts and increased the percentage of cells expressing the maturation-associated CD11b marker in primary cells isolated from bone marrow or peripheral blood of patients with acute myeloid leukemia. Collectively, these results provide a proof-of-concept that protein disulfide isomerase inhibition has potential as a therapeutic strategy for the treatment of acute myeloid leukemia and for the development of small-molecule inhibitors of protein disulfide isomerase.
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Diferenciación Celular/efectos de los fármacos , Dipéptidos/farmacología , Inhibidores Enzimáticos/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Metacrilatos/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Proteína Disulfuro Isomerasas/antagonistas & inhibidores , Femenino , Células HL-60 , Humanos , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/patología , Masculino , Proteínas de Neoplasias/metabolismo , Proteína Disulfuro Isomerasas/metabolismoRESUMEN
BACKGROUND: Accumulating evidence suggest that autophagy plays a pivotal role in various anticancer therapies, including photodynamic therapy (PDT), acting as a pro-death or pro-survival mechanism in a context-dependent manner. Therefore, we aimed to determine the role of autophagy in Photofrin-based PDT. METHODS: In vitro cytotoxic/cytostatic effects of PDT were evaluated with crystal violet cell viability assay. Autophagy induction was analyzed by immunoblotting and immunofluorescence using anti-LC3 antibody. Autophagy was inhibited by shRNA-mediated ATG5 knockdown or CRISPR/Cas9-mediated ATG5 knockout. Apoptosis was assessed by flow cytometry analysis of propidium iodide and anexin V-positive cells as well as by detection of cleaved PARP and caspase 3 proteins using immunoblotting. Protein carbonylation was evaluated by the 2,4-dinitrophenylhydrazine (DNPH) method. RESULTS: Photofrin-PDT leads to robust autophagy induction in two cancer cell lines, Hela and MCF-7. shRNA-mediated knockdown of ATG5 only partially blocks autophagic response and only marginally affects the sensitivity of Hela and MCF-7 cells to PDT. ATG5 knockout in HeLa cell line utilizing CRISPR/Cas9 genome editing results in increased PDT-mediated cytotoxicity, which is accompanied by an enhanced apoptotic response and increased accumulation of carbonylated proteins. CONCLUSIONS: Altogether, these observations imply that autophagy contributes to Photofrin-PDT resistance by enabling clearance of carbonylated and other damaged proteins. Therefore, autophagy inhibition may serve as a strategy to improve PDT efficacy.
Asunto(s)
Antineoplásicos/farmacología , Autofagia/efectos de los fármacos , Autofagia/efectos de la radiación , Éter de Dihematoporfirina/farmacología , Fármacos Fotosensibilizantes/farmacología , Autofagia/genética , Proteína 5 Relacionada con la Autofagia/genética , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Expresión Génica , Técnicas de Silenciamiento del Gen , Marcación de Gen , Humanos , Luz , Fotoquimioterapia , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
Downregulation of CD20, a molecular target for monoclonal antibodies (mAbs), is a clinical problem leading to decreased efficacy of anti-CD20-based therapeutic regimens. The epigenetic modulation of CD20 coding gene (MS4A1) has been proposed as a mechanism for the reduced therapeutic efficacy of anti-CD20 antibodies and confirmed with nonselective histone deacetylase inhibitors (HDACis). Because the use of pan-HDACis is associated with substantial adverse effects, the identification of particular HDAC isoforms involved in CD20 regulation seems to be of paramount importance. In this study, we demonstrate for the first time the role of HDAC6 in the regulation of CD20 levels. We show that inhibition of HDAC6 activity significantly increases CD20 levels in established B-cell tumor cell lines and primary malignant cells. Using pharmacologic and genetic approaches, we confirm that HDAC6 inhibition augments in vitro efficacy of anti-CD20 mAbs and improves survival of mice treated with rituximab. Mechanistically, we demonstrate that HDAC6 influences synthesis of CD20 protein independently of the regulation of MS4A1 transcription. We further demonstrate that translation of CD20 mRNA is significantly enhanced after HDAC6 inhibition, as shown by the increase of CD20 mRNA within the polysomal fraction, indicating a new role of HDAC6 in the posttranscriptional mechanism of CD20 regulation. Collectively, our findings suggest HDAC6 inhibition is a rational therapeutic strategy to be implemented in combination therapies with anti-CD20 monoclonal antibodies and open up novel avenues for the clinical use of HDAC6 inhibitors.
Asunto(s)
Antígenos CD20/genética , Antineoplásicos/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Linfoma no Hodgkin/tratamiento farmacológico , Rituximab/farmacología , Animales , Antígenos CD20/inmunología , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/patología , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Histona Desacetilasa 6 , Humanos , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/patología , Linfoma no Hodgkin/genética , Linfoma no Hodgkin/inmunología , Linfoma no Hodgkin/patología , Ratones Endogámicos BALB C , Ratones SCID , ARN Mensajero/genética , Células Tumorales Cultivadas , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Photodynamic therapy (PDT) has been shown to destroy tumour-associated lymphatic vessels. Therefore, we sought to investigate the functional outcomes of PDT-mediated damage to the lymphatic vessels. We observed that PDT with verteporfin, completely but transiently, blocks the functional lymphatic drainage in the orthotopic mammary tumour models. Sustained inhibition of lymphatic vessels regeneration induced by lenalidomide or the soluble form of vascular endothelial growth factor receptor 3 (sVEGFR3) that neutralises lymphangiogenic vascular endothelial growth factor C (VEGF-C), significantly impaired antitumour efficacy of PDT. Antilymphangiogenic compounds also significantly inhibited the ability of intratumourally inoculated dendritic cells (DCs) to translocate to local lymph nodes and diminished the number of tumour-infiltrating interferon-γ-secreting or tumour antigen-specific CD8+ T cells. Lenalidomide also abrogated antitumour effects of the combination immunotherapy with PDT and anti-programmed death-ligand 1 (PD-L1) antibodies. Altogether, these findings indicate that PDT-mediated damage to the lymphatic vessels negatively affects development of antitumour immunity, and that drugs that impair lymphatic vessel regeneration might not be suitable for the use in combination with PDT.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Puntos de Control del Ciclo Celular/efectos de los fármacos , Linfangiogénesis/efectos de los fármacos , Fotoquimioterapia , Porfirinas/metabolismo , Porfirinas/farmacología , Talidomida/análogos & derivados , Animales , Modelos Animales de Enfermedad , Femenino , Lenalidomida , Linfangiogénesis/efectos de la radiación , Vasos Linfáticos/efectos de los fármacos , Vasos Linfáticos/patología , Ratones , Ratones Endogámicos C57BL , Fármacos Fotosensibilizantes/farmacología , Talidomida/farmacología , Receptor 3 de Factores de Crecimiento Endotelial Vascular/farmacología , VerteporfinaRESUMEN
The genetic background of dilated cardiomyopathy is highly heterogeneous, with close to 100 known genes and a number of candidates described to date. Nebulin-related-anchoring protein (NRAP) is an actin-binding cytoskeletal protein expressed predominantly in striated and cardiac muscles, and is involved in myofibrillar assembly in the foetal heart and in force transmission in the adult heart. The homozygous NRAP truncating variant (rs201084642), which is predicted to introduce premature stop codon into all NRAP isoforms, was revealed in the dilated cardiomyopathy patient using whole exome sequencing. The same genotype was detected in the asymptomatic proband's brother. The expression of the NRAP protein was undetectable in the patient's heart muscle by the Western blot. Genotyping for rs201084642 in the ethnically matched cohort of 231 dilated cardiomyopathy patients did not reveal any additional subjects with this variant. Our findings suggest that the biallelic loss-of-function mutation in NRAP could constitute a relatively rare, low-penetrance genetic risk factor for dilated cardiomyopathy.