RESUMEN
Selective inhibition of Janus kinase 3 (JAK3) is a promising strategy for the treatment of autoimmune diseases. Based on the discovery of a hydrophobic pocket unutilized between the lead compound RB1 and the JAK3 protein, a series of covalent JAK3 inhibitors were prepared by introducing various aromatic fragments to RB1. Among them, J1b (JAK3 IC50 = 7.2 nM, other JAKs IC50 > 1000 nM) stood out because of its low toxicity (MTD > 2 g/kg) and superior anti-inflammatory activity in Institute of Cancer Research mice. Moreover, the acceptable bioavailability (F% = 31.69%) ensured that J1b displayed excellent immune regulation in collagen-induced arthritis mice, whose joints in the high-dose group were almost recovered to a normal state. Given its clear kinase selectivity (Bmx IC50 = 539.9 nM, other Cys909 kinases IC50 > 1000 nM), J1b was nominated as a highly selective JAK3 covalent inhibitor, which could be used to safely treat arthritis and other autoimmune diseases.
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Artritis Experimental , Artritis Reumatoide , Diseño de Fármacos , Janus Quinasa 3 , Inhibidores de Proteínas Quinasas , Animales , Janus Quinasa 3/antagonistas & inhibidores , Janus Quinasa 3/metabolismo , Ratones , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/inducido químicamente , Artritis Experimental/enzimología , Artritis Reumatoide/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Relación Estructura-Actividad , Ratones Endogámicos DBA , Humanos , Relación Dosis-Respuesta a Droga , Estructura Molecular , Masculino , Simulación del Acoplamiento MolecularRESUMEN
BACKGROUND: Signal regulatory protein ß1 (SIRPB1) is a signal regulatory protein member of the immunoglobulin superfamily and is capable of modulating receptor tyrosine kinase-coupled signaling. Copy number variations at the SIRPB1 locus were previously reported to associate with prostate cancer aggressiveness in patients, however, the role of SIRPB1 in prostate carcinogenesis is unknown. METHODS: Fluorescence in situ hybridization and laser-capture microdissection coupled with quantitative polymerase chain reaction was utilized to determine SIRPB1 gene amplification and messenger RNA expression in prostate cancer specimens. The effect of knockdown of SIRPB1 by RNA interference in PC3 prostate cancer cells on cell growth in colony formation assays and cell mobility in wound-healing, transwell assays, and cell cycle analysis was determined. Overexpression of SIPRB1 in C4-2 prostate cancer cells on cell migration, invasion, colony formation and cell cycle progression and tumor take rate in xenografts was also determined. Western blot assay of potential downstream SIRPB1 pathways was also performed. RESULTS: SIRPB1 gene amplification was detected in up to 37.5% of prostate cancer specimens based on in silico analysis of several publicly available datasets. SIRPB1 gene amplification and overexpression were detected in prostate cancer specimens. The knockdown of SIRPB1 significantly suppressed cell growth in colony formation assays and cell mobility. SIRPB1 knockdown also induced cell cycle arrest during the G0 /G1 phase and enhancement of apoptosis. Conversely, overexpression of SIPRB1 in C4-2 prostate cancer cells significantly enhanced cell migration, invasion, colony formation, and cell cycle progression and increased C4-2 xenograft tumor take rate in nude mice. Finally, this study presented evidence for SIRPB1 regulation of Akt phosphorylation and showed that Akt inhibition could abolish SIRPB1 stimulation of prostate cancer cell proliferation. CONCLUSIONS: These results suggest that SIRPB1 is a potential oncogene capable of activating Akt signaling to stimulate prostate cancer proliferation and could be a biomarker for patients at risk of developing aggressive prostate cancer.
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Moléculas de Adhesión de Célula Nerviosa/genética , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular/fisiología , Activación Enzimática , Amplificación de Genes , Xenoinjertos , Humanos , Masculino , Ratones , Ratones Desnudos , Moléculas de Adhesión de Célula Nerviosa/biosíntesis , Células PC-3 , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
Background: 2-Dodecyl-6-Methoxycyclohexa-2, 5-Diene-1,4-Dione (DMDD) was purified from the roots of Averrhoa carambola L. Previous research demonstrated that DMDD is a small molecular compound with significant therapeutic potential for tumors. However, the potential targets and pharmacological mechanism of DMDD to treat lung cancer has not been reported. Methods: We employed network pharmacology and experimental evaluation to reveal the pharmacological mechanism of DMDD against lung cancer. Potential therapeutic targets of DMDD were screened by PharmMapper. Differentially expressed genes (DEGs) in The Cancer Genome Atlas (TCGA) lung cancer data sets were extracted and analyzed by GEPIA2. The mechanism of DMDD against lung cancer was determined by PPI, gene ontology (GO) and KEGG pathway enrichment analysis. Survival analysis and molecular docking were employed to obtain the key targets of DMDD. Human lung cancer cell lines H1975 and PC9 were used to detect effects of DMDD treatment in vitro. The expression of key targets after DMDD treated was validated by Western Blot. Results: A total of 60 Homo sapiens potential therapeutic targets of DMDD and 3,545 DEGs in TCGA lung cancer datasets were identified. Gene ontology and pathway analysis revealed characteristic of the potential targets of DMDD and DEGs in lung cancer respectively. Cell cycle and pathways in cancer were overlapping with DMDD potential targets and lung cancer DEGs. Eight overlapping genes were found between DMDD potential therapeutic targets and lung cancer related DEGs. Survival analysis showed that high expression of DMDD potential targets CCNE1 and E2F1 was significantly related to poor patient survival in lung cancer. Molecular docking found that DMDD exhibited significant binding affinities within the active site of CCNE1 and E2F1. Further tests showed that DMDD inhibited the proliferation, migration and clone formation in lung cancer cell lines (H1975 and PC9) in a dose and time dependent manner. Mechanistically, DMDD treatment decreased the expression of CDK2, CCNE1, E2F1 proteins and induced cell cycle arrest at the G1/S phase in H1975 and PC9 cells. Conclusion: These results delineated that DMDD holds therapeutic potential that blocks tumorigenesis by cell cycle regulation in lung cancer, and may provide potential therapies for lung cancer.
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While microfluidic systems model aspects of metastasis, they are limited to artificially created tissues of limited complexity. We set out to develop an in vitro model of tumor cell migration from a primary tumor to a distant site that allows use of tissue. Accordingly, we created a macrofluidic model using culture plate wells connected with type I collagen-coated large bore tubing and has recirculating media. Green fluorescent protein-positive prostate carcinoma cells in a hydrogel or excised tumor xenografts from mice were placed into primary tumor sites and either human bone stromal cells (HS-5) in a hydrogel or human-derived bone chips were seeded into metastatic sites. Cells from the primary sites migrated to and grew in metastatic sites. Bone enhanced growth at metastatic sites and established a CXCL12 gradient that was higher in metastatic versus primary sites. AMD3100-mediated inhibition of CXCL12 function reduced the number of cells targeting the bone at the metastatic sites. In summary, we have developed a macrofluidic metastasis model that allows incorporation of tumor and metastatic microenvironment tissues and models chemotaxis. This system allows for incorporation of tumor heterogeneity and inclusion of an intact microenvironment. These features will facilitate identification of mechanisms and therapeutics for bone metastasis.
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Neoplasias Óseas/secundario , Hueso Esponjoso/metabolismo , Cabeza Femoral , Procedimientos Analíticos en Microchip/métodos , Neoplasias de la Próstata/patología , Células del Estroma/metabolismo , Animales , Movimiento Celular , Quimiocina CXCL12/metabolismo , Proteínas Fluorescentes Verdes/genética , Xenoinjertos , Humanos , Hidrogeles , Dispositivos Laboratorio en un Chip , Masculino , Ratones , Ratones Desnudos , Células PC-3 , Transducción Genética , Microambiente TumoralRESUMEN
Bone is the most frequent site of prostate cancer (PCa) metastasis; however, little is known about the role of the most common cell in bone, the osteocyte (OCy), in cancer biology. In this study we explored the crosstalk between PCa cells and OCys to determine if it contributes to PCa progression. PCa cells induced OCys to promote PCa proliferation, migration and invasion. A chemokine screen revealed that PCa cell induced OCys to produce growth-derived factor 15 (GDF15). Knockdown of GDF15 in OCys demonstrated that PCa cells conferred the ability on OCys to promote PCa proliferation, migration and invasion through GDF15. Consistent with this finding was the observation that the GDF15 receptor, GFRAL, was expressed on multiple PCa cell lines. Transcription factor array screening of PCa cells exposed to OCys with or without knockdown of GDF15 revealed that GDF15 in OCys promoted early growth response 1 (EGR1) expression in the PCa cells. Knockdown of EGR1 expression in PCa cells revealed it was required for the OCy-derived GDF15-mediated induction of in vitro PCa cell proliferation, migration and invasion. Subcutaneous co-injection of PCa cells and OCys into mice revealed that OCys promoted tumor growth in vivo, which was diminished by knockdown of GDF15 in the OCys. Knockdown of GDF15 in the tibiae diminished growth of PCa cancer cells injected into the tibiae, which was accompanied by decreased tumor cell proliferation and EGR1 expression. These results shed light on a novel mechanism through which PCa cells educate OCys to promote progression of PCa bone metastasis. They also suggest that targeting of GDF15-based and EGR1-based signaling pathways should be further explored for their potential to diminish progression of PCa bone metastasis.
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Neoplasias Óseas/metabolismo , Factor 15 de Diferenciación de Crecimiento/metabolismo , Osteocitos/metabolismo , Neoplasias de la Próstata/metabolismo , Animales , Neoplasias Óseas/secundario , Línea Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Progresión de la Enfermedad , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Invasividad Neoplásica , Metástasis de la Neoplasia , Neoplasias de la Próstata/patología , Transducción de SeñalRESUMEN
BACKGROUND: The chemoresistance of prostate cancer (PCa) is invariably associated with the aggressiveness and metastasis of this disease. New emerging evidence indicates that the epithelial-to-mesenchymal transition (EMT) may play pivotal roles in the development of chemoresistance and metastasis. As a hallmark of EMT, E-cadherin is suggested to be a key marker in the development of chemoresistance. However, the molecular mechanisms underlying PCa chemoresistance remain unclear. The current study aimed to explore the association between EMT and chemoresistance in PCa as well as whether changing the expression of E-cadherin would affect PCa chemoresistance. METHODS: Parental PC3 and DU145 cells and their chemoresistant PC3-TxR and DU145-TxR cells were analyzed. PC3-TxR and DU145-TxR cells were transfected with E-cadherin-expressing lentivirus to overexpress E-cadherin; PC3 and DU145 cells were transfected with small interfering RNA to silence E-cadherin. Changes of EMT phenotype-related markers and signaling pathways were assessed by Western blotting and quantitative real-time polymerase chain reaction. Tumor cell migration, invasion, and colony formation were then evaluated by wound healing, transwell, and colony formation assays, respectively. The drug sensitivity was evaluated using MTS assay. RESULTS: Chemoresistant PC3-TxR and DU145-TxR cells exhibited an invasive and metastatic phenotype that associated with EMT, including the down-regulation of E-cadherin and up-regulation of Vimentin, Snail, and N-cadherin, comparing with that of parental PC3 and DU145 cells. When E-cadherin was overexpressed in PC3-TxR and DU145-TxR cells, the expression of Vimentin and Claudin-1 was down-regulated, and tumor cell migration and invasion were inhibited. In particular, the sensitivity to paclitaxel was reactivated in E-cadherin-overexpressing PC3-TxR and DU145-TxR cells. When E-cadherin expression was silenced in parental PC3 and DU145 cells, the expression of Vimentin and Snail was up-regulated, and, particularly, the sensitivity to paclitaxel was decreased. Interestingly, Notch-1 expression was up-regulated in PC3-TxR and DU145-TxR cells, whereas the E-cadherin expression was down-regulated in these cells comparing with their parental cells. The use of γ-secretase inhibitor, a Notch signaling pathway inhibitor, significantly increased the sensitivity of chemoresistant cells to paclitaxel. CONCLUSION: The down-regulation of E-cadherin enhances PCa chemoresistance via Notch signaling, and inhibiting the Notch signaling pathway may reverse PCa chemoresistance.
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Cadherinas/genética , Cadherinas/metabolismo , Regulación hacia Abajo , Resistencia a Antineoplásicos , Paclitaxel/administración & dosificación , Neoplasias de la Próstata/tratamiento farmacológico , Receptores Notch/metabolismo , Animales , Antígenos CD , Línea Celular Tumoral , Movimiento Celular , Transición Epitelial-Mesenquimal/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Ratones , Paclitaxel/farmacología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A pot experiment was conducted to study the effects of plant growth regulator GA3 and metal chelate EDTA on enhancing the remediation of Pb contaminated soil, and the detoxification mechanism of Lolium perenne grown on Pb contaminated soil at 250 and 500 mg · kg(-1). The results showed that cell wall deposition and vacuolar compartmentalization played important roles in the detoxification of Pb in L. perenne shoot. The addition of EDTA alone increased Pb concentration in plants and Pb proportions in soluble fraction and organelles fraction, and enhanced the toxicity of Pb to plant, leading to the significant reduction of the plant biomass (P < 0.05). Foliar spray of lower concentration of GA3 (1 µmol · L(-1) or 10 µmol · L(-1)) alone significantly increased Pb accumulation by L. perenne (P < 0.05), but Pb proportions in soluble and organelles fraction were decreased, which alleviated the adverse effects of Pb on plant, thus improving the growth of plants (P < 0.05), with 1 µmol · L(-1) GA3 being the most effective. In contract, the addition of 100 µmol · L(-1) GA3 decreased Pb concentration in L. perenne, but increased the proportions of Pb in soluble fraction and organelles fraction, resulting in the reduction of plant biomass. Lower concen- tration of GA3 might alleviate the adverse effects of Pb and/or EDTA on plant, since the biomass amounts in the different treatments were in order of GA3 alone of lower concentration > GA3 of lower concentration + EDTA > EDTA alone. The combination application of low concentration of GA3 and EDTA showed a synergistic effect on the Pb accumulation in L. perenne (P < 0.05). Especially, Pb concentration in shoot and Pb extraction efficiency reached 1250.6 mg · kg(-1) and 1.1%, respec- tively, under the treatment of EDTA + 1 µmol L(-1) GA3 on the Pb 500 mg · kg(-1) soil. Therefore, the application of 1 µmol · L(-1) GA3 along with EDTA appeared to be a potential approach for phytoremediation of Pb contaminated soil.