RESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Piper longum L., an herbal medicine used in India and other Asian countries, is prescribed routinely for a range of diseases, including tumor. Piperlongumine, a natural product isolated from Piper longum L., has received widespread attention due to its various pharmacological activities, such as anti-inflammatory, antimicrobial, and antitumor effects. AIM OF THE STUDY: Chronic myelogenous leukemia (CML) is a hematopoietic disease caused by Bcr-Abl fusion gene, with an incidence of 15% in adult leukemias. Targeting Bcr-Abl by imatinib provides a successful treatment approach for CML. However, imatinib resistance is an inevitable issue for CML treatment. In particular, T315I mutant is the most stubborn of the Bcr-Abl point mutants associated with imatinib resistance. Therefore, it is urgent to find an alternative approach to conquer imatinib resistance. This study investigated the role of a natural product piperlongumine in overcoming imatinib resistance in CML. MATERIALS AND METHODS: Cell viability and apoptosis were evaluated by MTS assay and Annexin V/propidium iodide counterstaining assay, respectively. Levels of intracellular signaling proteins were assessed by Western blots. Mitochondrial membrane potential was reflected by the fluorescence intensity of rhodamine-123. The function of proteasome was detected using 20S proteasomal activity assay, proteasomal deubiquitinase activity assay, and deubiquitinase active-site-directed labeling. The antitumor effects of piperlongumine were assessed with mice xenografts. RESULTS: We demonstrate that (i) Piperlongumine inhibits proteasome function by targeting 20S proteasomal peptidases and 19S proteasomal deubiquitinases (USP14 and UCHL5) in Bcr-Abl-WT and Bcr-Abl-T315I CML cells; (ii) Piperlongumine inhibits the cell viability of CML cell lines and primary CML cells; (iii) Proteasome inhibition by piperlongumine leads to cell apoptosis and downregulation of Bcr-Abl; (iv) Piperlongumine suppresses the tumor growth of CML xenografts. CONCLUSIONS: These results support that blockade of proteasome activity by piperlongumine provides a new therapeutic strategy for treating imatinib-resistant CML.
Asunto(s)
Antineoplásicos , Productos Biológicos , Leucemia Mielógena Crónica BCR-ABL Positiva , Humanos , Ratones , Animales , Mesilato de Imatinib/farmacología , Mesilato de Imatinib/uso terapéutico , Complejo de la Endopetidasa Proteasomal/metabolismo , Resistencia a Antineoplásicos , Proliferación Celular , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Proteínas de Fusión bcr-abl/genética , Apoptosis , Enzimas Desubicuitinizantes/uso terapéutico , Productos Biológicos/uso terapéutico , Línea Celular Tumoral , Antineoplásicos/farmacología , Ubiquitina Tiolesterasa/uso terapéuticoRESUMEN
BACKGROUND: Chronic myeloid leukaemia (CML) is a haematological cancer featured by the presence of BCR-ABL fusion protein with abnormal tyrosine kinase activation. Classical tyrosine kinase inhibitor (TKI)-based therapies are available to patients with CML. However, acquired resistance to TKI has been a challenging obstacle, especially stubborn T315I mutation is the most common cause. Therefore, it is especially urgent to find more effective targets to overcome TKI resistance induced by BCR-ABLT315I . Proteasomal deubiquitinases (USP14 and UCHL5) have fundamental roles in the ubiquitin-proteasome system and possess multiple functions during cancer progression. METHODS: The human peripheral blood mononuclear cells were collected to measure the mRNA expression of USP14 and UCHL5, as well as to detect the toxicity effect of b-AP15. We explored the effect of b-AP15 on the activity of proteasomal deubiquitinases. We detected the effects of b-AP15 on BCR-ABLWT and BCR-ABLT315I CML cells in vitro and in the subcutaneous tumour model. We knocked down USP14 and/or UCHL5 by shRNA to explore whether these proteasomal deubiquitinases are required for cell proliferation of CML. RESULTS: In this study, we found that increased expression of the proteasomal deubiquitinase USP14 and UCHL5 in primary cancer cells from CML patients compared to healthy donors. b-AP15, an inhibitor of USP14 and UCHL5, exhibited potent tumour-killing activity in BCR-ABLWT and BCR-ABLT315I CML cell lines, as well as in CML xenografts and primary CML cells. Mechanically, pharmacological or genetic inhibition of USP14 and UCHL5 induced cell apoptosis and decreased the protein level of BCR-ABL in CML cells expressing BCR-ABLWT and BCR-ABLT315I . Moreover, b-AP15 synergistically enhanced the cytotoxic effect caused by TKI imatinib in BCR-ABLWT and BCR-ABLT315I CML cells. CONCLUSION: Collectively, our results demonstrate targeting USP14 and UCHL5 as a potential strategy for combating TKI resistance in CML.
Asunto(s)
Leucemia Mielógena Crónica BCR-ABL Positiva , Complejo de la Endopetidasa Proteasomal , Inhibidores de Proteínas Quinasas , Ubiquitina Tiolesterasa , Enzimas Desubicuitinizantes/genética , Enzimas Desubicuitinizantes/uso terapéutico , Resistencia a Antineoplásicos/genética , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Proteínas de Fusión bcr-abl/farmacología , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/patología , Piperidonas/metabolismo , Piperidonas/farmacología , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejo de la Endopetidasa Proteasomal/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Ubiquitina Tiolesterasa/genéticaRESUMEN
The ubiquitin-proteasome system is the crucial homeostatic mechanism responsible for the degradation and turnover of proteins. As such, alterations at this level are often associated with oncogenic processes, either through accumulation of undegraded pathway effectors or, conversely, excessive degradation of tumor-suppressing factors. Therefore, investigation of the ubiquitin- proteasome system has gained much attraction in recent years, especially in the context of hematological malignancies, giving rise to efficient therapeutics such as bortezomib for multiple myeloma. Current investigations are now focused on manipulating protein degradation via fine-tuning of the ubiquitination process through inhibition of deubiquitinating enzymes or development of PROTAC systems for stimulation of ubiquitination and protein degradation. On the other hand, the efficiency of Thalidomide derivates in myelodysplastic syndromes (MDS), such as Lenalidomide, acted as the starting point for the development of targeted leukemia-associated protein degradation molecules. These novel molecules display high efficiency in overcoming the limitations of current therapeutic regimens, such as refractory diseases. Therefore, in this manuscript we will address the therapeutic opportunities and strategies based on the ubiquitin-proteasome system, ranging from the modulation of deubiquitinating enzymes and, conversely, describing the potential of modern targeted protein degrading molecules and their progress into clinical implementation.
Asunto(s)
Leucemia Mieloide Aguda , Mieloma Múltiple , Síndromes Mielodisplásicos , Trastornos Mieloproliferativos , Humanos , Ubiquitina/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Complejo de la Endopetidasa Proteasomal/uso terapéutico , Lenalidomida/uso terapéutico , Talidomida/farmacología , Talidomida/uso terapéutico , Bortezomib/uso terapéutico , Descubrimiento de Drogas , Síndromes Mielodisplásicos/tratamiento farmacológico , Síndromes Mielodisplásicos/patología , Mieloma Múltiple/metabolismo , Enzimas Desubicuitinizantes/uso terapéuticoRESUMEN
Many diseases are driven by proteins that are aberrantly ubiquitinated and degraded. These diseases would be therapeutically benefited by targeted protein stabilization (TPS). Here we present deubiquitinase-targeting chimeras (DUBTACs), heterobifunctional small molecules consisting of a deubiquitinase recruiter linked to a protein-targeting ligand, to stabilize the levels of specific proteins degraded in a ubiquitin-dependent manner. Using chemoproteomic approaches, we discovered the covalent ligand EN523 that targets a non-catalytic allosteric cysteine C23 in the K48-ubiquitin-specific deubiquitinase OTUB1. We showed that a DUBTAC consisting of our EN523 OTUB1 recruiter linked to lumacaftor, a drug used to treat cystic fibrosis that binds ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR), robustly stabilized ΔF508-CFTR protein levels, leading to improved chloride channel conductance in human cystic fibrosis bronchial epithelial cells. We also demonstrated stabilization of the tumor suppressor kinase WEE1 in hepatoma cells. Our study showcases covalent chemoproteomic approaches to develop new induced proximity-based therapeutic modalities and introduces the DUBTAC platform for TPS.
Asunto(s)
Fibrosis Quística , Quimera/metabolismo , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Enzimas Desubicuitinizantes/metabolismo , Enzimas Desubicuitinizantes/uso terapéutico , Humanos , Ligandos , Ubiquitina/metabolismoRESUMEN
OBJECTIVES: Dysregulation of IL-12 and IL-23 is related to many autoimmune diseases including arthritis. The production of IL-12 and IL-23 were reported to be under the control of JMJD2D, whose activity and stability were promoted by the TRAF-binding domain (TRABID), a deubiquitinating enzyme that epigenetically modulated inflammatory gene expression. NSC1122002 is a novel inhibitor of TRABID, and this study aimed to examine the effects of NSC1122002 on the expression of IL-12 and IL-23 both in vitro and in vivo in the context of collagen-induced arthritis, consequently to evaluate its potential as a drug candidate for treating inflammatory disease. METHODS: Bone marrow cells were isolated to detect the effect of NSC1122002 on the development of innate immune cells and other precursor cells. Primary macrophages and osteoclasts were used to examine the impact of NSC1122002 on cytokine expression. Collagen-induced arthritis was established to determine the function of NSC1122002 in vivo. RESULTS: NSC112200 did not affect the development of innate immune cells, primary osteoclast, and haematopoietic stem cells. NSC112200 specifically downregulated the expression of IL-12 and IL-23 through promoting degradation of JMJD2D by directly inhibited the deubiquitinating activity of TRABID. Besides, NSC112200 significantly suppressed the induction of CIA in mice. CONCLUSIOINS: Our findings provided new insight into the pathological mechanism and intervention method for arthritis therapy and identified that NSC112200 could be a potential drug for treating autoimmune diseases.
Asunto(s)
Artritis Experimental , Enfermedades Autoinmunes , Animales , Artritis Experimental/inducido químicamente , Artritis Experimental/tratamiento farmacológico , Citocinas/metabolismo , Enzimas Desubicuitinizantes/uso terapéutico , Modelos Animales de Enfermedad , Interleucina-12 , Interleucina-23 , Ratones , Ratones Endogámicos DBA , Osteoclastos/metabolismoRESUMEN
The focus of basic and applied research on core stem cell transcription factors has paved the way to initial delineation of their characteristics, their regulatory mechanisms, and the applicability of their regulatory proteins for protein-induced pluripotent stem cells (protein-IPSC) generation and in further clinical settings. Striking parallels have been observed between cancer stem cells (CSCs) and stem cells. For the maintenance of stem cells and CSC pluripotency and differentiation, post translational modifications (i.e., ubiquitylation and deubiquitylation) are tightly regulated, as these modifications result in a variety of stem cell fates. The identification of deubiquitylating enzymes (DUBs) involved in the regulation of core stem cell transcription factors and CSC-related proteins might contribute to providing novel insights into the implications of DUB regulatory mechanisms for governing cellular reprogramming and carcinogenesis. Moreover, we propose the novel possibility of applying DUBs coupled with core transcription factors to improve protein-iPSC generation efficiency. Additionally, this review article further illustrates the potential of applying DUB inhibitors as a novel therapeutic intervention for targeting CSCs. Thus, defining DUBs as core pharmacological targets implies that future endeavors to develop their inhibitors may revolutionize our ability to regulate stem cell maintenance and differentiation, somatic cell reprogramming, and cancer stem cells.