Time-dependent Manner In Response Research Articles

CDK5RAP3 Inhibition by Hypoxia Activates P38MAPK to Facilitate Angiogenesis.

CDK5RAP3 is a recognized tumor suppressor that inhibits Chk1 and Chk2 and activates p53, all of which are involved with mediating toxin-induced apoptosis of cancer cells. CDK5RAP3 also inhibits p38MAPK phosphorylation and activity via mediating a p38 interaction with wild-type p53-induced phosphatase 1. This study aimed to investigate the antiangiogenic activity of CDK5RAP3 and its molecular mechanisms in human umbilical vein endothelial cells (HUVECs) under conditions of hypoxic conditions. Angiogenesis was induced in HUVECs mainly by vascular endothelial growth factor (VEGF). The CDK5RAP3 levels of HUVECs were reduced in a time-dependent manner in response to hypoxic treatment at 2% O2. The reduction of CDK5RAP3 was accompanied with increased p38MAPK phosphorylation and activation. Moderate hypoxia was found to significantly increase secreted VEGF concentrations, and the hypoxic conditioned medium (HCM) markedly enhanced proliferation, migration, and tube formation. Our findings indicate that moderate hypoxia facilitates angiogenesis by inhibiting CDK5RAP3. CDK5RAP3 exhibits a clear regulatory role in vascular regeneration, as downregulating its expression in endothelial cells enhances VEGF synthesis and subsequently improves cell migration and lumen formation capability. This study presents evidence indicating that moderate hypoxia facilitates angiogenesis by inhibiting CDK5RAP3, demonstrating the potential for CKD5RAP3 to be a potent antiangiogenic agent in angiogenesis regulation of cancer, ischemic diseases, and wound healing.

Long-term exposure to lead nitrate and zinc sulfate Nile tilapia impact the Aeromonas hydrophila treatment

BackgroundPollution with heavy metals (HMs) is time- and concentration-dependent. Lead and zinc pollute the aquatic environment, causing severe health issues in aquatic animals.Materials and methodsNile tilapia, the predominant cultured fish in Egypt, were experimentally exposed to 10% of LC50 of lead nitrate (PbNO3) and zinc sulfate (ZnSO4). Samples were collected in three different periods, 4, 6, and 8 weeks, in addition to a trial to treat the experimental fish infected with Aeromonas hydrophila, with an antibiotic (florfenicol).ResultsLiver enzymes were linearly upsurged in a time-dependent manner in response to HMs exposure. ALT was 92.1 IU/l and AST was 82.53 IU/l after eight weeks. In the eighth week of the HMs exposure, in the hepatic tissue, the levels of glutathione peroxidase (GPx), catalase (CAT), and metallothionein (MT) were increased to 117.8 U/mg prot, 72.2 U/mg prot, and 154.5 U/mg prot, respectively. On exposure to HMs, gene expressions of some cytokines were linearly downregulated in a time-dependent manner compared to the control. After four weeks of exposure to the HMs, the oxidative burst activity (OBA) of immune cells was decreased compared to the control 9.33 and 10.3 cells, respectively. Meanwhile, the serum bactericidal activity (SBA) significantly declined to 18.5% compared to the control 32.6% after eight weeks of exposure. Clinical signs of A. hydrophila infection were exaggerated in polluted fish, with a mortality rate (MR) of 100%. The re-isolation rate of A. hydrophila was decreased in fish treated with florfenicol regardless of the pollution impacts after eight weeks of HMs exposure.ConclusionIt could be concluded that the immune suppression and oxidative stress resulting from exposure to HMs are time-dependent. Clinical signs and post-mortem lesions in polluted fish infected with A. hydrophila were prominent. Infected-Nile tilapia had weak responses to florfenicol treatment due to HMs exposure.

Time-course transcriptome analysis of a double challenge bleomycin-induced lung fibrosis rat model uncovers ECM homoeostasis-related translationally relevant genes

BackgroundIdiopathic pulmonary fibrosis (IPF) is an irreversible disorder with a poor prognosis. The incomplete understanding of IPF pathogenesis and the lack of accurate animal models is limiting the development of...

Influence of the MUC1 Cell Surface Mucin on Gastric Mucosal Gene Expression Profiles in Response to Helicobacter pylori Infection in Mice.

The cell surface mucin MUC1 is an important host factor limiting Helicobacter pylori (H. pylori) pathogenesis in both humans and mice by providing a protective barrier and modulating mucosal epithelial and leukocyte responses. The aim of this study was to establish the time-course of molecular events in MUC1-modulated gene expression profiles in response to H. pylori infection in wild type (WT) and MUC1-deficient mice using microarray-determined mRNA expression, gene network analysis and Ingenuity Pathway Analysis (IPA). A time-course over the first 72 h of infection showed significantly higher mucosal loads of bacteria at 8 h of infection in Muc1−/− mice compared with WT, confirming its importance in the early stages of infection (P = 0.0003). Microarray analysis revealed 266 differentially expressed genes at one or more time-points over 72 h in the gastric mucosa of Muc1−/− mice compared with WT control using a threshold of 2-fold change. The SPINK1 pancreatic cancer canonical pathway was strongly inhibited in Muc1−/− mice compared with WT at sham and 8 h infection (P = 6.08E-14 and P = 2.25 E-19, respectively) but potently activated at 24 and 72 h post-infection (P = 1.38E-22 and P = 5.87E-13, respectively). The changes in this pathway are reflective of higher expression of genes mediating digestion and absorption of lipids, carbohydrates, and proteins at sham and 8 h infection in the absence of MUC1, but that this transcriptional signature is highly down regulated as infection progresses in the absence of MUC1. Uninfected Muc1−/− gastric tissue was highly enriched for expression of factors involved in lipid metabolism and 8 h infection further activated this network compared with WT. As infection progressed, a network of antimicrobial and anti-inflammatory response genes was more highly activated in Muc1−/− than WT mice. Key target genes identified by time-course microarrays were independently validated using RT-qPCR. These results highlight the dynamic interplay between the host and H. pylori, and the role of MUC1 in host defense, and provide a general picture of changes in cellular gene expression modulated by MUC1 in a time-dependent manner in response to H. pylori infection.

Temporal changes in postprandial blood transcriptomes reveal subject-specific pattern of expression of innate immunity genes after a high-fat meal

White blood cells are among the first responders to dietary components and their metabolites absorbed from the gut. The objective of this study was to determine the whole blood transcriptome response to high-fat challenge meals. A total of 45 fasting and postprandial (3-h and 6-h) whole blood transcriptomes from 5 subjects in a crossover intervention trial of a high-fat meal supplemented with placebo, blueberry powder or docosahexaenoic acid (DHA) were analyzed using RNA sequencing. Select target genes were validated by quantitative reverse-transcription polymerase chain reaction in 180 samples from 20 subjects. The largest contributor to variance was the subject (13,856 genes differentially expressed), followed by the subject on a specific day (2276 genes), followed by the subject's postprandial response (651 genes). After determining the nonsignificance of individual dietary treatments (blueberry, DHA, placebo), treatments were used as replicates to examine postprandial responses to a high-fat meal. The universal postprandial response (95 genes) was associated with lipid utilization, fatty acid beta-oxidation and circadian rhythms. Subject-specific postprandial responses were enriched for genes involved in the innate immune response, particularly those of pattern recognition receptors and their downstream signaling components. Genes involved in innate immune responses are differentially expressed in a subject-specific and time-dependent manner in response to the high-fat meals. These genes can serve as biomarkers to assess individual responsiveness to a high-fat diet in inducing postprandial inflammation. Furthermore, the dynamic temporal change in gene expression in postprandial blood suggests that monitoring these genes at multiple time points is necessary to reveal responders to dietary intervention.

Upregulation of sphingosine-1-phosphate receptor 3 on fibroblast-like synoviocytes is associated with the development of collagen-induced arthritis via increased interleukin-6 production.

BackgroundSphingosine-1-phosphate receptor 3 (S1P3) is one of five receptors for sphingosine-1-phosphate (S1P). S1P/S1P3 signaling is involved in numerous physiological and pathological processes including bone metabolism, sepsis, cancer, and immunity. In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLSs) are activated by several factors and promote abundant proinflammatory cytokine production and bone destruction. The aim of this study was to investigate whether S1P3 is associated with the development of autoimmune arthritis and the pathogenic function of FLSs.MethodsWild-type (WT) and S1P3 knockout (S1P3-KO) collagen-induced arthritis (CIA) mice were evaluated with respect to clinical and histological disease severity, along with the levels of anti-collagen antibodies and expression of tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6). S1P3 expression in the synovium was analyzed by real-time reverse-transcription polymerase chain reaction (RT-PCR) and immunofluorescence staining. FLSs isolated from CIA mice were activated with TNFα and S1P3 expression was analyzed by real-time RT-PCR. The role of S1P/S1P3 signaling in activated and non-activated FLSs was investigated by measuring cell proliferation and cyto/chemokine production by real-time RT-PCR and/or enzyme-linked immunosorbent assay.ResultsClinical and histological scores, and synovial IL-6 expression were significantly lower in S1P3-KO mice with CIA than in WT mice. Arthritic synovia had higher S1P3 expression than intact synovia and FLSs in arthritic joints expressed S1P3 in vivo. Primary cultured FLSs produced IL-6 in a time-dependent manner in response to S1P stimulation and exhibited higher levels of S1P3 expression after activation with TNFα. S1P3-induced production of IL-6 and MMP-3 was increased in FLSs pre-activated with TNFα.ConclusionIn this study, we demonstrated that S1P3 expression is associated with the development of autoimmune arthritis via inflammation-induced increases in S1P/S1P3 signaling that increase production of IL-6 in FLSs. Inhibition of S1P/S1P3 signaling could open the door to the development of new therapies for RA.

CAPA periviscerokinin-mediated activation of MAPK/ERK signaling through Gq-PLC-PKC-dependent cascade and reciprocal ERK activation-dependent internalized kinetics of Bom-CAPA-PVK receptor 2

Bombyx mori neuropeptide G protein-coupled receptor (BNGR)-A27 is a specific receptor for B. mori capability (CAPA) periviscerokinin (PVK), that is, Bom-CAPA-PVK receptor 2. Upon stimulation of Bom-CAPA-PVK-1 or -PVK-2, Bom-CAPA-PVK receptor 2 significantly increases cAMP-response element-controlled luciferase activity and Ca2+ mobilization in a Gq inhibitor-sensitive manner. However, the underlying mechanism(s) for CAPA/CAPA receptor system mediation of extracellular signal-regulated kinases1/2 (ERK1/2) activation remains to be explained further. Here, we discovered that Bom-CAPA-PVK receptor 2 stimulated ERK1/2 phosphorylation in a dose- and time-dependent manner in response to Bom-CAPA-PVK-1 or -PVK-2 with similar potencies. Furthermore, ERK1/2 phosphorylation can be inhibited by Gq inhibitor UBO-QIC, PLC inhibitor U73122, protein kinase C (PKC) inhibitor Go 6983, phospholipase D (PLD) inhibitor FIPI and Ca2+ chelators EGTA and BAPTA-AM. Moreover, Bom-CAPA-PVK-R2-induced activation of ERK1/2 was significantly attenuated by treatment with the Gβγ-specific inhibitors, phosphatidylinositol 3-kinase (PI3K)-specific inhibitor Wortmannin and Src-specific inhibitor PP2. Our data also demonstrate that receptor tyrosine kinase (RTK) transactivation pathways are involved in the mechanisms of Bom-CAPA-PVK receptor to ERK1/2 phosphorylation. In addition, β-arrestin1/2 is not involved in Bom-CAPA-PVK-R2-mediated ERK1/2 activation but required for the agonist-independent, ERK1/2 activation-dependent internalization of the G protein-coupled receptor (GPCR).

Retraction Note to "SDF-1/CXCR4 Axis Regulates Cell Cycle Progression and Epithelial-Mesenchymal Transition via Up-regulation of Survivin in Glioblastoma".

Stromal cell-derived factor 1 (SDF-1)/CXCR4 ligand-receptor axis is widely recommended as an attractive target for cancer therapy. Meanwhile, epithelial-mesenchymal transition (EMT) process is linked to disease pathophysiology. As one of inhibitors of apoptosis proteins, survivin is implicated in the onset and development of cancer. In the present study, we tried to determine the cause-effect associations between SDF-1/CXCR4 axis and survivin expression in glioblastoma U-251 cell line. Survivin activation and inhibition were induced with exogenous SDF-1 and survivin small interfering RNA (survivin siRNA), respectively. Western blot was used to detect relevant proteins in SDF-1/CXCR4 axis. Western blot analysis revealed that survivin expression in U-251 increased in a dose- and time-dependent manner in response to SDF-1 treatment. However, the interference with MEK/ERK and PI3K/AKT pathway prohibited SDF-1-induced survivin up-regulation. Importantly, survivin knockdown abrogated cell cycle progression and the expression of snail and N-cadherin, compared with non-transfectants. In conclusion, the present study shows that SDF-1 up-regulates survivin via MEK/ERK and PI3K/AKT pathway, leading to cell cycle progression and EMT occurrence dependent on survivin. The blockade of survivin will allow for the treatment of glioblastoma.

Agonist-mediated activation of Bombyx mori diapause hormone receptor signals to extracellular signal-regulated kinases 1 and 2 through Gq-PLC-PKC-dependent cascade

Diapause is a developmental strategy adopted by insects to survive in challenging environments such as the low temperatures of a winter. This unique process is regulated by diapause hormone (DH), which is a neuropeptide hormone that induces egg diapause in Bombyx mori and is involved in terminating pupal diapause in heliothis moths. An G protein-coupled receptor from the silkworm, B. mori, has been identified as a specific cell surface receptor for DH. However, the detailed information on the DH-DHR system and its mechanism(s) involved in the induction of embryonic diapause remains unknown. Here, we combined functional assays with various specific inhibitors to elucidate the DHR-mediated signaling pathways. Upon activation by DH, B. mori DHR is coupled to the Gq protein, leading to a significant increase of intracellular Ca2+ and cAMP response element-driven luciferase activity in an UBO-QIC, a specific Gq inhibitor, sensitive manner. B. mori DHR elicited ERK1/2 phosphorylation in a dose- and time-dependent manner in response to DH. This effect was almost completely inhibited by co-incubation with UBO-QIC and was also significantly suppressed by PLC inhibitor U73122, PKC inhibitors Gö6983 and the Ca2+ chelator EGTA. Moreover, DHR-induced activation of ERK1/2 was significantly attenuated by treatment with the Gβγ specific inhibitors gallein and M119K and the PI3K specific inhibitor Wortmannin, but not by the Src specific inhibitor PP2. Our data also demonstrates that the EGFR-transactivation pathway is not involved in the DHR-mediated ERK1/2 phosphorylation. Future efforts are needed to clarify the role of the ERK1/2 signaling pathway in the DH-mediated induction of B. mori embryonic diapause.

Gambogic acid potentiates clopidogrel-induced apoptosis and attenuates irinotecan-induced apoptosis through down-regulating human carboxylesterase 1 and -2

1. In this study, we report that gambogic acid (GA), a promising anticancer agent, potentiates clopidogrel-induced apoptosis and attenuates CPT-11-induced apoptosis by down-regulating human carboxylesterase (CES) 1 and -2 via ERK and p38 MAPK pathway activation, which provides a molecular explanation linking the effect of drug combination directly to the decreased capacity of hydrolytic biotransformation.2. The expression levels of CES1 and CES2 decreased significantly in a concentration- and time-dependent manner in response to GA in Huh7 and HepG2 cells; hydrolytic activity was also reduced.3. The results showed that pretreatment with GA potentiated clopidogrel-induced apoptosis by down-regulating CES1. Moreover, the GA-mediated repression of CES2 attenuated CPT-11-induced apoptosis.4. Furthermore, the ERK and p38 MAPK pathways were involved in the GA-mediated down-regulation of CES1 and CES2.5. Taken together, our data suggest that GA is a potent repressor of CES1 and CES2 and that combination with GA will affect the metabolism of drugs containing ester bonds.

Platelet-Derived Serotonin Stimulates TPO Production from Bone Marrow Mesenchymal Stromal Cells but Not Hepatocytes

In circulation thrombopoietin (TPO) level and platelet count is inversely related, but the precise controlling mechanism is still unknown. TPO is constitutively produced by the liver but can be induced to generate from bone marrow mesenchymal stromal cells (MSCs) in pathological conditions such as thrombocytopenia. Since serotonin (5-HT) can be released from the active platelets when the body undergoing severe thrombocytopenia or stress, it may function as an essential humoral mediator regulating TPO release. Our previous studies suggested that 5-HT enhances the growth of the colony-forming unit-fibroblast (CFU-F) from bone marrow, a progenitor of MSCs (Yang et al, Stem Cells, 2007; Ye et al, Stem Cells, 2014). However, the exact functions of 5-HT on MSCs and especially MSCs-derived TPO production are still under investigation. The bone marrow (BM) microenvironment may play an important role in the regulation of thrombopoiesis through release of thrombopoietic growth factor TPO. In this study, the expression of 5-HT2BR was identified in hMSCs and hepatocytic cell line MIHA by Q-PCR. 5-HT (200nM) promoted the proliferation of MSCs and MIHA cells by CCK-8 and MTT assay, respectively. This effect was abolished by 5-HT2BR blocker Ketanserin (2.0uM) in these cells. TPO RNA expression in hMSCs was enhanced in a time-dependent manner in response to the 5-HT treatment but not in MIHA cells. The maximum reaction peak was observed at 24 hrs with 1.5-fold increase compared to the untreated control in hMSCs. A significant increase TPO protein was found in the supernatants secreted by 5-HT-treated hMSCs. The changes measured by ELISA and cytokinearray and were 4.5-fold and 2.14-fold increased compared to the control sample. In addition to the evaluation of the effect of 5-HT on naked-TPO release from MSCs, we also investigate its impacts on the RNA expression of TPO contained within the MSC-derived microparticles (MPs). 5-HT was added to the hMSC culture for 24hrs, 36hrs and 48hrs respectively. It significantly stimulated MPs released from MSCs detected by flow cytometry. The maximal response time was observed at 36hrs. The AnnexinV positive population increased remarkably under 5-HT treatment. HtMSC Cell movement and MPs released were visualized under phase contrast microscopy. HtMSC cells with 5-HT treatment were stained with AnnexinV-PE and the plates were inspected under the Tirf microscopy to trace the shedding process of MPs. By using phase contrast microscopy, a notable generation of MPs was observed from approximate 6hrs after the addition of 5HT, while only a little amount of MPs were detected in the untreated control group. Budding MPs were observed in 5HT-treated MSCs under both SEM and TEM. The morphology of these MPs was compatible to those published in other studies. And these results were complementary to our previous data obtained by flow cytometry and Tirf microscopy. Our data proved that hMSCs-derived MPs contained inducible TPO mRNA, which was enhanced under 5-HT treatment with more than 10-fold increase compared to the untreated control. We further examined protein level of TPO in MPs using ELISA. The TPO protein level was increased more than 3-fold in 5-HT treated sample. Our findings suggested that 5-HT stimulated TPO released from MSCs in both dissociative and MP-bounded form, which indirectly promotes megakaryopoiesis and thrombopoiesis. DisclosuresYang:National Natural Science Foundation of China(81270580): Research Funding.

KPNA2 Contributes to the Inflammatory Processes in Synovial Tissue of Patients with Rheumatoid Arthritis and SW982 Cells.

Karyopherin-α2 (KPNA2) functions as an adaptor that transports several proteins to the nucleus. We investigated the function and possible mechanisms of KPNA2 involved in rheumatoid arthritis (RA). Western blotting and immunohistochemistry showed the protein expression of KPNA2 increased in synovial tissue of RA patients compared with the healthy controls. Double immunofluorescent staining indicated that KPNA2 co-localized with T cells, macrophage-like synoviocytes, fibroblast-like synoviocytes, and neutrophils in synovial tissue of RA patients. Moreover, the expression of KPNA2 in SW982 cells was increased in a time-dependent manner in response to TNFα stimulation. Both Western blotting and immunofluorescent staining assay revealed the co-localization of KPNA2 and P65 and their translocation from cytoplasma in TNFα-treated SW982 cells. Furthermore, knocking down the expression of KPNA2 by siRNA inhibited TNFα-induced expression of IL-6, MMP-1, and MMP-13 and, more importantly, decreased the P65 phosphorylation in SW982 cells. We therefore suggested that KPNA2 may play a key role in the inflammation process of RA via NF-κB P65 signal transduction pathway.

SDF-1/CXCR4 Axis Regulates Cell Cycle Progression and Epithelial-Mesenchymal Transition via Up-regulation of Survivin in Glioblastoma.

Stromal cell-derived factor 1 (SDF-1)/CXCR4 ligand-receptor axis is widely recommended as an attractive target for cancer therapy. Meanwhile, epithelial-mesenchymal transition (EMT) process is linked to disease pathophysiology. As one of inhibitors of apoptosis proteins, survivin is implicated in the onset and development of cancer. In the present study, we tried to determine the cause-effect associations between SDF-1/CXCR4 axis and survivin expression in glioblastoma U-251 cell line. Survivin activation and inhibition were induced with exogenous SDF-1 and survivin small interfering RNA (survivin siRNA), respectively. Western blot was used to detect relevant proteins in SDF-1/CXCR4 axis. Western blot analysis revealed that survivin expression in U-251 increased in a dose- and time-dependent manner in response to SDF-1 treatment. However, the interference with MEK/ERK and PI3K/AKT pathway prohibited SDF-1-induced survivin up-regulation. Importantly, survivin knockdown abrogated cell cycle progression and the expression of snail and N-cadherin, compared with non-transfectants. In conclusion, the present study shows that SDF-1 up-regulates survivin via MEK/ERK and PI3K/AKT pathway, leading to cell cycle progression and EMT occurrence dependent on survivin. The blockade of survivin will allow for the treatment of glioblastoma.

Abstract 1783: Puupehenol natural product inhibits STAT3 signaling and induces antitumor cell effects in vitro against human glioblastoma cells

Abstract Glioblastoma multiforme (GBM) is a malignant brain tumor that lacks effective therapeutics and has a high mortality rate. Constitutively-activated signal transducer and activator of transcription 3 (STAT3) protein is prevalent and implicated in GBM phenotype and represents a valid anti-GBM target. To discover novel STAT3 inhibitors suitable for GBM therapy, natural product extracts were investigated. Whole extracts from marine organisms were first screened for effects against cell viability using the human glioma model line, U251MG that harbors aberrantly-active STAT3 and normal fibroblasts that do not. The screening campaign identified the puupehenol, CC-1-2 from the whole extracts of leiodermatium sp as a bioactive compound that suppressed U251MG cell viability, with an IC50 4.1±0.1 µM, compared to an IC50 value of 7.3±0.3 µM against normal NIH3T3 mouse fibroblasts. Western blot analysis showed pSTAT3 levels in U251MG cells substantially decreased in a time-dependent manner in response to CC-1-2. Decreased pSTAT3 in treated cells occurred in parallel with partial suppression of both phospho- Janus kinase (Jak) 2 and pSrc levels and no induction of protein tyrosine phosphatase, PTP1B. Phosphorylated extracellular signal-regulated kinase (Erk) 1/2 levels also decreased in time-dependent manner, while pAkt remained unchanged in U251MG cells under the same treatment conditions. Furthermore, treatment of U251MG cells with CC-1-2 led to dramatic morphological changes that occurred in a dose- and time-dependent manner, compared to minimal changes observed for similarly-treated MCF-7 breast cancer cells that do not harbor aberrantly-active STAT3. Colony survival assay of U251MG cells revealed single treatment with 1-10 µM CC-1-2 decreased colony sizes and numbers in a dose-dependent manner, compared to moderate effects on MCF-7. In vitro wound-healing assay further showed reduced U251MG cell migration into the denuded area in response to CC-1-2 treatment. CC-1-2 further induced apoptosis in U251MG cells in a dose-dependent manner and suppressed the expression of c-Myc, Cyclin D1, and Bcl-2 levels, which are known STAT3-regulated genes. Studies together identify CC-1-2 as a natural product inhibitor of STAT3 that preferentially induces antitumor cell effects against human glioblastoma cells. Citation Format: Tyvette S. Hilliard, Christopher Chock, Philip Williams, James Turkson. Puupehenol natural product inhibits STAT3 signaling and induces antitumor cell effects in vitro against human glioblastoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1783. doi:10.1158/1538-7445.AM2014-1783

Acacetin (5,7-dihydroxy-4′-methoxyflavone) exhibits in vitro and in vivo anticancer activity through the suppression of NF-κB/Akt signaling in prostate cancer cells

Acacetin (5,7-dihydroxy-4'-methoxyflavone) is a flavonoid compound with antimutagenic, antiplasmodial, antiperoxidant, anti-inflammatory and anticancer effects. However, the molecular targets and pathways underlying the anticancer effects of acacetin are yet to be elucidated. In this study, we investigated whether acacetin induces apoptosis in the human prostate cancer cell line, DU145. The results of 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays revealed that cell viability decreased in a dose- and time-dependent manner in response to acacetin. 4',6-Diamidino-2-phenylindole (DAPI) staining revealed that chromatin condensation significantly increased in a dose-dependent manner. Flow cytometric analysis indicated that acacetin suppressed the viability of DU145 cells by inducing apoptosis. Western blot anlaysis of various markers of signaling pathways revealed that acacetin targets the Akt and nuclear factor (NF)-κB signaling pathways by inhibiting the phosphorylation of IκBα and NF-κB in a dose-dependent manner. Consistent with its ability to induce apoptosis, the acacetin-mediated inhibition of the pro-survival pathway, Akt, and of the NF-κB pathway was accompanied by a marked reduction in the levels of the NF-κB‑regulated anti-apoptotic proteins, Bcl-2 and X-linked inhibitor of apoptosis protein (XIAP), as well as of the proliferative protein, cyclooxygenase (COX)-2. We further evaluated the effects of acacetin on prostate cancer using mice subcutaneously injected with DU145 prostate cancer cells. The acacetin-treated nude mice bearing DU145 tumor xenografts exhibited significantly reduced tumor size and weight, due to the effects of acacetin on cancer cell apoptosis, as determined by terminal deoxyribonucleotide transferase-mediated dUTP nick end-labeling (TUNEL) assay. Our findings suggest that acacetin exerts antitumor effects by targeting the Akt/NF-κB signaling pathway. Rurther investigations on this flavonoid are warranted to evaluate its potential use in the prevention and therapy of prostate cancer.

Role of Cortactin in Dynamic Actin Remodeling Events in Gonadotrope Cells

GnRH induces marked activation of the actin cytoskeleton in gonadotropes; however, the physiological consequences and cellular mechanisms responsible have yet to be fully elucidated. The current studies focus on the actin scaffolding protein cortactin. Using the gonadotrope-derived αT3-1 cell line, we found that cortactin is phosphorylated at Y(421), S(405), and S(418) in a time-dependent manner in response to the GnRH agonist buserelin (GnRHa). GnRHa induced translocation of cortactin to the leading edge of the plasma membrane where it colocalizes with actin and actin-related protein 3 (Arp3). Incubation of αT3-1 cells with the c-src inhibitor phosphoprotein phosphatase 1, blocked tyrosine phosphorylation of cortactin, reduced cortactin association with Arp3, and blunted actin reorganization in response to GnRHa. Additionally, we used RNA silencing strategies to knock down cortactin in αT3-1 cells. Knockdown of cortactin blocked the ability of αT3-1 cells to generate filopodia, lamellipodia, and membrane ruffles in response to GnRHa. We show that lamellipodia and filopodia are capable of LHβ mobilization in primary pituitary culture after GnRHa treatment, and disruption of these structures using jasplakinolide reduces LH secretion. Collectively, our findings suggest that after GnRHa activation, src activity leads to tyrosine phosphorylation of cortactin, which facilitates its association with Arp3 to engage the actin cytoskeleton. The reorganization of actin by cortactin potentially underlies GnRHa-induced secretory events within αT3-1 cells.

Serotonin regulates TPO production from bone marrow mesenchymal stromal cells

The bone marrow (BM) microenvironment plays an important role in regulation of thrombopoiesis through release of thrombopoietic growth factor, among which thrombopoietin (TPO) is the most essential regulator. It has been realized that the TPO blood level and platelet count is inversely related, but the precise controlling mechanism is still under investigation. TPO is constitutively produced by the liver but can be induced to generate from BM mesenchymal stromal cells (MSCs) in pathological conditions such as thrombocytopenia. Since serotonin (5-HT) can be released from the active platelets when the body undergoing severe thrombocytopenia or stress, it may function as an essential humoral mediator regulating TPO production under this model. Our previous studies suggested that 5-HT enhances the growth of the colony-forming unit-fibroblast (CFU-F), a progenitor of MSCs (Yang M et al, 2007). However, the exact functions of 5-HT on MSCs and especially MSCs-derived TPO production are still under investigated. In this study, we showed that TPO RNA expression in hMSCs was enhanced in a time-dependent manner in response to the 5-HT treatment (200nM). The maximum reaction peak was observed at 24 hrs with 1.5-fold increase compared to the untreated control. A significant increase TPO protein was found in the supernatants secreted by 5-HT-treated hMSCs. The changes measured by ELISA and cytokinearray and were 4.5-fold (P=0.0372, n=4) and 2.14-fold increased compared to the control sample. Microparticles (MPs) are membrane-derived particles (0.1-1.0μm in size) produced from activated or apoptotic cells. They have been described as an important mediator in regulating a wide range of physiological and pathological response by transforming a spectrum of cytokines, signaling proteins, mRNAs and microRNAs. Recent studies have suggested that BM MSCs is one of the major sources for MPs production and that RNA contained in MSC-derived MPs can be isolated. Therefore, TPO mRNA released from MSCs could also be stored in MPs and released into the circulation or functions in a paracrine manner. In this study, in addition to the evaluation of the effect of 5-HT on naked-TPO release from MSCs, we also investigate its impacts on the RNA expression of TPO contained within the MSC-derived MPs. 5-HT was added to the hMSC culture for 24hrs, 36hrs and 48hrs respectively. It significantly stimulated MPs released from MSCs detected by flow cytometry. The maximal response time was observed at 36hrs. The AnnexinV positive population increased remarkably under 5-HT treatment (P=0.0007, n=4). HtMSC Cell movement and MPs released were visualized under phase contrast microscopy. HtMSC cells with 5-HT treatment were stained with AnnexinV-PE and the plates were inspected under the Tirf microscopy to trace the shedding process of MPs. By using phase contrast microscopy, a notable generation of MPs was observed from approximate 6hrs after the addition of 5HT, while only a little amount of MPs were detected in the untreated control group. Budding MPs were observed in 5HT-treated MSCs under both SEM and TEM. The morphology of these MPs was compatible to those published in other studies. And these results were complementary to our previous data obtained by flow cytometry and Tirf microscopy. Our data proved that hMSCs-derived MPs contained inducible TPO mRNA, which was enhanced under 5-HT treatment with more than 10-fold increase compared to the untreated control (P=0.0025, n=3). We further examined protein level of TPO in MPs using ELISA. The TPO protein level was increased more than 3-fold in 5-HT treated sample (P=0.0052, n=3). In summary, our findings suggested that 5-HT stimulated TPO released from MSCs in both dissociative and MP-bounded form, which indirectly promotes megakaryopoiesis and thrombopoiesis. Disclosures:No relevant conflicts of interest to declare.

MMP-1 overexpression induced by IL-1β: possible mechanism for inflammation in degenerative lumbar facet joint

BackgroundMore and more attention has been focused on the inflammation or degeneration caused by biochemical factors in radiculopathy during lumbar facet joint degeneration. This study was designed to examine the expression and relationship of MMP-1/TIMP-1 and interleukin-1β (IL-1β), and to analyze the possible mechanism in degenerative lumbar facet joint disease. MethodsLumbar facet joint cartilage and synovial tissues in 36 cases of posterior lumbar surgery were harvested to investigate IL-1β and MMP-1/TIMP-1 by immunohistochemistry and Western blot analysis. Double labeling immunofluorescence and real-time PCR, respectively, were used to assess the relationship between IL-1β and MMP-1. ResultsIL-1β and MMP-1 were low in the lumbar disc herniation (LDH) group, and increased markedly in the lumbar spinal canal stenosis (LSCS) group (P<0.05). However, there is no significant difference of TIMP-1 between LDH group and LSCS group (P>0.05). Double staining results indicated that IL-1β overlapped with MMP-1 in the LSCS group. Moreover, real-time PCR results showed that MMP-1 mRNA in chondrocytes in vitro was affected in a dose- and time-dependent manner in response to IL-1β stimulation. ConclusionsOverexpression of MMP-1, induced by IL-1β, plays an important role in the inflammatory process of lumbar facet joint degeneration.

Pathological Cyclic Strain-Induced Apoptosis in Human Periodontal Ligament Cells through the RhoGDIα/Caspase-3/PARP Pathway

AimHuman periodontal ligament (PDL) cells incur changes in morphology and express proteins in response to cyclic strain. However, it is not clear whether cyclic strain, especially excessive cyclic strain, induces PDL cell apoptosis and if so, what mechanism(s) are responsible. The aim of the present study was to elucidate the molecular mechanisms by which pathological levels of cyclic strain induce human PDL cell apoptosis.Materials and MethodsHuman PDL cells were obtained from healthy premolar tissue. After three to five passages in culture, the cells were subjected to 20% cyclic strain at a frequency of 0.1 Hz for 6 or 24 h using an FX-5000T system. Morphological changes of the cells were assessed by inverted phase-contrast microscopy, and apoptosis was detected by fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide staining followed by flow cytometry. Protein expression was evaluated by Western blot analysis.ResultsThe number of apoptotic human PDL cells increased in a time-dependent manner in response to pathological cyclic strain. The stretched cells were oriented parallel to each another with their long axes perpendicular to the strain force vector. Cleaved caspase-3 and poly-ADP-ribose polymerase (PARP) protein levels increased in response to pathological cyclic strain over time, while Rho GDP dissociation inhibitor alpha (RhoGDIα) decreased. Furthermore, knock-down of RhoGDIα by targeted siRNA transfection increased stretch-induced apoptosis and upregulated cleaved caspase-3 and PARP protein levels. Inhibition of caspase-3 prevented stretch-induced apoptosis, but did not change RhoGDIα protein levels.ConclusionThe overall results suggest that pathological-level cyclic strain not only influenced morphology but also induced apoptosis in human PDL cells through the RhoGDIα/caspase-3/PARP pathway. Our findings provide novel insight into the mechanism of apoptosis induced by pathological cyclic strain in human PDL cells.

Time-course microarrays reveal modulation of developmental, lipid metabolism and immune gene networks in intrascapular brown adipose tissue during the development of diet-induced obesity

The aim of this study was to establish the time-course of molecular events in intrascapular brown adipose tissue (iBAT) during the development of diet-induced obesity using microarrays and molecular network analysis. C57BL/6J male inbred mice were fed a high-fat diet (HFD) or normal diet (ND) and killed at multiple time-points over 24 weeks. Global transcriptional changes in iBAT were determined by time-course microarrays of pooled RNA (n=6, pools per time-point) at 2, 4, 8, 20 and 24 weeks using Illumina MouseWG-6 v2.0 Beadchips. Molecular networks were constructed using the Ingenuity knowledgebase based on differentially expressed genes at each time-point. Body weight and subcutaneous adipose were progressively increased over 24 weeks, whereas iBAT was significantly increased between 6 and 12 weeks in HFD-fed C57BL/6J mice compared with controls. Blood glucose and insulin levels were increased between 16 and 24 weeks. Time-course microarrays, revealed 155 differentially expressed genes at one or more time-points over 24 weeks in the iBAT of HFD-fed mice compared with controls. Time-course network analysis revealed a network of skeletal muscle development genes that was activated between 2 and 4 weeks, subsequently a network of immune trafficking genes was activated at 8 weeks. After 20 and 24 weeks, multiple lipid metabolism and immune response networks were activated. Several target genes identified by time-course microarrays were independently validated using RT-qPCR. Tnnc1 was upregulated early between 2 and 4 weeks, later Cd68 and Col1a1 were upregulated between 20 and 24 weeks, whereas 11β-hydroxysteroid dehydrogenase (Hsd11b1) was consistently downregulated during the development of diet-induced obesity. Molecular networks in iBAT are modulated in a time-dependent manner in response to a HFD. A broad range of gene targets exists to alter molecular changes within iBAT during the development of diet-induced obesity.