RESUMO
The fourth member of the leucine-rich repeat-containing GPCR family (LGR4, frequently referred to as GPR48) and its cognate ligands, R-spondins (RSPOs) play crucial roles in the development of multiple organs as well as the survival of adult stem cells by activation of canonical Wnt signaling. Wnt/ß-catenin signaling acts to regulate breast cancer; however, the molecular mechanisms determining its spatiotemporal regulation are largely unknown. In this study, we identified LGR4 as a master controller of Wnt/ß-catenin signaling-mediated breast cancer tumorigenesis, metastasis, and cancer stem cell (CSC) maintenance. LGR4 expression in breast tumors correlated with poor prognosis. Either Lgr4 haploinsufficiency or mammary-specific deletion inhibited mouse mammary tumor virus (MMTV)- PyMT- and MMTV- Wnt1-driven mammary tumorigenesis and metastasis. Moreover, LGR4 down-regulation decreased in vitro migration and in vivo xenograft tumor growth and lung metastasis. Furthermore, Lgr4 deletion in MMTV- Wnt1 tumor cells or knockdown in human breast cancer cells decreased the number of functional CSCs by â¼90%. Canonical Wnt signaling was impaired in LGR4-deficient breast cancer cells, and LGR4 knockdown resulted in increased E-cadherin and decreased expression of N-cadherin and snail transcription factor -2 ( SNAI2) (also called SLUG), implicating LGR4 in regulation of epithelial-mesenchymal transition. Our findings support a crucial role of the Wnt signaling component LGR4 in breast cancer initiation, metastasis, and breast CSCs.-Yue, Z., Yuan, Z., Zeng, L., Wang, Y., Lai, L., Li, J., Sun, P., Xue, X., Qi, J., Yang, Z., Zheng, Y., Fang, Y., Li, D., Siwko, S., Li, Y., Luo, J., Liu, M. LGR4 modulates breast cancer initiation, metastasis, and cancer stem cells.
Assuntos
Neoplasias da Mama/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Animais/metabolismo , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Receptores Acoplados a Proteínas G/biossíntese , Via de Sinalização Wnt , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Xenoenxertos , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Knockout , Camundongos Nus , Metástase Neoplásica , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Células-Tronco Neoplásicas/patologia , Receptores Acoplados a Proteínas G/genéticaRESUMO
CD38 is a multifunctional enzyme expressed in a variety of mammalian tissues, its catalytic activity was involved in a wide range of physiological processes. Based on the reported inhibitor of human CD38 NADase, 33 purine derivatives were designed and synthesized. The biological activity assay showed that compounds 20 and 38 exhibited almost the same extent of inhibitory activities on human CD38 NADase as the lead compound H2. The results also revealed that small substituents at C-6 of purine ring gave no obvious effect on inhibitory activity, but phenylpropionyl moiety at N-2 could affect the binding mode of the compound with CD38. This study provides a reliable basis for future rational design of inhibitors for CD38.
Assuntos
ADP-Ribosil Ciclase 1/antagonistas & inibidores , Inibidores Enzimáticos/química , Purinas/química , Inibidores Enzimáticos/síntese química , Humanos , Purinas/síntese químicaRESUMO
Microplastics (MPs) pollution and seawater acidification have increasingly become huge threats to the ocean ecosystem. Their impacts on microalgae are of great importance, since microalgae are the main primary producers and play a critical role in marine ecosystems. However, the impact of microplastics and acidification on unicellular red algae, which have a unique phycobiliprotein antenna system, remains unclear. Therefore, the impacts of polystyrene-MPs alone and the combined effects of MPs and seawater acidification on the typical unicellular marine red algae Porphyridium purpureum were investigated in the current study. The result showed that, under normal seawater condition, microalgae densities were increased by 17.75-41.67 % compared to the control when microalgae were exposed to small-sized MPs (0.1 µm) at concentrations of 5-100 mg L-1. In addition, the photosystem II and antioxidant enzyme system were not subjected to negative effects. The large-sized MPs (1 µm) boosted microalgae growth at a low concentration of MPs (5 mg L-1). However, it was observed that microalgae growth was significantly inhibited when MPs concentration increased up to 50 and 100 mg L-1, accompanied by the remarkably reduced Fv/Fm value and the elevated levels of SOD, CAT enzymes, phycoerythrin (PE), and extracellular polysaccharide (EPS). Compared to the normal seawater condition, microalgae densities were enhanced by 52.11-332.56 % under seawater acidification, depending on MPs sizes and concentrations, due to the formed CO2-enrichment condition and appropriate pH range. PE content in microalgal cells was significantly enhanced, but SOD and CAT activities as well as EPS content markedly decreased under acidification conditions. Overall, the impacts of seawater acidification were more pronounced than MPs impacts on microalgae growth and physiological responses. These findings will contribute to a substantial understanding of the effects of MPs on marine unicellular red microalgae, especially in future seawater acidification scenarios.
Assuntos
Microplásticos , Fotossíntese , Rodófitas , Água do Mar , Poluentes Químicos da Água , Água do Mar/química , Fotossíntese/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Rodófitas/efeitos dos fármacos , Rodófitas/química , Concentração de Íons de Hidrogênio , Microplásticos/toxicidade , Microalgas/efeitos dos fármacos , Antioxidantes/metabolismo , Matriz Extracelular de Substâncias Poliméricas/efeitos dos fármacos , Porphyridium/efeitos dos fármacos , Acidificação dos OceanosRESUMO
Therapeutics targeting osteoclasts are commonly used treatments for bone metastasis; however, whether and how osteoclasts regulate premetastatic niche and bone tropism are largely unknown. In this study, we report that osteoclast precursors (OPs) can function as a premetastatic niche component that facilitates breast cancer (BCa) bone metastasis at early stages. At the molecular level, unbiased GPCR ligand/agonist screening in BCa cells suggested that R-spondin 2 (RSPO2) and RANKL, through interaction with their receptor LGR4, promoted osteoclastic premetastatic niche formation and enhanced BCa bone metastasis. This was achieved by RSPO2/RANKL-LGR4 signal modulating the WNT inhibitor DKK1 through Gαq and ß-catenin signaling. DKK1 directly facilitated OP recruitment through suppression of its receptor LDL receptor-related protein 5 (LRP5) but not LRP6, upregulating Rnasek expression via inhibition of canonical WNT signaling. In clinical samples, RSPO2, LGR4, and DKK1 expression showed a positive correlation with BCa bone metastasis. Furthermore, soluble LGR4 extracellular domain (ECD) protein, acting as a decoy receptor for RSPO2 and RANKL, significantly alleviated bone metastasis and osteolytic lesions in a mouse bone metastasis model. These findings provide unique insights into the functional role of OPs as key components of the premetastatic niche for BCa bone metastasis and identify RSPO2/RANKL-LGR4 signaling as a promising target for inhibiting BCa bone metastasis.
Assuntos
Neoplasias Ósseas/metabolismo , Neoplasias da Mama/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Neoplasias/metabolismo , Osteoclastos/metabolismo , Ligante RANK/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Microambiente Tumoral , Animais , Neoplasias Ósseas/genética , Neoplasias da Mama/genética , Neoplasias da Mama/secundário , Linhagem Celular Tumoral , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica , Proteínas de Neoplasias/genética , Ligante RANK/genética , Receptores Acoplados a Proteínas G/genéticaRESUMO
OBJECTIVE: To extract and analyze fatty acids in leaves of L. chinense Oliver and L. chinense var. rubrum Yieh. METHODS: The fatty acids were extracted with Soxhlet extractor and identified by the GC-MS method. RESULTS: 31, 26 and 30 components were identified from the leaves of L. chinense, the green leaves and red leaves of L. chinense var. rubrum. There were 19, 14 and 16 kinds of fatty acid in the respective leaves, which accounted for 99.09%, 95.31% and 98.83% of the total extraction, respectively. The main components in the extraction were oleic acid (30.77%, 47.01%, 75.28%), linoleic acid (48.49%, 29.26%, 0%), hexadecanoic acid (9.83%, 8.87%, 11.71%), octadecanoic acid (3.86%, 3.18%, 4.88%) and docosanoic acid (2.37%, 2.17%, 2.58%). CONCLUSION: Many kinds of unsaturated fatty acids exist in leaves of L. chinense Oliver and L. chinense var. rubrum Yieh. The leaves of these two plants can be used as the traditional Chinese medicine.
Assuntos
Ácidos Graxos/análise , Hamamelidaceae/química , Folhas de Planta/química , Ácidos Graxos/química , Ácidos Graxos Insaturados/análise , Cromatografia Gasosa-Espectrometria de Massas , Hamamelidaceae/classificação , Ácido Linoleico/análise , Ácido Oleico/análise , Ácido Palmítico/análiseRESUMO
cADPR is a well-recognized signaling molecule by modulating the RyRs, but considerable debate exists regarding whether cADPR can bind to and gate the TRPM2 channel, which mediates oxidative stress signaling in diverse physiological and pathological processes. Here, we show that purified cADPR evoked TRPM2 channel currents in both whole-cell and cell-free single-channel recordings and specific binding of cADPR to the purified NUDT9-H domain of TRPM2 by surface plasmon resonance. Furthermore, by combining computational modeling with electrophysiological recordings, we show that the TRPM2 channels carrying point mutations at H1346, T1347, L1379, S1391, E1409, and L1484 possess distinct sensitivity profiles for ADPR and cADPR. These results clearly indicate cADPR is a bona fide activator at the TRPM2 channel and clearly delineate the structural basis for cADPR binding, which not only lead to a better understanding in the gating mechanism of TRPM2 channel but also shed light on a cADPR-induced RyRs-independent Ca2+ signaling mechanism.
Assuntos
ADP-Ribosil Ciclase/metabolismo , Adenosina Difosfato Ribose/metabolismo , Cálcio/metabolismo , Pirofosfatases/metabolismo , Canais de Cátion TRPM/metabolismo , ADP-Ribosil Ciclase/química , ADP-Ribosil Ciclase/genética , Sítios de Ligação , Células HEK293 , Humanos , Mutação Puntual , Conformação Proteica , Pirofosfatases/química , Pirofosfatases/genética , Canais de Cátion TRPM/química , Canais de Cátion TRPM/genéticaRESUMO
Activation of the transient receptor potential melastatin 2 (TRPM2) channel occurs during the response to oxidative stress under physiological conditions as well as in pathological processes such as ischemia and diabetes. Accumulating evidence indicates that adenosine diphosphate ribose (ADPR) is the most important endogenous ligand of TRPM2. However, although it is known that ADPR binds to the NUDT9 homology (NUDT9-H) domain in the intracellular C-terminal region, the molecular mechanism underlying ADPR binding and activation of TRPM2 remains unknown. In this study, we generate a structural model of the NUDT9-H domain and identify the binding pocket for ADPR using induced docking and molecular dynamics simulation. We find a subset of 11 residues-H1346, T1347, T1349, L1379, G1389, S1391, E1409, D1431, R1433, L1484, and H1488-that are most likely to directly interact with ADPR. Results from mutagenesis and electrophysiology approaches support the predicted binding mechanism, indicating that ADPR binds tightly to the NUDT9-H domain, and suggest that the most significant interactions are the van der Waals forces with S1391 and L1484, polar solvation interaction with E1409, and electronic interactions (including π-π interactions) with H1346, T1347, Y1349, D1431, and H1488. These findings not only clarify the roles of a range of newly identified residues involved in ADPR binding in the TRPM2 channel, but also reveal the binding pocket for ADPR in the NUDT9-H domain, which should facilitate structure-based drug design for the TRPM2 channel.
Assuntos
Adenosina Difosfato Ribose/farmacologia , Simulação de Acoplamento Molecular , Canais de Cátion TRPM/química , Adenosina Difosfato Ribose/química , Substituição de Aminoácidos , Sítios de Ligação , Células HEK293 , Humanos , Ligação Proteica , Pirofosfatases/química , Homologia de Sequência , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismoRESUMO
In the past decade, the discovery, synthesis, and evaluation for hundreds of CD38 covalent and non-covalent inhibitors has been reported sequentially by our group and partners; however, a systematic structure-based guidance is still lacking for rational design of CD38 inhibitor. Here, we carried out a comparative analysis of pharmacophore features and quantitative structure-activity relationships for CD38 inhibitors. The results uncover that the essential interactions between key residues and covalent/non-covalent CD38 inhibitors include (i) hydrogen bond and hydrophobic interactions with residues Glu226 and Trp125, (ii) electrostatic or hydrogen bond interaction with the positively charged residue Arg127 region, and (iii) the hydrophobic interaction with residue Trp189. For covalent inhibitors, besides the covalent effect with residue Glu226, the electrostatic interaction with residue Arg127 is also necessary, while another hydrogen/non-bonded interaction with residues Trp125 and Trp189 can also be detected. By means of the SYBYL multifit alignment function, the best CoMFA and CoMSIA with CD38 covalent inhibitors presented cross-validated correlation coefficient values (q(2)) of 0.564 and 0.571, and non-cross-validated values (r(2)) of 0.967 and 0.971, respectively. The CD38 non-covalent inhibitors can be classified into five groups according to their chemical scaffolds, and the residues Glu226, Trp189, and Trp125 are indispensable for those non-covalent inhibitors binding to CD38, while the residues Ser126, Arg127, Asp155, Thr221, and Phe222 are also important. The best CoMFA and CoMSIA with the F12 analogues presented cross-validated correlation coefficient values (q(2)) of 0.469 and 0.454, and non-cross-validated values (r(2)) of 0.814 and 0.819, respectively.