Zerumbone,an electrophilic sesquiterpene,induces cellular proteo-stress leading to activation of ubiquitin–proteasome system and autophagy

Zerumbone, a sesquiterpene present in Zingiber zerumbet Smith, has been implicated as a promising chemopreventive agent. Interestingly, a number of studies have revealed that its potent bioactivities are dependent on the electrophilic moiety of its α,β-unsaturated carbonyl group, while our recent findings showed its chemical potential for binding to cellular proteins through a Michael reaction. In the present study, modifications of proteins by zerumbone led to their insolubilization in vitro. In living cell models, zerumbone induced ubiquitination and aggregation of cellular proteins, which demonstrated its substantial proteo-toxicity. On the other hand, it was also revealed that zerumbone possesses potential for activating intracellular proteolysis mechanisms of the ubiquitin–proteasome system and autophagy. Furthermore, it up-regulated expressions of pro-autophagic genes including p62, which is known as a cargo receptor of aggrephagy, the selective autophagic process for protein aggregates. Pretreatment of Hepa1c1c7 cells with zerumbone conferred a phenotype resistant to cytotoxicity and protein modifications by 4-hydroxy-2-nonenal, an endogenous lipid peroxidation product, in a p62-dependent manner. Together, these results suggest that protein modifications by zerumbone cause mild proteo-stress, thereby activating intracellular proteolysis machineries to maintain protein homeostasis. We consider these effects on proteolysis mechanisms to be hormesis, which provides beneficial functions through mild biological stresses.     

RICK regulates the odontogenic differentiation of dental pulp stem cells through activation of TNF-α via the ERK and not through NF-κB signaling pathway

Dental pulp stem cells (DPSCs) are capable of both self-renewal and multilineage differentiation, which play a positive role in dentinogenesis. Studies have shown that tumor necrosis factor-α (TNF-α) is involved in the differentiation of DPSCs under pro-inflammatory stimuli, but the mechanism of action of TNF-α is unknown. Rip-like interacting caspase-like apoptosis-regulatory protein kinase (RICK) is a biomarker of an early inflammatory response that plays a key role in modulating cell differentiation, but the role of RICK in DPSCs is still unclear. In this study, we identified that RICK regulates TNF-α-mediated odontogenic differentiation of DPSCs via the ERK signaling pathway. The expression of the biomarkers of odontogenic differentiation dental matrix protein-1 (DMP-1), dentin sialophosphoprotein (DSPP), biomarkers of odontogenic differentiation, increased in low concentration (1–10 ng/ml) of TNF-α and decreased in high concentration (50–100 ng/ml). Odontogenic differentiation increased over time in the odontogenic differentiation medium. In the presence of 10 ng/L TNF-α, the expression of RICK increased gradually over time, along with odontogenic differentiation. Genetic silencing of RICK expression reduced the expression of odontogenic markers DMP-1 and DSPP. The ERK, but not the NF-κB signaling pathway, was activated during the odontogenic differentiation of DPSCs. ERK signaling modulators decreased when RICK expression was inhibited. PD98059, an ERK inhibitor, blocked the odontogenic differentiation of DPSCs induced by TNF-α. These results provide a further theoretical and experimental basis for the potential use of RICK in targeted therapy for dentin regeneration.     

Ischemia induces phospholamban dephosphorylation via activation of calcineurin, PKC-α, and protein phosphatase 1, thereby inducing calcium overload in reperfusion

Cardiac sarcoplasmic reticulum (SR) Ca²⁺ ATPase (SERCA2a) promotes Ca²⁺ uptake in the SR. Dephosphorylated phospholamban (PLB) inhibits SERCA2a activity. We found a distinct dephosphorylation of PLB at Thr¹⁷ and Ser¹⁶ after 20-30 min of ischemia produced by coronary artery occlusion in rats. The aim of the study was to investigate how PLB is dephosphorylated in ischemia and to determine whether PLB dephosphorylation causes myocardial hypercontraction and calpain activation through Ca²⁺ overload in reperfusion. Protein inhibitor-1 (I-1) specifically inhibits protein phosphatase 1 (PP1), the predominant PLB phosphatase in heart. A Ca²⁺-dependent phosphatase calcineurin may also induce PLB dephosphorylation. Ischemia for 30 min induced PKC-α translocation, resulting in inactivation of I-1 through PKC-α-dependent phosphorylation at Ser⁶⁷. The PP1 activation following I-1 inactivation was thought to induce PLB dephosphorylation in ischemia. Ischemia for 30 min activated calcineurin, and pre-treatment with a calcineurin inhibitor, cyclosporine A (CsA), inhibited PKC-α translocation, I-1 phosphorylation at Ser⁶⁷, and PLB dephosphorylation in ischemia. Reperfusion for 5 min following 30 min of ischemia induced spreading of contraction bands (CBs) and proteolysis of fodrin by calpain. Both CsA and an anti-PLB antibody that inhibits binding of PLB to SERCA2a reduced the CB area and fodrin breakdown after reperfusion. These results reveal a novel pathway via which ischemia induces calcineurin-dependent activation of PKC-α, inactivation of I-1 through PKC-α-dependent phosphorylation at Ser⁶⁷, and PP1-dependent PLB dephosphorylation. The pathway contributes to the spreading of CBs and calpain activation through Ca²⁺ overload in early reperfusion.     

Palmitate induces SHIP2 expression via the ceramide-mediated activation of NF-κB,and JNK in skeletal muscle cells

Aims

Elevated plasma free fatty acids impair the insulin signaling by induction of the expression of protein phosphatases. However, the effect of palmitate on SH2-containing inositol 5′-phosphatase 2 (SHIP2) expression has not been investigated. Here we investigated the effects of palmitate on SHIP2 expression and elucidated the underlying mechanisms in skeletal muscle cells.

Main methods

SHIP2 mRNA and protein levels were measured in C2C12 myotubes exposed to palmitate. Specific inhibitors were used to identify the signaling pathways involved in SHIP2 expression.

Key findings

The results showed that 0.5 mM palmitate significantly upregulates the mRNA and protein levels of SHIP2 in C2C12 cells. To address the role of palmitate intracellular metabolites in SHIP2 expression, the myotubes were treated with palmitate in the presence of ceramide and diacylglycerol synthesis inhibitors. The results demonstrated that only ceramide synthesis inhibition could prevent palmitate-induced SHIP2 expression in these cells. In addition, the incubation of muscle cells with different concentrations of C2-ceramide dose-dependently enhanced SHIP2 expression. Furthermore, the inhibition of both JNK and NF-κB pathways could prevent ceramide-induced SHIP2 expression in myotubes.

Significance

These findings suggest that palmitate contributes to SHIP2 overexpression in skeletal muscle via the mechanisms involving the activation of ceramide-JNK and NF-κB pathways.     

Neu1 desialylation of sialyl α-2,3-linked β-galactosyl residues of TOLL-like receptor 4 is essential for receptor activation and cellular signaling

The ectodomain of TOLL-like receptors (TLR) is highly glycosylated with several N-linked gylcosylation sites located in the inner concave surface. The precise role of these sugar N-glycans in TLR receptor activation is unknown. Recently, we have shown that Neu1 sialidase and not Neu2, -3 and -4 forms a complex with TLR-2, -3 and -4 receptors on the cell-surface membrane of naïve and activated macrophage cells (Glycoconj J DOI 10.1007/s10719-009-9239-8). Activation of Neu1 is induced by TLR ligands binding to their respective receptors. Here, we show that endotoxin lipopolysaccharide (LPS)-induced MyD88/TLR4 complex formation and subsequent NFκB activation is dependent on the removal of α-2,3-sialyl residue linked to β-galactoside of TLR4 by the Neu1 activity associated with LPS-stimulated live primary macrophage cells, macrophage and dendritic cell lines but not with primary Neu1-deficient macrophage cells. Exogenous α-2,3 sialyl specific neuraminidase (Streptoccocus pneumoniae) and wild-type T. cruzi trans-sialidase (TS) but not the catalytically inactive mutant TS?Asp98-Glu mediate TLR4 dimerization to facilitate MyD88/TLR4 complex formation and NFκB activation similar to those responses seen with LPS. These same TLR ligand-induced NFκB responses are not observed in TLR deficient HEK293 cells, but are re-established in HEK293 cells stably transfected with TLR4/MD2, and are significantly inhibited by α-2,3-sialyl specific Maackia amurensis (MAL-2) lectin, α-2,3-sialyl specific galectin-1 and neuraminidase inhibitor Tamiflu but not by α-2,6-sialyl specific Sambucus nigra lectin (SNA). Taken together, the findings suggest that Neu1 desialylation of α-2,3-sialyl residues of TLR receptors enables in removing a steric hinderance to receptor association for TLR activation and cellular signaling.     

Adiponectin induces dendritic cell activation via PLCγ/JNK/NF-κB pathways, leading to Th1 and Th17 polarization

Adiponectin (APN) is a crucial regulator for many inflammatory processes, but its effect on Th cell-mediated responses has not been fully understood. Thus, we investigated the immune-modulatory effects of APN on dendritic cells (DCs) controlling Th cell polarization. APN induced maturation and activation of DCs, as demonstrated by the increased expression of MHC class II, costimulatory molecules in both mouse and human DCs, and it significantly enhanced production of proinflammatory cytokines. APN triggered degradation of IκB proteins, nuclear translocation of NF-κB p65 subunit, and phosphorylation of MAPKs in DCs. Pretreatment with a phospholipase C (PLC)γ inhibitor and a JNK inhibitor suppressed IL-12 production and NF-κB binding activity. Additionally, PLCγ inhibitor downregulated phosphorylation of JNK, indicating that PLCγ and JNK may be upstream molecules of NF-κB. Importantly, APN-treated DCs significantly induced both Th1 and Th17 responses in allogeneic CD4(+) T cells. The addition of a neutralizing anti-IL-12 mAb to the cocultures abolished the secretion of IFN-γ, whereas the blockage of IL-23 and IL-1β suppressed APN-induced IL-17 production. Immunization of mice with OVA-pulsed, APN-treated DCs efficiently led to Ag-specific Th1 and Th17 cell responses. Taken together, these results demonstrated that APN effectively induced activation of DCs through PLCγ/JNK/NF-κB-signaling pathways, leading to enhanced Th1 and Th17 responses.     

N-Formyl-3,4-methylenedioxy-benzylidene-γ-butyrolaetam, KNK437 induces caspase-3 activation through inhibition of mTORC1 activity in Cos-1 cells

The mammalian target of rapamycin complex 1 (mTORC1: mTOR-raptor interaction) and heat shock protein 70 (Hsp70) regulate various cellular processes and are crucial for the progression of many cancers and metabolic diseases. In the recent study, we reported that interaction of Hsp70 with tuberous sclerosis complex 1 (TSC1) regulated apoptosis. This study was designed to elucidate the underlying mechanism in Cos-1 cells. Here, we show that N-formyl-3,4-methylenedioxy-benzylidene-γ-butyrolaetam (KNK437), which inhibits the expression level of Hsp70, abrogated phosphorylation of mTOR and S6K in response to insulin, and inhibited mTORC1 activity via disruption of an interaction between mTOR and raptor. In addition, KNK437 did not alter TSC1/2 complex formation. Furthermore, KNK437 inhibited the mTOR-raptor interaction on the outer membrane of the mitochondria and triggered caspase-3 activation. A reduction in the level of Hsp70 could result in the inhibition of the mTORC1 signaling pathway, thereby inducing apoptosis.     

Iron overload induces apoptosis of osteoblast cells via eliciting ER stress-mediated mitochondrial dysfunction and p-eIF2α/ATF4/CHOP pathway in vitro

Iron is an essential element for crucial biological function; whereas excess iron sedimentation impairs the main functions of tissues or organs. Cumulative researches have shown that the disturbances in iron metabolism, especially iron overload is closely concatenating with bone loss. Nevertheless, the specific process of iron overload-induced apoptosis in osteoblasts has not been thoroughly studied. In this study, our purpose is to elucidate the mechanism of osteoblast apoptosis induced by iron overload via the MC3T3-E1 cell line. Ferric ammonium citrate (FAC) was utilized to simulate iron overload conditions in vitro. These results showed that treatment with FAC dose-dependently induced the apoptosis of MC3T3-E1 cells at 48 h, dysfunction of iron metabolism, and increased intracellular reactive oxygen species (ROS) levels. Following, FAC does-dependently caused the calcium dyshomeostasis, decreased the calcium concentration in endoplasmic reticulum (ER), but increased the crosstalk between ER and mitochondria, and calcium concentration in the mitochondria. Moreover, FAC dose-dependently decreased mitochondrial membrane potential (MMP) and enhanced the expression of apoptosis related proteins (Bax, Cyto-C and C-caspase3). We furthermore revealed that FAC treatment activated the ER-mediated cell apoptosis via p-eIF2α/ATF4/CHOP pathway in MC3T3-E1 osteoblasts cells. In addition, pretreatment with the N-acetylcysteine (NAC) or Tauroursodeoxycholate Sodium (TUDC) attenuated cell apoptosis, ROS levels, mitochondria fragmentation and ER stress-related protein expression, and recovered the protein expression related to iron metabolism. In conclusion, our finding suggested that iron overload induced apoptosis via eliciting ER stress, which resulted in mitochondrial dysfunction and activated p-eIF2α/ATF4/CHOP pathway.     

Purification of a peptide from seahorse,that inhibits TPA-induced MMP,iNOS and COX-2 expression through MAPK and NF-κB activation,and induces human osteoblastic and chondrocytic differentiation

Ongoing efforts to search for naturally occurring, bioactive substances for the amelioration of arthritis have led to the discovery of natural products with substantial bioactive properties. The seahorse (Hippocampus kuda Bleeler), a telelost fish, is one source of known beneficial products, yet has not been utilized for arthritis research. In the present work, we have purified and characterized a bioactive peptide from seahorse hydrolysis. Among the hydrolysates tested, pronase E-derived hydrolysate exhibited the highest alkaline phosphatase (ALP) activity, a phenotype marker of osteoblast and chondrocyte differentiation. After its separation from the hydrolysate by several purification steps, the peptide responsible for the ALP activity was isolated and its sequence was identified as LEDPFDKDDWDNWK (1821 Da). We have shown that the isolated peptide induces differentiation of osteoblastic MG-63 and chondrocytic SW-1353 cells by measuring ALP activity, mineralization and collagen synthesis. Our results indicate that the peptide acts during early to late stages of differentiation in MG-63 and SW-1353 cells. We also assessed the concentration dependence of the peptide's inhibition of MMP (-1, -3 and -13), iNOS and COX-2 expression after treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a common form of phorbol ester. The peptide also inhibited NO production in MG-63 and SW-1353 cells. To elucidate the mechanisms by which the peptide acted, we examined its effects on TPA-induced MAPKs/NF-κB activation and determined that the peptide treatment significantly reduced p38 kinase/NF-κB in MG-63 cells and MAPKs/NF-κB in SW-1353 cells.     

Differential involvement of α4β2, α7 and α9α10 nicotinic acetylcholine receptors in B lymphocyte activation in vitro

Mouse B lymphocytes express several nicotinic acetylcholine receptor (nAChR) subtypes, their exact functions being not clearly understood. Here we show that α7 nAChR was present in about 60%, while α4β2 and α9(α10) nAChRs in about 10% and 20% of mouse spleen B lymphocytes, respectively; Balb/c and C57Bl/6 mice possessed different relative amounts of these nAChR subtypes. α4β2 and α7, but not α9(α10) nAChRs, were up-regulated upon B lymphocyte activation in vitro. Flow cytometry and sandwich ELISA studies demonstrated that α7 and α9(α10) nAChRs are coupled to CD40, whereas α4β2 nAChR is coupled to IgM. B lymphocytes of both α7(-/-) and β2(-/-) mice responded to anti-CD40 stronger than those of the wild-type mice, whereas the cells of β2(-/-) mice responded to anti-IgM worse than those of the wild-type or α7(-/-) mice. Inhibition of α7 and α9(α10) nAChRs with methyllicaconitine resulted in considerable augmentation of CD40-mediated B lymphocyte proliferation in cells of all genotypes; stimulation of α4β2 nAChRs with epibatidine increased the IgM-mediated proliferation of the wild-type and α7(-/-), but not β2(-/-) cells. Inhibition of α9(α10) nAChRs with α-conotoxin PeAI exerted weak stimulating effect on CD40-mediated proliferation. This nAChR subtype was up-regulated in α7(-/-) B-cells. α7 nAChRs were found recruited to immune synapses between human T and B lymphocytes, both of which produced acetylcholine. It is concluded that α7 nAChR fulfills inhibitory CD40-related mitogenic function, α4β2 nAChR produces a stimulatory IgM-related effect, while α9α10 nAChR is a "reserve" receptor, which partly compensates the absence of α7 nAChR in α7(-/-) cells. Acetylcholine is an additional mediator to modulate activation of interacting T and B lymphocytes.     

Cannabinoid receptor 1 induces a biphasic ERK activation via multiprotein signaling complex formation of proximal kinases PKCε, Src, and Fyn in primary neurons

Cannabinoid receptors 1 (CB1Rs) play important roles in the regulation of dendritic branching, synapse density, and synaptic transmission through multiple G-protein-coupled signaling systems, including the activation of the extracellular signal-regulated kinases ERK1/2. The proximal signaling interactions leading to ERK1/2 activation by CB1R in CNS remain, however, unclear. Here, we present evidence that the CB1R agonist methanandamide induced a biphasic and sustained activation of ERK1/2 in primary neurons derived from E7 telencephalon. We show that E7 neurons natively express high levels of CB1R message and protein, the majority of which associates with PKC? at basal conditions. We now demonstrate that the first peak of ERK activation by CB1R was mediated by the sequential activation of G(q), PLC, and PKC?, selectively, and that the CB1R-activated PKC? acutely formed transient signaling modules containing activated Src and Fyn. A second pool of CB1Rs, coupled to PTX-sensitive activation of G(i/o), utilized as effectors additional Src and Fyn molecules to generate a second, additional wave of ERK activation at 15 min. Concurrently to these intermolecular signaling interactions, cytoskeleton-associated proteins MARCKS and p120catenin were drastically modified by phosphorylation of PKC and Src, respectively. These receptor-proximal signaling events correlated well with induction of neuritic outgrowth in the long term. Our data provide evidence for multiprotein signaling complex formation in the coupling of CB1R to activation of ERK in CNS neurons, and may elucidate several of the less understood acute effects of cannabinoid drugs.     

GW1929 inhibits α7 nAChR expression through PPARγ-independent activation of p38 MAPK and inactivation of PI3-K/mTOR: The role of Egr-1

Studies demonstrated that peroxisome proliferator-activated receptor gamma (PPARγ) ligands reduce nicotine-induced non small cell lung carcinoma (NSCLC) cell growth through inhibition of nicotinic acetylcholine receptor (nAChR) mediated signaling pathways. However, the mechanisms by which PPARγ ligands inhibited nAChR expression remain elucidated. Here, we show that GW1929, a synthetic PPARγ ligand, not only inhibited but also antagonized the stimulatory effect of acetylcholine on NSCLC cell proliferation. Interestingly, GW1929 inhibited α7 nAChR expression, which was not blocked by GW9662, an antagonist of PPARγ, or by PPARγ siRNA, but was abrogated by the p38 MPAK inhibitor SB239063. GW1929 reduced the promoter activity of α7 nAChR and induced early growth response-1 (Egr-1) protein expression, which was overcame by SB239063, but enhanced by inhibitors of PI3-K and mTOR. Silencing of Egr-1 blocked, while overexpression of Egr-1 enhanced, the effect of GW1929 on α7 nAChR expression and promoter activity. Finally, GW1929 induced Egr-1 bound to specific DNA areas in the α7 nAChR gene promoter. Collectively, these results demonstrate that GW1929 not only inhibits but also antagonizes Ach-induced NSCLC cell growth by inhibition of α7 nAChR expression through PPARγ-independent signals that are associated with activation of p38 MPAK and inactivation of PI3-K/mTOR, followed by inducing Egr-1 protein and Egr-1 binding activity in the α7 nAChR gene promoter. By downregulation of the α7 nAchR, GW1929 blocks cholinergic signaling and inhibits NSCLC cell growth.     

Contribution of type 1 diabetes to rat liver dysfunction and cellular damage via activation of NOS,PARP, IκBα/NF-κB,MAPKs, and mitochondria-dependent pathways: Prophylactic role of arjunolic acid

Diabetic mellitus, a chronic metabolic disorder, is one of the most important health problems in the world, especially in developing countries. Our earlier investigations reported the beneficial action of arjunolic acid (AA) against streptozotocin-mediated type 1 hyperglycemia. We have demonstrated that AA possesses protective roles against drug- and chemical- (environmental toxins) induced hepatotoxicity. Liver is the main organ of detoxification. The purpose of this study was to explore whether AA plays any protective role against hyperglycemic hepatic dysfunctions and, if so, what molecular pathways it utilizes for the mechanism of its protective action. In experimental rats, type 1 hyperglycemia was induced by streptozotocin. AA was administered orally at a dose of 20 mg/kg body wt both before and after diabetic induction. An insulin-treated group was included in the study as a positive control for type 1 diabetes. Hyperglycemia caused a loss in body weight, reduction in serum insulin level, and increased formation of HbA_1C as well as advanced glycation end products (AGEs). Elevated levels of serum ALT and ALP, increased production of ROS and RNS, increased lipid peroxidation, increased 8-OHdG/2-dG ratio, and decreased GSH content and cellular antioxidant defense established the hyperglycemic liver dysfunction. Activation of iNOS, IκBα/NF-κB, and MAPK pathways as well as signals from mitochondria were found to be involved in initiating apoptotic cell death. Hyperglycemia caused overexpression of PARP, reduction in intracellular NAD as well as ATP level, and increased DNA fragmentation in the liver tissue of the diabetic animals. Results of immunofluorescence (using anti-caspase-3 and anti-Apaf-1 antibodies), DAPI/PI staining, and DNA ladder formation and information obtained from FACS analysis confirmed the apoptotic cell death in diabetic liver tissue. Histological studies also support the experimental findings. AA treatment prevented or ameliorated the diabetic liver complications and apoptotic cell death. The effectiveness of AA in preventing the formation of ROS, RNS, HbA_1C, AGEs, and oxidative stress signaling cascades and protecting against PARP-mediated DNA fragmentation can speak about its potential uses for diabetic patients.     

CCN4 induces IL-6 production through αvβ5 receptor,PI3K,Akt, and NF-κB singling pathway in human synovial fibroblasts

Introduction

Osteoarthritis (OA) is the most common degenerative joint disease that is involved in the degradation of articular cartilage. The exact etiology of OA is not completely understood. CCN4 is related to up-regulation in the cartilage of patients with osteoarthritis. Previous studies have shown that CCN4 might be associated with the pathogenesis of OA, but the exact signaling pathways in CCN4-mediated IL-6 expression in synovial fibroblasts (SF) are largely unknown. Therefore, we explored the intracellular signaling pathway involved in CCN4-induced IL-6 production in human synovial fibroblast cells.

Methods

CCN4-induced IL-6 production was assessed with quantitative real-time qPCR and ELISA. The mechanisms of action of CCN4 in different signaling pathways were studied by using Western blotting. Neutralizing antibodies of integrin were used to block the integrin signaling pathway. Luciferase assays were used to study IL-6 and NF-κB promoter activity. Immunocytochemistry was used to examine the translocation activity of p65.

Results

Osteoarthritis synovial fibroblasts (OASFs) showed significant expression of CCN4 and the expression was higher than in normal SFs. OASF stimulation with CCN4 induced concentration- and time-dependent increases in IL-6 production. Pretreatment of OASFs with αvβ5 but not α5β1 and αvβ3 integrin antibodies reduced CCN4-induced IL-6 production. CCN4-mediated IL-6 production was attenuated by PI3K inhibitor ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and Wortmannin), Akt inhibitor (Akti), and NF-κB inhibitor (PDTC and TPCK). Stimulation of cells with CCN4 also increased PI3K, Akt, and NF-κB activation.

Conclusions

Our results suggest that CCN4 activates αvβ5 integrin, PI3K, Akt, and NF-κB pathways, leading to up-regulation of IL-6 production. According to our results, CCN4 may be an appropriate target for drug intervention in OA in the future.     

High glucose induces rat mesangial cells proliferation and MCP-1 expression via ROS-mediated activation of NF-κB pathway,which is inhibited by eleutheroside E

Glomerular hypertrophy and extracellular matrix accumulation are early features of diabetic nephropathy (DN). High glucose-induced oxidative stress is implicated in the etiology of DN. This study aims to investigate the effect of eleutheroside E (EE) on high glucose mediated rat mesangial cells (MCs) proliferation and monocyte chemoattractant protein-1 (MCP-1) expression and the underlying mechanism. MCs proliferation was assessed by MTT assay. Reactive oxygen species (ROS) level and MCP-1 expression were evaluated by ELISA kit. The protein expression of p47, NF-κB p65, p-NF-κB p65, IκBα, p-IκBα, IKKβ and p-IKKβ were determined by Western blot. The results showed that treatment with EE markedly attenuated high glucose induced MCs proliferation and in a dose-dependent manner. Intervention with EE also significantly blocked high glucose induced intracellular ROS production by decreasing NADPH oxidase activity. Meanwhile, EE administration could effectively alleviate the high glucose-stimulated activation of NF-κB, the degradation of IκBα and the expression of MCP-1. These results demonstrate that high glucose enhances MCs proliferation and MCP-1 expression by activating the ROS/NF-κB pathway and can be inhibited by EE. Our findings provide a new perspective for the clinical treatment of DN.     

Cell cycle arrest or survival signaling through αv integrins,activation of PKC and ERK1/2 lead to anoikis resistance of ovarian cancer spheroids

Ovarian cancer is the most lethal gynecologic cancer mainly due to spheroids organization of cancer cells that disseminate within the peritoneal cavity. We have investigated the molecular mechanisms by which ovarian cancer spheroids resist anoikis, choosing as models the 2 well-characterized human ovarian cancer cell lines IGROV1 and SKOV3. These cell lines have the propensity to float as clusters, and were isolated from tumor tissue and ascites, respectively. To form spheroids, IGROV1 and SKOV3 ovarian adenocarcinoma cells were maintained under anchorage-independent culture conditions, in which both lines survive at least a week. A short apoptotic period prior to a survival signaling commitment was observed for IGROV1 cells whereas SKOV3 cells entered G0/G1 phase of the cell cycle. This difference in behavior was due to different signals. With regard to SKOV3 cells, activation of p38 and an increase in p130/Rb occurred once anchorage-independent culture was established. Analyses of the survival signaling pathway switched on by IGROV1 cells showed that activation of ERK1/2 was required to evade apoptosis, an effect partly dependent on PKC activation and αv integrins. αv-integrin expression is essential for survival through activation of ERK1/2 phosphorylation.