Increased expression of NLRP3 associated with elevated levels of HMGB1 in children with febrile seizures: a case-control study.

BACKGROUND: High mobility group box-1 (HMGB1) is an endogenous danger signal that mediates activation of the innate immune response including NLR pyrin domain containing 3 (NLRP3) inflammasome activation and proinflammatory cytokine release. Although HMGB1 and NLRP3 have been implicated in the pathophysiology of seizures, the correlation between HMGB1 and NLRP3 expression has not been determined in children with febrile seizures (FS). To explore the relationship between extra-cellular HMGB1 and NLRP3 in children with FS, we analyzed serum HMGB1, NLRP3, caspase-1, and proinflammatory cytokines in patients with FS. METHODS: Thirty children with FS and thirty age-matched febrile controls were included in this study. Blood was obtained from the children with FS within 1 h of the time of the seizure; subsequently, the serum contents of HMGB1, NLRP3, caspase-1, interleukin (IL)-1ß, interleukin (IL)-6, and tumour necrosis factor-α (TNF-α) were determined by enzyme-linked immunosorbent assay. The Mann‒Whitney U test was used to compare serum cytokine levels between FS patients and controls. Spearman's rank correlation coefficient was calculated to detect significant correlations between cytokine levels. RESULTS: Serum levels of HMGB1, NLRP3, caspase-1, IL-1ß, IL-6, and TNF-α were significantly higher in FS patients than in febrile controls (p < 0.05). Serum levels of HMGB1 were significantly correlated with levels of NLRP3 and caspase-1 (both, p < 0.05). Serum levels of caspase-1 were significantly correlated with levels of IL-1ß (p < 0.05). Serum levels of IL-1ß were significantly correlated with levels of IL-6 and TNF-α (p < 0.05). CONCLUSIONS: HMGB1 is up-regulated in the peripheral serum of FS patients, which may be responsible, at least in part, for the increased expression of NLRP3 and Caspase-1. Increased expression of caspase-1 was significantly associated with elevated serum levels of IL-1ß. Given that activated Caspase-1 directly regulates the expression of mature IL-1ß and positively correlates with activation of the NLRP3 inflammasome, our data suggest that increased levels of peripheral HMGB1 possibly mediate IL-1ß secretion through the activation of the NLRP3 inflammasome in children with FS. Thus, both HMGB1 and NLRP3 might be potential targets for preventing or limiting FS.

Cancer-associated fibroblasts contribute to cancer metastasis and apoptosis resistance in human ovarian cancer via paracrine SDF-1a

Background Cancer-associated fibroblasts (CAFs), one of the main members of stromal cells in tumor microenvironment are proposed to play a central role in promoting tumor metastasis. It is unclear whether and how CAFs mediates tumor metastasis or chemoresistance in human ovarian cancer. Methods CAFs were extracted from human ovarian cancer tissues (OCs) of patients with different kinds of histological types. Results We found that CAFs showed more aggressive potency than those tumor cells, both of which were isolated from the same ovarian cancer specimen. Moreover, when co-cultured with CAFs, cell migration abilities of ovarian cancer cells (SKOV3, OVCAR3 and HEY) were significantly increased. Next, we preliminarily detected a higher CAFs density in sections of metastatic lesions than those in primary tumor site of primary OCs clinically. However, no significant difference of stromal derived factors-1α (SDF-1α) production from CAFs was found between primary and metastatic lesions. Additionally, in contrast with tumor cells, CAFs exhibited obvious apoptosis resistance when treated with cisplatin. Furthermore, we found that cisplatin-induced cytotoxicity and apoptosis were significantly inhibited by co-cultured with recombinant human SDF-1α in SKOV3 in a time and dose-dependent manner, and this effect was suppressed by the CXCR4 antagonist AMD3100. Conclusions CAFs might be involved in the malignant metastasis in human ovarian cancer through promoting cell migration in tumor cells. And their resistance to cytotoxic agents might be mediated by paracrine SDF-1α/CXCR4 signaling in ovarian cancer (AU)

Cancer-associated fibroblasts contribute to cancer metastasis and apoptosis resistance in human ovarian cancer via paracrine SDF-1α.

BACKGROUND: Cancer-associated fibroblasts (CAFs), one of the main members of stromal cells in tumor microenvironment are proposed to play a central role in promoting tumor metastasis. It is unclear whether and how CAFs mediates tumor metastasis or chemoresistance in human ovarian cancer. METHODS: CAFs were extracted from human ovarian cancer tissues (OCs) of patients with different kinds of histological types. RESULTS: We found that CAFs showed more aggressive potency than those tumor cells, both of which were isolated from the same ovarian cancer specimen. Moreover, when co-cultured with CAFs, cell migration abilities of ovarian cancer cells (SKOV3, OVCAR3 and HEY) were significantly increased. Next, we preliminarily detected a higher CAFs density in sections of metastatic lesions than those in primary tumor site of primary OCs clinically. However, no significant difference of stromal derived factors-1α (SDF-1α) production from CAFs was found between primary and metastatic lesions. Additionally, in contrast with tumor cells, CAFs exhibited obvious apoptosis resistance when treated with cisplatin. Furthermore, we found that cisplatin-induced cytotoxicity and apoptosis were significantly inhibited by co-cultured with recombinant human SDF-1α in SKOV3 in a time and dose-dependent manner, and this effect was suppressed by the CXCR4 antagonist AMD3100. CONCLUSIONS: CAFs might be involved in the malignant metastasis in human ovarian cancer through promoting cell migration in tumor cells. And their resistance to cytotoxic agents might be mediated by paracrine SDF-1α/CXCR4 signaling in ovarian cancer.

YWHAG Mutations Cause Childhood Myoclonic Epilepsy and Febrile Seizures: Molecular Sub-regional Effect and Mechanism.

YWHAG, which encodes an adapter protein 14-3-3γ, is highly expressed in the brain and regulates a diverse range of cell signaling pathways. Previously, eight YWHAG mutations have been identified in patients with epileptic encephalopathy (EE). In this study, using trios-based whole exome sequencing, we identified two novel YWHAG mutations in two unrelated families with childhood myoclonic epilepsy and/or febrile seizures (FS). The identified mutations included a heterozygous truncating mutation (c.124C>T/p.Arg42Ter) and a de novo missense mutation (c.373A>G/p.Lys125Glu). The two probands experienced daily myoclonic seizures that were recorded with ictal generalized polyspike-slow waves, but became seizure-free with simple valproate treatment. The other affected individuals presented FS. The truncating mutation was identified in the family with six individuals of mild phenotype, suggesting that YWHAG mutations of haploinsufficiency are relatively less pathogenic. Analysis on all missense mutations showed that nine mutations were located within 14-3-3γ binding groove and another mutation was located at residues critical for dimerization, indicating a molecular sub-regional effect. Mutation Arg132Cys, which was identified recurrently in five patients with EE, would have the strongest influence on binding affinity. 14-3-3γ dimers supports target proteins activity. Thus, a heterozygous missense mutation would lead to majority dimers being mutants; whereas a heterozygous truncating mutation would lead to only decreasing the number of wild-type dimer, being one of the explanations for phenotypical variation. This study suggests that YWHAG is potentially a candidate pathogenic gene of childhood myoclonic epilepsy and FS. The spectrum of epilepsy caused by YWHAG mutations potentially range from mild myoclonic epilepsy and FS to severe EE.

Roles of Mitogen-Activating Protein Kinase Kinase Kinase Kinase-3 (MAP4K3) in Preterm Skeletal Muscle Satellite Cell Myogenesis and Mammalian Target of Rapamycin Complex 1 (mTORC1) Activation Regulation.

BACKGROUND Preterm skeletal muscle genesis is a paradigm for myogenesis. The role of mitogen-activating protein kinase kinase kinase kinase-3 (MAP4K3) in preterm skeletal muscle satellite cells myogenesis or its relationship to mammalian target of rapamycin complex 1 (mTORC1) activity have not been previously elaborated. MATERIAL AND METHODS Small interfering RNA (siRNA) interference technology was used to inhibit MAP4K3 expression. Leucine stimulation experiments were performed following MAP4K3-siRNA interference. The differentiation of primary preterm skeletal muscle satellite cells was observed after siRNA-MAP4K3 interference. Western blot analysis was used to determine the expression of MAP4K3, MyHC, MyoD, myogenin, p-mTOR, and p-S6K1. The immunofluorescence fusion index of MyHC and myogenin were detected. MAP4K3 effects on preterm rat satellite cells differentiation and its relationship to mTORC1 activity are reported. RESULTS MAP4K3 siRNA knockdown inhibited myotube formation and both MyoD and myogenin expression in primary preterm rat skeletal muscle satellite cells, but MAP4K3 siRNA had no effect on the activity of mTORC1. In primary preterm rat skeletal muscle satellite cells, MAP4K3 knockdown resulted in significantly weaker, but not entirely blunted, leucine-induced mTORC1 signaling. CONCLUSIONS MAP4K3 positively regulates preterm skeletal muscle satellite cell myogenesis, but may not regulate mTORC1 activity. MAP4K3 may play a role in mTORC1 full activation in response to leucine.

Leucine Promotes Proliferation and Differentiation of Primary Preterm Rat Satellite Cells in Part through mTORC1 Signaling Pathway.

Signaling through the mammalian target of rapamycin (mTOR) in response to leucine modulates many cellular and developmental processes. However, in the context of satellite cell proliferation and differentiation, the role of leucine and mTORC1 is less known. This study investigates the role of leucine in the process of proliferation and differentiation of primary preterm rat satellite cells, and the relationship with mammalian target of rapamycin complex 1 (mTORC1) activation. Dissociation of primary satellite cells occurred with type I collagenase and trypsin, and purification, via different speed adherence methods. Satellite cells with positive expression of Desmin were treated with leucine and rapamycin. We observed that leucine promoted proliferation and differentiation of primary satellite cells and increased the phosphorylation of mTOR. Rapamycin inhibited proliferation and differentiation, as well as decreased the phosphorylation level of mTOR. Furthermore, leucine increased the expression of MyoD and myogenin while the protein level of MyoD decreased due to rapamycin. However, myogenin expressed no affect by rapamycin. In conclusion, leucine may up-regulate the activation of mTORC1 to promote proliferation and differentiation of primary preterm rat satellite cells. We have shown that leucine promoted the differentiation of myotubes in part through the mTORC1-MyoD signal pathway.

The C3G/Rap1 pathway promotes secretion of MMP-2 and MMP-9 and is involved in serous ovarian cancer metastasis.

Complete resection is pivotal to improve survival to epithelial ovarian cancer (EOC). Crk SH3-domain-binding guanine nucleotide-releasing factor (C3G) is involved in multiple signaling pathways and it has opposite roles in different cancers. The present study aimed to identify C3G expression in ovarian tissue samples from patients with EOC and to explore its association with tumor grade. Eighty-seven archival paraffin-embedded, formalin-fixed, ovarian cancer tissues with serous histology were stained for C3G by immunohistochemistry. To evaluate the contribution of C3G to Rap1 activity, 36 patients with serous ovarian cancer (SOC) were investigated. Additionally, C3G was knocked down in SKOV3 and HEY cells. C3G regulated Rap1 activity and high Rap1 activity was correlated with poor differentiation, advanced FIGO stage, and unsuccessful cytoreductive surgery of SOC. Knockdown of C3G suppressed cell invasion, intravasation and extravasation, and reduced Rap1 activity and secretion of matrix metalloproteinase (MMP)-2 and MMP-9. C3G-mediated activation of Rap1 could direct the tumor pattern of human SOC by promoting the secretion of MMP-2 and MMP-9. These results suggest that C3G is involved in the metastatic spread of EOC.

Elmo1 helps dock180 to regulate Rac1 activity and cell migration of ovarian cancer.

OBJECTIVE: Engulfment and cell motility 1 (Elmo1) has been reported to cooperate with dedicator of cytokinesis 1 (Dock180) and to be linked to the invasive phenotype of cancer cells through activating small G-protein Rac. We aimed to study the role of Elmo1 in the malignant migration of ovarian cancer. METHODS: Engulfment and cell motility 1 expression was evaluated in specimens from 93 patients with serous ovarian cancer (SOC) by immunohistochemical staining. Next, Elmo1-RNAi cells were established by validated small interference RNAs. Cell proliferation and cell motility were observed and compared with Dock180-RNAi cells. To confirm their synergetic contribution to forming focal adhesion and activating Rac1, Rac1-GTP level was measured by GST pull-down assay and immunofluorescence was used to observe focal adhesion formation both in Elmo1-RNAi and Dock180-RNAi cells. RESULTS: Engulfment and cell motility 1 was mainly overexpressed in high-grade SOC tissues. Western blot analysis demonstrated that both Elmo1 and Dock180 expressions were hampered in Elmo1-RNAi cells. Compared with the negative control, decreased colony formation and cell invasion were observed in Elmo1-RNAi cells and Dock180-RNAi cells. Consistently, both exhibited reduced Rac1-GTP level and inhibited focal adhesion formation. CONCLUSIONS: Engulfment and cell motility 1 presents with synergetic action in helping Dock180 to activate Rac1 and promote cell motility, and thus promote untoward expansion and aggressiveness of SOC.