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BACKGROUND: Despite advancements in chronic myeloid leukemia (CML) therapy with tyrosine kinase inhibitors (TKIs), resistance and intolerance remain significant challenges. Leukemia stem cells (LSCs) and TKI-resistant cells rely on altered mitochondrial metabolism and oxidative phosphorylation. Targeting rewired energy metabolism and inducing non-apoptotic cell death, along with the release of damage-associated molecular patterns (DAMPs), can enhance therapeutic strategies and immunogenic therapies against CML and prevent the emergence of TKI-resistant cells and LSC persistence. METHODS: Transcriptomic analysis was conducted using datasets of CML patients' stem cells and healthy cells. DNA damage was evaluated by fluorescent microscopy and flow cytometry. Cell death was assessed by trypan blue exclusion test, fluorescent microscopy, flow cytometry, colony formation assay, and in vivo Zebrafish xenografts. Energy metabolism was determined by measuring NAD+ and NADH levels, ATP production rate by Seahorse analyzer, and intracellular ATP content. Mitochondrial fitness was estimated by measurements of mitochondrial membrane potential, ROS, and calcium accumulation by flow cytometry, and morphology was visualized by TEM. Bioinformatic analysis, real-time qPCR, western blotting, chemical reaction prediction, and molecular docking were utilized to identify the drug target. The immunogenic potential was assessed by high mobility group box (HMGB)1 ELISA assay, luciferase-based extracellular ATP assay, ectopic calreticulin expression by flow cytometry, and validated by phagocytosis assay, and in vivo vaccination assay using syngeneic C57BL/6 mice. RESULTS: Transcriptomic analysis identified metabolic alterations and DNA repair deficiency signatures in CML patients. CML patients exhibited enrichment in immune system, DNA repair, and metabolic pathways. The gene signature associated with BRCA mutated tumors was enriched in CML datasets, suggesting a deficiency in double-strand break repair pathways. Additionally, poly(ADP-ribose) polymerase (PARP)1 was significantly upregulated in CML patients' stem cells compared to healthy counterparts. Consistent with the CML patient DNA repair signature, treatment with the methylated indolequinone MAC681 induced DNA damage, mitochondrial dysfunction, calcium homeostasis disruption, metabolic catastrophe, and necroptotic-like cell death. In parallel, MAC681 led to PARP1 degradation that was prevented by 3-aminobenzamide. MAC681-treated myeloid leukemia cells released DAMPs and demonstrated the potential to generate an immunogenic vaccine in C57BL/6 mice. MAC681 and asciminib exhibited synergistic effects in killing both imatinib-sensitive and -resistant CML, opening new therapeutic opportunities. CONCLUSIONS: Overall, increasing the tumor mutational burden by PARP1 degradation and mitochondrial deregulation makes CML suitable for immunotherapy.
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Myelomonocytic and monocytic acute myeloid leukemia (AML) subtypes are intrinsically resistant to venetoclax-based regimens. Identifying targetable vulnerabilities would limit resistance and relapse. We previously documented the synergism of venetoclax and cardiac glycoside (CG) combination in AML. Despite preclinical evidence, the repurposing of cardiac glycosides (CGs) in cancer therapy remained unsuccessful due to a lack of predictive biomarkers. We report that the ex vivo response of AML patient blasts and the in vitro sensitivity of established cell lines to the hemi-synthetic CG UNBS1450 correlates with the ATPase Na+/K+ transporting subunit alpha 1 (ATP1A1)/BCL2 like 1 (BCL2L1) expression ratio. Publicly available AML datasets identify myelomonocytic/monocytic differentiation as the most robust prognostic feature, along with core-binding factor subunit beta (CBFB), lysine methyltransferase 2A (KMT2A) rearrangements, and missense Fms-related receptor tyrosine kinase 3 (FLT3) mutations. Mechanistically, BCL2L1 protects from cell death commitment induced by the CG-mediated stepwise triggering of ionic perturbation, protein synthesis inhibition, and MCL1 downregulation. In vivo, CGs showed an overall tolerable profile while impacting tumor growth with an effect ranging from tumor growth inhibition to regression. These findings suggest a predictive marker for CG repurposing in specific AML subtypes.
Assuntos
Glicosídeos Cardíacos , Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Sulfonamidas/farmacologia , Tirosina Quinase 3 Semelhante a fms/metabolismo , Linhagem Celular Tumoral , ATPase Trocadora de Sódio-Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/uso terapêutico , Proteína bcl-X/metabolismoRESUMO
OBJECTIVE: Alterations in lipid metabolism are associated with aging and age-related diseases. Chaperone-mediated autophagy (CMA) is a lysosome-dependent process involved in specific protein degradation. Heat shock cognate 71 kDa protein (Hsc70) recognizes cytosolic proteins with KFERQ motif and allows them to enter the lysosome via lysosome-associated membrane glycoprotein 2 isoform A (LAMP2A). CMA deficiency is associated with dysregulated lipid metabolism in the liver. In this study, we examined the effect of CMA on lipid metabolism in the aged liver. METHODS: 12-week-old and 88-week-old mice were employed to assess the effect of aging on hepatic CMA activity. We generated CMA-deficient mouse primary hepatocytes using siRNA for Lamp2a and liver-specific LAMP2A knockdown mice via adeno-associated viruses expressing short hairpin RNAs to investigate the influence of CMA on lipid metabolism. RESULTS: We noted aging-induced progression toward fatty liver and a decrease in LAMP2A levels in total protein and lysosomes. The expression of genes associated with fatty acid oxidation was markedly downregulated in the aged liver, as verified in CMA-deficient mouse primary hepatocytes. In addition, the aged liver accumulated nuclear receptor corepressor 1 (NCoR1), a negative regulator of peroxisome proliferator-activated receptor α (PPARα). We found that Hsc70 binds to NCoR1 via the KFERQ motif. Lamp2a siRNA treatment accumulated NCoR1 and decreased the fatty acid oxidation rate. Pharmacological activation of CMA by AR7 treatment increased LAMP2A expression, leading to NCoR1 degradation. A liver-specific LAMP2A knockdown via adeno-associated viruses expressing short hairpin RNAs caused NCoR1 accumulation, inactivated PPARα, downregulated the expression of fatty acid oxidation-related genes and significantly increased liver triglyceride levels. CONCLUSIONS: Our results elucidated a novel PPARα regulatory mechanism involving CMA-mediated NCoR1 degradation during aging. These findings demonstrate that CMA dysregulation is crucial for the progression of aging-related fatty liver diseases.
Assuntos
Autofagia Mediada por Chaperonas , Animais , Camundongos , Autofagia , PPAR alfa/genética , Envelhecimento , Fígado , Metabolismo dos Lipídeos , Ácidos Graxos/farmacologiaRESUMO
Despite genetic perturbations resulting in embryo lethality for most mitotic kinases, loss of the histone H3 mitotic kinase HASPIN reveals no adverse effect in mice models, establishing HASPIN as a promising target for anticancer therapy. However, developing a HASPIN inhibitor from conventional pharmacophores poses a technical challenge as this atypical kinase shares slight similarities with eukaryotic protein kinases. Chemically modifying a cytotoxic 4'-thioadenosine analogue through high genotoxicity yielded several novel nongenotoxic kinase inhibitors. In silico apporoaches utilizing transcriptomic and chemical similarities with known compounds and KINOMEscan profiles unveiled the HASPIN inhibitor LJ4827. LJ4827's specificity and potency as a HASPIN inhibitor were verified through in vitro kinase assay and X-ray crystallography. HASPIN inhibition by LJ4827 reduced histone H3 phosphorylation and impeded Aurora B recruitment in cancer cell centromeres but not in noncancer cells. Through transcriptome analysis of lung cancer patients, PLK1 was determined as a druggable synergistic partner to complement HASPIN inhibition. Chemical or genetic PLK1 perturbation with LJ4827 effectuated pronounced lung cancer cytotoxicity in vitro and in vivo. Therefore, LJ4827 is a novel anticancer therapeutic for selectively impeding cancer mitosis through potent HASPIN inhibition, and simultaneous HASPIN and PLK1 interference is a promising therapeutic strategy for lung cancer.
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The heterodimer of human ubiquitin fusion degradation 1 (hUfd1) and human nuclear protein localization 4 (hNpl4) is a major cofactor of human p97 adenosine triphosphatase (ATPase). The p97-Ufd1-Npl4 complex translocates the ubiquitin-conjugated proteins from the endoplasmic reticulum membrane to the cytoplasm. Ubiquitinated proteins are then degraded by the proteasome. The structures of Npl4 and Ufd1-Npl4 (UN) complex in Saccharomyces cerevisiae have been recently reported; however, the structures of hNpl4 and the human UN complex remain unknown. Here, we report the crystal structures of the human UN complex at a resolution of 2.7 Å and hNpl4 at a resolution of 3.0 Å. We also present atomic details and characterization of the human UN complex. Crystallographic studies and site-directed mutagenesis of the hUfd1 residues involved in the interaction with hNpl4 revealed the atomic details of the two proteins.
Assuntos
Adenosina Trifosfatases , Proteínas de Saccharomyces cerevisiae , Humanos , Ligação Proteica , Adenosina Trifosfatases/química , Proteínas Nucleares/metabolismo , Ubiquitina/metabolismo , Retículo Endoplasmático/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteína com Valosina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
While failure in resolution of inflammation is considered to increase the risk of tumorigenesis, there is paucity of experimental as well as clinical evidence supporting this association. Resolvin D1 (RvD1) is a representative pro-resolving lipid mediator that is endogenously generated from docosahexaenoic acid for the resolution of inflammation. Here, we report a decreased level of RvD1 in the blood from colorectal cancer patients and mice having inflammation-induced colon cancer, suggesting plasma RvD1 as a potential biomarker for monitoring colorectal cancer. Administration of RvD1 attenuated dextran sodium sulfate (DSS)-induced colitis and azoxymethane (AOM) plus DSS-induced colorectal carcinogenesis by suppressing the production of interleukin-6 (IL-6) and IL-6-mediated chromosomal instability. The protective effect of RvD1 against chromosomal instability is associated with downregulation of IL-6-induced Cyclin D1 expression, which appears to be mediated by blocking the Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) axis. RvD1 inhibited the STAT3 signaling pathway by interfering with the binding of IL-6 to its receptor (IL-6R), suggesting the novel function of RvD1 as a putative IL-6R antagonist. Together, our findings suggest that RvD1-mediated blockade of IL-6 signal transmission may contribute to inhibition of chromosomal instability and tumorigenesis.
Assuntos
Carcinogênese/patologia , Colite/complicações , Neoplasias do Colo/tratamento farmacológico , Neoplasias Colorretais/tratamento farmacológico , Ácidos Docosa-Hexaenoicos/farmacologia , Interleucina-6/farmacologia , Fuso Acromático/efeitos dos fármacos , Animais , Carcinogênese/metabolismo , Estudos de Casos e Controles , Colite/induzido quimicamente , Colite/patologia , Neoplasias do Colo/etiologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Neoplasias Colorretais/etiologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Fuso Acromático/patologiaRESUMO
Rationale: Cancer stem cells (CSCs) are known to cause tumor recurrence and drug resistance. The heat shock protein (HSP) system plays a major role in preserving expression and function of numerous oncoproteins, including those involved in the CSC activities. We explored novel anticancer drugs, especially those targeting HSP components required for the functional role of CSCs. Methods: Investigation of the role of the HSP system in CSCs and screening of a natural product chemical library were performed by utilizing cancer cell lines, primary cultures of patient-derived xenografts (PDXs), and their putative CSC subpopulations (i.e., those grown under sphere-forming conditions, stably transfected with reporter vectors carrying NANOG or POUSF1 promoters, or carrying high ALDH activity) in vitro and PDX and KrasG12D/+-driven tumor models in vivo. Regulation of the HSP system was investigated by immunoprecipitation, drug affinity responsive target stability assay, binding experiments using ATP-agarose beads and biotinylated drug, and docking analysis. Results: The HSP system was activated in CSCs via transcriptional upregulation of the HSP system components, especially HSP70. Evodiamine (Evo) was identified to induce apoptosis in both CSC and bulk non-CSC populations in human lung, colon, and breast cancer cells and their sublines with chemoresistance. Evo administration decreased the multiplicity, volume, and load of lung tumors in KrasG12D/+ transgenic mice and the growth of cancer cell line- and PDX-derived tumors without detectable toxicity. Mechanistically, Evo disrupted the HSP system by binding the N-terminal ATP-binding pocket of HSP70 and causing its ubiquitin-mediated degradation. Conclusions: Our findings illustrate HSP70 as a potential target for eliminating CSCs and Evo as an effective HSP70-targeting anticancer drug eradicating both CSCs and non-CSCs with a minimal toxicity.
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Antineoplásicos/farmacologia , Proteínas de Choque Térmico HSP70/metabolismo , Neoplasias/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Quinazolinas/farmacologia , Células A549 , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Células HCT116 , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Neoplasias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Peroxisome proliferator-activated receptor gamma (PPARγ) has recently been revealed to regulate tumor microenvironments. In particular, genetic alterations of PPARγ found in various cancers have been reported to play important roles in tumorigenesis by affecting PPARγ transactivation. In this study, we found that helix H3 of the PPARγ ligand-binding domain (LBD) has a number of sites that are mutated in cancers. To uncover underlying molecular mechanisms between helix H3 mutations and tumorigenesis, we performed structureâfunction studies on the PPARγ LBDs containing helix H3 mutations found in cancers. Interestingly, PPARγ Q286E found in bladder cancer induces a constitutively active conformation of PPARγ LBD and thus abnormal activation of PPARγ/RXRα pathway, which suggests tumorigenic roles of PPARγ in bladder cancer. In contrast, other helix H3 mutations found in various cancers impair ligand binding essential for transcriptional activity of PPARγ. These data indicate that cancer-associated mutations clustered in helix H3 of PPARγ LBD exhibit differential effects in PPARγ-mediated tumorigenesis and provide a basis for the development of new biomarkers targeting tumor microenvironments.
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MINERVA (melanoma invasion by ERK), also known as FAM129B, is a member of the FAM129 protein family, which is only present in vertebrates. MINERVA is involved in key signaling pathways regulating cell survival, proliferation and apoptosis and found upregulated in many types of cancer promoting invasion. However, the exact function of the protein remains elusive. X-ray crystallographic methods were implemented to determine the crystal structure of MINERVAΔC, lacking C-terminal flexible region. Trypsin digestion was required before crystallization to obtain diffraction-quality crystals. While the N-terminal pleckstrin homology (PH) domain exhibits the typical fold of PH domains, lipid binding assay indicates specific affinity towards phosphatidic acid and inositol 3-phosphate. A helix-rich domain that constitutes the rest of the molecule demonstrates a novel L-shaped fold that encompasses the PH domain. The overall structure of MINERVAΔC with binding assays and cell-based experiments suggest plasma membrane association of MINERVA and its function seem to be tightly regulated by various motifs within the C-terminal flexible region. Elucidation of MINERVAΔC structure presents a novel fold for an α-helix bundle domain that would provide a binding platform for interacting partners.
Assuntos
Membrana Celular/metabolismo , Cristalografia por Raios X/métodos , Melanoma/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Humanos , Melanoma/patologia , Modelos Moleculares , Fosfoproteínas/isolamento & purificação , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
Despite the discovery of tyrosine kinase inhibitors (TKIs) for the treatment of breakpoint cluster region-Abelson (BCR-ABL)+ cancer types, patients with chronic myeloid leukemia (CML) treated with TKIs develop resistance and severe adverse effects. Combination treatment, especially with a histone deacetylase (HDAC) 6 inhibitor (HDAC6i), appears to be an attractive option to prevent TKI resistance, considering the potential capacity of an HDAC6i to diminish BCR-ABL expression. We first validated the in vivo anti-cancer potential of the compound 7b by significantly reducing the tumor burden of BALB/c mice xenografted with K-562 cells, without notable organ toxicity. Here, we hypothesize that the HDAC6i compound 7b can lead to BCR-ABL downregulation in CML cells and sensitize them to TKI treatment. The results showed that combination treatment with imatinib and 7b resulted in strong synergistic caspase-dependent apoptotic cell death and drastically reduced the proportion of leukemia stem cells, whereas this treatment only moderately affected healthy cells. Ultimately, the combination significantly decreased colony formation in a semisolid methylcellulose medium and tumor mass in xenografted zebrafish compared to each compound alone. Mechanistically, the combination induced BCR-ABL ubiquitination and downregulation followed by disturbance of key proteins in downstream pathways involved in CML proliferation and survival. Taken together, our results suggest that an HDAC6i potentiates the effect of imatinib and could overcome TKI resistance in CML cells.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Fusão bcr-abl/metabolismo , Desacetilase 6 de Histona/antagonistas & inibidores , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Ubiquitinação/efeitos dos fármacos , Animais , Caspases/efeitos dos fármacos , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Células K562 , Camundongos , Camundongos Endogâmicos BALB C , Ensaio Tumoral de Célula-Tronco , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Genome-wide association studies of gastric cancer (GC) cases have revealed common gastric cancer susceptibility loci with low effect size. We investigated rare variants with high effect size via whole-exome sequencing (WES) of subjects with familial clustering of gastric cancer. WES of DNAs from the blood of 19 gastric cancer patients and 36 unaffected family members from 14 families with two or more gastric cancer patients were tested. Linkage analysis combined with association tests were performed using Pedigree Variant Annotation, Analysis, and Search Tool (pVAAST) software. Based on the logarithm of odds (LOD) and permutation-based composite likelihood ratio test (CLRT) from pVAAST, MUC4 was identified as a predisposing gene (LOD P-value = 1.9×10-5; permutation-based P-value of CLRT ≤ 9.9×10-9). In a larger cohort consisting of 597 GC patients and 9,759 healthy controls genotyped with SNP array, we discovered common variants in MUC4 regions (rs148735556, rs11717039, and rs547775645) significantly associated with GC supporting the association of MUC4 with gastric cancer. And the MUC4 variants were found in higher frequency in The Cancer Genome Atlas Study (TCGA) germline samples of patients with multiple cancer types. Immunohistochemistry indicated that MUC4 was downregulated in the noncancerous gastric mucosa of subjects with MUC4 germline missense variants, suggesting that loss of the protective function of MUC4 predisposes an individual to gastric cancer. Rare variants in MUC4 can be novel gastric cancer susceptibility loci in Koreans possessing the familial clustering of gastric cancer.
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Sequenciamento do Exoma , Ligação Genética , Predisposição Genética para Doença , Variação Genética , Mucina-4/genética , Estudos de Coortes , Família , Feminino , Células Germinativas/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Mucina-4/química , Linhagem , Reprodutibilidade dos Testes , Estômago/patologia , Neoplasias Gástricas/genéticaRESUMO
BACKGROUND: Chronic myeloid leukemia (CML) pathogenesis is mainly driven by the oncogenic breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 (BCR-ABL) fusion protein. Since BCR-ABL displays abnormal constitutive tyrosine kinase activity, therapies using tyrosine kinase inhibitors (TKis) such as imatinib represent a major breakthrough for the outcome of CML patients. Nevertheless, the development of TKi resistance and the persistence of leukemia stem cells (LSCs) remain barriers to cure the disease, justifying the development of novel therapeutic approaches. Since the activity of histone deacetylase (HDAC) is deregulated in numerous cancers including CML, pan-HDAC inhibitors may represent promising therapeutic regimens for the treatment of CML cells in combination with TKi. RESULTS: We assessed the anti-leukemic activity of a novel hydroxamate-based pan-HDAC inhibitor MAKV-8, which complied with the Lipinski's "rule of five," in various CML cells alone or in combination with imatinib. We validated the in vitro HDAC-inhibitory potential of MAKV-8 and demonstrated efficient binding to the ligand-binding pocket of HDAC isoenzymes. In cellulo, MAKV-8 significantly induced target protein acetylation, displayed cytostatic and cytotoxic properties, and triggered concomitant ER stress/protective autophagy leading to canonical caspase-dependent apoptosis. Considering the specific upregulation of selected HDACs in LSCs from CML patients, we investigated the differential toxicity of a co-treatment with MAKV-8 and imatinib in CML versus healthy cells. We also showed that beclin-1 knockdown prevented MAKV-8-imatinib combination-induced apoptosis. Moreover, MAKV-8 and imatinib co-treatment synergistically reduced BCR-ABL-related signaling pathways involved in CML cell growth and survival. Since our results showed that LSCs from CML patients overexpressed c-MYC, importantly MAKV-8-imatinib co-treatment reduced c-MYC levels and the LSC population. In vivo, tumor growth of xenografted K-562 cells in zebrafish was completely abrogated upon combined treatment with MAKV-8 and imatinib. CONCLUSIONS: Collectively, the present findings show that combinations HDAC inhibitor-imatinib are likely to overcome drug resistance in CML pathology.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Inibidores de Histona Desacetilases/uso terapêutico , Mesilato de Imatinib/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Acetilação/efeitos dos fármacos , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/química , Apoptose/efeitos dos fármacos , Proteína Beclina-1/genética , Sítios de Ligação , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Simulação por Computador , Resistencia a Medicamentos Antineoplásicos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/metabolismo , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/química , Humanos , Mesilato de Imatinib/farmacologia , Isoenzimas/química , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Simulação de Acoplamento Molecular , Células-Tronco Neoplásicas/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The proviral integration site for Moloney murine leukemia virus (PIM) family of serine/threonine-specific kinases consist of three isoforms, that regulate proliferation, apoptosis, metabolism, invasion, and metastasis of cancer cells. Among these, abnormally elevated kinase activity of PIM-1 contributes to the progression of gastric cancer and predicts poor prognosis and a low survival rate in gastric cancer patients. In the present study, we found that resveratrol, one of the representative chemopreventive and anticarcinogenic phytochemicals, directly binds to PIM-1 and thereby inhibits its catalytic activity in human gastric cancer SNU-601 cells. This resulted in suppression of phosphorylation of the proapoptotic Bad, a known substrate of PIM-1. Resveratrol, by inactivating PIM-1, also inhibited anchorage-independent growth and proliferation of SNU-601 cells. To understand the molecular interaction between resveratrol and PIM-1, we conducted docking simulation and found that resveratrol directly binds to the PIM-1 at the ATP-binding pocket. In conclusion, the proapototic and anti-proliferative effects of resveratrol in gastric cancer cells are likely to be mediated through suppression of PIM-1 kinase activity, which may represent a novel mechanism underlying its chemopreventive and anticarcinogenic actions.
Assuntos
Proliferação de Células/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Resveratrol/farmacologia , Neoplasias Gástricas/tratamento farmacológico , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Neoplasias Gástricas/metabolismoRESUMO
Adiponectin is an adipocyte-derived cytokine having an insulin-sensitizing activity. During the phenotypic screening of secondary metabolites derived from the marine fungus Aspergillusterreus, a poly cyclin-dependent kinase (CDK) inhibitor butyrolactone I affecting CDK1 and CDK5 was discovered as a potent adiponectin production-enhancing compound in the adipogenesis model of human bone marrow-derived mesenchymal stem cells (hBM-MSCs). CDK5 inhibitors exhibit insulin-sensitizing activities by suppressing the phosphorylation of peroxisome proliferator-activated receptor γ (PPARγ). However, the adiponectin production-enhancing activities of butyrolactone I have not been correlated with the potency of CDK5 inhibitor activities. In a target identification study, butyrolactone I was found to directly bind to PPARγ. In the crystal structure of the human PPARγ, the ligand-binding domain (LBD) in complex with butyrolactone I interacted with the amino acid residues located in the hydrophobic binding pockets of the PPARγ LBD, which is a typical binding mode of the PPARγ partial agonists. Therefore, the adiponectin production-enhancing effect of butyrolactone I was mediated by its polypharmacological dual modulator activities as both a CDK5 inhibitor and a PPARγ partial agonist.
Assuntos
4-Butirolactona/análogos & derivados , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , PPAR gama/agonistas , Inibidores de Proteínas Quinases/farmacologia , 4-Butirolactona/química , 4-Butirolactona/farmacologia , Adipogenia/efeitos dos fármacos , Adiponectina/biossíntese , Sítios de Ligação/fisiologia , Células da Medula Óssea , Células Cultivadas , Cristalografia por Raios X , Quinase 5 Dependente de Ciclina/química , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , PPAR gama/química , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Estrutura Terciária de ProteínaRESUMO
The N-Myc downstream-regulated gene (NDRG) family belongs to the α/ß-hydrolase fold and is known to exert various physiologic functions in cell proliferation, differentiation, and hypoxia-induced cancer metabolism. In particular, NDRG3 is closely related to proliferation and migration of prostate cancer cells, and recent studies reported its implication in lactate-triggered hypoxia responses or tumorigenesis. However, the underlying mechanism for the functions of NDRG3 remains unclear. Here, we report the crystal structure of human NDRG3 at 2.2 Å resolution, with six molecules in an asymmetric unit. While NDRG3 adopts the α/ß-hydrolase fold, complete substitution of the canonical catalytic triad residues to non-reactive residues and steric hindrance around the pseudo-active site seem to disable the α/ß-hydrolase activity. While NDRG3 shares a high similarity to NDRG2 in terms of amino acid sequence and structure, NDRG3 exhibited remarkable structural differences in a flexible loop corresponding to helix α6 of NDRG2 that is responsible for tumor suppression. Thus, this flexible loop region seems to play a distinct role in oncogenic progression induced by NDRG3. Collectively, our studies could provide structural and biophysical insights into the molecular characteristics of NDRG3.
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Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Sequência de Aminoácidos/genética , Diferenciação Celular/genética , Proliferação de Células/genética , Humanos , Masculino , Proteínas do Tecido Nervoso/genética , Neoplasias da Próstata/genética , Conformação Proteica , Proteínas Supressoras de Tumor/metabolismo , Difração de Raios X/métodosRESUMO
The N-degron pathway is a proteolytic system in which a single N-terminal amino acid acts as a determinant of protein degradation. Especially, degradation signaling of N-terminal asparagine (Nt-Asn) in eukaryotes is initiated from its deamidation by N-terminal asparagine amidohydrolase 1 (NTAN1) into aspartate. Here, we have elucidated structural principles of deamidation by human NTAN1. NTAN1 adopts the characteristic scaffold of CNF1/YfiH-like cysteine hydrolases that features an α-ß-ß sandwich structure and a catalytic triad comprising Cys, His, and Ser. In vitro deamidation assays using model peptide substrates with varying lengths and sequences showed that NTAN1 prefers hydrophobic residues at the second-position. The structures of NTAN1-peptide complexes further revealed that the recognition of Nt-Asn is sufficiently organized to produce high specificity, and the side chain of the second-position residue is accommodated in a hydrophobic pocket adjacent to the active site of NTAN1. Collectively, our structural and biochemical analyses of the substrate specificity of NTAN1 contribute to understanding the structural basis of all three amidases in the eukaryotic N-degron pathway.
Assuntos
Amidoidrolases/metabolismo , Asparagina/metabolismo , Peptídeos/metabolismo , Proteólise , Amidoidrolases/química , Asparagina/análise , Domínio Catalítico , Cristalografia por Raios X , Humanos , Modelos Moleculares , Peptídeos/química , Especificidade por SubstratoRESUMO
In the present study, a series of 6-substituted aminoindazole derivatives were designed, synthesized, and evaluated for bio-activities. The compounds were initially designed as indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors based on the structural feature of five IDO1 inhibitors, which are currently on clinical trials, and the important anticancer activity of the indazole scaffold. One of them, compound N-(4-fluorobenzyl)-1,3-dimethyl-1H-indazol-6-amine (36), exhibited a potent anti-proliferative activity with an IC50 value of 0.4 ± 0.3 µM in human colorectal cancer cells (HCT116). This compound also remarkably suppressed the IDO1 protein expression. In the cell-cycle studies, the suppressive activity of compound 36 in HCT116 cells was related to the G2/M cell cycle arrest. Altogether, the current findings demonstrate that compound 36 would be promising for further development as a potential anticancer agent.
RESUMO
Glutathione (GSH) degradation plays an essential role in GSH homeostasis, which regulates cell survival, especially in cancer cells. Among human GSH degradation enzymes, the ChaC2 enzyme acts on GSH to form 5-l-oxoproline and Cys-Gly specifically in the cytosol. Here, we report the crystal structures of ChaC2 in two different conformations and compare the structural features with other known γ-glutamylcyclotransferase enzymes. The unique flexible loop of ChaC2 seems to function as a gate to achieve specificity for GSH binding and regulate the constant GSH degradation rate. Structural and biochemical analyses of ChaC2 revealed that Glu74 and Glu83 play crucial roles in directing the conformation of the enzyme and in modulating the enzyme activity. Based on a docking study of GSH to ChaC2 and binding assays, we propose a substrate-binding mode and catalytic mechanism. We also found that overexpression of ChaC2, but not mutants that inhibit activity of ChaC2, significantly promoted breast cancer cell proliferation, suggesting that the GSH degradation by ChaC2 affects the growth of breast cancer cells. Our structural and functional analyses of ChaC2 will contribute to the development of inhibitors for the ChaC family, which could effectively regulate the progression of GSH degradation-related cancers.
Assuntos
Glutationa/metabolismo , gama-Glutamilciclotransferase/química , gama-Glutamilciclotransferase/metabolismo , Domínio Catalítico , Proliferação de Células , Células HEK293 , Humanos , Células MCF-7 , Simulação de Acoplamento Molecular , Mutação , Multimerização Proteica , Estrutura Quaternária de Proteína , Alinhamento de Sequência , gama-Glutamilciclotransferase/genéticaRESUMO
Imatinib is an effective anticancer drug for the treatment of leukemia. Interestingly, when an FDA-approved drug library was tested for agents that block peroxisome proliferator-activated receptor γ (PPARγ) phosphorylation at Ser245 to evaluate possibilities of antidiabetic drug repositioning, imatinib was determined as a PPARγ antagonist ligand. However, it is not well understood how imatinib binds to PPARγ or would improve insulin sensitivity without classical agonism. Here, we report the crystal structure of the PPARγ R288A mutant in complex with imatinib. Imatinib bound to Arm2 and Arm3 regions in the ligand-binding domain (LBD) of PPARγ, of which the Arm3 region is closely related to the inhibition of PPARγ phosphorylation at Ser245. The binding of imatinib in LBD induced a stable conformation of helix H2' and the Ω loop compared with the ligand-free state. In contrast, imatinib does not interact with Tyr473 on PPARγ helix H12, which is important for the classical agonism associated with side effects. Our study provides new structural insights into the PPARγ regulation by imatinib and may contribute to the development of new antidiabetic drugs targeting PPARγ while minimizing known side effects.
Assuntos
Hipoglicemiantes/farmacologia , Mesilato de Imatinib/farmacologia , PPAR gama/química , PPAR gama/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Reposicionamento de Medicamentos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Hipoglicemiantes/química , Mesilato de Imatinib/química , Modelos Moleculares , Mutação , PPAR gama/genética , Fosforilação/efeitos dos fármacos , Domínios Proteicos , Estrutura Secundária de Proteína , Serina/metabolismoRESUMO
Treatment of acute myeloid leukemia (AML) patients is still hindered by resistance and relapse, resulting in an overall poor survival rate. Recently, combining specific B-cell lymphoma (Bcl)-2 inhibitors with compounds downregulating myeloid cell leukemia (Mcl)-1 has been proposed as a new effective strategy to eradicate resistant AML cells. We show here that 1(R), 6(S), 1'(R), 6'(S), 11(R), 17(S)-fistularin-3, a bromotyrosine compound of the fistularin family, isolated from the marine sponge Suberea clavata, synergizes with Bcl-2 inhibitor ABT-199 to efficiently kill Mcl-1/Bcl-2-positive AML cell lines, associated with Mcl-1 downregulation and endoplasmic reticulum stress induction. The absolute configuration of carbons 11 and 17 of the fistularin-3 stereoisomer was fully resolved in this study for the first time, showing that the fistularin we isolated from the marine sponge Subarea clavata is in fact the (+)-11(R), 17(S)-fistularin-3 stereoisomer keeping the known configuration 1(R), 6(S), 1'(R), and 6'(S) for the verongidoic acid part. Docking studies and in vitro assays confirm the potential of this family of molecules to inhibit DNA methyltransferase 1 activity.