RESUMEN
BACKGROUND: The long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a cancer biomarker. Furthermore, fusion of the MALAT1 gene with glioma-associated oncogene 1 (GLI1) is a diagnostic marker of plexiform fibromyxoma and gastroblastoma; however, the function of this fusion gene remains unexplored. METHOD: In this study, we elucidate the structure and function of the MALAT1::GLI1 fusion gene. To this end, we determined a transcriptional start site (TSS) and promoter region for truncated GLI1 expression using rapid amplification of the 5' cDNA end and a luciferase reporter assay in cultured cells transfected with a plasmid harboring the MALAT1::GLI1 fusion gene. RESULTS: We found that the TATA box, ETS1 motif, and TSS were located in MALAT1 and that MALAT1 exhibited transcriptional activity and induced expression of GLI1 from the MALAT1::GLI1 fusion gene. Truncated GLI1, lacking SUMOylation and SUFU binding sites and located in the nucleus, upregulated mRNA expression of GLI1 target genes in the hedgehog signaling pathway. CONCLUSIONS: We demonstrate a distinct and alternative function of MALAT1 as a transcriptional promoter for expression of the MALAT1::GLI1 fusion gene. Our findings will aid future research on MALAT1 and its fusion gene partners.
Asunto(s)
ARN Largo no Codificante , Neoplasias Gástricas , Humanos , Proteínas Hedgehog/genética , Regiones Promotoras Genéticas , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Neoplasias Gástricas/patología , Factores de Transcripción/genética , Proteína con Dedos de Zinc GLI1/genética , Proteína con Dedos de Zinc GLI1/metabolismoRESUMEN
The hepatotoxin microcystin-LR is a strong inhibitor of serine/threonine protein phosphatase (PP) 1 and PP2A. The onset of its cytotoxicity depends on its selective uptake via the hepatocyte uptake transporters, organic anion transporting polypeptide (OATP) 1B1 and OATP1B3. Understanding and preventing the cytotoxicity of microcystin-LR is crucial to maintain human health. This chemoprevention study demonstrates that the herbal plant extract of iwajisha (20 µg/mL) reduced microcystin-LR cytotoxicity in OATP1B3-expressing cells by approximately six times. In addition, 20 µM acteoside, which is one of the major compounds in iwajisha, reduced microcystin-LR cytotoxicity by approximately 7.4 times. Acteoside could also reduce the cytotoxicity of other compounds, such as okadaic acid and nodularin, which are both substrates of OATP1B3 and inhibitors of PP1/PP2A. To investigate the mechanism by which the cytotoxicity of microcystin-LR is attenuated by acteosides, microcystin-LR and microcystin-LR-binding proteins in cells were examined after microcystin-LR and acteosides were co-exposed. Thus, acteoside noncompetitively inhibited microcystin-LR uptake by OATP1B3-expressing cells. Furthermore, acteoside inhibited the intracellular interaction of microcystin-LR with its binding protein(s), including the 22 kDa protein. Furthermore, using immunoblot analysis, acteoside induced the phosphorylation of extracellular signal-regulated kinase (ERK), which is one of the survival signaling molecules. These results suggest that acteoside reduces microcystin-LR cytotoxicity through several mechanisms, including the inhibition of microcystin-LR uptake via OATP1B3, and decreased interaction between microcystin-LR and its binding protein(s), and that ERK signaling activation contributes to the attenuation effect of acteoside against microcystin-LR cytotoxicity.
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Transportadores de Anión Orgánico Sodio-Independiente , Transportadores de Anión Orgánico , Humanos , Miembro 1B3 de la Familia de los Transportadores de Solutos de Aniones Orgánicos , Microcistinas/metabolismo , Microcistinas/toxicidad , Transportadores de Anión Orgánico/metabolismo , Fenoles/farmacologíaRESUMEN
The circadian rhythm-related genes BHLHE40/DEC1 and BHLHE41/DEC2 have various functions under different cell and tissue conditions. BHLHE41/DEC2 has been reported to be both a cancer-suppressive and an oncogenic gene during cancer development. The effects of BHLHE41/DEC2 on differentiation have been examined using Bhlhe41/Dec2 knockout mice and/or in vitro differentiation models, and research has been conducted using genetic analysis of tumor cells, in vitro analysis of cancer cell lines, and immunohistochemical studies of the clinical samples. We summarize some of these studies, detail several problems, and consider possible reasons for contradictory results and the needs for further research.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Neoplasias Pulmonares , Animales , Ratones , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Línea Celular , Ritmo Circadiano/fisiología , Neoplasias Pulmonares/genética , HumanosRESUMEN
Nucleolar stress response is caused by perturbations in ribosome biogenesis, induced by the inhibition of ribosomal RNA processing and synthesis, as well as ribosome assembly. This response induces p53 stabilization and activation via ribosomal protein L11 (RPL11), suppressing tumor progression. However, anticancer agents that kill cells via this mechanism, and their relationship with the therapeutic efficiency of these agents, remain largely unknown. Here, we sought to investigate whether topoisomerase inhibitors can induce nucleolar stress response as they reportedly block ribosomal RNA transcription. Using rhabdomyosarcoma and rhabdoid tumor cell lines that are sensitive to the nucleolar stress response, we evaluated whether nucleolar stress response is associated with sensitivity to topoisomerase inhibitors ellipticine, doxorubicin, etoposide, topotecan, and anthracyclines. Cell proliferation assay indicated that small interfering RNA-mediated RPL11 depletion resulted in decreased sensitivity to topoisomerase inhibitors. Furthermore, the expression of p53 and its downstream target proteins via western blotting showed the suppression of p53 pathway activation upon RPL11 knockdown. These results suggest that the sensitivity of cancer cells to topoisomerase inhibitors is regulated by RPL11-mediated nucleolar stress responses. Thus, RPL11 expression may contribute to the prediction of the therapeutic efficacy of topoisomerase inhibitors and increase their therapeutic effect of topoisomerase inhibitors.
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Neoplasias , Proteína p53 Supresora de Tumor , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Ribosómicas/metabolismo , Nucléolo Celular/metabolismo , Línea Celular Tumoral , Antibióticos Antineoplásicos/farmacología , ARN Ribosómico/genética , Inhibidores de Topoisomerasa II/farmacología , Inhibidores de Topoisomerasa II/metabolismo , Antraciclinas/farmacología , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Neoplasias/metabolismoRESUMEN
Lung cancer constitutes a threat to human health. BHLHE41 plays important roles in circadian rhythm and cell differentiation as a negative regulatory transcription factor. This study investigates the role of BHLHE41 in lung cancer progression. We analyzed BHLHE41 function via in silico and immunohistochemical studies of 177 surgically resected non-small cell lung cancer (NSCLC) samples and 18 early lung squamous cell carcinoma (LUSC) cases. We also examined doxycycline (DOX)-inducible BHLHE41-expressing A549 and H2030 adenocarcinoma cells. BHLHE41 expression was higher in normal lung than in lung adenocarcinoma (LUAD) tissues and was associated with better prognosis for the overall survival (OS) of patients. In total, 15 of 132 LUAD tissues expressed BHLHE41 in normal lung epithelial cells. Staining was mainly observed in adenocarcinoma in situ and the lepidic growth part of invasive cancer tissue. BHLHE41 expression constituted a favorable prognostic factor for OS (p = 0.049) and cause-specific survival (p = 0.042) in patients with LUAD. During early LUSC, 7 of 18 cases expressed BHLHE41, and this expression was inversely correlated with the depth of invasion. DOX suppressed cell proliferation and increased the autophagy protein LC3, while chloroquine enhanced LC3 accumulation and suppressed cell death. In a xenograft model, DOX suppressed tumor growth. Our results indicate that BHLHE41 expression prevents early lung tumor malignant progression by inducing autophagic cell death in NSCLC.
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Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/patología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Células A549 , Adulto , Anciano , Anciano de 80 o más Años , Animales , Muerte Celular Autofágica/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Doxiciclina/farmacología , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Pronóstico , Modelos de Riesgos Proporcionales , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Citrin deficiency is an autosomal recessive disorder caused by loss-of-function mutations in SLC25A13, encoding the liver-specific mitochondrial aspartate/glutamate transporter. It has a broad spectrum of clinical phenotypes, including life-threatening neurological complications. Conventional protein replacement therapy is not an option for these patients because of drug delivery hurdles, and current gene therapy approaches (e.g., AAV) have been hampered by immunogenicity and genotoxicity. Although dietary approaches have shown some benefits in managing citrin deficiency, the only curative treatment option for these patients is liver transplantation, which is high-risk and associated with long-term complications because of chronic immunosuppression. To develop a new class of therapy for citrin deficiency, codon-optimized mRNA encoding human citrin (hCitrin) was encapsulated in lipid nanoparticles (LNPs). We demonstrate the efficacy of hCitrin-mRNA-LNP therapy in cultured human cells and in a murine model of citrin deficiency that resembles the human condition. Of note, intravenous (i.v.) administration of the hCitrin-mRNA resulted in a significant reduction in (1) hepatic citrulline and blood ammonia levels following oral sucrose challenge and (2) sucrose aversion, hallmarks of hCitrin deficiency. In conclusion, mRNA-LNP therapy could have a significant therapeutic effect on the treatment of citrin deficiency and other mitochondrial enzymopathies with limited treatment options.
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Citrulinemia/tratamiento farmacológico , Citrulinemia/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Terapia Genética/métodos , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , ARN Mensajero/uso terapéutico , Animales , Conducta Animal/efectos de los fármacos , Modelos Animales de Enfermedad , Técnicas de Inactivación de Genes , Glucosafosfato Deshidrogenasa/genética , Células HeLa , Células Hep G2 , Humanos , Lípidos/química , Mutación con Pérdida de Función , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Nanopartículas/química , Sistemas de Lectura Abierta/genética , ARN Mensajero/síntesis química , ARN Mensajero/química , ARN Mensajero/genética , Transfección , Resultado del TratamientoRESUMEN
BACKGROUND: Glioblastoma multiforme (GBM), the most common brain malignancy in adults, is generally aggressive and incurable, even with multiple treatment modalities and agents. Filamins (FLNs) are a group of actin-binding proteins that regulate the actin cytoskeleton in cells. However, the role of FLNs in malignancies-particularly in GBM-is unclear. METHODS: The relation between FLNC expression and overall survival in GBM was evaluated by the Kaplan-Meier analysis using GBM patients from the Kagoshima University Hospital (n = 90) and data from the Cancer Genome Atlas (TCGA) (n = 153). To assess FLNC function in GBM, cell migration and invasion were examined with Transwell and Matrigel invasion assays using FLNC-overexpressing U251MG and LN299 GBM cells, and ShRNA-mediated FLNC knocked-down KNS81 and U87MG cells. The gelatin zymography assay was used to estimate matrix metalloproteinase (MMP) 2 activity. RESULTS: In silico analysis of GBM patient data from TCGA and immunohistochemical analyses of clinical GBM specimens revealed that increased FLNC expression was associated with poor patient prognosis. FLNC overexpression in GBM cell lines was positively correlated with enhanced invasiveness, but not migration, and was accompanied by upregulation of MMP2. CONCLUSIONS: FLNC is a potential therapeutic target and biomarker for GBM progression.
Asunto(s)
Biomarcadores de Tumor/genética , Filaminas/genética , Glioblastoma/genética , Invasividad Neoplásica/genética , Citoesqueleto de Actina/genética , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Glioblastoma/epidemiología , Glioblastoma/patología , Humanos , Estimación de Kaplan-Meier , Masculino , Metaloproteinasa 2 de la Matriz/genética , Invasividad Neoplásica/patologíaRESUMEN
Undifferentiated pleomorphic sarcoma (UPS) is the second most frequent soft tissue sarcoma. Because of its resistance to chemotherapy, UPS patients are treated with surgical resection and complementary radiotherapy. However, since standard chemotherapy has not been established, unresectable or metastatic cases result in a poor prognosis. Therefore, the identification of a more effective therapy for UPS patients is needed. The development and progression of malignant tumors involve epigenetic alterations, and histone deacetylases (HDAC) have become a promising chemotherapeutic target. In this study, we investigated the potential effects and mechanisms of an HDAC inhibitor, LBH589, in UPS cells. We confirmed that LBH589 exhibits potent antitumor activities in four human UPS cell lines (GBS-1, TNMY-1, Nara-F, and Nara-H) and IC50 values ranged from 7 to 13 nM. A mouse xenograft model showed that LBH589 treatment effectively suppressed tumor growth. FACS analysis showed that LBH589 induced apoptosis and G2/M cell cycle arrest. Among apoptosis-related proteins, the expressions of Bcl-2 and Bcl-xL were decreased and the expression of Bak and Bim increased. Among cell cycle-related proteins, reductions of CDK1, p-CDK1, cyclin B1, Aurora A, and Aurora B were observed after LBH589 treatment. RNA microarray identified the FOS-like antigen 1 (FOSL1) gene as a downregulated gene in response to LBH589 in UPS cells. While knockdown of FOSL1 decreased UPS cell proliferation, overexpression induced cell proliferation. Our results show that LBH589 could be a promising chemotherapeutic agent in the treatment of UPS and downregulation of the FOSL1 gene could be the new molecular target of UPS treatment.
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Regulación hacia Abajo , Inhibidores de Histona Desacetilasas/administración & dosificación , Panobinostat/administración & dosificación , Proteínas Proto-Oncogénicas c-fos/genética , Sarcoma/tratamiento farmacológico , Animales , Proteínas Reguladoras de la Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Concentración 50 Inhibidora , Ratones , Panobinostat/farmacología , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tyrosine kinase inhibitors (TKIs) are used as the first choice for chronic myeloid leukemia (CML) pharmacotherapeutics. Some patients taking these drugs showed good therapeutic reactivity despite the disappearance of drugs from blood. We investigated whether these drugs have sustained effects even after their disappearance and whether their effects depend on their amounts of intracellular accumulation. Cell proliferation after exposure of K562 cells or Multidrug resistance-1 (MDR-1)-transfected K562 cells was determined by a cell counting kit-8 assay. The intracellular accumulation amount of the drug showing a sustained cytostatic effect was measured by ultra high performance liquid chromatography mass spectrometry. Cell viability decreased in a culture time-dependent manner after washing out nilotinib and dasatinib. The sustained cytostatic effect of dasatinib, but not that of nilotinib, correlated with the intracellular accumulation level. In contrast, imatinib showed continuous a cytostatic effect after drug washout for long-term exposure but not after drug washout for short-term exposure. These results suggest that a good response in patients with a low serum concentration of imatinib, nilotinib or dasatinib may be due to the cytostatic effect of that drug continues even after its disappearance in plasma.
Asunto(s)
Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Antineoplásicos/farmacología , Línea Celular Tumoral , Dasatinib/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Mesilato de Imatinib/farmacología , Células K562 , Pirimidinas/farmacologíaRESUMEN
Glioblastoma multiforme (GBM), the most common primary malignant brain tumor in adults, is characterized by rapid proliferation, aggressive migration, and invasion into normal brain tissue. Formin proteins have been implicated in these processes. However, the role of formin-like 1 (FMNL1) in cancer remains unclear. We studied FMNL1 expression in glioblastoma samples using immunohistochemistry. We sought to analyze the correlation between FMNL1 expression, clinicopathologic variables, and patient survival. Migration and invasion assays were used to verify the effect of FMNL1 on glioblastoma cell lines. Microarray data were downloaded from The Cancer Genome Atlas and analyzed using gene set enrichment analysis (GSEA). FMNL1 was an independent predictor of poor prognosis in a cohort of 217 glioblastoma multiforme cases (p < 0.001). FMNL1 expression was significantly higher in the mesenchymal subtype. FMNL1 upregulation and downregulation were associated with mesenchymal and proneural markers in the GSEA, respectively. These data highlight the important role of FMNL1 in the neural-to-mesenchymal transition. Conversely, FMNL1 downregulation suppressed glioblastoma multiforme cell migration and invasion via DIAPH1 and GOLGA2, respectively. FMNL1 downregulation also suppressed actin fiber assembly, induced morphological changes, and diminished filamentous actin. FMNL1 is a promising therapeutic target and a useful biomarker for GBM progression.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Forminas/metabolismo , Glioblastoma/metabolismo , Mesodermo/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Femenino , Forminas/genética , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/patología , Humanos , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mesodermo/patología , Pronóstico , Interferencia de ARN , Análisis de SupervivenciaRESUMEN
Thymidine phosphorylase (TP) is a rate-limiting enzyme in thymidine catabolism. TP has several important roles in biological and pharmacological mechanisms; importantly TP acts as an angiogenic factor and one of metabolic enzymes of fluoro-pyrimidine anticancer agents and modifies inflammation. Improving our understanding of the characteristics and functions of TP has led to the development of novel TP-based anticancer therapies. We recently reported that TP-dependent thymidine catabolism contributes to tumour survival in low nutrient conditions and the pathway from thymidine to the glycolysis cascade is affected in the context of physiological and metabolic conditions. In this review, we describe recent advancement in our understanding of TP, with a focus on cancer cell biology and the pharmacology of pyrimidine analogue anticancer agents. This review provides comprehensive understanding of the molecular mechanism of TP function in cancer.
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Neoplasias/patología , Timidina Fosforilasa/metabolismo , Animales , Resistencia a Antineoplásicos , Humanos , FN-kappa B/metabolismo , Neoplasias/metabolismo , Neovascularización PatológicaRESUMEN
Mice carrying simultaneous homozygous mutations in the genes encoding citrin, the mitochondrial aspartate-glutamate carrier 2 (AGC2) protein, and mitochondrial glycerol-3-phosphate dehydrogenase (mGPD), are a phenotypically representative model of human citrin (a.k.a., AGC2) deficiency. In this study, we investigated the voluntary oral intake and preference for sucrose, glycerol or ethanol solutions by wild-type, citrin (Ctrn)-knockout (KO), mGPD-KO, and Ctrn/mGPD double-KO mice; all substances that are known or suspected precipitating factors in the pathogenesis of human citrin deficiency. The double-KO mice showed clear suppressed intake of sucrose, consuming less with progressively higher concentrations compared to the other mice. Similar observations were made when glycerol or ethanol were given. The preference of Ctrn-KO and mGPD-KO mice varied with the different treatments; essentially no differences were observed for sucrose, while an intermediate intake or similar to that of the double-KO mice was observed for glycerol and ethanol. We next examined the hepatic glycerol 3-phosphate, citrate, citrulline, lysine, glutamate and adenine nucleotide levels following forced enteral administration of these solutions. A strong correlation between the simultaneous increased hepatic glycerol 3-phosphate and decreased ATP or total adenine nucleotide content and observed aversion of the mice during evaluation of their voluntary preferences was found. Overall, our results suggest that the aversion observed in the double-KO mice to these solutions is initiated and/or mediated by hepatic metabolic perturbations, resulting in a behavioral response to increased hepatic cytosolic NADH and a decreased cellular adenine nucleotide pool. These findings may underlie the dietary predilections observed in human citrin deficient patients.
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Citrulinemia/metabolismo , Sacarosa en la Dieta/administración & dosificación , Etanol/administración & dosificación , Glicerol/administración & dosificación , Hígado/química , Adenosina Trifosfato/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/genética , Animales , Antiportadores/genética , Modelos Animales de Enfermedad , Glicerolfosfato Deshidrogenasa/genética , Glicerofosfatos/metabolismo , Humanos , Ratones , Ratones NoqueadosRESUMEN
Gemcitabine is widely used for pancreatic, lung, and bladder cancer. However, drug resistance against gemcitabine is a large obstacle to effective chemotherapy. Nucleoside transporters, nucleoside and nucleotide metabolic enzymes, and efflux transporters have been reported to be involved in gemcitabine resistance. Although most of the resistant factors are supposed to be related to each other, it is unclear how one factor can affect the other one. In this study, we established gemcitabine-resistant pancreatic cancer cell lines. Gemcitabine resistance in these cells is caused by two major processes: a decrease in gemcitabine uptake and overexpression of ribonucleotide reductase large subunit (RRM1). Knockdown of RRM1, but not the overexpression of concentrative nucleoside transporter 1 (CNT1), could completely overcome the gemcitabine resistance. RRM1 knockdown in gemcitabine-resistant cells could increase the intracellular accumulation of gemcitabine by increasing the nucleoside transporter expression. Furthermore, a synergistic effect was observed between hydroxyurea, a ribonucleotide reductase (RR) inhibitor, and gemcitabine on the gemcitabine-resistant cells. Here we indicate that RR is one of the most promising targets to overcome gemcitabine resistance in gemcitabine-resistant cells with dual resistant factors.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Desoxicitidina/análogos & derivados , Resistencia a Antineoplásicos/genética , Inhibidores Enzimáticos/farmacología , Neoplasias Pancreáticas/patología , Ribonucleótido Reductasas/antagonistas & inhibidores , Ribonucleótido Reductasas/fisiología , Desoxicitidina/metabolismo , Desoxicitidina/farmacología , Inhibidores Enzimáticos/metabolismo , Humanos , Neoplasias Pancreáticas/metabolismo , Células Tumorales Cultivadas , GemcitabinaRESUMEN
A series of 1-aryl-3,4-substituted-1H-pyrazol-5-ol derivatives was synthesized and evaluated as prostate cancer antigen-1 (PCA-1/ALKBH3) inhibitors to obtain a novel anti-prostate cancer drug. After modifying 1-(1H-benzimidazol-2-yl)-3,4-dimethyl-1H-pyrazol-5-ol (1), a hit compound found during random screening using a recombinant PCA-1/ALKBH3, 1-(1H-5-methylbenzimidazol-2-yl)-4-benzyl-3-methyl-1H-pyrazol-5-ol (35, HUHS015), was obtained as a potent PCA-1/ALKBH3 inhibitor both in vitro and in vivo. The bioavailability (BA) of 35 was 7.2% in rats after oral administration. As expected, continuously administering 35 significantly suppressed the growth of DU145 cells, which are human hormone-independent prostate cancer cells, in a mouse xenograft model without untoward effects.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Antineoplásicos/farmacología , Diseño de Fármacos , Neoplasias de la Próstata/tratamiento farmacológico , Pirazoles/farmacología , Administración Oral , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/síntesis química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Masculino , Neoplasias de la Próstata/metabolismo , Pirazoles/administración & dosificación , Pirazoles/síntesis química , Ratas , Relación Estructura-ActividadRESUMEN
The present study established a primary hepatocyte culture for the small Indian mongoose (Herpestes auropunctatus). To determine the suitable medium for growing the primary hepatic cells of this species, we compared the condition of cells cultured in three media that are frequently used for mammalian cell culture: Dulbecco's Modified Eagle's Medium, RPMI-1640, and William's E. Of these, William's E medium was best suited for culturing the hepatic cells of this species. Using periodic acid-Schiff staining and ultrastructural observations, we demonstrated the cells collected from mongoose livers were hepatocytes. To evaluate the distribution of mercury (Hg) in the liver tissue, we carried out autometallography staining. Most of the Hg compounds were found in the central region of hepatic lobules. Smooth endoplasmic reticulum, which plays a role inxenobiotic metabolism, lipid/cholesterol metabolism, and the digestion and detoxification of lipophilic substances is grown in this area. This suggested that Hg colocalized with smooth endoplasmic reticulum. The results of the present study could be useful to identify the detoxification systems of wildlife with high Hg content in the body, and to evaluate the susceptibility of wildlife to Hg toxicity.
Asunto(s)
Hepatocitos/citología , Herpestidae , Hígado/metabolismo , Mercurio/metabolismo , Cultivo Primario de Células , Animales , Medios de Cultivo , Retículo Endoplásmico Liso/metabolismo , Femenino , Hepatocitos/metabolismo , Hepatocitos/ultraestructura , Hígado/citología , MasculinoRESUMEN
Heart function is highly dependent on mitochondria, which not only produce energy but also regulate many cellular functions. Therefore, mitochondria are important therapeutic targets in heart failure. Abcb10 is a member of the ABC transporter superfamily located in the inner mitochondrial membrane and plays an important role in haemoglobin synthesis, biliverdin transport, antioxidant stress, and stabilization of the iron transporter mitoferrin-1. However, the mechanisms underlying the impairment of mitochondrial transporters in the heart remain poorly understood. Here, we generated mice with cardiomyocyte-specific loss of Abcb10. The Abcb10 knockouts exhibited progressive worsening of cardiac fibrosis, increased cardiovascular risk markers and mitochondrial structural abnormalities, suggesting that the pathology of heart failure is related to mitochondrial dysfunction. As the mitochondrial dysfunction was observed early but mildly, other factors were considered. We then observed increased Hif1α expression, decreased NAD synthase expression, and reduced NAD+ levels, leading to lysosomal dysfunction. Analysis of ABCB10 knockdown HeLa cells revealed accumulation of Fe2+ and lipid peroxides in lysosomes, leading to ferroptosis. Lipid peroxidation was suppressed by treatment with iron chelators, suggesting that lysosomal iron accumulation is involved in ferroptosis. We also observed that Abcb10 knockout cardiomyocytes exhibited increased ROS production, iron accumulation, and lysosomal hypertrophy. Our findings suggest that Abcb10 is required for the maintenance of cardiac function and reveal a novel pathophysiology of chronic heart failure related to lysosomal function and ferroptosis.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Ferroptosis , Lisosomas , Mitocondrias Cardíacas , Miocitos Cardíacos , Animales , Humanos , Ratones , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Ferroptosis/genética , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Células HeLa , Hierro/metabolismo , Peroxidación de Lípido , Lisosomas/metabolismo , Ratones Noqueados , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/patología , Mitocondrias Cardíacas/genética , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Dysregulated growth and motility of vascular smooth muscle cells (VSMC) play important role in obstructive vascular diseases. We previously reported that gene transfer of thymidine phosphorylase (TP) into rat VSMC inhibits cell proliferation and attenuates balloon injury induced neointimal hyperplasia; however, the mechanism remains unclear. The current study identified a signaling pathway that mediates effect of TP inhibited VSMC proliferation with a TP activity-dependent manner. Rat VSMC overexpressing human TP gene (C2) or control empty vector (PC) were used. Serum stimulation induced constitutive STAT3 phosphorylation at tyrosine705 in C2 cell but not in PC, which was independent of JAK2 signaling pathway. Inhibition of Src family kinases activity inhibited STAT3 phosphorylation in C2 cells. Lyn activity was higher in C2 cell than in PC. SiRNA based gene knockdown of Lyn significantly decreased serum induced STAT3 phosphorylation in C2 and dramatically increased proliferation of this cell, suggesting that Lyn plays a pivotal role in TP inhibited VSMC proliferation. Unphosphorylated STAT3 (U-STAT3) expression was significantly increased in C2 cells, which may be due to the increased STAT3 transcription. Gene transfection of mouse wild-type or Y705F mutant STAT3 into PC cell or mouse primary cultured VSMC significantly reduced proliferation of these cells, suggesting that overexpression of U-STAT3 inhibits VSMC proliferation. We conclude that Lyn mediates TP induced STAT3 activation, which subsequently contributes to upregulate expression of U-STAT3. The U-STAT3 plays a critical role in inhibiting VSMC proliferation.
Asunto(s)
Proliferación Celular , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/fisiología , Factor de Transcripción STAT3/genética , Timidina Fosforilasa/fisiología , Animales , Células Cultivadas , Humanos , Janus Quinasa 2/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Miocitos del Músculo Liso/enzimología , Fosforilación , Procesamiento Proteico-Postraduccional , Ratas Sprague-Dawley , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Factor de Transcripción STAT3/metabolismo , Timidina Fosforilasa/biosíntesis , Timidina Fosforilasa/genética , Regulación hacia Arriba , Familia-src Quinasas/metabolismoRESUMEN
Snail, a repressor of E-cadherin gene transcription, induces epithelial-to-mesenchymal transition and is involved in tumor progression. Snail also mediates resistance to cell death induced by serum depletion. By contrast, we observed that snail-expressing MDCK (MDCK/snail) cells undergo cell death at a higher rate than control (MDCK/neo) cells in low-glucose medium. Therefore, we investigated whether snail expression influences cell metabolism in MDCK cells. Although gylcolysis was not affected in MDCK/snail cells, they did exhibit reduced pyruvate dehydrogenase (PDH) activity, which controls pyruvate entry into the tricarboxylic acid (TCA) cycle. Indeed, the activity of multiple enzymes involved in the TCA cycle was decreased in MDCK/snail cells, including that of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDH2), succinate dehydrogenase (SDH), and electron transport Complex II and Complex IV. Consequently, lower ATP content, lower oxygen consumption and increased survival under hypoxic conditions was also observed in MDCK/snail cells compared to MDCK/neo cells. In addition, the expression and promoter activity of pyruvate dehydrogenase kinase 1 (PDK1), which phosphorylates and inhibits the activity of PDH, was increased in MDCK/snail cells, while expression levels of glutaminase 2 (GLS2) and ATP-citrate lyase (ACLY), which are involved in glutaminolysis and fatty acid synthesis, were decreased in MDCK/snail cells. These results suggest that snail modulates cell metabolism by altering the expression and activity of key enzymes. This results in enhanced glucose dependency and leads to cell death under low-glucose conditions. On the other hand, the reduced requirements for oxygen and nutrients from the surrounding environment, might confer the resistance to cell death induced by hypoxia and malnutrition.
Asunto(s)
Redes y Vías Metabólicas/genética , Factores de Transcripción/metabolismo , Aconitato Hidratasa/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Apoptosis/genética , Cadherinas/genética , Supervivencia Celular/genética , Perros , Glucosa/metabolismo , Glucólisis/genética , Isocitrato Deshidrogenasa/metabolismo , Ácido Láctico/metabolismo , Células de Riñón Canino Madin Darby , Potencial de la Membrana Mitocondrial/genética , Consumo de Oxígeno/genética , Factores de Transcripción de la Familia Snail , Succinato DeshidrogenasaRESUMEN
Our understanding of metabolic reprogramming in cancer has tremendously improved along with the technical progression of metabolomic analysis. Metabolic changes in cancer cells proved much more complicated than the classical Warburg effect. Previous studies have approached metabolic changes as therapeutic and/or chemopreventive targets. Recently, several clinical trials have reported anti-cancer agents associated with metabolism. However, whether cancer cells are dependent on metabolic reprogramming or favor suitable conditions remains nebulous. Both scenarios are possibly intertwined. Identification of downstream molecules and the understanding of mechanisms underlying reprogrammed metabolism can improve the effectiveness of cancer therapy. Here, we review several examples of the metabolic reprogramming of cancer cells and the therapies targeting the metabolism-related molecules as well as discuss practical approaches to improve the next generation of cancer therapies focused on the metabolic reprogramming of cancer.
Asunto(s)
Antineoplásicos , Neoplasias , Humanos , Glucólisis , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Metabolismo Energético , Antineoplásicos/farmacología , Antineoplásicos/uso terapéuticoRESUMEN
Approximately 20% of pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) relapse or are refractory to chemotherapy despite the low frequency of TP53 mutations. The nucleolar stress response is a P53-activating mechanism via MDM2 inhibition by ribosomal protein L11 (RPL11). We analyzed the role of the nucleolar stress response using BCP-ALL cell lines and patient samples by drug sensitivity tests, Western blotting, and reverse transcription polymerase chain reaction. We revealed that the nucleolar stress response works properly in TP53 wild-type human BCP-ALL cell lines. Next, we found that 6-mercaptopurine, methotrexate, daunorubicin, and cytarabine had anti-leukemic effects via the nucleolar stress response within BCP-ALL treatment. Comparing the samples at onset and relapse in children with BCP-ALL, RPL11 mRNA expression decreased at relapse in seven of nine cases. Furthermore, leukemia cells with relapse acquired resistance to these four drugs and suppressed P53 and RPL11 expression. Our findings suggest that the nucleolar stress response is a novel anti-leukemia mechanism in BCP-ALL. As these four drugs are key therapeutics for BCP-ALL treatment, dysfunction of the nucleolar stress response may be related to clinical relapse or refractoriness. Nucleolar stress response may be a target to predict and improve the chemotherapy effect for pediatric BCP-ALL.