Biomedical subjects

Min Qui

Publications and source records attributed to Min Qui.

9 recordsLinked to original sources

PubMed2006

Inhibition of inducible Nitric Oxide Synthase by a mustard gas analog in murine macrophages.

BACKGROUND: 2-Chloroethyl ethyl sulphide (CEES) is a sulphur vesicating agent and an analogue of the chemical warfare agent 2,2'-dichlorodiethyl sulphide, or sulphur mustard gas (HD). Both CEES and HD are alkylating agents that influence cellular thiols and are highly toxic. In a previous publication, we reported that lipopolysaccharide (LPS) enhances the cytotoxicity of CEES in murine RAW264.7 macrophages. In the present investigation, we studied the influence of CEES on nitric oxide (NO) production in LPS stimulated RAW264.7 cells since NO signalling affects inflammation, cell death, and wound healing. Murine macrophages stimulated with LPS produce NO almost exclusively via inducible nitric oxide synthase (iNOS) activity. We suggest that the influence of CEES or HD on the cellular production of NO could play an important role in the pathophysiological responses of tissues to these toxicants. In particular, it is known that macrophage generated NO synthesised by iNOS plays a critical role in wound healing. RESULTS: We initially confirmed that in LPS stimulated RAW264.7 macrophages NO is exclusively generated by the iNOS form of nitric oxide synthase. CEES treatment inhibited the synthesis of NO (after 24 hours) in viable LPS-stimulated RAW264.7 macrophages as measured by either nitrite secretion into the culture medium or the intracellular conversion of 4,5-diaminofluorescein diacetate (DAF-2DA) or dichlorofluorescin diacetate (DCFH-DA). Western blots showed that CEES transiently decreased the expression of iNOS protein; however, treatment of active iNOS with CEES in vitro did not inhibit its enzymatic activity CONCLUSION: CEES inhibits NO production in LPS stimulated macrophages by decreasing iNOS protein expression. Decreased iNOS expression is likely the result of CEES induced alteration in the nuclear factor kappa B (NF-kappaB) signalling pathway. Since NO can act as an antioxidant, the CEES induced down-regulation of iNOS in LPS-stimulated macrophages could elevate oxidative stress. Since macrophage generated NO is known to play a key role in cutaneous wound healing, it is possible that this work has physiological relevance with respect to the healing of HD induced skin blisters.

Animals↗

PubMed2006

Comparative effects of RRR-alpha- and RRR-gamma-tocopherol on proliferation and apoptosis in human colon cancer cell lines.

BACKGROUND: Mediterranean societies, with diets rich in vitamin E isoforms, have a lower risk for colon cancer than those of northern Europe and the Americas. Vitamin E rich diets may neutralize free radicals generated by fecal bacteria in the gut and prevent DNA damage, but signal transduction activities can occur independent of the antioxidant function. The term vitamin E represents eight structurally related compounds, each differing in their potency and mechanisms of chemoprevention. The RRR-gamma-tocopherol isoform is found primarily in the US diet, while RRR-alpha-tocopherol is highest in the plasma. METHODS: The effectiveness of RRR-alpha- and RRR-gamma-tocopherol at inhibiting cell growth and inducing apoptosis in colon cancer cell lines with varying molecular characteristics (SW480, HCT-15, HCT-116 and HT-29) and primary colon cells (CCD-112CoN, nontransformed normal phenotype) was studied. Colon cells were treated with and without RRR-alpha- or RRR-gamma-tocopherol using varying tocopherol concentrations and time intervals. Cell proliferation and apoptosis were measured using the trypan blue assay, annexin V staining, DNA laddering and caspase activation. RESULTS: Treatment with RRR-gamma-tocopherol resulted in significant cell death for all cancer cell lines tested, while RRR-alpha-tocopherol did not. Further, RRR-gamma-tocopherol treatment showed no cytotoxicity to normal colon cells CCD-112CoN at the highest concentration and time point tested. RRR-gamma-tocopherol treatment resulted in cleavage of PARP, caspase 3, 7, and 8, but not caspase 9. Differences in the percentage cell death and apoptosis were observed in different cell lines suggesting that molecular differences in these cell lines may influence the ability of RRR-gamma-tocopherol to induce cell death. CONCLUSION: This is the first study to demonstrate that multiple colon cancer cell lines containing varying genetic alterations will under go growth reduction and apoptosis in the presence of RRR-gamma-tocopherol without damage to normal colon cells. The amount growth reduction was dependent upon the molecular signatures of the cell lines. Since RRR-gamma-tocopherol is effective at inhibition of cell proliferation at both physiological and pharmacological concentrations dietary RRR-gamma-tocopherol may be chemopreventive, while pharmacological concentrations of RRR-gamma-tocopherol may aid chemotherapy without toxic effects to normal cells demonstrated by most chemotherapeutic agents.

Antioxidants↗

PubMed2006

Assays for nitric oxide expression.

The purpose of this chapter is to review the analytical methodology specifically associated with studying the role of nitric oxide (NO) in mast cell physiology and biochemistry. The methodology for measuring cellular secretion of nitric oxide with Griess Reagent will be described in detail, as well as the use of 4,5-diaminofluorescein diacetate for continuous monitoring of nitric oxide production in live cells. We will point out the limitations of the analytical techniques and also indicate areas in which promising analytical techniques have not yet been applied to the study of mast cell physiology, that is, new research opportunities. In addition to reviewing the methodology associated with measuring NO itself, we will briefly touch upon some analytical methods important in characterizing the biochemical products formed from nitric oxide (e.g., 3-nitrotyrosine).

Animals↗

PubMed2004

Tocopherols and the treatment of colon cancer.

UNLABELLED: Colorectal cancer is the second most common cause of cancer deaths in the United States. Vitamin E (VE) and other antioxidants may help prevent colon cancer by decreasing the formation of mutagens arising from the free radical oxidation of fecal lipids or by "non-antioxidant" mechanisms. VE is not a single molecule, but refers to at least eight different molecules, that is, four tocopherols and four tocotrienols. METHODS: Both animal models and human colon cancer cell lines were used to evaluate the chemopreventive potential of different forms of VE. Rats were fed diets deficient in tocopherols or supplemented with either alpha-tocopherol or gamma-tocopherol. Half the rats in each of these groups received normal levels of dietary Fe and the other half Fe at eight times the normal level. In our cell experiments, we looked at the role of gamma-tocopherol in upregulating peroxisome proliferator-activated receptor-gamma (PPAR-gamma) in the SW 480 human cell line. RESULTS: Rats fed the diets supplemented with alpha-tocopherol had higher levels of VE in feces, colonocytes, plasma, and liver than did rats fed diets supplemented with gamma-tocopherol. Dietary Fe levels did not influence tocopherol levels in plasma, liver, or feces. For colonocytes, high dietary Fe decreased tocopherol levels. Rats fed the gamma-tocopherol-supplemented diets had lower levels of fecal lipid hydroperoxides than rats fed the alpha-tocopherol-supplemented diets. Ras-p21 levels were significantly lower in rats fed the gamma-tocopherol-supplemented diets compared with rats fed the alpha-tocopherol-supplemented diets. High levels of dietary Fe were found to promote oxidative stress in feces and colonocytes. Our data with the SW480 cells suggest that both alpha- and gamma-tocopherol upregulate PPAR-gamma mRNA and protein expression. gamma-tocopherol was, however, found to be a better enhancer of PPAR-gamma expression than alpha-tocopherol at the concentrations tested.

Animals↗

PubMed2003

Gamma (gamma) tocopherol upregulates peroxisome proliferator activated receptor (PPAR) gamma (gamma) expression in SW 480 human colon cancer cell lines.

BACKGROUND: Tocopherols are lipid soluble antioxidants that exist as eight structurally different isoforms. The intake of gamma-tocopherol is higher than alpha-tocopherol in the average US diet. The clinical results of the effects of vitamin E as a cancer preventive agent have been inconsistent. All published clinical trials with vitamin E have used alpha-tocopherol. Recent epidemiological, experimental and molecular studies suggest that gamma-tocopherol may be a more potent chemopreventive form of vitamin E compared to the more-studied alpha-tocopherol. Gamma-tocopherol exhibits differences in its ability to detoxify nitrogen dioxide, growth inhibitory effects on selected cancer cell lines, inhibition of neoplastic transformation in embryonic fibroblasts, and inhibition of cyclooxygenase-2 (COX-2) activity in macrophages and epithelial cells. Peroxisome proliferator activator receptor gamma (PPARgamma) is a promising molecular target for colon cancer prevention. Upregulation of PPARgamma activity is anticarcinogenic through its effects on downstream genes that affect cellular proliferation and apoptosis. The thiazolidine class of drugs are powerful PPARgamma ligands. Vitamin E has structural similarity to the thiazolidine, troglitazone. In this investigation, we tested the effects of both alpha and gamma tocopherol on the expression of PPARgamma mRNA and protein in SW 480 colon cancer cell lines. We also measured the intracellular concentrations of vitamin E in SW 480 colon cancer cell lines. RESULTS: We have discovered that the alpha and gamma isoforms of vitamin E upregulate PPARgamma mRNA and protein expression in the SW480 colon cancer cell lines. gamma-Tocopherol is a better modulator of PPARgamma expression than alpha-tocopherol at the concentrations tested. Intracellular concentrations increased as the vitamin E concentration added to the media was increased. Further, gamma-tocopherol-treated cells have higher intracellular tocopherol concentrations than those treated with the same concentrations of alpha-tocopherol. CONCLUSION: Our data suggest that both alpha and gamma tocopherol can upregulate the expression of PPARgamma which is considered an important molecular target for colon cancer chemoprevention. We show that the expression of PPARgamma mRNA and protein are increased and these effects are more pronounced with gamma-tocopherol. Gamma-tocopherol's ability to upregulate PPARgamma expression and achieve higher intracellular concentrations in the colonic tissue may be relevant to colon cancer prevention. We also show that the intracellular concentrations of gamma-tocopherol are several fold higher than alpha-tocopherol. Further work on other colon cancer cell lines are required to quantitate differences in the ability of these forms of vitamin E to induce apoptosis, suppress cell proliferation and act as PPAR ligands as well as determine their effects in conjunction with other chemopreventive agents. Upregulation of PPARgamma by the tocopherols and in particular by gamma-tocopherol may have relevance not only to cancer prevention but also to the management of inflammatory and cardiovascular disorders.

Cell Line, Tumor↗

PubMed2003

Lipopolysaccharide enhances the cytotoxicity of 2-chloroethyl ethyl sulfide.

BACKGROUND: The bacterial endotoxin, lipopolysaccharide (LPS), is a well-characterized inflammatory factor found in the cell wall of Gram-negative bacteria. In this investigation, we studied the cytotoxic interaction between 2-chloroethyl ethyl sulfide (CEES or ClCH2CH2SCH2CH3) and LPS using murine RAW264.7 macrophages. CEES is a sulfur vesicating agent and is an analog of 2,2'-dichlorodiethyl sulfide (sulfur mustard). LPS is a ubiquitous natural agent found in the environment. The ability of LPS and other inflammatory agents (such as TNF-alpha and IL-1beta) to modulate the toxicity of CEES is likely to be an important factor in the design of effective treatments. RESULTS: RAW 264.7 macrophages stimulated with LPS were found to be more susceptible to the cytotoxic effect of CEES than unstimulated macrophages. Very low levels of LPS (20 ng/ml) dramatically enhanced the toxicity of CEES at concentrations greater than 400 microM. The cytotoxic interaction between LPS and CEES reached a maximum 12 hours after exposure. In addition, we found that tumor necrosis factor-alpha (TNF-alpha) and interleukin-1-beta (IL-1-beta) as well as phorbol myristate acetate (PMA) also enhanced the cytotoxic effects of CEES but to a lesser extent than LPS. CONCLUSION: Our in vitro results suggest the possibility that LPS and inflammatory cytokines could enhance the toxicity of sulfur mustard. Since LPS is a ubiquitous agent in the natural environment, its presence is likely to be an important variable influencing the cytotoxicity of sulfur mustard toxicity. We have initiated further experiments to determine the molecular mechanism whereby the inflammatory process influences sulfur mustard cytotoxicity.

Adjuvants, Immunologic↗

PubMed2003

Regulation of nitric oxide production from macrophages by lipopolysaccharide and catecholamines.

Catecholamines are elaborated in stress responses to mediate vasoconstriction, and elevate systemic vascular resistance and blood pressure. They are elaborated in disorders such as sepsis, cocaine abuse, and cardiovascular disease. The aim of the study was to determine whether catecholamines affect nitric oxide (NO) production, as NO is a vasodilator and counteracts the harmful effects of catecholamines. RAW264.7 macrophage cells were cultured with lipopolysaccharide (LPS)+/-epinephrine, norepinephrine, and dopamine at 5x10(-6)M concentrations for 24h. Supernatants were harvested for measuring NO by spectrophotometry using the Greiss reagent and cells were harvested for detecting inducible NO synthase (iNOS) by Western blot. NO production in RAW 264.7 macrophages was increased significantly by addition of LPS (0.5-10ng/ml) in a dose-dependent fashion. The NO production induced by LPS was further enhanced by epinephrine and norepinephrine, and to a lesser extent by dopamine. These increases in NO correlated with expression of iNOS protein in these cells. The enhancing effect of iNOS synthesis by epinephrine and norepinephrine on LPS-induced macrophages was down regulated by beta-adrenoceptor antagonist, propranolol, and dexamethasone. The results suggest that catecholamines have a synergic effect on LPS in induction of iNOS synthesis and NO production, and this may mediate some of the vascular effects of infection. These data support a novel role for catecholamines in disorders such as septic shock and cocaine use, and indicate that beta-adrenoceptor antagonists and glucocorticoids may be used therapeutically for modulation of the catecholamine-NO axis in disease states.

Adrenergic beta-Antagonists↗

PubMed2002

The uptake of tocopherols by RAW 264.7 macrophages.

BACKGROUND: Alpha-Tocopherol and gamma-tocopherol are the two major forms of vitamin E in human plasma and the primary lipid soluble antioxidants. The dietary intake of gamma-tocopherol is generally higher than that of alpha-tocopherol. However, alpha-tocopherol plasma levels are about four fold higher than those of gamma-tocopherol. Among other factors, a preferential cellular uptake of gamma-tocopherol over alpha-tocopherol could contribute to the observed higher plasma alpha-tocopherol levels. In this investigation, we studied the uptake and depletion of both alpha-tocopherol and gamma-tocopherol (separately and together) in cultured RAW 264.7 macrophages. Similar studies were performed with alpha-tocopheryl quinone and gamma-tocopheryl quinone, which are oxidation products of tocopherols. RESULTS: RAW 264.7 macrophages showed a greater uptake of gamma-tocopherol compared to alpha-tocopherol (with uptake being defined as the net difference between tocopherol transported into the cells and loss due to catabolism and/or in vitro oxidation). Surprisingly, we also found that the presence of gamma-tocopherol promoted the cellular uptake of alpha-tocopherol. Mass balance considerations suggest that products other than quinone were formed during the incubation of tocopherols with macrophages. CONCLUSION: Our data suggests that gamma-tocopherol could play a significant role in modulating intracellular antioxidant defence mechanisms. Moreover, we found the presence of gamma-tocopherol dramatically influenced the cellular accumulation of alpha-tocopherol, i.e., gamma-tocopherol promoted the accumulation of alpha-tocopherol. If these results could be extrapolated to in vivo conditions they suggest that gamma-tocopherol is selectively taken up by cells and removed from plasma more rapidly than alpha-tocopherol. This could, in part, contribute to the selective maintenance of alpha-tocopherol in plasma compared to gamma-tocopherol.

Journal Article↗

PubMed2002

The influence of dietary iron and tocopherols on oxidative stress and ras-p21 levels in the colon.

The purpose of this investigation was to determine how dietary levels of alpha-tocopherol, gamma-tocopherol and iron influence oxidative stress and ras-p21 levels in the colon. Rats were fed diets deficient in tocopherols (-E) or supplemented with either 0.156 mmol of alpha-tocopherol (AE)/kg diet or 0.156 mmol of gamma-tocopherol (GE)/kg of diet. Half the rats in each of these three groups received dietary iron at a level of 35 mg/kg diet and the other half at eight times this level (280 mg/kg diet). Rats fed the AE diets had higher levels of Vitamin E in feces, colonocytes, plasma and liver than did rats fed the GE diets. Dietary iron levels did not influence tocopherol levels in plasma, liver or feces. For colonocytes, high dietary iron decreased tocopherol levels. The ratio of gamma-tocopherol (in the GE groups) to alpha-tocopherol (in the AE groups) was 0.13 for plasma, 0.11 for liver, 0.28 for colonocytes and 0.51 for feces. The plasma ratio is not, therefore, predictive of the ratio in colonocytes and feces. High levels of dietary iron increased levels of fecal lipid hydroperoxides. Moreover, rats fed the GE diets had lower levels of fecal lipid hydroperoxides than rats fed the AE diets. The levels of ras-p21 were significantly lower in rats fed the GE diets compared with rats fed the AE diets. The gamma-tocopherol may, therefore, play a significant role in preventing colon cancer. High levels of dietary iron were found to promote oxidative stress in feces and colonocytes.

Animals↗