Alpha Phosphorylation Research Articles

EIF2 alpha phosphorylation alleviates UVA-induced HO-1 expression in mouse epidermal cells

Ultraviolet A (UVA) irradiation is a potential environmental stressor, which contributes to inflammation, photoaging, and carcinogenesis. UVA causes endoplasmic reticulum stress, hence phosphorylates the α subunit of eIF2. Meanwhile, UVA also induces expression of haem oxygenase-1 (HO-1) and nuclear factor erythroid-derived two related factor 2 (Nrf2) in human skin cells. In mouse JB6 cell, we found high dose UVA could change cell morphology, cause cell viability loss. UVA irradiation activated phosphorylation of eIF2α and Nrf2-HO-1 pathway in a dose-dependent manner. Besides, modulation of eIF2α phosphorylation status could alter expression pattern of Nrf2-HO-1 signalling. Salubrinal, a selective inhibitor of eIF2α dephosphorylation, increased the S phase in cell cycle of JB6 cells after UVA irradiation, suggesting phosphorylation status of eIF2α may affect cellular homeostasis under UVA irradiation. The study directed to further acknowledge about the relationship of UVA-induced eIF2α phosphorylation and Nrf2-HO-1 pathway, which may play a role in phototherapy and photo protection.

Red ginseng-derived saponin fraction suppresses the obesity-induced inflammatory responses via Nrf2-HO-1 pathway in adipocyte-macrophage co-culture system.

The aim of this study was to investigate the effect of saponin fraction (SF) from red ginseng on obesity-induced inflammatory response in a co-culture system of 3T3-L1 and RAW264.7 cells. HPLC analysis showed that SF contains more than 50% ginsenosides, and Rb1 was the most abundant ginsenoside [135.31 μg/mg (extract)]. The production of nitric oxide and cytokines, induced by adipocyte-conditioned medium (3T3-CM), was significantly decreased by SF. SF (100 μg/mL) suppressed the abundance of tumor necrosis factor-alpha (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and interleukin-6 (IL-6) by 78%, 40%, and 22%, respectively. This SF-mediated reduction in inflammatory cytokines was due to the suppression of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) phosphorylation, and translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) into the nucleus. SF also regulated adipokine expression in adipocytes, which were stimulated by macrophage-conditioned medium (RAW-CM); adiponectin expression was upregulated (> 2-fold), while resistin was downregulated (40%). In the contact system of adipocytes and macrophages, SF significantly decreases MCP-1 (37%) and IL-6 (25%) production. In the transwell system, SF (100 μg/mL) significantly increased the abundance of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and its target protein, hemoxygenase-1 (HO-1) by 1.5∼3.5-fold and 2.8∼3.6-fold, respectively, thus increasing Nrf2 translocation into nucleus. However, SF-mediated inhibitory effect on the release of IL-6 and MCP-1 cytokines was reversed in the Nrf2 or HO-1 knockdown condition. This result indicated that SF-mediated inhibition of obesity-induced inflammation was dependent on Nrf2 activation.

Effects of eicosapentaenoic acid intake on denervation-induced mitochondrial adaptation in mouse skeletal muscle

Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated fatty acid that is abundant in fish oil, and has anti-inflammatory or anti-obesity effects. However, the effects of EPA supplementation on mitochondrial content and dynamics (fusion and fission) in skeletal muscle has not been elucidated. We investigated the effects of EPA intake for 4 weeks on denervation-induced mitochondrial adaptation in mice skeletal muscle. ICR mice (male, 8 weeks old) were daily administrated olive oil (control) or EPA at a dose of 300 mg/kg body weight by gavage for 4 weeks. After 2 weeks of oil intake, mice underwent unilateral sciatic nerve transection surgery. The hindlimb without surgery served as the sham-operated control. Body and skeletal muscle weights did not differ between the control and the EPA groups. Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) and oxidative phosphorylation (OXPHOS) proteins were significantly decreased by denervation surgery. Denervation also induced the reduction of mitochondrial fusion molecules and the increase of mitochondrial fission molecules. Expression levels of PGC-1α and mitochondrial respiration protein MTCO1 were higher in the EPA group than in the control (olive oil) group. In addition, the EPA group contained higher levels of mitochondrial fusion protein OPA1. Our results suggest that EPA intake prevents the reduction of mitochondrial content and fusion proteins in denervated skeletal muscle.

Sodium selenite induces apoptosis via ROS-mediated NF-κB signaling and activation of the Bax-caspase-9-caspase-3 axis in 4T1 cells.

Sodium selenite (SSE), a source of inorganic selenium, has been widely used as a clinical cancer treatment, but the precise molecular mechanisms of SSE remain to be elucidated. Our in vitro experiments have confirmed that SSE treatment causes a transient increase in intracellular reactive oxygen species (ROS) levels, resulting in the inhibition of nuclear transcription factor-κB (NF-κB) signaling and p65 and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha phosphorylation levels in 4T1 cells. The inhibition of NF-κB subsequently increased the expression of the apoptosis gene B-cell lymphoma-2-associated X (Bax) and downregulated the transcription of antiapoptosis genes, such as B-cell lymphoma-2, cellular inhibitor of apoptosis 1, and X-linked inhibitor of apoptosis. Additionally, the accumulation of ROS caused mitochondrial dysfunction, leading to the activation of caspase-9 and -3, thereby resulting in apoptosis. However, modulation of the ROS level by the chemical inhibitor N-acetyl-cysteine reversed these events. Similarly, in vitro murine syngeneic breast tumor models showed that SSE inhibits tumor growth by promoting apoptosis. These results indicate that SSE induces apoptosis via ROS-mediated inhibition of NF-κB signaling and activation of the Bax-caspase-9-caspase-3 axis.

Panax notoginseng Saponins Regulate Macrophage Polarization under Hyperglycemic Condition via NF-κB Signaling Pathway.

Panax notoginseng saponins (PNS), the principal constituents derived from Panax notoginseng, have been extensively used for treating cardiocerebral vascular diseases in China and other Asian countries. The main effects of PNS were anti-inflammatory properties, inhibition of platelet aggregation, improvement of blood flow and insulin resistance, and so on. This study was carried out to explore the effects of PNS on macrophage polarization under hyperglycemic conditions. Human acute monocyte leukemia cell line THP-1 cells were induced into macrophages with Phorbol ester (PMA). Macrophages were then divided into five groups as follows: control (5.5mMol/l glucose), hyperglycemia group (15mMol/l glucose), hyperglycemia plus low-dose PNS (20ug/ml), hyperglycemia plus moderate-dose PNS (40ug/ml), and hyperglycemia plus high-dose PNS (60ug/ml). After 48-hour cell culture, the percentages of M1- and M2-polarized macrophages were measured by flow cytometry analysis. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was used to evaluate the Ym1 and arginase 1 expression in macrophages. Protein expression of arginase 1, NF-κB p50, p65, and inhibitor of κB (IκB) alpha phosphorylation in macrophages was identified with Western blotting. PNS, especially the high-dose PNS, remarkably increased M2 phenotype ratio in macrophages cultured with hyperglycemia, and the mRNA expression of Ym1 and arginase 1 in macrophages was also upregulated. Meanwhile, PNS remarkably increased the protein expression of arginase 1 and decreased IκB-alpha phosphorylation and subunits of NF-κB p50 and p65 from macrophages in culture medium with hyperglycemia. Taken together, our work demonstrated that PNS promote macrophages to transform M2 phenotype under hyperglycemic conditions through downregulating NF-κB signaling pathway.

Glycine Relieves Intestinal Injury by Maintaining mTOR Signaling and Suppressing AMPK, TLR4, and NOD Signaling in Weaned Piglets after Lipopolysaccharide Challenge

This study was conducted to envaluate whether glycine could alleviate Escherichia coli lipopolysaccharide (LPS)-induced intestinal injury by regulating intestinal epithelial energy status, protein synthesis, and inflammatory response via AMPK, mTOR, TLR4, and NOD signaling pathways. A total of 24 weanling piglets were randomly allotted to 1 of 4 treatments: (1) non-challenged control; (2) LPS-challenged control; (3) LPS + 1% glycine; (4) LPS + 2% glycine. After 28 days feeding, piglets were injected intraperitoneally with saline or LPS. The pigs were slaughtered and intestinal samples were collected at 4 h postinjection. The mRNA expression of key genes in these signaling pathways was measured by real-time PCR. The protein abundance was measured by Western blot analysis. Supplementation with glycine increased jejunal villus height/crypt depth ratio. Glycine also increased the jejunal and ileal protein content, RNA/DNA ratio, and jejunal protein/DNA ratio. The activities of citroyl synthetase in ileum, and α-ketoglutarate dehydrogenase complex in jejunum, were increased in the piglets fed diets supplemented with glycine. In addition, glycine decreased the jejunal and ileal phosphorylation of AMPKα, and increased ileal phosphorylation of mTOR. Furthermore, glycine downregulated the mRNA expression of key genes in inflammatory signaling. Meanwhile, glycine increased the mRNA expression of negative regulators of inflammatory signaling. These results indicate that glycine supplementation could improve energy status and protein synthesis by regulating AMPK and mTOR signaling pathways, and relieve inflammation by inhibiting of TLR4 and NOD signaling pathways to alleviate intestinal injury in LPS-challenged piglets.

Metformin Activates AMPK Signaling but Inhibits Mitophagy in the Mouse Heart

Metformin, a commonly prescribed first-line drug for the treatment of type 2 diabetes mellitus, has been shown to reduce cardiac injury under diabetic and other pathological conditions. However, the mechanisms that mediate the cardioprotective effects of metformin remain controversial. One prevailing hypothesis suggests that metformin activates AMPK-autophagy pathway which eliminates protein aggregates and damaged mitochondria, thus protecting the heart from injury. In the present study, we tested this hypothesis using both in vivo and in vitro models. FVB/N mice were administered 200 mg/kg of metformin through gavage once a day for two days. As expected, metformin activated AMPK signaling in the heart as shown by the increased phosphorylation of AMPK alpha at Thr172 and its downstream effecter Acetyl-CoA Carboxylase at Ser79. Autophagy and mitophagy were determined by Western blot analysis of LC3-II, a well-established marker of autophagic vesicles. Surprisingly, however, metformin did not affect LC3-II levels in total tissue lysates and significantly reduced LC3-II in mitochondrial fractions, suggesting that metformin actually inhibited rather stimulated mitophagy, contrary to the current belief. This was confirmed by measuring mitophagy flux using lysosomal inhibitors pepstatin A and E64d. The ability of metformin to inhibit mitophagy was also corroborated by a novel dual fluorescent mitophagy reporter showing reduced degradation rate of mitochondria in the lysosomes. All these results were reproduced in H9c2 rat cardiac myoblast cells. Collectively, our results show that metformin is able to activate AMPK signaling but paradoxically inhibits mitophagy in the heart, suggesting that the cardioprotective benefit of metformin observed in many different models may be mediated through mechanisms other than autophagy and mitophagy. Future studies will explore the mechanisms by which metformin inhibits mitophagy and protects the heart. Disclosure Y. Zhang: None. M.M. Chang: None. P.R. Pinkhasova: None. F. Zhao: None. T. Kobayashi: None. Y. Huang: None. S. Kobayashi: None. Q. Liang: None.

Selenium ameliorates Staphylococcus aureus-induced inflammation in bovine mammary epithelial cells by inhibiting activation of TLR2, NF-\u03baB and MAPK signaling pathways

BackgroundStaphylococcus aureus (S. aureus) internalization into bovine mammary epithelial cells (bMECs) is considered an important pathogenic mechanism for the establishment of mastitis. Given the interesting link between selenium (Se) status and mastitis, our objective was to prove that Se was essential to suppress pro-inflammatory mediators, in part, by modulation of Toll-like receptor2 (TLR2), nuclear factor kappaB (NF-κB) and mitogen activated protein kinase (MAPK) signal transduction pathway in bMECs.ResultsResults showed that Se (0~ 16 μM) did not affect the growth of bMECs. The mRNA expression of TLR2, Myeloid differentiation factor 88 (Myd88), Interleukin-1 receptor-associated kinase4 (Irak4), Interleukin-1 receptor-associated kinase1 (Irak1) and TNF receptor-associated factor6 (Traf6) in TLR2 signal pathway were increased or significantly increased by S. aureus. Se played an important role in regulating the genes expression of TLR2, Myd88, Traf6 but not in controlling the expression of Irak4 and Irak1. In addition, Se exerted strong inhibitory effects on the genes expression of tumor necrosis factor-alpha (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) induced by S. aureus. To further investigate the possible signaling mechanisms involved in the processes, we analyzed the role of MAPK and NF-κB signaling pathway in inflammation response in S. aureus-stimulated bMECs in vitro. Results showed that the phosphorylation of inhibitory kappaB alpha (IκBα), p65, p38 and extracellular regulated protein kinase (Erk) were significantly increased in S. aureus-stimulated bMECs. It indicated that S. aureus activated NF-κB and MAPK signaling pathway. We also examined the effects of Se on the phosphorylation of IκBα, p65, p38 and Erk in NF-κB and MAPK signaling pathway, which have well been proved to control the synthesis and release of pro-inflammatory mediators during inflammation. The findings are exciting, that pretreatment with Se (4, 8 μM) significantly suppressed the phosphorylation of IκBα, p65, p38 and Erk.ConclusionsThese results suggest that Se down-regulates inflammatory mediators TNF-α, IL-1β and IL-6 gene expressions via TLR2, NF-κB and MAPK signaling pathway in S. aureus-stimulated bMECs, which may be responsible for the anti-inflammatory effect of Se.

VS-5584 as a PI3K/mTOR inhibitor enhances apoptotic effects of subtoxic dose arsenic trioxide via inhibition of NF-κB activity in B cell precursor-acute lymphoblastic leukemia

Activation of the phosphoinositide 3-kinase (PI3K)/Akt/mTOR pathway as a survival signaling cascade is a prominent feature of cancers such as acute lymphoblastic leukemia (ALL). In patients with B-cell precursor-ALL (BCP-ALL), the high activity of the pathway correlates with the weak response to anti-leukemic drugs and relapse as a result of downstream prosurvival pathway activation, such as nuclear factor kappa B (NF-κB). Recent targeted therapy (PI3K/mTOR inhibitors) in combination with a multifunctional conventional chemotherapeutic drug may be useful for treatment of BCP-ALL patients. In the current study, the potential of a subtoxic dose (0.2 μM) of arsenic trioxide (ATO) in combination with VS-5584 (a highly potent PI3K/mTOR dual inhibitor) was tested for blocking of the PI3K/Akt/mTOR pathway, inhibition of NF-κB activation and induction of apoptosis and cell-cycle arrest. The data indicate that VS-5584 as a PI3K/mTOR inhibitor inhibited cell proliferation and induced apoptosis in NALM-6 cells by means of NF-κB transcriptional activity suppression. This apoptotic process markedly increased 72 h after administration of the subtoxic dose of ATO. We also showed that concomitant treatment of VS-5584 and the subtoxic dose of ATO significantly inhibited phosphorylation of NF-κB inhibitor alpha (IκBα) and S6 ribosomal protein (S6) as the downstream proteins of the PI3K/Akt/mTOR pathway. Combining VS-5584 and a subtoxic dose of ATO also resulted in down expression of the NF-κB target genes involved in cell proliferation and survival. These results indicate that incorporation of VS-5584/ATO combination into BCP-ALL therapeutic protocols can improve treatment and the survival of patients.

The protective autophagy activated by GANT-61 in MYCN amplified neuroblastoma cells is mediated by PERK.

The proto-oncogene MYC can trigger the unfolded protein response (UPR). The double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK), one of three primary branches of the UPR, is a key regulator of autophagy, promoting tumorigenesis. Upon activation of PERK, there is an increase in phosphorylation of the eukaryotic initiation factor-2 alpha (eIF2α), which in turn, activates the transcription factor-4 (ATF4), responsible for an increased expression of LC3, a common autophagy marker. PERK is repressed upon GLI1 and GLI2 induction. GANT-61 is an inhibitor of GLI1 and GLI2, known to reduce autophagy in MYCN non-amplified, but not in MYCN amplified neuroblastoma (NB) cells. In our study, we tested the effect of the joint administration of a PERK inhibitor (GSK2606414) and the GLI inhibitor GANT-61 to MYCN amplified and MYCN non-amplified NB cells. Our results suggest that inhibition of PERK impairs GANT-61 induced autophagy in NB cells with MYCN amplification, but had no effect on the MYCN non-amplified NB cells. In summary, PERK seems to be a good therapeutic target for NB. Inhibition of PERK reduces autophagy in MYCN amplified NB cells, thus amplifying the efficacy of the GLI inhibitor GANT-61 in reducing proliferation of this type of cancer cells.

Protein Disulfide Isomerase Silence Inhibits Inflammatory Functions of Macrophages by Suppressing Reactive Oxygen Species and NF-κB Pathway.

Macrophages play an essential role in inflammation. Protein disulfide isomerase (PDI) is central to the redox system, which is closely linked with the inflammatory function of macrophages. However, the relationship between PDI and inflammation is still unknown. In this study, we tested the effects of PDI on inflammatory responses in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS). Using CRISPR/Cas9 system, we found that PDI knockout suppressed migration, M1 polarization, and secretion of tumor necrosis factor-α (TNF-α) and interluekin-6 (IL-6). The repression of these inflammatory processes was accompanied by decreased production of reactive oxygen species (ROS). PDI ablation also inactivated the phosphorylation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and activated the phosphorylation of NF-κB inhibitor alpha (IκBα). These findings demonstrate that PDI knockout inhibits the inflammatory function of macrophages by decreasing ROS production and inactivating NF-κB pathway.

The role of cysteine string protein alpha phosphorylation at SER10 and SER34 by PKCGAMMA for the presynaptic maintenance

Background: Protein kinase C (PKC) has been implicated in the control of neuronal cell survival. PKCγ-KO animal models showed symptoms of parkinsonian syndrome, including the dopaminergic neuronal loss in the Substantia Nigra. However, the PKCγ substrates responsible for the dopaminergic neuronal cell survival in vivo have not yet been elucidated. We previously found 10 phosphoproteins including Cysteine string protein alpha (CSPα) that had decreased phosphorylation levels in the striatum of PKCγ-KO mice.

Activation of the unfolded protein response in sarcoma cells treated with rapamycin or temsirolimus.

Activation of the unfolded protein response (UPR) in eukaryotic cells represents an evolutionarily conserved response to physiological stress. Here, we report that the mTOR inhibitors rapamycin (sirolimus) and structurally related temsirolimus are capable of inducing UPR in sarcoma cells. However, this effect appears to be distinct from the classical role for these drugs as mTOR inhibitors. Instead, we detected these compounds to be associated with ribosomes isolated from treated cells. Specifically, temsirolimus treatment resulted in protection from chemical modification of several rRNA residues previously shown to bind rapamycin in prokaryotic cells. As an application for these findings, we demonstrate maximum tumor cell growth inhibition occurring only at doses which induce UPR and which have been shown to be safely achieved in human patients. These results are significant because they challenge the paradigm for the use of these drugs as anticancer agents and reveal a connection to UPR, a conserved biological response that has been implicated in tumor growth and response to therapy. As a result, eIF2 alpha phosphorylation and Xbp-1 splicing may serve as useful biomarkers of treatment response in future clinical trials using rapamycin and rapalogs.

Development of 1-((1,4-trans)-4-Aryloxycyclohexyl)-3-arylurea Activators of Heme-Regulated Inhibitor as Selective Activators of the Eukaryotic Initiation Factor 2 Alpha (eIF2α) Phosphorylation Arm of the Integrated Endoplasmic Reticulum Stress Response.

Heme-regulated inhibitor (HRI), an eukaryotic translation initiation factor 2 alpha (eIF2α) kinase, plays critical roles in cell proliferation, differentiation, adaptation to stress, and hemoglobin disorders. HRI phosphorylates eIF2α, which couples cellular signals, including endoplasmic reticulum (ER) stress, to translation. We previously identified 1,3-diarylureas and 1-((1,4-trans)-4-aryloxycyclohexyl)-3-arylureas (cHAUs) as specific activators of HRI that trigger the eIF2α phosphorylation arm of ER stress response as molecular probes for studying HRI biology and its potential as a druggable target. To develop drug-like cHAUs needed for in vivo studies, we undertook bioassay-guided structure-activity relationship studies and tested them in the surrogate eIF2α phosphorylation and cell proliferation assays. We further evaluated some of these cHAUs in endogenous eIF2α phosphorylation and in the expression of the transcription factor C/EBP homologous protein (CHOP) and its mRNA, demonstrating significantly improved solubility and/or potencies. These cHAUs are excellent candidates for lead optimization for development of investigational new drugs that potently and specifically activate HRI.

Sesn2 gene ablation enhances susceptibility to gentamicin-induced hair cell death via modulation of AMPK/mTOR signaling

The process of gentamicin-induced hair cell damage includes the activation of oxidative stress processes. Sestrins, as stress-responsive proteins, protect cells against oxidative stress. Sestrins, particularly Sestrin-2, suppress excessive reactive oxygen species (ROS) accumulation and inhibit mammalian target of rapamycin complex 1 (mTORC1). Thus, we addressed the role of Sestrin-2 in the regulation of sensory hair cell survival after gentamicin exposure. Here, we show that Sestrins were expressed in the inner ear tissues, and Sestrin-2 immunolocalized in sensory hair cells and spiral ganglion (SG). The expression of Sestrin-2 was unchanged, and later downregulated, in gentamicin-treated explants from wild-type mice in vitro. Compared with wild-type mice, Sestrin-2 knockout mice exhibited significantly greater hair cell loss in gentamicin-treated cochlear explants. Significant downregulation of phosphorylation of AMP-activated protein kinase alpha (AMPKα) and upregulation of the 70-kDa ribosomal protein S6 kinase (p70S6K) were measured in wild-type cochlear explants exposed to gentamicin compared with their untreated controls. Such regulatory effect was not observed between explants from untreated and gentamicin-treated knockout mice. The gentamicin effect on mTOR signaling was rapamycin-sensitive. Thus, our data provide evidence that Sestrin-2 plays an important role in the protection of hair cells against gentamicin, and the mTOR signaling pathway appears to be modulated by gentamicin during hair cell death.

Effects of hyperbaric oxygen therapy in enhancing expressions of e-NOS, TNF-α and VEGF in wound healing

Wound healing is a physiological process that occurs progressively through overlapping phases. Tissue oxygenation is an important part of the complex regulation for wound healing. Hyperbaric Oxygen (HBO) therapy is a method of increasing oxygen delivery to tissues. The therapy improves tissue oxygenation and stimulates the formation of H2O2 as a secondary messenger for Tumour Necrosis Factor alpha (TNF α), e-NOS, VEGF and Nuclear Factor Kappa Beta phosphorylation (NF-Kb) which play an important role in the rapid transcription of a wide variety of genes in response to extracellular stimuli. This study aims to determine the effects of Hyperbaric Oxygen therapy in enhancing the expressions of e-NOS, TNF-α, VEGF and wound healing. This study is an animal study with a ‘randomized control group of pre-test and post test design’ on 28 Wistar rats. Randomly, the rats were divided into 4 groups with 7 rats in each group. The HBO treatment group 1 received 5 sessions of HBO 2.4 ATA in 3 × 30 minutes; the HBO treatment group 2 received 10 sessions of HBO 2.4 ATA in 3 × 30 minutes; and each of the control groups were without HBO. Each of the 28 male rats were given a full thickness excisional wound of 1 × 1cm. Examinations of e-NOS, TNF-α, VEGF expressions and wound healing were performed on day-0 (pre-HBO) and day-5 HBO or on day-0 (pre-HBO) and day-10 HBO. The resultsshowthat the Hyperbaric Oxygen therapy can improve e-NOS (p=0.02), TNF-α (p= 0.02), VEGF expression (p=0.02) and wound healing (p=0.002) significantly in the provision of HBO 2.4 ATA for 3 × 30 minutes in 5 sessions over 5 consecutive days. While the 10 sessions of HBO 2.4 ATA for 3 × 30 minutes over 10 consecutive days only increase e-NOS (p=0.02), TNF-α (p=0.04), VEGF expression significantly (p=0.03) but do not improve wound healing significantly (p=0.3) compared with no HBO. The study concludes that HBO can improve the expressions of e-NOS, TNF-α, VEGF and wound healing in the provision of HBO 2.4 ATA for 3 × 30 minutes in 5 sessions, while the 10 sessions of HBO 2.4 ATA for 3 × 30 minutes only increase e-NOS, TNF-α, VEGF expression but do not improve wound healing.

Ponatinib promotes a G1 cell-cycle arrest of merlin/NF2-deficient human schwann cells.

Neurofibromatosis type 2 (NF2) is a genetic syndrome that predisposes individuals to multiple benign tumors of the central and peripheral nervous systems, including vestibular schwannomas. Currently, there are no FDA approved drug therapies for NF2. Loss of function of merlin encoded by the NF2 tumor suppressor gene leads to activation of multiple mitogenic signaling cascades, including platelet-derived growth factor receptor (PDGFR) and SRC in Schwann cells. The goal of this study was to determine whether ponatinib, an FDA-approved ABL/SRC inhibitor, reduced proliferation and/or survival of merlin-deficient human Schwann cells (HSC). Merlin-deficient HSC had higher levels of phosphorylated PDGFRα/β, and SRC than merlin-expressing HSC. A similar phosphorylation pattern was observed in phospho-protein arrays of human vestibular schwannoma samples compared to normal HSC. Ponatinib reduced merlin-deficient HSC viability in a dose-dependent manner by decreasing phosphorylation of PDGFRα/β, AKT, p70S6K, MEK1/2, ERK1/2 and STAT3. These changes were associated with decreased cyclin D1 and increased p27Kip1levels, leading to a G1 cell-cycle arrest as assessed by Western blotting and flow cytometry. Ponatinib did not modulate ABL, SRC, focal adhesion kinase (FAK), or paxillin phosphorylation levels. These results suggest that ponatinib is a potential therapeutic agent for NF2-associated schwannomas and warrants further in vivo investigation.

The Root Extract of Pueraria lobata and Its Main Compound, Puerarin, Prevent Obesity by Increasing the Energy Metabolism in Skeletal Muscle.

Radix Pueraria lobata (RP) has been reported to prevent obesity and improve glucose metabolism; however, the mechanism responsible for these effects has not been elucidated. The mechanism underlying anti-obesity effect of RP was investigated in high-fat diet (HFD) induced obese mice and skeletal muscle cells (C2C12). Five-week-old C5BL/6 mice were fed a HFD containing or not containing RP (100 or 300 mg/kg) or metformin (250 mg/kg) for 16 weeks. RP reduced body weight gain, lipid accumulation in liver, and adipocyte and blood lipid levels. In addition, RP dose-dependently improved hyperglycemia, insulinemia, and glucose tolerance, and prevented the skeletal muscle atrophy induced by HFD. Furthermore, RP increased the peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) expression and phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) in skeletal muscle tissues. RP and its main component, puerarin, increased mitochondrial biogenesis and myotube hypertrophy in C2C12 cells. The present study demonstrates that RP can prevent diet-induced obesity, glucose tolerance, and skeletal muscle atrophy in mouse models of obesity. The mechanism responsible for the effect of RP appears to be related to the upregulation of energy metabolism in skeletal muscle, which at the molecular level may be associated with PGC-1α and AMPK activation.

Emodin Increases Expression of Insulin-Like Growth Factor Binding Protein 1 through Activation of MEK/ERK/AMPKα and Interaction of PPARγ and Sp1 in Lung Cancer

Background: Emodin has anti-neoplastic activities on multiple tumors. However, the molecular mechanisms underlying this effect still remain to be fully understood. Methods: Cell viability and cell cycle distribution were measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays and flow cytometry, respectively. Cell invasion and migration were examined by transwell invasion and wound healing assays. Western blot analysis was performed to examine the phosphorylation and protein expression of AMP-activated protein kinase alpha (AMPKα), extracellular signaling-regulated kinase 1/2 (ERK1/2), peroxisome proliferators-activated receptor gamma (PPARγ), insulin-like growth factor (IGF) binding protein 1 (IGFBP1) and the transcription factor Sp1. QRT-PCR was used to examine the mRNA levels of the IGFBP1 gene. Small interfering RNAs (siRNAs) were used to knockdown PPARγ and IGFBP1 genes. Exogenously expression of IGFBP1 and Sp1 was determined by transient transfection assays. IGFBP1 promoter activity was measured by Secrete-Pair Dual Luminescence Assay Kit. In vivo nude mice xenograft model and bioluminescent imaging system were used to confirm the findings. Results: We showed that emodin induced cell cycle arrest of NSCLC cells. Emodin increased PPARγ protein and luciferase reporter activity, which were abolished by inhibitors of MAPK extracellular signaling-regulated kinase (ERK) kinase (MEK)/ERK and AMPK. Silencing of PPARγ abrogated emodin-inhibited cell growth and cell cycle arrest. Furthermore, emodin elevated IGFBP1 mRNA, protein, and promoter activity through activation of PPARγ. Intriguingly, overexpressed Sp1 attenuated emodin-induced IGFBP1 expression, which was not observed in cells with silenced PPARγ gene. Moreover, silencing of IGFBP1 gene blunted emodin-induced inhibition of cell growth and cell cycle arrest. On the contrary, overexpressed IGFBP1 enhanced emodin-induced phosphorylation of AMPKα and ERK1/2, and restored emodin-inhibited growth in cells with silenced endogenous IGFBP1 gene. Emodin also inhibited growth of lung xenograft tumors and Sp1, and increased IGFBP1 and PPARγ protein expressions In vivo. Conclusion: Collectively, our results show that emodin inhibits growth of non-small-cell lung cancer (NSCLC) cells through ERK and AMPKα-mediated induction of PPARγ, followed by reduction of Sp1. This in turn induces IGFBP1 gene expression. Thus, the signaling cascades, positive feedback loop and cooperative interplay between transcription factors-induced the expression of IGFBP1 gene contribute to the overall responses of emodin. This study provides a novel mechanism by which emodin inhibits growth of human lung cancer cells.

Phosphorylation of Translation Initiation Factor 2-Alpha in Leishmania donovani under Stress Is Necessary for Parasite Survival.

The transformation of Leishmania donovani from a promastigote to an amastigote during mammalian host infection displays the immense adaptability of the parasite to survival under stress. Induction of translation initiation factor 2-alpha (eIF2α) phosphorylation by stress-specific eIF2α kinases is the basic stress-perceiving signal in eukaryotes to counter stress. Here, we demonstrate that elevated temperature and acidic pH induce the phosphorylation of Leishmania donovani eIF2α (LdeIF2α). In vitro inhibition experiments suggest that interference of LdeIF2α phosphorylation under conditions of elevated temperature and acidic pH debilitates parasite differentiation and reduces parasite viability (P < 0.05). Furthermore, inhibition of LdeIF2α phosphorylation significantly reduced the infection rate (P < 0.05), emphasizing its deciding role in successful invasion and infection establishment. Notably, our findings suggested the phosphorylation of LdeIF2α under H2O2-induced oxidative stress. Inhibition of H2O2-induced LdeIF2α phosphorylation hampered antioxidant balance by impaired redox homeostasis gene expression, resulting in increased reactive oxygen species accumulation (P < 0.05) and finally leading to decreased parasite viability (P < 0.05). Interestingly, exposure to sodium antimony glucamate and amphotericin B induces LdeIF2α phosphorylation, indicating its possible contribution to protection against antileishmanial drugs in common use. Overall, the results strongly suggest that stress-induced LdeIF2α phosphorylation is a necessary event for the parasite life cycle under stressed conditions for survival.