Network Pharmacology and Molecular Docking Study on the Potential Mechanism of Yi-Qi-Huo-Xue-Tong-Luo Formula in Treating Diabetic Peripheral Neuropathy.

OBJECTIVE: To investigate the potential mechanism of action of Yi-Qi-Huo-Xue-Tong-Luo formula (YQHXTLF) in the treatment of diabetic peripheral neuropathy (DPN). METHODS: Network pharmacology and molecular docking techniques were used in this study. Firstly, the active ingredients and the corresponding targets of YQHXTLF were retrieved using the Traditional Chinese Medicine Systems Pharmacology (TCMSP) platform; subsequently, the targets related to DPN were retrieved using GeneCards, Online Mendelian Inheritance in Man (OMIM), Pharmgkb, Therapeutic Target Database (TTD) and Drugbank databases; the common targets of YQHXTLF and DPN were obtained by Venn diagram; afterwards, the "YQHXTLF Pharmacodynamic Component-DPN Target" regulatory network was visualized using Cytoscape 3.6.1 software, and Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed on the potential targets using R 3.6.3 software. Finally, molecular docking of the main chemical components in the PPI network with the core targets was verified by Autodock Vina software. RESULTS: A total of 86 active ingredients and 229 targets in YQHXTLF were screened, and 81 active ingredients and 110 targets were identified to be closely related to diabetic peripheral neuropathy disease. PPI network mapping identified TP53, MAPK1, JUN, and STAT3 as possible core targets. KEGG pathway analysis showed that these targets are mostly involved in AGE-RAGE signaling pathway in diabetic complications, TNF signaling pathway, and MAPK signaling pathway. The molecular docking results showed that the main chemical components of YQHXTLF have a stable binding activity to the core pivotal targets. CONCLUSION: YQHXTLF may act on TP53, MAPK1, JUN, and STAT3 to regulate inflammatory response, apoptosis, or proliferation as a molecular mechanism for the treatment of diabetic peripheral neuropathy, reflecting its multitarget and multipathway action, and providing new ideas to further uncover its pharmacological basis and mechanism of action.

Fourth-ventricle leptin infusions dose-dependently activate hypothalamic signal transducer and activator of transcription 3.

Previous studies have shown that very low-dose infusions of leptin into the third or the fourth ventricle alone have little effect on energy balance, but simultaneous low-dose infusions cause rapid weight loss and increased phosphorylation of STAT3 (p-STAT3) in hypothalamic sites that express leptin receptors. Other studies show that injecting high doses of leptin into the fourth ventricle inhibits food intake and weight gain. Therefore, we tested whether fourth-ventricle leptin infusions that cause weight loss are associated with increased leptin signaling in the hypothalamus. In a dose response study 14-day infusions of increasing doses of leptin showed significant hypophagia, weight loss, and increased hypothalamic p-STAT3 in rats receiving at least 0.9 µg leptin/day. In a second study 0.6 µg leptin/day transiently inhibited food intake and reduced carcass fat, but had no significant effect on energy expenditure. In a final study, we identified the localization of STAT3 activation in the hypothalamus of rats receiving 0, 0.3, or 1.2 µg leptin/day. The high dose of leptin, which caused weight loss in the first experiment, increased p-STAT3 in the ventromedial, dorsomedial, and arcuate nuclei of the hypothalamus. The low dose that increased brown fat UCP1 but did not affect body composition in the first experiment had little effect on hypothalamic p-STAT3. We propose that hindbrain leptin increases the precision of control of energy balance by lowering the threshold for leptin signaling in the forebrain. Further studies are needed to directly test this hypothesis.

Picroside II Inhibits the MEK-ERK1/2-COX2 Signal Pathway to Prevent Cerebral Ischemic Injury in Rats.

The objective of this study is to explore the neuroprotective effect and mechanism of picroside II on ERK1/2-COX2 signal transduction pathway after cerebral ischemic injury in rats. Focal cerebral ischemic models were established by inserting monofilament threads into the middle cerebral artery in 200 Wistar rats. Twenty four rats were randomly selected into control group, while the other rats were randomly divided into six groups: model group, picroside group, lipopolysaccharide (LPS) with picroside group, U0126 with picroside group, LPS group, and U0126 group with each group containing three subgroups with ischemia at 6, 12, and 24 h. Neurobehavioral function in the rats was evaluated by modified neurological severity score points (mNSS) test; structure of neurons was observed using hematoxylin-eosin (HE) staining; apoptotic cells were counted using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay; expressions of phosphorylated mitogen/extracellular signal-regulated kinase kinas1/2 (pMEK1/2), phosphorylated extracellular signal-regulated protein kinase1/2 (pERK1/2), and cyclooxygenase (COX2) in the cortex were determined using immunohistochemistry (IHC) and Western blot (WB); and real-time PCR was used to determine the level of COX2 mRNA. The neurological behavioral malfunction appeared in all rats with middle cerebral artery occlusion (MCAO). In the model group, neuron damage was extensive, while the neurobehavioral function score, apoptotic cell index, expression of pMEK1/2, pERK1/2, and COX2 and the level of COX2 mRNA increased significantly when compared to the control group. The peak COX2 mRNA level was in ischemia 12 h, prior to the peak in COX2 protein expression. In the picroside and U0126 groups, the neurological behavioral function was improved, and the number of apoptotic cells and the expression of pMEK1/2, pERK1/2, and COX2 decreased significantly when compared to the model group. In the LPS with picroside group, at ischemia 6 h neuron damage was extensive, and pMEK1/2, pERK1/2, and COX2 expression were much higher than in the model group. But at ischemia 12 and 24 h, the expression of pMEK1/2, pERK1/2, and COX2 decreased slightly, and the neurobehavioral function also improved slightly. In LPS group, neuron damage was extensive, pMEK1/2, pERK1/2, and COX2 expression was still at a high level, and COX2 mRNA peak arrived at ischemic 12 h. Picroside II downregulates COX2 expression after MCAO by inhibiting MEK-ERK1/2 in rats to protect neurons from apoptosis and inflammation.

Inhibition of interleukin 1ß-stimulated interleukin-6 production by cranberry components in human gingival epithelial cells: effects on nuclear factor κB and activator protein 1 activation pathways.

BACKGROUND AND OBJECTIVE: In periodontitis, gingival epithelial cells can produce interleukin (IL)-6, a regulator of osteoclastic bone resorption, in response to IL-1ß. IL-1ß regulates cytokine expression via signaling pathways, including nuclear factor (NF)-κB and mitogen activated protein kinase (MAPK)/activator protein (AP)-1. Cranberry proanthocyanidins (PACs) inhibit IL-1ß-stimulated IL-6 production, but specific mechanisms are unclear. The objectives of this study were to determine effects of cranberry PACs on NF-κB and MAPK/AP-1 activation of IL-1ß-stimulated IL-6 production in gingival epithelial cells. MATERIAL AND METHODS: Cranberry high molecular weight non-dialyzable material (NDM), rich in PACs, was derived from cranberry juice. Human gingival epithelial cells [Smulow-Glickman (S-G)] were incubated with IL-1ß in the presence or absence of NDM or inhibitors of NF-κB, [nemo-binding domain (NBD) peptide] or AP-1 (SP600125), and IL-6 levels were measured by ELISA. Effects of NDM on IL-1ß-activated NF-κB and AP-1 and phosphorylated intermediates in both pathways were measured in cell extracts via binding to specific oligonucleotides and specific sandwich ELISAs, respectively. Data were analyzed using ANOVA and Scheffe's F procedure for post hoc comparisons. RESULTS: IL-1ß (≥ 0.1 nm) caused a time- and dose-dependent stimulation of S-G epithelial cell IL-6 production (p < 0.005). This was significantly decreased in a dose-dependent manner by NBD peptide or SP600125 [maximum inhibition ~30-40% (p < 0.02)], and together, the two inhibitors decreased IL-6 by ~80%, similar to the inhibition caused by NDM (p < 0.001). IL-1ß stimulated NF-κB and AP-1 activation (p < 0.003), which was inhibited by NDM (p < 0.0001). NDM did not significantly affect IL-1ß-stimulated levels of phosphorylated intermediates in the NF-κB pathway (IκBα) or the AP-1 pathway (c-Jun, ERK1/2). CONCLUSION: In S-G epithelial cells, IL-1ß appeared to upregulate IL-6 production via activation of both NF-κB and MAPK/AP-1 signaling pathways because cranberry NDM decreased nuclear levels of IL-1ß-activated NF-κB (p65) and AP-1 (phospho-c-Jun) and strongly inhibited IL-6 production. Lack of inhibition of phosphorylation of IκBα, c-Jun or ERK1/2 suggested that NDM might affect both pathways downstream from those points in S-G cells, such as ubiquitination and proteosomal degradation of IκBα, or inhibition of nuclear activity of c-Jun and/or ERK1/2. Defining these points of inhibition precisely may help identify molecular targets of cranberry polyphenols.

Inhibitory effect of Zingiber cassumunar extracts on lipopolysaccharide-induced cyclooxygenase-2 and matrix metalloproteinase expression in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Lipopolysaccharides (LPS) induce the production of proinflammatory mediators such as prostaglandins and matrix metalloproteinases (MMPs) in human gingival fibroblasts (HGFs). Zingiber cassumunar is a medicinal plant that possesses anti-inflammatory properties. The aim of this study was to determine the effects of the Z. cassumunar extract on the expression of cyclooxygenase (COX)-1, COX-2 and MMP-2 in HGFs challenged with LPS. MATERIAL AND METHODS: HGFs were treated with LPS in the presence or absence of Z. cassumunar extracts. The levels of expression of COX-1, COX-2 and MMP-2 mRNAs and of COX-1, COX-2 and MMP-2 proteins were detected by reverse transcription-polymerase chain reaction and western blotting, respectively. MMP-2 activities in cell-culture supernatants were determined using gelatin zymography. MAPK activation was evaluated by western blotting. RESULTS: LPS treatment of HGFs resulted in the activation of ERK1/2, p38 and JNK. Z. cassumunar extracts significantly inhibited the phosphorylation of ERK1/2 and JNK in HGFs stimulated with LPS. A lesser inhibitory effect was observed for the phosphorylation of p38. RT-PCR and western blot analyses showed that Z. cassumunar extracts inhibited the LPS-induced expression of COX-2 mRNA and COX-2 protein, respectively, but not of COX-1 mRNA or COX-1 protein. Pretreatment of HGFs with Z. cassumunar also attenuated the induction of MMP-2 with LPS. CONCLUSION: Our results indicate that Z. cassumunar extracts inhibit COX-2 and MMP-2 production by LPS-activated human gingival fibroblasts through blocking the proinflammatory signaling pathway involving ERK1/2, JNK and p38.

Shikonin derivatives inhibited LPS-induced NOS in RAW 264.7 cells via downregulation of MAPK/NF-kappaB signaling.

AIM OF THE STUDY: Shikonin/alkannin (SA) derivatives, analogs of naphthoquinone pigments, are the major components of root extracts of the Chinese medicinal herb (Lithospermum erythrorhizon; LE) and widely distributed in several folk medicines. In the present study, the effect and the underline molecular mechanism of shikonin derivatives isolated from root extracts of Lithospermum euchroma on lipopolysaccharide (LPS)-induced inflammatory response were investigated. MATERIALS AND METHODS: Effects of five SA derivatives, including SA, acetylshikonin, beta,beta-dimethylacrylshikonin, 5,8-dihydroxy-1.4-naphthoquinone, and 1,4-naphthoquinone on LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in mouse macrophage RAW264.7 cells were examined. RESULTS: Data suggested that SA derivatives inhibited LPS-induced NO and PGE(2) production, and iNOS protein expression. RT-PCR analysis showed that SA derivatives diminished LPS-induced iNOS mRNA expression. Moreover, the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 in LPS-stimulated RAW 264.7 cells was concentration-dependently suppressed by SA derivatives. SA inhibited NF-kappaB activation by prevention of the degradation of inhibitory factor-kappaB and p65 level in nuclear fractions induced by LPS. CONCLUSIONS: Taken together, these results suggest that the anti-inflammatory properties of SA derivatives might result from inhibition of iNOS protein expression through the downregulation of NF-kappaB activation via suppression of phosphorylation of ERK, in LPS-stimulated RAW 264.7 cells.

Inhibitory mechanisms of dihydroginsenoside Rg3 in platelet aggregation: critical roles of ERK2 and cAMP.

Ginsenoside Rg3, a single ginseng saponin, is known to be a major anti-platelet component of protopanaxadiol that is isolated from Korean red ginseng. In this study, we investigated whether dihydroginsenoside Rg3, a stable chemical derivative of ginsenoside Rg3, also demonstrated anti-platelet activity. Dihydroginsenoside Rg3 inhibited thrombin-induced platelet aggregation in a concentration-dependent manner with an IC50 (concentration producing 50% inhibition) of 18.8 +/- 0.4 microM. Ginsenoside Rg3 inhibited platelet aggregation which was induced by thrombin (0.1 U mL(-1)) with an IC50 of 40.2 +/- 0.9 microM. We next determined whether dihydroginsenoside Rg3 affected different types of ligand-induced platelet aggregation. We found that dihydroginsenoside Rg3 inhibited collagen-induced platelet aggregation with an IC50 of 20.0 +/- 0.9 microM. To elucidate the inhibitory mechanism of dihydroginsenoside Rg3 on aggregation, we analysed its downstream signalling pathway. It was interesting to note that dihydroginsenoside Rg3 elevated cyclic AMP production in resting platelets, but did not affect cyclic GMP production. In addition, we found that dihydroginsenoside Rg3 potently suppressed phosphorylation of extracellular signal-regulated kinase 2 (ERK2), which was stimulated by collagen (2.5 microg mL(-1)), but not of p38 mitogen-activated protein kinase. Taken together, our results indicate that dihydroginsenoside Rg3 potently inhibited platelet aggregation via the modulation of downstream signalling components such as cAMP and ERK2.

Anti-inflammatory effects of the methanol extract of Sedum telephium ssp. maximum in lipopolysaccharide- stimulated rat peritoneal macrophages.

Sedum telephium ssp. maximum is a medicinal plant that possesses anti-inflammatory, analgesic and keratolytic properties. We investigated the anti-inflammatory activity of its methanolic extract (STME) in rat peritoneal macrophages (MPhis) stimulated with lipopolysaccharide from Salmonella enteritidis. After stimulation with 10 microg/ml of LPS, MPhis were coincubated with different doses of STME (8, 16 and 32 microg/ml) or RPMI medium alone using different times of incubation. STME reduced levels of tumor necrosis factor-alpha, both mRNA and its protein, and significantly decreased IL-1beta and IL-6 production. Moreover, STME inhibited inducible nitric oxide synthase expression and blunted nitrite release and inhibited both extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase activation in lipopolysaccharide-stimulated MPhis. Data show that STME might be useful as a potential anti-inflammatory agent.

Extracts of Liriopsis tuber protect AMPA induced brain damage and improve memory with the activation of insulin receptor and ERK I/II.

The brain insulin receptor and ERK I/II are known to play an important role in memory formation and neuroprotection. A series of experiments was designed to explore if Liriopsis tuber (LT) extracts could exhibit neuroprotection and memory enhancing actions. LT was extracted with 70% methanol and subsequently fractionated into chloroform (fraction C), chloroform/methanol-(3:1) (fraction CM), methanol-soluble (fraction M) and methanol-insoluble, water-soluble fractions (fraction A). The LT fractions (T, C, M, A) significantly inhibited the cortical depolarization induced by AMPA in cortical slices of rats. In addition, these fractions were also effective in promoting memory in the passive avoidance test in mice. To gain insight into the mechanism of memory enhancing effects by Liriopsis tuber extracts, the activities of hippocampal insulin receptors and ERK I/II were tested in rats. Extract of LT (T) dramatically stimulated tyrosine phosphorylation of the insulin receptor, while fraction C of LT also significantly stimulated the same. In addition, ERK I/II were stimulated and cholinesterase activities were inhibited by fractions T, C, M and A in the rat hippocampus. These results suggest that Liriopsis tuber extracts may exert neuroprotection and memory enhancing effects via activation of the insulin receptor and ERK I/II as well as inhibiting cholinesterase.

Astragali Radix elicits anti-inflammation via activation of MKP-1, concomitant with attenuation of p38 and Erk.

Although Astragali Radix (Astragalus, AR), the root of Astragalus membranaceus (Fisch) Bunge, is widely used in oriental medicine for tonifying the immune response and improving circulation, the underlying mechanism(s) by which these effects are induced remains unclear. Here, we report that AR displays anti-inflammatory effects in zymosan air-pouch mice by reducing the expression of iNOS, COX-2, IL-6, IL-1beta and TNF-alpha and by decreasing the production of nitric oxide (NO). In a similar manner, AR reduces the expression of IL-6, iNOS, and COX-2 in lipopolysaccharide (LPS)-treated Raw 264.7 cells. We further demonstrate that AR attenuates the activity of p38 and Erk1/2 and stimulates mitogen-activated protein kinase phosphatase-1 (MKP-1) in LPS-treated Raw 264.7 cells. Additionally, AR interferes with the translocation of NFkappaB to the nucleus, subsequently resulting in NFkappaB-dependent transcriptional repression. Taken together, these data reveal that AR has an anti-inflammatory effect that is mediated by the MKP-1-dependent inactivation of p38 and Erk1/2 and inhibition of NFkappaB-mediated transcription. These results imply that the AR herb has a potential anti-inflammatory activity.

Danggui buxue tang--a Chinese herbal decoction activates the phosphorylations of extracellular signal-regulated kinase and estrogen receptor alpha in cultured MCF-7 cells.

Danggui buxue tang (DBT), a Chinese herbal decoction used to treat ailments in women, contains radix Astragali (Huangqi; RA) and radix Angelicae Sinensis (Danggui; RAS). The weight ratio of RA to RAS used in DBT must be 5:1 as stipulated as early as AD 1247; however, DBT's mechanism of action has never been described. Here, the estrogenic effects of DBT were investigated by determining the phosphorylations of estrogen receptor alpha (ER alpha) and extracellular signal-regulated kinase 1/2 (Erk1/2) in cultured MCF-7 cells. The application of DBT triggered the phosphorylation of ER alpha and Erk1/2 in a time-dependent manner. In contrast to the effect of estrogen, DBT triggered ER alpha phosphorylation at both S118 and S167. This DBT-specific phosphorylation was not triggered by an extract of one of the individual herbs, or by mixing the extracts of RA and RAS. DBT-induced downstream signals are described here. These signals suggest the uniqueness of this Chinese herbal decoction that requires a well-defined formulation.

Position of Src tyrosine kinases in the interaction between angiotensin II and endothelin in in vivo vascular protein synthesis.

OBJECTIVES: Endothelin is a necessary intermediate in the trophic action of angiotensin II during hypertension-induced resistance artery remodeling in vivo. Since Src tyrosine kinases can be activated by both agonists, we studied their role in the trophic action of angiotensin II, endothelin and their interaction in rat small mesenteric arteries. METHODS AND RESULTS: Twenty-six hour infusion of high-dose angiotensin II (400 ng/kg per min) or endothelin (5 pmol/kg per min) via osmotic pumps significantly enhanced vascular protein synthesis in vivo. When angiotensin II was used as the trophic stimulus, treatment with a Src tyrosine kinase inhibitor (PP2, 0.5 mg/kg, starting at 21 h of the 26-h stimulation) produced a significant attenuation of extracellular regulated kinase 1 (ERK 1) phosphorylation and of protein synthesis. However, PP2 administered at 21 h or throughout the 26-h infusion did not abrogate the elevation of protein synthesis induced by endothelin. Moreover, endothelin did not enhance the phosphorylation of ERK 1/2 in small mesenteric arteries. We confirmed that angiotensin II stimulated the expression of prepro-endothelin mRNA in small mesenteric arteries in a Src-dependent manner, as the response was inhibited by PP2. To support the specific inhibitory activity of PP2 on Src tyrosine kinases in vivo, angiotensin II-induced phosphorylation of cortactin, a Src-specific substrate, was inhibited by PP2. CONCLUSION: Src tyrosine kinases represent an important signaling element in angiotensin II-induced endothelin production in small arteries in vivo. However, Src tyrosine kinases did not appear to contribute to the trophic signaling of endothelin, suggesting that they lie upstream of endothelin in the angiotensin II-endothelin-protein synthesis cascade.

Inhibition of mitogen-mediated proliferation of rat vascular smooth muscle cells by labedipinedilol-A through PKC and ERK 1/2 pathway.

Labedipinedilol-A is a novel 1, 4-dihydropyridine type calcium antagonist with alpha-receptor blocking activity. This study investigates the effects of labedipinedilol-A on mitogen-induced proliferation of rat vascular smooth muscle cells (VSMCs). Labedipinedilol-A's inhibition on cell proliferation was measured by the tetrazolium salt (XTT) test. Labedipinedilol-A dose-dependently inhibited mitogen-induced DNA synthesis, determined by the incorporation of 5-bromo-2'-deoxyuridine (BrdU). Labedipinedilol-A was also found capable of inhibiting the migration of VSMCs induced by PDGF-BB with an IC50 value of 5.6 microM. In accordance with these findings, labedipinedilol-A revealed blocking of the FBS-inducible progression through G0/G1 to S phase of the cell cycle in synchronized cells. Labedipinedilol-A appeared to cause inhibition of mitogens-induced PKC translocation, suggesting the probable involvement of protein kinase C (PKC) in this cellular response. Labedipinedilol-A reduced both intracellular Ca and the phosphorylation of extracellular signal-regulated protein kinase 1/2 in PDGF-BB-stimulated VSMCs. It also suppressed the levels of proliferative cell nuclear antigen (PCNA) in VSMCs both time- and dose-dependently. These results indicate that labedipinedilol-A may inhibit cell proliferation by attenuating activation of the ERK 1/2 pathway, which is regulated by PKC and Ca, suggesting that it may have great potential in the prevention of progressive atherosclerosis.

In vivo activation of insulin receptor tyrosine kinase by melatonin in the rat hypothalamus.

Melatonin is the pineal hormone that acts via a pertussis toxin-sensitive G-protein to inhibit adenylate cyclase. However, the intracellular signalling effects of melatonin are not completely understood. Melatonin receptors are mainly present in the suprachiasmatic nucleus (SCN) and pars tuberalis of both humans and rats. The SCN directly controls, amongst other mechanisms, the circadian rhythm of plasma glucose concentration. In this study, using immunoprecipitation and immunoblotting, we show that melatonin induces rapid tyrosine phosphorylation and activation of the insulin receptor beta-subunit tyrosine kinase (IR) in the rat hypothalamic suprachiasmatic region. Upon IR activation, tyrosine phosphorylation of IRS-1 was detected. In addition, melatonin induced IRS-1/PI3-kinase and IRS-1/SHP-2 associations and downstream AKT serine phosphorylation and MAPK (mitogen-activated protein kinase) phosphorylation, respectively. These results not only indicate a new signal transduction pathway for melatonin, but also a potential cross-talk between melatonin and insulin.

Synergistic interaction of estradiol and insulin-like growth factor-I in the activation of PI3K/Akt signaling in the adult rat hypothalamus.

Estradiol and insulin-like growth factor-I (IGF-I) interact in the hypothalamus to regulate neuronal function, synaptic plasticity and neuroendocrine events. However, the molecular mechanisms involved in these interactions are still unknown. In the present study, the effect of estradiol on the signaling pathways of IGF-I receptor has been assessed in the hypothalamus of young adult ovariectomized rats, using specific antibodies for the phosphorylated forms of extracellular-signal regulated kinase (ERK) 1 and ERK2 and Akt/protein kinase B (Akt/PKB). Estradiol treatment resulted, between 6 and 24 h after systemic administration, in dose-dependent effects on the phosphorylation of ERK and Akt/PKB. Estradiol did not modify the level of ERK phosphorylation induced by intracerebroventricular administration of IGF-I. However, both hormones had a synergistic effect on the phosphorylation of Akt/PKB. These findings suggest that estrogen effects in the hypothalamus may be mediated in part by the activation of the signaling pathways of the IGF-I receptor.

Induction of caspase-mediated apoptosis and cell-cycle G1 arrest by selenium metabolite methylselenol.

Previous work based on mono-methyl selenium compounds that are putative precursors of methylselenol has strongly implicated this metabolite in the induction of caspase-mediated apoptosis of human prostate carcinoma and leukemia cells and G1 arrest in human vascular endothelial and cancer epithelial cells. To test the hypothesis that methylselenol itself is responsible for exerting these cellular effects, we examined the apoptotic action on DU145 human prostate cancer cells and the G1 arrest effect on the human umbilical vein endothelial cells (HUVECs) of methylselenol generated with seleno-L-methionine as a substrate for L-methionine-alpha-deamino-gamma-mercaptomethane lyase (EC4.4.1.11, also known as methioninase). Exposure of DU145 cells to methylselenol so generated in the sub-micromolar range led to caspase-mediated cleavage of poly(ADP-ribose) polymerase, nucleosomal DNA fragmentation, and morphologic apoptosis and resulted in a profile of biochemical effects similar to that of methylseleninic acid (MSeA) exposure as exemplified by the inhibition of phosphorylation of protein kinase AKT and extracellularly regulated kinases 1/2. In HUVEC, methylselenol exposure recapitulated the G1 arrest action of MSeA in mitogen-stimulated G1 progression during mid-G1 to late G1. This stage specificity was mimicked by inhibitors of phosphatidylinositol 3-kinase. The results support methylselenol as an active selenium metabolite for inducing caspase-mediated apoptosis and cell-cycle G1 arrest. This cell-free methylselenol-generation system is expected to have significant usefulness for studying the biochemical and molecular targeting mechanisms of this critical metabolite and may constitute the basis of a novel therapeutic approach for cancer, using seleno-L-methionine as a prodrug.

Cytotoxic action of acetyl-11-keto-beta-boswellic acid (AKBA) on meningioma cells.

Acetyl-11-keto-beta-boswellic acid (AKBA) is a naturally occurring pentacyclic triterpene isolated from the gum resin exudate of the tree Boswellia serrata (frankincense). Because pentacyclic triterpenes have antiproliferative and cytotoxic effects against different tumor types, we investigated whether AKBA would act in a similar fashion on primary human meningioma cell cultures. Primary cell cultures were established from surgically removed meningioma specimens. The number of viable cells in the absence/presence of AKBA was determined by the non-radioactive cell proliferation assay. The activation status of the proliferative cell marker, extracellular signal-regulated kinase-1 and -2 (Erk-1 and Erk-2) was determined by immunoblotting with the antibody that recognizes the activated form of these proteins. Treatment of meningioma cells by AKBA revealed a potent cytotoxic activity with half-maximal inhibitory concentrations in the range of 2 - 8 microM. At low micromolar concentrations, AKBA rapidly and potently inhibited the phosphorylation of Erk-1/2 and impaired the motility of meningioma cells stimulated with platelet-derived growth factor BB. The cytotoxic action of AKBA on meningioma cells may be mediated, at least in part, by the inhibition of the Erk signal transduction pathway. Because of the central role the Erk pathway plays in signal transduction and tumorigenesis, further investigation into the potential clinical use for AKBA and related boswellic acids is warranted.

Roles of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase in apoptosis of human monoblastic leukemia U937 cells by lectin-II isolated from Korean mistletoe.

The mitogen-activated protein kinase (MAPK) family members have been implicated in cell survival. We have previously demonstrated that cytotoxic lectin-II isolated from Korean mistletoe induces apoptotic cell death in the human monoblastic leukemia cell line, U937, via the activation of the stress-activated protein kinases/c-Jun N-terminal kinase (SAPK/JNK). In the present study, the roles of extracellular signal-regulated kinases (ERK1/2) and p38 MAPK in lectin-II-induced apoptosis have been investigated. Treatment of U937 cells with lectin-II resulted in apoptotic DNA fragmentation, which was preceded by the activation of ERK1/2, p38 MAPK and SAPK/JNK. This lectin-II-induced DNA fragmentation was significantly enhanced when ERK1/2 activation was selectively inhibited by PD098059. 12-O-tetradecanoylphorbol-13-acetate, which stimulates ERK activity in U937 cells, markedly reduced lectin-II-induced DNA fragmentation. Inhibition of p38 MAPK activity with p38-specific inhibitor, SB203580, partially inhibited lectin-II-induced DNA fragmentation. These results suggest that ERK1/2 and p38 MAPK may have opposite effects on cell survival in response to cytotoxic mistletoe lectin-II, which may contribute to the modulation of lectin-II-mediated cytotoxic activity.

Effects of Ginkgo biloba extract (EGb 761) and quercetin on lipopolysaccharide-induced signaling pathways involved in the release of tumor necrosis factor-alpha.

Administration of bacterial lipopolysaccharide (LPS) to laboratory animals and cultured macrophages induces tumor necrosis factor-alpha (TNF-alpha), a pro-inflammatory cytokine. Pretreatment with Ginkgo biloba extract (EGb 761) inhibited the in vivo production of TNF-alpha (measured by ELISA) after challenge with LPS. To begin to understand the mechanism of this inhibition, we evaluated the in vitro effects of EGb 761 and its flavonoid component, quercetin, on LPS-treated RAW 264.7 macrophages. Pretreatment with EGb 761 or quercetin concentration-dependently inhibited TNF-alpha release, as measured by the L929 fibroblast assay. Northern blotting demonstrated that quercetin inhibited LPS-induced TNF-alpha mRNA, but did not alter its half-life. Activation of mitogen-activated protein kinases (MAPKs) and the redox-sensitive transcription factors, nuclear factor-kappaB (NF-kappaB) and activator protein 1 (AP-1), are key events in the signal transduction pathways mediating TNF-alpha induction. Phosphorylation of extracellular signal-related kinases 1 and 2 (ERK 1/2), p38 MAPK, and Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), members of the MAPK family, was analyzed by western blotting. Our results suggest that quercetin is unique in its ability to inhibit TNF-alpha transcription by inhibiting the phosphorylation and activation of JNK/SAPK and, therefore, suppressing AP-1-DNA binding [assessed by electrophoretic mobility shift analysis (EMSA)]. Results from western analysis, EMSA, and transient transfections suggest that EGb 761 diminishes LPS-induced NF-kappaB but has no effect on LPS-induced TNF-alpha transcription. Both EGb 761 and quercetin inhibited ERK1/2 phosphorylation and p38 MAPK activity, which are important in the post-transcriptional regulation of TNF-alpha mRNA.

Mitogen-activated protein kinase regulates normal transition from metaphase to interphase following parthenogenetic activation in porcine oocytes.

The decrease in maturation-promoting factor (MPF) activity precedes that in mitogen-activated protein kinase (MAPK) activity after egg activation, but the cellular functions of this delayed inactivation of MAPK are still unclear. The present study was conducted to examine the essential role of MAPK activity for supporting the transition from metaphase to interphase in porcine oocytes matured in vitro. The increases in the phosphorylated forms of MAPK and the activities of MAPK and histone H1 kinase (H1K) were shown in oocytes arrested at the metaphase II (MII) stage. After additional incubation of MII-arrested oocytes in medium with added U0126, a specific inhibitor of MAPK kinase, 24% of oocytes completed the second meiotic division and underwent entry into interphase with pronucleus (PN) formation, but not second polar body (PB-2) emission. The intensities of the phosphorylated forms of MAPK and the activities of MAPK and H1K in matured oocytes treated with U0126 were significantly decreased by the treatment with U0126. Electrostimulation to induce artificial activation caused both H1K and MAPK inactivation; the inactivation of H1K preceded the inactivation of MAPK and sustained high levels of MAPK activity were detected during the period of PB-2 emission. However, the time sequence required for MAPK inactivation was significantly reduced by the addition of U0126 to the culture medium following electrostimulation, resulting in the dramatic inactivation of MAPK distinct from that of H1K. In these oocytes, PB-2 emission was markedly inhibited but little difference was found in the time course of PN formation compared with oocytes not treated with U0126. These findings suggest that the decrease in MAPK activity is partly involved in driving matured oocytes out of metaphase to induce PN development, and that the delayed MAPK inactivation after the onset of MPF inactivation in activated oocytes has a crucial role for PB-2 emission to accomplish the transition from meiosis to mitosis.