Dysregulation of Gene Expression of Key Signaling Mediators in PBMCs from People with Type 2 Diabetes Mellitus.

Diabetes is currently the fifth leading cause of death by disease in the USA. The underlying mechanisms for type 2 Diabetes Mellitus (DM2) and the enhanced susceptibility of such patients to inflammatory disorders and infections remain to be fully defined. We have recently shown that peripheral blood mononuclear cells (PBMCs) from non-diabetic people upregulate expression of inflammatory genes in response to proteasome modulators, such as bacterial lipopolysaccharide (LPS) and soybean lectin (LEC); in contrast, resveratrol (RES) downregulates this response. We hypothesized that LPS and LEC will also elicit a similar upregulation of gene expression of key signaling mediators in (PBMCs) from people with type 2 diabetes (PwD2, with chronic inflammation) ex vivo. Unexpectedly, using next generation sequencing (NGS), we show for the first time, that PBMCs from PwD2 failed to elicit a robust LPS- and LEC-induced gene expression of proteasome subunit LMP7 (PSMB8) and mediators of T cell signaling that were observed in non-diabetic controls. These repressed genes included: PSMB8, PSMB9, interferon-γ, interferon-λ, signal-transducer-and-activator-of-transcription-1 (STAT1), human leukocyte antigen (HLA DQB1, HLA DQA1) molecules, interleukin 12A, tumor necrosis factor-α, transporter associated with antigen processing 1 (TAP1), and several others, which showed a markedly weak upregulation with toxins in PBMCs from PwD2, as compared to those from non-diabetics. Resveratrol (proteasome inhibitor) further downregulated the gene expression of these inflammatory mediators in PBMCs from PwD2. These results might explain why PwD2 may be susceptible to infectious disease. LPS and toxins may be leading to inflammation, insulin resistance, and thus, metabolic changes in the host cells.

Reprograming of Gene Expression of Key Inflammatory Signaling Pathways in Human Peripheral Blood Mononuclear Cells by Soybean Lectin and Resveratrol.

Inflammation is linked to several human diseases like microbial infections, cancer, heart disease, asthma, diabetes, and neurological disorders. We have shown that the prototype inflammatory agonist LPS modulates the activity of Ubiquitin-Proteasome System (UPS) and regulates transcription factors such as NF-κB, leading to inflammation, tolerance, hypoxia, autophagy, and apoptosis of cells. We hypothesized that proteasome modulators resveratrol and soybean lectin would alter the gene expression of mediators involved in inflammation-induced signaling pathways, when administered ex vivo to human peripheral blood mononuclear blood cells (PBMCs) obtained from normal healthy controls. To test this hypothesis, analysis of RNA derived from LPS-treated human PBMCs, with or without resveratrol and soybean lectin, was carried out using Next Generation Sequencing (NGS). Collectively, the findings described herein suggest that proteasome modulators, resveratrol (proteasome inhibitor) and lectins (proteasome activator), have a profound capacity to modulate cytokine expression in response to proteasome modulators, as well as expression of mediators in multiple signaling pathways in PBMCs of control subjects. We show for the first-time that resveratrol downregulates expression of mediators involved in several key signaling pathways IFN-γ, IL-4, PSMB8 (LMP7), and a subset of LPS-induced genes, while lectins induced IFN-γ, IL-4, PSMB8, and many of the same genes as LPS that are important for innate and adaptive immunity. These findings suggest that inflammation may be influenced by common dietary components and this knowledge may be used to prevent or reverse inflammation-based diseases.

δ-Tocotrienol feeding modulates gene expression of EIF2, mTOR, protein ubiquitination through multiple-signaling pathways in chronic hepatitis C patients.

BACKGROUND: δ-Tocotrienol is a naturally occurring proteasome inhibitor, which has the capacity to inhibit proliferation and induce apoptosis in several cancer cells obtained from several organs of humans, and other cancer cell lines. Moreover, results of plasma total mRNAs after δ-tocotrienol feeding to hepatitis C patients revealed significant inhibition in the expression of pro-inflammatory cytokines (TNF-α, VCAM1, proteasome subunits) and induction in the expression of ICAM1 and IFN-γ after post-treatment. This down-regulation of proteasome subunits leads to autophagy, apoptosis of immune cells and several genes. The present study describes RNA-sequence analysis of plasma total mRNAs obtained from δ-tocotrienol treatment of hepatitis C patients on gene expression regulated by proteasome. METHODS: Pooled specimens of plasma total mRNAs of pre-dose versus post-dose of δ-tocotrienol treatment of hepatitis C patients were submitted to RNA-sequence analyses. The data based on > 1 and 8-fold expression changes of 2136 genes were uploaded into "Ingenuity Pathway Analyses (IPA)" for core analysis, which describes possible canonical pathways, upstream regulators, diseases and functional metabolic networks. RESULTS: The IPA of "molecules" indicated fold change in gene expression of 953 molecules, which covered several categories of biological biomarkers. Out of these, gene expression of 220 related to present study, 12 were up-regulated, and 208 down-regulated after δ-tocotrienol treatment. The gene expression of transcription regulators (ceramide synthase 3 and Mohawk homeobox) were up-regulated, and gene expression of 208 molecules were down-regulated, involved in several biological functions (HSP90AB1, PSMC3, CYB5R4, NDUFB1, CYP2R1, TNFRF1B, VEGFA, GPR65, PIAS1, SFPQ, GPS2, EIF3F, GTPBP8, EIF4A1, HSPA14, TLR8, TUSSC2). IPA of "causal network" indicated gene regulators (676), in which 76 down-regulated (26 s proteasomes, interleukin cytokines, and PPAR-ligand-PPA-Retinoic acid-RXRα, PPARγ-ligand-PPARγ-Retinoic acid-RARα, IL-21, IL-23) with significant P-values. The IPA of "diseases and functions" regulators (85) were involved with cAMP, STAT2, 26S proteasome, CSF1, IFNγ, LDL, TGFA, and microRNA-155-5p, miR-223, miR-21-5p. The IPA of "upstream analysis" (934) showed 57 up-regulated (mainly 38 microRNAs) and 64 gene regulators were down-regulated (IL-2, IL-5, IL-6, IL-12, IL-13, IL-15, IL-17, IL-18, IL-21, IL-24, IL-27, IL-32), interferon ß-1a, interferon γ, TNF-α, STAT2, NOX1, prostaglandin J2, NF-κB, 1κB, TCF3, and also miRNA-15, miRNA-124, miRNA-218-5P with significant activation of Z-Score (P < 0.05). CONCLUSIONS: This is first report describing RNA-sequence analysis of δ-tocotrienol treated plasma total mRNAs obtained from chronic hepatitis C patients, that acts via multiple-signaling pathways without any side-effects. These studies may lead to development of novel classes of drugs for treatment of chronic hepatitis C patients.

Proteasome inhibitors modulate anticancer and anti-proliferative properties via NF-kB signaling, and ubiquitin-proteasome pathways in cancer cell lines of different organs.

BACKGROUND: Cancer is second most common cause of death in the United State. There are over 100 different types of cancer associated with different human organs, predominantly breast, liver, pancreas, prostate, colon, rectum, lung, and stomach. We have recently reported properties of pro-inflammatory (for treatment of various types of cancers), and anti-inflammatory (for cardiovascular disease and diabetes) compounds. The major problem associated with development of anticancer drugs is their lack of solubility in aqueous solutions and severe side effects in cancer patients. Therefore, the present study was carried out to check anticancer properties of selected compounds, mostly aqueous soluble, in cancer cell lines from different organs. METHODS: The anticancer properties, anti-proliferative, and pro-apoptotic activity of novel naturally occurring or FDA approved, nontoxic, proteasome inhibitors/activators were compared. In addition to that, effect of δ-tocotrienol on expression of proteasome subunits (X, Y, Z, LMP7, LMP2, LMP10), ICAM-1, VCAM-1, and TNF-α using total RNAs derived from plasmas of hepatitis C patients was investigated. RESULTS: Our data demonstrated that following compounds are very effective in inducing apoptosis of cancer cells: Thiostrepton, dexamethasone, 2-methoxyestradiol, δ-tocotrienol, quercetin, amiloride, and quinine sulfate have significant anti-proliferation properties in Hela cells (44% - 87%) with doses of 2.5-20 µM, compared to respective controls. Anti-proliferation properties of thiostrepton, 2-methoxyestradiol, δ-tocotrienol, and quercetin were 70% - 92%. However, thiostrepton, dexamethasone, 2-methoxyestradiol, δ-tocotrienol, quercetin, and quinine sulphate were effective in pancreatic, prostate, breast, lungs, melanoma, Β-lymphocytes, and T-cells (Jurkat: 40% to 95%) compared to respective controls. In lung cancer cells, these compounds were effective between 5 and 40 µM. The IC50 values of anti-proliferation properties of thiostrepton in most of these cell lines were between doses of 2.5-5 µM, dexamethasone 2.5-20 µM, 2-methoxyestradiol 2.5-10 µM, δ-tocotrienol 2.5-20 µM, quercetin 10-40 µM, and (-) Corey lactone 40-80 µM. In hepatitis C patients, δ-tocotrienol treatment resulted in significant decrease in the expression of pro-inflammatory cytokines. CONCLUSIONS: These data demonstrate effectiveness of several natural-occurring compounds with anti-proliferative properties against cancer cells of several organs of humans. Thiostrepton, dexamethasone, 2-methoxyestradiol, δ-tocotrienol and quercetin are very effective for apoptosis of cancer cells in liver, pancreas, prostate, breast, lung, melanoma, Β-lymphocytes and T-cells. The results have provided an opportunity to test these compounds either individually or in combination as dietary supplements in humans for treatment of various types of cancers.

Resveratrol Downregulates Biomarkers of Sepsis Via Inhibition of Proteasome's Proteases.

Lipopolysaccharide (LPS) is the main agonist of gram-negative bacteria and initiates inflammation. We recently reported that plasmas from sepsis patients revealed increased levels of following group of biomarkers; VCAM-1, ICAM1, CRP, resistin, and proteasome LMP subunits. Our objective here was to compare effects of resveratrol (shown to be a nonspecific proteasome inhibitor by us) and a known LMP7 inhibitor (ONX-0914, specific inhibitor) on proteasome's activities, as well as on inflammatory markers mentioned above in human blood monocytes. Using fluorescence-based assays on blood monocytes purified proteasomes, resveratrol (0-100 µM) inhibited all three protease activities, predominantly LMP7. Similarly, resveratrol inhibited all three protease activities using cell-based luminescence assay. In contrast, ONX-0914 was more selective and potent for LMP7 activity. Resveratrol and ONX-0914, both significantly inhibited expression of LPS-induced biomarkers mentioned above in CD14 monocytes. Moreover, resveratrol itself, as well as in combination with LPS, accumulated pIκBα in CD14 monocytes. Collectively, our data suggest that resveratrol is a less potent inhibitor of all three; CT-like (predominantly LMP7), T-like and PA protease activities and is less toxic to human monocytes than ONX-0914 (a selector inhibitor of only LMP7) as observed by an autophagy detection kit. Also, resveratrol reduces LPS-induced inflammatory cytokine expression by decreasing the translocation of NF-κB due to an increase in inhibitor pIκBα. Therefore, resveratrol can be used to curb inflammation in diseased states like sepsis and other disorders.

Of Mice and Men: Proteasome's Role in LPS-Induced Inflammation and Tolerance.

The molecular basis responsible for tolerance following inflammatory response to lipopolysaccharide (LPS) is not well understood. We hypothesized that inflammation/tolerance in monocytes/ macrophages is dependent on the proteases of proteasome. To test our hypothesis, first, we examined the expression of different proteasome subunits in different human and mouse monocytes/macrophages. Secondly, we investigated the effect of proteasome subunits/ proteases on LPS-induced expression of tumor necrosis factor-α (TNF-α) and nitric oxide (NO) during inflammation and tolerance using mouse RAW 264.7 macrophages, THP1 cells, and cluster of differentiation 14 positive (CD14) human monocytes. We found that RAW 264.7 cells (XYZ), mouse peritoneal resident, thioglycollate-elicited macrophages, primed RAW 264.7 (XYZ, LMP), and human monocytes (LMP) expressed different types of proteasome subunits/activities. Cells containing predominantly either LMP subunits (such as THP-1 and human monocytes), or only X, Y, Z subunits (RAW 264.7 cells not primed) could only induce TNF-α, but not NO, while cells containing all five to six subunits (XYZ, LMP) of the proteasome could induce both mediators in response to LPS. Distinct states of inflammation/tolerance in LPS treated cells, strongly correlated with an upregulation or downregulation of proteasome's subunits (proteases), respectively. Moreover, interferon-γ treatment of tolerant cells caused robust induction of proteasome's subunit expression in mouse macrophages and human monocytes, and cells regained their ability to respond to LPS. These studies are vital for understanding function of proteasome's subunits during inflammation/tolerance in mouse and human cells, and for design of therapeutic strategies for all diseases based on inflammation.

Evaluation of Pharmacokinetics, and Bioavailability of Higher Doses of Tocotrienols in Healthy Fed Humans.

BACKGROUND: Tocotrienols has been known to lower serum lipid parameters below 500 mg/d, while increase lipid parameters at higher dose of 750 mg/d. δ-Tocotrienol has a novel inflammatory property of concentration-dependent inhibition and activation. Therefore, inhibition (anti-inflammatory) property of tocotrienols at low doses is useful for cardiovascular disease, whereas, activation (pro-inflammatory) property using high dose is found effective for treatments of various types of cancer. We have recently described plasma bioavailability of 125 mg/d, 250 mg/d and 500 mg/d doses of δ-tocotrienol in healthy fed subjects, which showed dose-dependent increases in area under the curve (AUC) and maximum concentration (Cmax). Hence, in the current study, higher doses of tocotrienols have used to analyze its effect on plasma pharmacokinetic parameters. AIMS: To evaluate the safety and bioavailability of higher doses (750 mg and 1000 mg) of annatto-based tocotrienols in healthy fed subjects. All four isomers (α-, ß-, γ-, δ-) of tocols (tocotrienols and tocopherols) present in the plasmas of subjects were quantified and analyzed for various pharmacokinetic parameters. STUDY DESIGN: An open-label, randomized study was performed to analyze pharmacokinetics and bioavailability of δ-tocotrienol in 6 healthy fed subjects. All subjects (3/dose) were randomly assigned to one of each dose of 750 mg or 1000 mg. Blood samples were collected at 0, 1, 2, 4, 6, 8 h intervals and all isomers of α-,ß-,γ-,δ-tocotrienols, and tocopherols in plasmas were quantified by HPLC. RESULTS: Oral administration of 750 and 1000 mg/d of tocotrienols resulted in dose-dependent increases in plasmas (ng/ml) AUCt0-t8 6621, 7450; AUCt0-∞ 8688, 9633; AUMC t0-∞ 52497, 57199; MRT 6.04, 5.93; Cmax 1444, 1592 (P<0.05), respectively, of δ-tocotrienol isomer. Moreover, both doses also resulted in plasmas Tmax 3.33-4 h; elimination half-life (t1/2 h) 2.74, 2.68; time of clearance (Cl-T, l/h) 0.086, 0.078; volume of distribution (Vd/f, mg/h) 0.34, 0.30; and elimination rate constant (ke; h-1) 0.25, 0.17, respectively of δ- tocotrienol isomer. Similar results of these parameters were reported for γ-tocotrienol, ß- tocotrienol, α-tocotrienol, δ-tocopherol, γ-tocopherol, and ß-tocopherol, except for α- tocopherol. CONCLUSIONS: This study has described pharmacokinetics using higher doses of 750 mg/d and 1000 mg/d of δ-tocotrienol. These results confirmed earlier findings that Tmax was 3-4 h for all isomers of tocotrienols and tocopherols except for α-tocopherol (6 h). These higher doses of tocotrienols were found safe in humans and may be useful for treatments of various types of cancer, diabetes, and Alzheimer's disease.

Mammalian Lipopolysaccharide Receptors Incorporated into the Retroviral Envelope Augment Virus Transmission.

The orally transmitted retrovirus mouse mammary tumor virus (MMTV) requires the intestinal microbiota for persistence. Virion-associated lipopolysaccharide (LPS) activates Toll-like receptor 4 (TLR4), stimulating production of the immunosuppressive cytokine IL-10 and MMTV evasion of host immunity. However, the mechanisms by which MMTV associates with LPS remain unknown. We find that the viral envelope contains the mammalian LPS-binding factors CD14, TLR4, and MD-2, which, in conjunction with LPS-binding protein (LBP), bind LPS to the virus and augment transmission. MMTV isolated from infected mice lacking these LBPs cannot engage LPS or stimulate TLR4 and have a transmission defect. Furthermore, MMTV incorporation of a weak agonist LPS from Bacteroides, a prevalent LPS source in the gut, significantly enhances the ability of this LPS to stimulate TLR4, suggesting that MMTV intensifies these immunostimulatory properties. Thus, an orally transmitted retrovirus can capture, modify, and exploit mammalian receptors for bacterial ligands to ensure successful transmission.

Nutritional Supplement-5 with a Combination of Proteasome Inhibitors (Resveratrol, Quercetin, δ-Tocotrienol) Modulate Age-Associated Biomarkers and Cardiovascular Lipid Parameters in Human Subjects.

BACKGROUND: Age-associated altered redox imbalances and dysregulated immune function, contribute to the development of a variety of age associated diseases. Inflammatory markers and lipid profiles are useful prognostic indicators of a variety of age-associated and cardiovascular diseases. We have previously studied the impact of several proteasome inhibitors on several markers of inflammation and lipid profiles in vitro, in vivo, in cell lines, animal models, and in human subjects. The current study represents an extension of this work. Our main hypothesis is that a combination of various naturally-occurring proteasome inhibitors, which inhibits nitric oxide (NO), and C-reactive protein (CRP) mediated inflammation, will have better efficacy in the prevention and treatment of age-associated disorders including cardiovascular disease. METHODS: Two double blind, randomized, placebo-controlled cross-over trials were conducted to determine the impact of a mixture of NS-5 (resveratrol, pterostilbene, quercetin, δ-tocotrienol, nicotinic acid) on serum NO, CRP, γ-glutamyl-transferase (γ-GT) activity, total antioxidant status (TAS), total cholesterol, LDL-cholesterol, HDL-cholesterol and triglycerides levels. Healthy seniors (Group-1; n = 32) free-living (A, B; 16/group), and hypercholesterolemic (Group-2; n = 64) subjects on AHA-Step-1-diet were divided into two groups (C, D; 32/group). Baseline levels were established for parameters as mentioned above. Groups A, C were administered 4-capsules/d of NS-5 and groups B, D, placebo (starch) for 6-weeks. Groups were crossed-over, followed by a 2-week wash-out period. Groups A, C were given 4-capsules/d of placebo and groups B, D, 4-capsules/d of NS-5 for 6-weeks. Groups C, D were continued on AHA-Step-1-diet. RESULTS: All the subjects completed each phase in both studies without any complaints. There were significant ( P < 0.01 - 0.05) decreases in the serum levels of NO (30%, 26%), CRP (29%, 21%), γ-GT activity (14%, 17%), and blood pressure (systolic/diastolic, 3/6%, 3/3%) of Groups A and B, respectively, of free-living healthy seniors without affecting the total, HDL-, LDL-cholesterol or triglycerides compared to their respective baseline values. However, serum levels of NO (36%, 43%), CRP (31%, 48%), γ-GT (17%, 20%), total cholesterol (19%, 15%), LDL-cholesterol (28%, 20%), triglycerides (11%, 18%) of Groups C and D were significantly ( P < 0.01-0.05) decreased with NS-5 treatment of hypercholesterolemic subjects compared to baseline values, without affecting the serum HDL-cholesterol levels. The serum levels of total antioxidant status (TAS) were increased (10%, 14%; P < 0.05) in Groups A and B, increased (19%, 24%; P < 0.02), and blood pressure (systolic/diastolic, 5/6%, 3/5%) in Groups C and D with NS-5 treatment, compared to respective baseline values. CONCLUSIONS: The consumption of NS-5 mixture decreased significantly serum NO, CRP and γ-GT levels, improved TAS and lipid profiles at risk cardiovascular and hold promise for delaying onset of age-associated diseases.

Suppression of Nitric Oxide Production and Cardiovascular Risk Factors in Healthy Seniors and Hypercholesterolemic Subjects by a Combination of Polyphenols and Vitamins.

BACKGROUND: Dysregulated immune function associated with ageing has been implicated in a variety of human diseases. We have demonstrated the anti-inflammatory properties of resveratrol, pterostilbene, morin hydrate, quercetin, δ-tocotrienol, riboflavinin a variety of experimental animal models, and determined that these compounds act by inhibiting proteasome activity. AIMS: To determine whether serum nitric oxide (NO) levels increase with age in humans, and whether the combined cholesterol-lowering and inflammation-reducing properties of resveratrol, pterostilbene, Morin hydrate, quercetin, δ-tocotrienol, riboflavin, and nicotinic acid would reduce cardiovascular risk factors in humans when used as nutritional supplements with, or without, other dietary changes. METHODS: Elderly human subjects were stratified into two groups based on total serum cholesterol levels. Initial total serum cholesterol levels were normal and elevated in Group 1 and 2 subjects, respectively. Baseline serum NO, C-reactive protein (CRP), γ-glutamyltransferase (γ-GT) activity, uric acid, total antioxidant status (TAS), total cholesterol, HDL-cholesterol, LDL-cholesterol, and triglycerides levels were established over a four week period. Group 1 subjects subsequently received nutritional supplementation with one of two different combinations (NS-7 = 25 mg of each, resveratrol, pterostilbene, quercetin, δ-tocotrienol, nicotinic acid, morin hydrate or NS-6 = morin hydrate replaced with quercetin, 50 mg/capsule). Group 2 subjects also received these nutritional supplements (two capsules/d), but an AHA Step-1 diet was also implemented. After these interventions were administered for four weeks, the above parameters were re-measured and changes from baseline levels determined. Nitric acid (NO) levels in children, young adults, and seniors were also compared. RESULTS: The key results of the current study were: 1) that serum NO levels were significantly increased in seniors compared to both children (~80%) and young adults (~65%); 2) that the intake of two capsules/d of NS-7 or NS-6 for four weeks significantly (P < 0.05) decreased serum NO (39%, 24%), CRP (19%, 21%), uric acid (6%, 12%) levels, and γ-GT activity (8%, 6%), respectively in free-living healthy seniors; 3) that serum NO (36%, 29%), CRP (29%, 20%), uric acid (6%, 9%) γ-GT activity (9%, 18%), total cholesterol (8%, 11%), LDL-cholesterol (10%, 13%), and triglycerides (16%, 23%) levels were significantly (P < 0.02) decreased in hypercholesterolemic subjects restricted to AHA Step-1 diet plus intake of SN-7 or SN-6 (two capsules/d), respectively; 4) that TAS was increased (3%, 9%; P < 0.05) in free-living healthy seniors receiving NS-7 or NS-6 alone, and in hypercholesterolemic subjects plus AHA Step-1 diet (20%, 12%; P < 0.02) with either of the combinations tested. CONCLUSIONS: Serum NO levels are elevated in elderly humans compared to children or young adults. Diet supplementation with combinations of resveratrol, pterostilbene, morin hydrate, quercetin, δ-tocotrienol, riboflavin, and nicotinic acid reduce cardiovascular risk factors in humans when used as nutritional supplements with, or without, other dietary changes.

Inhibition of nitric oxide and inflammatory cytokines in LPS-stimulated murine macrophages by resveratrol, a potent proteasome inhibitor.

BACKGROUND: Altered immune function during ageing results in increased production of nitric oxide (NO) and other inflammatory mediators. Recently, we have reported that NO production was inhibited by naturally-occurring proteasome inhibitors (quercetin, δ-tocotrienol, and riboflavin) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, and thioglycolate-elicited peritoneal macrophages from C57BL/6 mice. In a continuous effort to find more potent, non-toxic, commercially available, naturally-occurring proteasome inhibitors that suppress inflammation, the present study was carried out to describe the inhibition of NF-κB activation and NO, TNF-α, IL-6, IL-1ß, and iNOS expression by trans-resveratrol, trans-pterostilbene, morin hydrate, and nicotinic acid in LPS-induced RAW 264.7 cells and thioglycolate-elicited peritoneal macrophages from C57BL/6 and BALB/c mice. RESULTS: The present results indicate that resveratrol, pterostilbene, and morin hydrate caused significant inhibition (>70% to 90%; P < 0.02) in the activities of chymotrypsin-like, trypsin-like, and post-acidic (post-glutamase) proteasome sites in RAW 264.7 cells at a dose of only 20 µM. These compounds also inhibited the production of NO by RAW-264.7 cells stimulated with LPS alone (>40%; P < 0.05), or LPS + interferon-γ (IFN-γ; >60%; P < 0.02). Furthermore, resveratrol, pterostilbene, morin hydrate, and quercetin suppressed secretion of TNF-α (>40%; P < 0.05) in LPS-stimulated RAW 264.7 cells, and suppressed NF-κB activation (22% to 45%; P < 0.05) in LPS-stimulated HEK293T cells. These compounds also significantly suppressed LPS-induced expression of TNF-α, IL-1ß, IL-6, and iNOS genes in RAW 264.7 cells, and also in thioglycolate-elicited peritoneal macrophages from C57BL/6 and BALB/c mice. CONCLUSIONS: The present results clearly demonstrate that resveratrol and pterostilbene are particularly potent proteasome inhibitors that suppress expression of genes, and production of inflammatory products in LPS-stimulated RAW 264.7 cells, and macrophages from C57BL/6 and BALB/c mice. Resveratrol and pterostilbene which are present in grapes, blueberries, and red wine, have been implicated as contributing factors to the lower incidence of cardiovascular disease in the French population, despite their relatively high dietary fat intake. Consequently, it appears likely that the beneficial nutritional effects of resveratrol and pterostilbene are due at least in part, to their ability to inhibit NF-κB activation by the proteasome, thereby suppressing activation of pro-inflammatory cytokines and iNOS genes, resulting in decreased secretion of TNF-α, IL-1ß, IL-6, and NO levels, in response to inflammatory stimuli. This is the first report demonstrating that resveratrol and pterostilbene act as proteasome inhibitors, thus providing a mechanism for their anti-inflammatory effects.

Proteasome protease mediated regulation of cytokine induction and inflammation.

We have previously demonstrated that proteasome serves as a central regulator of inflammation and macrophage function. Until recently, proteasomes have generally been considered to play a relatively passive role in the regulation of cellular activity, i.e., any ubiquitinated protein was considered to be in discriminatively targeted for degradation by the proteasome. We have demonstrated, however, by using specific proteasome protease inhibitors and knockout mice lacking specific components of immunoproteasomes, that proteasomes (containing X, Y, and Z protease subunits) and immunoproteasomes (containing LMP7, LMP2, and LMP10 protease subunits) have well-defined functions in cytokine induction and inflammation based on their individual protease activities. We have also shown that LPS-TLR mediated signaling in the murine RAW 264.7 macrophage cell line results in the replacement of macrophage immunoproteasomal subunits. Such modifications serve as pivotal regulators of LPS-induced inflammation. Our findings support the relatively novel concept that defects in structure/function of proteasome protease subunits caused by genetic disorders, aging, diet, or drugs may well have the potential to contribute to modulation of proteasome activity. Of particular relevance, we have identified quercetin and resveratrol, significant constituents present in berries and in red wine respectively, as two novel proteasome inhibitors that have been previously implicated as disease-modifying natural products. We posit that natural proteasome inhibitors/activators can potentially be used as therapeutic response modifiers to prevent/treat diseases through pathways involving the ubiquitin-proteasome pathway (UP-pathway), which likely functions as a master regulator involved in control of overall inflammatory responses. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.

Methamphetamine increases LPS-mediated expression of IL-8, TNF-α and IL-1ß in human macrophages through common signaling pathways.

The use of methamphetamine (MA) has increased in recent years, and is a major health concern throughout the world. The use of MA has been associated with an increased risk of acquiring HIV-1, along with an increased probability of the acquisition of various sexually transmitted infections. In order to determine the potential effects of MA exposure in the context of an infectious agent, U937 macrophages were exposed to various combinations of MA and bacterial lipopolysaccharide (LPS). Treatment with MA alone caused significant increases in the levels of TNF-α, while treatment with both MA and LPS resulted in significant increases in TNF-α, IL-1ß and the chemokine IL-8. The increases in cytokine or chemokine levels seen when cells were treated with both LPS and MA were generally greater than those increases observed when cells were treated with only LPS. Treatment with chemical inhibitors demonstrated that the signal transduction pathways including NF-kB, MAPK, and PI3-Akt were involved in mediating the increased inflammatory response. As discussed in the paper, these pathways appear to be utilized by both MA and LPS, in the induction of these inflammatory mediators. Since these pathways are involved in the induction of inflammation in response to other pathogens, this suggests that MA-exacerbated inflammation may be a common feature of infectious disease in MA abusers.

A critical role for the inducible proteasomal subunits LMP7 and MECL1 in cytokine production by activated murine splenocytes.

BACKGROUND AND PURPOSE: The proteasome is a multi-subunit complex that proteolytically cleaves proteins. The replacement of the constitutive proteasome subunits ß1, ß2, and/or ß5 with the IFNγ-inducible subunits LMP2, MECL1, and/or LMP7 results in the 'immunoproteasome'. The inducible subunits change the cleavage specificities of the proteasome, but it is unclear whether they have functions in addition to this. The purpose of the present study was to determine the role of the proteasome in general, as well as LMP7 and MECL1 specifically, with regard to cytokine production by activated primary splenocytes. METHODS: A LMP7/MECL1-null mouse was engineered to determine the roles of these subunits in cytokine production. Isolated splenocytes from wild-type and LMP7/MECL1-/- mice were treated with lactacystin and activated with PMA and ionomycin and subsequently cytokine mRNA levels were quantified. RESULTS: The present study demonstrates that LMP7/MECL1 regulates the expression of IFNγ, IL4, IL10, IL2Rß, GATA3, and t-bet. In contrast, the regulation of IL2, IL13, TNFα, and IL2Rα by the proteasome appears to occur independently of LMP7/MECL1. CONCLUSIONS: Collectively, the present study demonstrates that LMP7 and MECL1 regulate cytokine expression, suggesting this system represents a novel mechanism for the regulation of cytokines and cytokine signaling.

A variety of novel lipid A structures obtained from Francisella tularensis live vaccine strain.

F. tularensis is a Gram-negative coccobacillus that causes tularemia. Its LPS has nominal biological activity. Currently, there is controversy regarding the structure of the lipid A obtained from F. tularensis live vaccine strain (LVS). Therefore, to resolve this controversy, the purification and structural identification of this LPS was crucial. To achieve this, LPS from F. tularensis LVS was acid hydrolyzed to obtain crude lipid A that was methylated and purified by HPLC and the fractions were analyzed by MALDI-TOF MS. The structure of the major lipid A species was composed of a glucosamine disaccharide backbone substituted with four fatty acyl groups and a phosphate (1-position) with a molecular mass of 1505. The major lipid A component contained 18:0[3-O(16:0)] in the distal subunit and two 18:0(3-OH) fatty acyl chains at the 2- or 3-positions of the reducing subunit. Additional variations in the lipid A species include: heterogeneity in fatty acyl groups, a phosphate or a phosphoryl galactosamine at the 1-position, and a hexose at the 4' or 6' position, some of which have not been previously described for F. tularensis LVS. This analysis revealed that lipid A from F. tularensis LVS is far more complex than originally believed.

Inhibition of nitric oxide in LPS-stimulated macrophages of young and senescent mice by δ-tocotrienol and quercetin.

BACKGROUND: Changes in immune function believed to contribute to a variety of age-related diseases have been associated with increased production of nitric oxide (NO). We have recently reported that proteasome inhibitors (dexamethasone, mevinolin, quercetin, δ-tocotrienol, and riboflavin) can inhibit lipopolysaccharide (LPS)-induced NO production in vitro by RAW 264.7 cells and by thioglycolate-elicited peritoneal macrophages derived from four strains of mice (C57BL/6, BALB/c, LMP7/MECL-1(-/-) and PPAR-α(-/-) knockout mice). The present study was carried out in order to further explore the potential effects of diet supplementation with naturally-occurring inhibitors (δ-tocotrienol and quercetin) on LPS-stimulated production of NO, TNF-α, and other pro-inflammatory cytokines involved in the ageing process. Young (4-week-old) and senescent mice (42-week old) were fed control diet with or without quercetin (100 ppm), δ-tocotrienol (100 ppm), or dexamethasone (10 ppm; included as positive control for suppression of inflammation) for 4 weeks. At the end of feeding period, thioglycolate-elicited peritoneal macrophages were collected, stimulated with LPS, LPS plus interferon-ß (IFN-ß), or LPS plus interferon-γ (IFN-γ), and inflammatory responses assessed as measured by production of NO and TNF-α, mRNA reduction for TNF-α, and iNOS genes, and microarray analysis. RESULTS: Thioglycolate-elicited peritoneal macrophages prepared after four weeks of feeding, and then challenged with LPS (10 ng or 100 ng) resulted in increases of 55% and 73%, respectively in the production of NO of 46-week-old compared to 8-week-old mice fed control diet alone (respective control groups), without affecting the secretion of TNF-α among these two groups. However, macrophages obtained after feeding with quercetin, δ-tocotrienol, and dexamethasone significantly inhibited (30% to 60%; P < 0.02) the LPS-stimulated NO production, compared to respective control groups. There was a 2-fold increase in the production of NO, when LPS-stimulated macrophages of quercetin, δ-tocotrienol, or dexamethasone were also treated with IFN-ß or IFN-γ compared to respective control groups. We also demonstrated that NO levels and iNOS mRNA expression levels were significantly higher in LPS-stimulated macrophages from senescent (0.69 vs 0.41; P < 0.05), compared to young mice. In contrast, age did not appear to impact levels of TNF-α protein or mRNA expression levels (0.38 vs 0.35) in LPS-stimulated macrophages. The histological analyses of livers of control groups showed lesions of peliosis and microvesicular steatosis, and treated groups showed Councilman body, and small or large lymphoplasmacytic clusters. CONCLUSIONS: The present results demonstrated that quercetin and δ-tocotrienols inhibit the LPS-induced NO production in vivo. The microarray DNA analyses, followed by pathway analyses indicated that quercetin or δ-tocotrienol inhibit several LPS-induced expression of several ageing and pro-inflammatory genes (IL-1ß, IL-1α, IL-6, TNF-α, IL-12, iNOS, VCAM1, ICAM1, COX2, IL-1RA, TRAF1 and CD40). The NF-κB pathway regulates the production of NO and inhibits the pro-inflammatory cytokines involved in normal and ageing process. These ex vivo results confirmed the earlier in vitro findings. The present findings of inhibition of NO production by quercetin and δ-tocotrienol may be of clinical significance treating several inflammatory diseases, including ageing process.

Suppression of nitric oxide induction and pro-inflammatory cytokines by novel proteasome inhibitors in various experimental models.

BACKGROUND: Inflammation has been implicated in a variety of diseases associated with ageing, including cancer, cardiovascular, and neurologic diseases. We have recently established that the proteasome is a pivotal regulator of inflammation, which modulates the induction of inflammatory mediators such as TNF-α, IL-1, IL-6, and nitric oxide (NO) in response to a variety of stimuli. The present study was undertaken to identify non-toxic proteasome inhibitors with the expectation that these compounds could potentially suppress the production of inflammatory mediators in ageing humans, thereby decreasing the risk of developing ageing related diseases. We evaluated the capacity of various proteasome inhibitors to suppress TNF-α, NO and gene suppression of TNF-α, and iNOS mRNA, by LPS-stimulated macrophages from several sources. Further, we evaluated the mechanisms by which these agents suppress secretion of TNF-α, and NO production. Over the course of these studies, we measured the effects of various proteasome inhibitors on the RAW 264.7 cells, and peritoneal macrophages from four different strains of mice (C57BL/6, BALB/c, proteasome double subunits knockout LMP7/MECL-1-/-, and peroxisome proliferator-activated receptor-α,-/- (PPAR-α,-/-) knockout mice. We also directly measured the effect of these proteasome inhibitors on proteolytic activity of 20S rabbit muscle proteasomes. RESULTS: There was significant reduction of chymotrypsin-like activity of the 20S rabbit muscle proteasomes with dexamethasone (31%), mevinolin (19%), δ-tocotrienol (28%), riboflavin (34%), and quercetin (45%; P < 0.05). Moreover, quercetin, riboflavin, and δ-tocotrienol also inhibited chymotrypsin-like, trypsin-like and post-glutamase activities in RAW 264.7 whole cells. These compounds also inhibited LPS-stimulated NO production and TNF-α, secretion, blocked the degradation of P-IκB protein, and decreased activation of NF-κB, in RAW 264.7 cells. All proteasome inhibitors tested also significantly inhibited NO production (30% to 60% reduction) by LPS-induced thioglycolate-elicited peritoneal macrophages derived from all four strains of mice. All five compounds also suppressed LPS-induced TNF-α, secretion by macrophages from C57BL/6 and BALB/c mice. TNF-α, secretion, however, was not suppressed by any of the three proteasome inhibitors tested (δ-tocotrienol, riboflavin, and quercetin) with LPS-induced macrophages from LMP7/MECL-1-/- and PPAR-α,-/- knockout mice. Results of gene expression studies for TNF-α, and iNOS were generally consistent with results obtained for TNF-α, protein and NO production observed with four strains of mice. CONCLUSIONS: Results of the current study demonstrate that δ-tocotrienol, riboflavin, and quercetin inhibit NO production by LPS-stimulated macrophages of all four strains of mice, and TNF-α, secretion only by LPS-stimulated macrophages of C57BL/6 and BALB/c mice. The mechanism for this inhibition appears to be decreased proteolytic degradation of P-IκB protein by the inhibited proteasome, resulting in decreased translocation of activated NF-κB to the nucleus, and depressed transcription of gene expression of TNF-α, and iNOS. Further, these naturally-occurring proteasome inhibitors tested appear to be relatively potent inhibitors of multiple proteasome subunits in inflammatory proteasomes. Consequently, these agents could potentially suppress the production of inflammatory mediators in ageing humans, thereby decreasing the risk of developing a variety of ageing related diseases.

Tocotrienols-induced inhibition of platelet thrombus formation and platelet aggregation in stenosed canine coronary arteries.

BACKGROUND: Dietary supplementation with tocotrienols has been shown to decrease the risk of coronary artery disease. Tocotrienols are plant-derived forms of vitamin E, which have potent anti-inflammatory, antioxidant, anticancer, hypocholesterolemic, and neuroprotective properties. Our objective in this study was to determine the extent to which tocotrienols inhibit platelet aggregation and reduce coronary thrombosis, a major risk factor for stroke in humans. The present study was carried out to determine the comparative effects of α-tocopherol, α-tocotrienol, or tocotrienol rich fraction (TRF; a mixture of α-+γ-+δ-tocotrienols) on in vivo platelet thrombosis and ex vivo platelet aggregation (PA) after intravenous injection in anesthetized dogs, by using a mechanically stenosed circumflex coronary artery model (Folts' cyclic flow model). RESULTS: Collagen-induced platelet aggregation (PA) in platelet rich plasma (PRP) was decreased markedly after treatment with α-tocotrienol (59%; P<0.001) and TRF (92%; P<0.001). α-Tocopherol treatment was less effective, producing only a 22% (P<0.05) decrease in PA. Adenosine diphosphate-induced (ADP) PA was also decreased after treatment with α-tocotrienol (34%; P<0.05) and TRF (42%; P<0.025). These results also indicate that intravenously administered tocotrienols were significantly better than tocopherols in inhibiting cyclic flow reductions (CFRs), a measure of the acute platelet-mediated thrombus formation. Tocotrienols (TRF) given intravenously (10 mg/kg), abolished CFRs after a mean of 68 min (range 22 -130 min), and this abolition of CFRs was sustained throughout the monitoring period (50-160 min).Next, pharmacokinetic studies were carried out and tocol levels in canine plasma and platelets were measured. As expected, α-Tocopherol treatment increased levels of total tocopherols in post- vs pre-treatment specimens (57 vs 18 µg/mL in plasma, and 42 vs 10 µg/mL in platelets). However, treatment with α-tocopherol resulted in slightly decreased levels of tocotrienols in post- vs pre-treatment samples (1.4 vs 2.9 µg/mL in plasma and 2.3 vs 2.8 µg/mL in platelets). α-Tocotrienol treatment increased levels of both tocopherols and tocotrienols in post- vs pre-treatment samples (tocopherols, 45 vs 10 µg/mL in plasma and 28 vs 5 µg/mL in platelets; tocotrienols, 2.8 vs 0.9 µg/mL in plasma and 1.28 vs 1.02 µg/mL in platelets). Treatment with tocotrienols (TRF) also increased levels of tocopherols and tocotrienols in post- vs pre-treatment samples (tocopherols, 68 vs 20 µg/mL in plasma and 31.4 vs 7.9 µg/mL in platelets; tocotrienols, 8.6 vs 1.7 µg/mL in plasma and 3.8 vs 3.9 µg/mL in platelets). CONCLUSIONS: The present results indicate that intravenously administered tocotrienols inhibited acute platelet-mediated thrombus formation, and collagen and ADP-induced platelet aggregation. α-Tocotrienols treatment induced increases in α-tocopherol levels of 4-fold and 6-fold in plasma and platelets, respectively. Interestingly, tocotrienols (TRF) treatment induced a less pronounced increase in the levels of tocotrienols in plasma and platelets, suggesting that intravenously administered tocotrienols may be converted to tocopherols. Tocotrienols, given intravenously, could potentially prevent pathological platelet thrombus formation and thus provide a therapeutic benefit in conditions such as stroke and myocardial infarction.

The immunoproteasomes regulate LPS-induced TRIF/TRAM signaling pathway in murine macrophages.

We have proposed the novel concept that the macrophage ubiquitin-proteasome pathway functions as a key regulator of Lipopolysaccharide (LPS)-induced inflammation signaling. These findings suggest that proteasome-associated protease subunits X, Y, and Z are replaced by LMP subunits after LPS treatment of RAW 264.7 cells. The objective here was to determine the contribution of selective LMP proteasomal subunits to LPS-induced nitric oxide (NO) and TNF-α production in primary murine macrophages. Accordingly, thioglycollate-elicited macrophages from LMP7, LMP2, LMP10 (MECL-1), and LMP7/MECL-1 double knockout mice were stimulated in vitro with LPS, and were found to generate markedly reduced NO levels compared to wild-type (WT) mice, whereas TNF-α levels responses were essentially unaltered relative to wild-type responses. The recent studies suggest that the TRIF/TRAM pathway is defective in LMP knockouts which may explain why iNOS/NO are not robustly induced in LPS-treated macrophages from knockouts. Treating these macrophages with IFN-γ and LPS, however, reverses this defect, leading to robust NO induction. TNF-α is induced by LPS in the LMP knockout macrophages because IκB and IRAK are degraded normally via the MyD88 pathway. Collectively, these findings strongly support the concept that LMP7/MECL-1 proteasomes subunits actively function to regulate LPS-induced NO production by affecting the TRIF/TRAM pathway.