Search

Hepatocellular carcinoma (HCC) is one of the leading global causes of cancer mortality. MicroRNAs (miRNAs) are small interfering RNAs that alleviate the levels of protein expression by suppressing translation, inducing mRNA cleavage, and promoting mRNA degradation. miR-122 is the most abundant miRNA in the liver and is responsible for several liver-specific functions, including metabolism, cellular growth and differentiation, and hepatitis virus replication. Recent studies have shown that aberrant regulation of miR-122 is a key factor contributing to the development of HCC. In this review, the signaling pathways and the molecular targets of miR-122 involved in the progression of HCC have been summarized, and the importance of miR-122 in therapy has been discussed. [ABSTRACT FROM AUTHOR]

Protein kinase C‐δ (PKCδ) is a diacylglycerol‐dependent, calcium‐independent novel PKC isoform that is engaged in various cell signaling pathways, such as cell proliferation, apoptosis, inflammation, and oxidative stress. In this study, we searched for proteins that bind PKCδ using a yeast two‐hybrid assay and identified murine arrest‐defective 1 (mARD1) as a binding partner. The interaction between PKCδ and mARD1 was confirmed by glutathione S‐transferase pull‐down and co‐immunoprecipitation assays. Furthermore, recombinant PKCδ phosphorylated full‐length mARD1 protein. The NetPhos online prediction tool suggested PKCδ phosphorylates Ser80, Ser108, and Ser114 residues of mARD1 with the highest probability. Based on these results, we synthesized peptides containing these sites and examined their phosphorylations using recombinant PKCδ. Autoradiography confirmed these sites were efficiently phosphorylated. Consequent mass spectrometry and peptide sequencing in combination with MALDI‐TOF MS/MS confirmed that Ser80 and Ser108 were major phosphorylation sites. The alanine mutations of Ser80 and Ser108 abolished the phosphorylation of mARD1 by PKCδ in 293T cells supporting these observations. In addition, kinase assays using various PKC isotypes showed that Ser80 of ARD1 was phosphorylated by PKCβI and PKCζ isotypes with the highest selectivity, while Ser108 and/or Ser114 were phosphorylated by PKCγ with activities comparable to that of the PKCδ isoform. Overall, these results suggest the possibility that PKCδ transduces signals by regulating phosphorylation of ARD1. [ABSTRACT FROM AUTHOR]

RhoA is a key player in actin cytoskeleton reorganization and exerts most of its effect through the RhoA-ROCKs signaling pathway. Although recent studies have stressed the roles of ROCKs as regulators of glucose metabolism, little is known of the roles played by RhoA, the upstream regulators of ROCKs and other isotypes of Rho small-GTPases. This study was undertaken to determine whether Rho isotypes modulate glucose transport and insulin signaling in insulin-sensitive cell models, that is, 3T3-L1 adipocytes and L6 myoblasts. Glucose uptake assays showed that RhoA knockdown using siRNA reduced insulin-stimulated glucose transport in both cell types, whereas knockdown of RhoB or RhoC did not. Furthermore, RhoA overexpression increased insulin-stimulated glucose transport. Interestingly, the insulin-stimulated PI3K-Akt signaling pathway was unaffected under RhoA-depleted or -overexpressed conditions, which suggested RhoA might regulate glucose transport via an Akt-independent pathway. Interestingly, an immunoblot assay of signaling molecules related to actin-myosin cytoskeletal remodeling showed that unlike RhoA or RhoC, RhoA regulated ERM phosphorylation. Our results suggest that RhoA, but not RhoB or RhoC, mediates glucose transport by regulating the vesicle trafficking machinery in an Akt-independent manner. Image 1 • RhoA coordinates insulin-stimulated glucose transport adipocytes and myoblasts whereas RhoB or RhoC does not. • RhoA contributes to glucose transport by influencing ERM cytoskeleton-modulating proteins. • PI3K-Akt signaling pathway is not involved in RhoA-mediated glucose transport in adipocytes and myoblasts. [ABSTRACT FROM AUTHOR]

Abstract: ARD1 protein is a mammalian gene product homologous to a yeast Ard1p (Arrest defective 1 protein) acetyltransferase. Although two alternative splicing products of ARD1, ARD1235 and ARD1225, were reported in mouse, only ARD1235 orthologue was reported in humans. Here we show that ARD1225 is not expressed in humans, suggesting that factors regulating alternative splicing of ARD1 may have evolved differently between species. In human cells, hARD1235 is shown to be present in both nucleus and cytoplasm. However, in mouse cells, mARD1235 and mARD1225 proteins are localized to the nucleus and cytoplasm, respectively. Moreover, during apoptosis, ARD1235 and ARD1225 isoforms are destabilized by different mechanisms in a species-specific manner and dependent on destabilizing reagents. These results indicate that ARD1235 and ARD1225 isoforms may have different activities and function in different subcellular compartments of mammalian cells. [Copyright &y& Elsevier]

Abstract: Cyclin-dependent kinase 2 (Cdk2) activity is thought to be involved in cell death-associated chromatin condensation and other manifestations of apoptotic death. Here we show that during TNFα-induced apoptosis, PKCδ is activated in a caspase-3-dependent manner and phosphorylates p21WAF1/CIP1, a specific cyclin-dependent kinase inhibitor, on 146Ser. This residue is located near a cyclin-binding motif (Cy2) that plays an important role in the interaction between p21WAF1/CIP1 and Cdk2, and its phosphorylation modulates the ability of p21WAF1/CIP1 to associate with Cdk2. The phosphorylation of p21WAF1/CIP1 is temporally related to the activation kinetics of Cdk2 activity during the apoptosis. We propose that during TNFα-induced apoptosis, PKCδ-mediated phosphorylation of p21WAF1/CIP1 at 146Ser attenuates the Cdk2 binding of p21WAF1/CIP1 and thereby upregulates Cdk2 activity. [Copyright &y& Elsevier]

Abstract: Ginsenoside-Rh2 (G-Rh2) has been shown to induce apoptosis in a variety of cell types. In this study, we show that G-Rh2-induced apoptosis is accompanied by the mitochondrial release of cytochrome c and activation of caspase-3 in the human hepatoma cell line, SK-HEP-1. Furthermore, protein kinase C delta (PKCδ) activity was markedly up-regulated in a lipid activator-independent manner with kinetics similar to those of PKCδ and PARP cleavages during the apoptotic progression. Pre-treatment of cells with the caspase-3 specific inhibitor (z-DEVD-fmk) effectively prevented the G-Rh2-induced proteolytic activation of PKCδ. Moreover, rottlerin, a specific PKCδ inhibitor blocked G-Rh2-induced proapoptotic effects on the cells including the release of cytochrome c, activation of caspase-3 activity, and proteolytic cleavage and activation of PKCδ. These results suggest that G-Rh2-induced apoptosis is functionally linked to mitochondrial dysfunction and caspase-3 activity is regulated by positive feedback with PKCδ via the mitochondrial pathway. [Copyright &y& Elsevier]

Here we show that the PEP8-TAT2 peptide is effectively transduced into HeLa cells and that it inhibits cellular cyclin-Cdk2 activity. Like the PEP8 peptide, the PEP8-TAT2 peptide inhibits Cdk2 activity in vitro with an IC50 value of 5 nM, as determined by an immuno-complex kinase assay. It also inhibits DNA synthesis in and proliferation of cultured HeLa cells by arresting cell cycle at the G1/S transition. Further, the PEP8-TAT2 peptide inhibits cell death-associated Cdk2 activity and thereby prevents apoptotic progression in paclitaxel-treated cells. We propose that this inhibitor peptide is an effective agent to suppress the proliferation of human cancer cells, as well as apoptotic progression, by blocking cellular cyclin-dependent Cdk kinase activity. [Copyright &y& Elsevier]

We provide here evidence that c-Jun N-terminal protein kinase 1 (JNK1) activity is differentially up-regulated during apoptosis of SK-HEP-1 cells after treatment with ginsenoside Rh2 (G-Rh2). The G-Rh2-mediated JNK1 activation that occurred for the first 10–30 min was associated with SEK1 activity, but thereafter, the sustained activation was associated not with SEK1 activity, but with proteolytic cleavage of JNK1-associated p21WAF1/CIP1. Supporting this is that the expression of the dominant negative SEK1 mutant effectively blocked the early JNK1 activation phase but did not alter the sustained activation phase or apoptosis. Furthermore, expression of p21D112N, an uncleavable mutant of p21WAF1/CIP1, suppressed the later JNK1 activation. Moreover, the stable overexpression of ectopic JNK1 suppressed apoptosis while expression of the dominant negative JNK1 mutant promoted it. We propose that the early SEK1-associated JNK1 activation phase acts to prolong cell survival in response to apoptosis-inducing agents, thereby serving as an intervening checkpoint prior to the commitment to apoptosis. [Copyright &y& Elsevier]

KD025, a ROCK2 isoform-specific inhibitor, has an anti-adipogenic activity which is not mediated by ROCK2 inhibition. To identify the target, we searched binding targets of KD025 by using the KINOMEscanTM screening platform, and we identified casein kinase 2 (CK2) as a novel target. KD025 showed comparable binding affinity to CK2α (Kd = 128 nM). By contrast, CK2 inhibitor CX-4945 and ROCK inhibitor fasudil did not show such cross-reactivity. In addition, KD025 effectively inhibited CK2 at a nanomolar concentration (IC50 = 50 nM). We examined if the inhibitory effect of KD025 on adipocyte differentiation is through the inhibition of CK2. Both CX-4945 and KD025 suppressed the generation of lipid droplets and the expression of proadipogenic genes Pparg and Cebpa in 3T3-L1 cells during adipocyte differentiation. Fasudil exerted no significant effect on the quantity of lipid droplets, but another ROCK inhibitor Y-27632 increased the expression of Pparg and Cebpa. Both CX-4945 and KD025 acted specifically in the middle stage (days 1–3) but were ineffective when treated at days 0–1 or the late stages, indicating that CX-4945 and KD025 may regulate the same target, CK2. The mRNA and protein levels of CK2α and CK2β generally decreased in 3T3-L1 cells at day 2 but recovered thereafter. Other well-known CK2 inhibitors DMAT and quinalizarin inhibited effectively the differentiation of 3T3-L1 cells. Taken together, the results of this study confirmed that KD025 inhibits ROCK2 and CK2, and that the inhibitory effect on adipocyte differentiation is through the inhibition of CK2. [ABSTRACT FROM AUTHOR]

• KD025 is a ROCK2 selective inhibitor under clinical trials. • GSE8686 microarray dataset was analyzed to discover novel KD025's roles. • DEG analysis indicated the implication of KD025 in neutrophil biology. • GSEA and hierarchical heatmap analysis extracted enriched functions of KD025. • Unreported novel roles of KD025 were efficiently discovered. Rho-associated protein kinases (ROCKs) have various cellular functions, which include actin cytoskeleton remodeling and vesicular trafficking, and there are two major mammalian ROCK isotypes, namely, ROCK1 (ROKβ) and ROCK2 (ROKα). The ROCK2-specific inhibitor KD025 (SLx-2119) is currently undergoing phase II clinical trials, but its cellular functions have not been fully explored. In this study, we investigated the functions of KD025 at the genomics level by bioinformatics analysis using the GSE8686 microarray dataset from the NCBI GEO database, in three different primary human cell lines. An initial microarray analysis conducted by Boerma et al. focused on the effects of KD025 on cell adhesion and blood coagulation, but did not provide comprehensive information on the functions of KD025. Our analysis of differentially expressed genes (DEGs) showed ~70% coincidence with Boerma et al.'s findings, and newly identified that CCND1, CXCL2, NT5E , and SMOX were differentially expressed by KD025. However, due to low numbers of co-regulated DEGs, we were unable to extract the functions of KD025 with significance. To overcome this limitation, we used gene set enrichment analysis (GSEA) and the heatmap hierarchical clustering method. We confirmed KD025 regulated inflammation and adipogenesis pathways, as previously reported experimentally. In addition, we found KD025 has novel regulatory functions on various pathways, including oxidative phosphorylation, WNT signaling, angiogenesis, and KRAS signaling. Further studies are required to systematically characterize these newly identified functions of KD025. [ABSTRACT FROM AUTHOR]

Depression is twice as prevalent in women as in men, however, most preclinical studies of depression have used male rodent models. This study aimed to examine how stress affects metabolic profiles depending on sex using a rodent depression model: sub-chronic variable stress (SCVS). The SCVS model of male and female mice was established in discovery and validation sets. The stress-induced behavioral phenotypic changes were similar in both sexes, however, the metabolic profiles of female plasma and brain became substantially different after stress, whereas those of males did not. Four stress-differential plasma metabolites—β-hydroxybutyric acid (BHB), L-serine, glycerol, and myo-inositol—could yield biomarker panels with excellent performance to discern the stressed individuals only for females. Disturbances in BHB, glucose, 1,5-anhydrosorbitol, lactic acid, and several fatty acids in the plasma of stressed females implied a systemic metabolic shift to β-oxidation in females. The plasma levels of BHB and corticosterone only in stressed females were observed not only in SCVS but also in an acute stress model. These results collectively suggest a sex difference in the metabolic responses by stress, possibly involving the energy metabolism shift to β-oxidation and the HPA axis dysregulation in females. [ABSTRACT FROM AUTHOR]

Disuse muscle atrophy is characterized by a decrease in muscle mass and strength and an increase in glycolytic muscle fiber type. Although Schisandra chinensis extract has beneficial effects on muscle atrophy induced by various conditions (e.g., dexamethasone and aging), the effect of gomisin G, a lignan component of S. chinensis , on disuse muscle atrophy is unclear. Here, we induced disuse muscle atrophy through wire immobilization of the hind legs in mice followed by the oral administration of gomisin G. The cross-sectional area and muscle strength in disuse muscle atrophic mice were increased by gomisin G; however, the total muscle mass did not increase. Gomisin G decreased the expression of muscle atrophic factors (myostatin, atrogin-1, and MuRF1) but increased the expression of protein synthesis factors (mTOR and 4E-BP1). In H 2 O 2 -treated C2C12 myotubes, the level of puromycin incorporation (as a marker of protein synthesis) gradually increased in a dose-dependent manner by gomisin G. Furthermore, gomisin G induced a muscle fiber switch from fast-type glycolytic fibers (type 2B) to slow-type oxidative fibers (type I, 2A) in the gastrocnemius (GA) muscle as proved a decrease in the expression of TnI-FS and an increase in the expression of TnI-SS. Gomisin G increased mitochondrial DNA content and ATP levels in the GA muscle and COX activity in H 2 O 2 -treated C2C12 myotubes, improving mitochondrial function. Mechanistically, mitochondrial biogenesis is regulated by gomisin G via the Sirt 1/PGC-1α signaling pathway, targeting NRF1 and TFAM. These data suggest that gomisin G has a potential therapeutic effect on disuse muscle atrophy. [Display omitted] • Gomisin G improves grip strength in disuse muscle atrophic mice. • Gomisin G increases muscle synthesis rate in H2O2-treated C2C12 myotubes. • Gomisin G increases slow-type oxidative fiber contents in muscle of disuse mice. • Gomisin G induces mitochondrial biogenesis via the Sirt 1/PGC-1α signaling pathway. [ABSTRACT FROM AUTHOR]

Naïve and primed pluripotent stem cells recapitulate the peri- and post-implantation development, respectively. Thus, investigation of distinct traits between each pluripotent stem cell type would shed light on early embryonic processes. Herein, by screening a fluorescent probe library, we found that intracellular glycogen led to specific reactivity to CDg4, a glycogen fluorescence sensor, in both human and mouse naïve embryonic stem cells (ESCs). The requirement of constant inhibition of Gsk3β as well as high oxidative phosphorylation (OxPHOS) in naïve compared to primed ESCs was closely associated to high level of intracellular glycogen in naïve ESCs. Both capacity of OxPHOS and stored glycogen, rescued naïve ESCs by transient inhibition of glycolysis, which selectively eliminated primed ESCs. Additionally, naïve ESCs with active OxPHOS were enriched from a mixture with primed ESCs by high reactivity to ATP-Red1, a mitochondrial ATP fluorescence probe. These results indicate the active OxPHOS and high intracellular glycogen as a novel "biomarker" delineating metabolic remodeling during the transition of naïve pluripotency. • High intracellular glycogen in naïve ESCs upon Gsk3β inhibition. • Live isolation of naïve ESCs from specificity of CDg4 fluorescence probe. • Live isolation of naïve ESCs from ATP-Red1 due to high OxPHOS. • Tolerance of naïve ESCs to temporal inhibition of glycolysis due to distinct glucose metabolism. [ABSTRACT FROM AUTHOR]

Previous observations demonstrated that cimetidine decreased the clearance of procainamide (PA) and/or N-acetylprocainamide (NAPA; the primary metabolite of PA) resulting in the increased systemic exposure and the decrease of urinary excretion. Despite an abundance of in vitro and in vivo data regarding pharmacokinetic interactions between PA/NAPA and cimetidine, however, a mechanistic approach to elucidate these interactions has not been reported yet. The primary objective of this study was to construct a physiological model that describes pharmacokinetic interactions between PA/NAPA and cimetidine, an inhibitor of rat organic cation transporter 2 (rOCT2) and rat multidrug and toxin extrusion proteins (rMATE1), by performing extensive in vivo and in vitro pharmacokinetic studies for PA and NAPA performed in the absence or presence of cimetidine in rats. When a single intravenous injection of PA HCl (10 mg/kg) was administered to rats, co-administration of cimetidine (100 mg/kg) significantly increased systemic exposure and decreased the systemic (CL) and renal (CLR) clearance of PA, and reduced its tissue distribution. Similarly, cimetidine significantly decreased the CLR of NAPA formed by the metabolism of PA and increased the AUC of NAPA. Considering that these drugs could share similar renal secretory pathways (e.g., via rOCT2 and rMATE1), a physiologically-based pharmacokinetic (PBPK) model incorporating semi-mechanistic kidney compartments was devised to predict drug-drug interactions (DDIs). Using our proposed PBPK model, DDIs between PA/NAPA and cimetidine were successfully predicted for the plasma concentrations and urinary excretion profiles of PA and NAPA observed in rats. Moreover, sensitivity analyses of the pharmacokinetics of PA and NAPA showed the inhibitory effects of cimetidine via rMATE1 were probably important for the renal elimination of PA and NAPA in rats. The proposed PBPK model may be useful for understanding the mechanisms of interactions between PA/NAPA and cimetidine in vivo. [ABSTRACT FROM AUTHOR]

Rho-associated kinases (ROCKs) have been reported to antagonize adipocyte differentiation, and inhibition of ROCKs by small molecules promotes adipogenesis. Surprisingly, our recent study revealed that the ROCK2-specific inhibitor KD025 (SLx-2119), suppresses differentiation at the intermediate stage in 3T3-L1 preadipocytes. To address whether the anti-adipogenic activity of KD025 is a generalizable property, we examined the effect of KD025 in human adipose-derived stem cells (hADSCs). KD025 significantly suppressed the adipocyte differentiation of hADSCs with downregulation of the protein and mRNA expression of various adipogenic and lipogenic markers, including PPARγ, C/EBPα, SREBP-1c, Glut4 and FABP4. Notably, we observed that adipocyte differentiation is effectively suppressed by exposure to KD025 during the mid-to-late period of adipogenesis but not at the earlier stages, showing stage-specificity. Contrary to expectations, KD025 upregulated the insulin signaling, as confirmed by the increased phosphorylation levels of Akt and GSK-3α/β, and the differentiation-promoting activity of insulin signaling was observed to be overwhelmed by the inhibitory activity. In addition, we observed that other ROCK inhibitors (Y-27632, fasudil, and H-1152P) did not suppress but promoted adipocyte differentiation. These results indicate that KD025 suppresses adipocyte differentiation by modulation of key factors activated at the intermediate stage of differentiation, and not by inhibition of ROCK2. [ABSTRACT FROM AUTHOR]

In compliance with green chemistry, metallic nanoparticles can be biogenically synthesized using plant extracts. Herein, three different deep eutectic solvents (DESs) were used instead of water to prepare green tea extracts (GTEs). The resultant DES-based GTEs exerted different beneficial effects on the biosynthesis of silver nanoparticles. Compared with the water-based GTE (W-GTE), the DES-based GTEs contained higher levels of catechins (up to 235% more) that are natural reducing and capping agents and showed higher synthesis efficiency. In particular, two DESs, which consist of glycerol and betaine with a common urea molecule and are designated as GU and BU, respectively, could produce silver nanoparticles with superior properties to water. Specifically, GU- and BU-GTE-based silver nanoparticles showed uniformly reduced sizes, 39.12 (±5.33) nm and 43.11 (±6.42) nm, respectively and homogenous face-centered cubic crystallinity. DESs added to W-GTEs lead to synthesis of silver nanoparticles with improved dispersion. The DES-GTE-based silver nanoparticles at 60 μg/mL displayed in vitro anti-cancer activity comparable to doxorubicin at 12.5 μg/mL. These results suggest that the tested DESs could serve as efficient extraction solvents for phytochemicals as well as effective surface modifiers without posing adverse effects for subsequent applications of the synthesized nanoparticles. The favorable effects of GU and BU were found to partially involve ammonia, which was formed during DES preparation at high temperatures (≥80 °C). This study shows that DESs can promote the biogenic synthesis of silver nanoparticles by playing diverse roles depending on the unique properties of the DESs and their constituents. • DES-based green tea extracts were used to synthesize AgNPs for the first time. • DESs exert constructive effects on synthesis efficiency, particle size, and dispersion. • DESs can efficiently extract phytochemicals (reducing and end capping agents). • DESs can also serve as effective surface modifiers of AgNPs. • Ammonia unintentionally produced in DESs can affect the AgNP biosynthesis. [ABSTRACT FROM AUTHOR]

In compliance with green chemistry, metallic nanoparticles can be biogenically synthesized using plant extracts. Herein, three different deep eutectic solvents (DESs) were used instead of water to prepare green tea extracts (GTEs). The resultant DES-based GTEs exerted different beneficial effects on the biosynthesis of silver nanoparticles. Compared with the water-based GTE (W-GTE), the DES-based GTEs contained higher levels of catechins (up to 235% more) that are natural reducing and capping agents and showed higher synthesis efficiency. In particular, two DESs, which consist of glycerol and betaine with a common urea molecule and are designated as GU and BU, respectively, could produce silver nanoparticles with superior properties to water. Specifically, GU- and BU-GTE-based silver nanoparticles showed uniformly reduced sizes, 39.12 (±5.33) nm and 43.11 (±6.42) nm, respectively and homogenous face-centered cubic crystallinity. DESs added to W-GTEs lead to synthesis of silver nanoparticles with improved dispersion. The DES-GTE-based silver nanoparticles at 60 μg/mL displayed in vitro anti-cancer activity comparable to doxorubicin at 12.5 μg/mL. These results suggest that the tested DESs could serve as efficient extraction solvents for phytochemicals as well as effective surface modifiers without posing adverse effects for subsequent applications of the synthesized nanoparticles. The favorable effects of GU and BU were found to partially involve ammonia, which was formed during DES preparation at high temperatures (≥80 °C). This study shows that DESs can promote the biogenic synthesis of silver nanoparticles by playing diverse roles depending on the unique properties of the DESs and their constituents. • DES-based green tea extracts were used to synthesize AgNPs for the first time. • DESs exert constructive effects on synthesis efficiency, particle size, and dispersion. • DESs can efficiently extract phytochemicals (reducing and end capping agents). • DESs can also serve as effective surface modifiers of AgNPs. • Ammonia unintentionally produced in DESs can affect the AgNP biosynthesis. [ABSTRACT FROM AUTHOR]

Cordyceps militaris is a medicinal mushroom and its bioactive compound, cordycepin, is reported to have many pharmacological activities. The goal of this study was to investigate the effects of C. militaris extract (CME) and cordycepin on osteoclast differentiation in vitro and on an inflammatory bone loss in vivo. In RAW 264.7 cells, CME and cordycepin showed dose-dependent inhibition of receptor activator of the nuclear factor kappa B (NF- κB) ligand (RANKL)-induced osteoclast differentiation by TRAP (tartrate-resistant acid phosphatase) staining. Moreover, the mRNA expression of osteoclastogenesis-related genes (TRAP, cathepsin K, MMP-9, and NFATc1) was also inhibited by CME and cordycepin. Also, cordycepin significantly inhibited RANKL-induced phosphorylation of p38 and NF- κB, but not that of other members of mitogen-activated protein kinase families. To examine the effect of CME on bone loss in vivo, we used a mouse model of lipopolysaccharide (LPS)-mediated bone loss. Micro-CT analysis of the femurs showed that LPS treatment caused bone loss. However, bone loss was significantly attenuated in mice treated with CME. These results suggest that cordycepin or/and CME have inhibitory effects on osteoclast differentiation in vitro and that they suppress inflammatory bone loss in vivo. [ABSTRACT FROM AUTHOR]

Paradols are non-pungent and biotransformed metabolites of shogaols and reduce inflammatory responses as well as oxidative stress as shogaols. Recently, shogaol has been noted to possess therapeutic potential against several central nervous system (CNS) disorders, including cerebral ischemia, by reducing neuroinflammation in microglia. Therefore, paradol could be used to improve neuroinflammation-associated CNS disorders. Here, we synthesized paradol derivatives (2- to 10-paradols). Through the initial screening for anti-inflammatory activities using lipopolysaccharide (LPS)-stimulated BV2 microglia, 6-paradol was chosen to be the most effective compound without cytotoxicity. Pretreatment with 6-paradol reduced neuroinflammatory responses in LPS-stimulated BV2 microglia by a concentration-dependent manner, which includes reduced NO production by inhibiting iNOS upregulation and lowered secretion of proinflammatory cytokines (IL-6 and TNF-α). To pursue whether the beneficial in vitro effects of 6-paradol leads towards in vivo therapeutic effects on transient focal cerebral ischemia characterized by neuroinflammation, we employed middle cerebral artery occlusion (MCAO)/reperfusion (M/R). Administration of 6-paradol immediately after reperfusion significantly reduced brain damage in M/R-challenged mice as assessed by brain infarction, neurological deficit, and neural cell survival and death. Furthermore, as observed in cultured microglia, 6-paradol administration markedly reduced neuroinflammation in M/R-challenged brains by attenuating microglial activation and reducing the number of cells expressing iNOS and TNF-α, both of which are known to be produced in microglia following M/R challenge. Collectively, this study provides evidences that 6-paradol effectively protects brain after cerebral ischemia, likely by attenuating neuroinflammation in microglia, suggesting it as a potential therapeutic agent to treat cerebral ischemia. [ABSTRACT FROM AUTHOR]

Abstract: AKAP12 (A-Kinase anchoring protein 12) is a protein kinase C substrate and a potential tumor suppressor. AKAP12 is down-regulated by several oncogenes and strongly suppressed in various cancers including prostate, ovarian and breast cancers. AKAP12 acts as a regulator of mitogenesis by anchoring key signal proteins such as PKA, PKC, and cyclins. In this study, AKAP12 was found to suppress tumor cell viability by inducing apoptosis via caspase-3 in HT1080 cells. This AKAP12-induced apoptosis was associated with a decreased expression of Bcl-2 and increased expression of Bax. Moreover, AKAP12-transfectant strongly induced the expression of Cip1/p21 and Kip1/p27, but resulted in a decrease in cyclin D1 involved in G1 progression. Accordingly, these results suggest that AKAP12 may play an important role in tumor growth suppression by inducing apoptosis with the regulation of multiple molecules in the cell cycle progression. [Copyright &y& Elsevier]

We show that caspase-3 cleaves Cdc6 at D290/S and D442/G sites, producing p32-tCdc6 (truncated Cdc6) and p49-tCdc6, respectively, during etoposide- or tumor necrosis factor (TNF)-α-induced apoptosis. The expression of these tCdc6 proteins, p32- and p49-tCdc6, promotes etoposide-induced apoptosis. The expression of tCdc6 perturbs the loading of Mcm2 but not Orc2 onto chromatin and activates ataxia telangiectasia mutated (ATM) and ATM and Rad-3 related (ATR) kinase activities with kinetics similar to that of the phosphorylation of Chk1/2. The activation kinetics are consistent with elevated cellular levels of p53 and mitochondrial levels of Bax. The tCdc6-induced effects are all suppressed to control levels by expressing a Cdc6 mutant that cannot be cleaved by caspase-3 (Cdc6-UM). Cdc6-UM expression attenuates the TNF-α-induced activation of ATM and caspase-3 activities. When ATM or ATR is down-expressed by using the small interfering RNA technique, the TNF-α- or tCdc6-induced activation of caspase-3 activities is suppressed in the cells. These results suggest that tCdc6 proteins act as dominant-negative inhibitors of replication initiation and that they disrupt chromatin structure and/or induce DNA damage, leading to the activation of ATM/ATR kinase activation and p53-Bax-mediated apoptosis. [ABSTRACT FROM AUTHOR]

Abstract: Mouse ARD1 (mARD1) has been reported to negatively regulate the hypoxia-inducible factor 1α (HIF-1α) protein by acetylating a lysine residue and enhancing HIF-1α ubiquitination and degradation. However, it was recently reported that human ARD1 (hARD1) does not affect HIF-1α stability. To further explore the activities of the two orthologs, three mouse (mARD1198, mARD1225, mARD1235) and two human (hARD1131, hARD1235) variants were identified and characterized. Among these, mARD1225 was previously reported as a novel negative regulator of HIF-1α. Amino acid sequence analysis showed that the C-terminal region (aa 158–225) of mARD1225 completely differs from those of mouse and human ARD1235, although all three proteins share a well-conserved N-acetyltransferase domain (aa 45–130). The effects of ARD1 variants were evaluated with respect to HIF-1α stability and acetylation activity. Interestingly, mARD1225 strongly decreased the level of HIF-1α and increased the extent of acetylation, whereas mARD1235 and hARD1235 variants had a much weaker effect on HIF-1α stability and acetylation. These results suggest that ARD1 variants might have different effects on HIF-1α stability and acetylation, which may reflect diverse biological functions that remain to be determined. [Copyright &y& Elsevier]

KD025 (SLx-2119), the first specific Rho-associated protein kinase 2 (ROCK2) inhibitor, is a potential new drug candidate currently undergoing several phase 2 clinical trials for psoriasis, idiopathic pulmonary fibrosis, chronic graft-versus-host disease, and systemic sclerosis. In this study, a bio-analytical method was developed and fully validated for the quantification of KD025 in rat plasma and for application in pharmacokinetic studies. KD025 and GSK429286A (the internal standard) in rat plasma samples were analyzed by high-performance liquid chromatography-tandem mass spectrometry with m/z transition values of 453.10 → 366.10 and 433.00 → 178.00, respectively. The method was fully validated according to the United State Food and Drug Administration guidelines in terms of selectivity, linearity, accuracy, precision, sensitivity, matrix effects, extraction recovery, and stability. The method enabled the quantification of KD025 levels in rat plasma following oral administration of 5 mg/kg KD025 and intravenous administration of 2 mg/kg KD025 to rats, respectively. Our findings suggest that the developed method is practical and reliable for pharmacokinetic studies of KD025 in preclinical animals. [ABSTRACT FROM AUTHOR]

Metabolomics was applied to a C57BL/6N mouse model of chronic unpredictable mild stress (CMS). Such mice were treated with two antidepressants from different categories: fluoxetine and imipramine. Metabolic profiling of the hippocampus was performed using gas chromatography-mass spectrometry analysis on samples prepared under optimized conditions, followed by principal component analysis, partial least squares-discriminant analysis, and pair-wise orthogonal projections to latent structures discriminant analyses. Body weight measurement and behavior tests including an open field test and the forced swimming test were completed with the mice as a measure of the phenotypes of depression and antidepressive effects. As a result, 23 metabolites that had been differentially expressed among the control, CMS, and antidepressant-treated groups demonstrated that amino acid metabolism, energy metabolism, adenosine receptors, and neurotransmitters are commonly perturbed by drug treatment. Potential predictive markers for treatment effect were identified: myo-inositol for fluoxetine and lysine and oleic acid for imipramine. Collectively, the current study provides insights into the molecular mechanisms of the antidepressant effects of two widely used medications. [ABSTRACT FROM AUTHOR]

Chronic inflammation is fundamental for the induction of insulin resistance in the muscle tissue of vertebrates. Although several miRNAs are thought to be involved in the development of insulin resistance, the role of miRNAs in the association between inflammation and insulin resistance in muscle tissue is poorly understood. Herein, we investigated the aberrant expression of miRNAs by conducting miRNA microarray analysis of TNF-α-treated mouse C2C12 myotubes. We identified two miRNAs that were upregulated and six that were downregulated by a >1.5-fold change compared to normal cells. Among the findings, qRT-PCR analysis confirmed that miR-494 is consistently upregulated by TNF-α-induced inflammation. Overexpression of miR-494 in CHOIR/IRS1 and C2C12 myoblasts suppressed insulin action by down-regulating phosphorylations of GSK-3α/β, AS160 and p70S6K, downstream of Akt. Moreover, overexpression of miR-494 did not regulate TNF-α-mediated inflammation . Among genes bearing the seed site for miR-494, RT-PCR analysis showed that the expression of Stxbp5, an inhibitor of glucose transport, was downregulated following miR-494 inhibition. In contrast, the expression of PTEN decreased in the cells analyzed, thus showing that both positive and negative regulators of insulin action may be simultaneously controlled by miR-494. To investigate the overall effect of miR-494 on insulin signaling, we performed a PCR array analysis containing 84 genes related to the insulin signaling pathway, and we observed that 25% of genes were downregulated (P<0.05) and 11% were upregulated (P<0.05). These results confirm that miR-494 might contribute to insulin sensitivity by positive and negative regulation of the expression of diverse genes. Of note, PCR array data showed downregulation of Slc2A4, a coding gene for Glut4. Altogether, the present study concludes that the upregulation of miR-494 expression by TNF-α-mediated inflammation exacerbates insulin resistance. Therefore, we suggest that miR-494 could prove an important target for the diagnosis and therapy of inflammation-mediated insulin resistance in muscle. [ABSTRACT FROM AUTHOR]