Dunne Fong

In vivo inhibition of nitric oxide synthase gene expression by curcumin, a cancer preventive natural product with anti-inflammatory properties.

Curcumin is a naturally occurring, dietary polyphenolic phytochemical that is under preclinical trial evaluation for cancer preventive drug development and whose working pharmacological actions include anti-inflammation. With respect to inflammation, in vitro, it inhibits the activation of free radical-activated transcription factors, such as nuclear factor kappaB (NFkappaB) and AP-1, and reduces the production of pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF alpha), interleukin-1beta (IL-1beta), and interleukin-8. Inducible nitric oxide synthase (iNOS) is an inflammation-induced enzyme that catalyzes the production of nitric oxide (NO), a molecule that may lead to carcinogenesis. Here, we report that in ex vivo cultured BALB/c mouse peritoneal macrophages, 1-20 microM of curcumin reduced the production of iNOS mRNA in a concentration-dependent manner. Furthermore, we demonstrated that, in vivo, two oral treatments of 0.5 mL of a 10-microM solution of curcumin (92 ng/g of body weight) reduced iNOS mRNA expression in the livers of lipopolysaccharide(LPS)-injected mice by 50-70%. Although many hold that curcumin needs to be given at dosages that are unattainable through diet to produce an in vivo effect, we were able to obtain potency at nanomoles per gram of body weight. This efficacy is associated with two modifications in our preparation and feeding regimen: 1) an aqueous solution of curcumin was prepared by initially dissolving the compound in 0.5 N NaOH and then immediately diluting it in PBS; and 2) mice were fed curcumin at dusk after fasting. Inhibition was not observed in mice that were fed ad lib., suggesting that food intake may interfere with the absorption of curcumin.

Inhibition of inducible nitric oxide synthase gene expression and enzyme activity by epigallocatechin gallate, a natural product from green tea

Chronic inflammation has been implicated as the underlying factor in the pathogenesis of many disorders. In the past decade, inflammation-related endogenous production of reactive nitrogen species, similar to oxygen free radicals, has also been suggested as a risk factor for cancer, in addition to the well-studied exogenous nitroso compounds. Epidemiological, in vitro, and animal model studies have implicated green tea to be protective against nitroso compound-induced and inflammation-related cancer. Therefore, we investigated the effect of epigallocatechin-3-gallate (EGCG), one of the known biologically active catechins contained in green tea, on the production of nitric oxide (NO.). We have shown previously that EGCG reduces NO. production as measured by nitrite accumulation in the culture medium. Expanding on this finding, in this report we show that EGCG may do so by two mechanisms: reduction of inducible nitric oxide synthase (iNOS) gene expression and inhibition of enzyme activity. Addition of 1-10 microM EGCG to lipopolysaccharide- and interferon-gamma-activated mouse peritoneal cells reduced iNOS mRNA expression concentration dependently, to 82-14%, as measured by relative reverse transcription-polymerase chain reaction. Addition of 50-750 microM EGCG, in a concentration-dependent manner, inhibited the enzyme activity of iNOS, to 85-14%, and neuronal nitric oxide synthase (nNOS), to 93-56%, as measured by citrulline formation. EGCG competitively inhibited binding of arginine and tetrahydrobiopterin, and the gallate structure is important for this action.

Synergy between ethanol and grape polyphenols, quercetin, and resveratrol, in the inhibition of the inducible nitric oxide synthase pathway.

In atherosclerosis and tumor initiation, inducible nitric oxide synthase (iNOS) has been implicated in the damage of vascular walls and DNA, respectively. Moderate consumption of red wine has been ascribed as a preventive for coronary heart disease; however, there has been much debate over whether the beneficial effect is from grape polyphenolic components or ethanol. We studied the interaction of grape compounds on nitric oxide (NO) production by macrophages, mediators of blood vessel damage in atherosclerosis. For the murine macrophage cell line RAW 264.7, stimulation with lipopolysaccharide and interferon-gamma led to expression of the iNOS gene and production of NO. The polyphenols quercetin and resveratrol at a micromolar range suppressed iNOS gene expression and NO production, as determined by reverse transcription-polymerase chain reaction and nitrite assay. The polyphenols were also found to be scavengers of NO in an acellular system using sodium nitroprusside under physiological conditions. Ethanol, at a moderate level, did not produce any appreciable level of reduction of iNOS or NO activity. However, its presence at 0.1 to 0.75% enhanced the effect of grape polyphenols concentration-dependently. Thus, the interaction between these components plays a significant role in the health effects of red wine, especially with respect to their effect on the NO pathway.

Inhibition of growth and sensitization to cisplatin-mediated killing of ovarian cancer cells by polyphenolic chemopreventive agents

The polyphenolic compounds curcumin and quercetin increased sensitivity of ovarian cancer cells (CAOV3 and SKOV3) to cisplatin. The effect was obtained when the compounds were added simultaneously with cisplatin, as well as when they were added 24 h before. High serum levels of certain cytokines, for example interleukin‐6 (IL‐6), have been associated with poor prognosis and cisplatin resistance in various forms of cancer. Furthermore, it has been hypothesized that cytokines may increase proliferation, metastasis, and stimulate production of detoxification enzymes and multi‐drug resistant proteins. Curcumin inhibits the production of many cytokines. The two ovarian cell lines differ significantly in IL‐6 production, and correspondingly the high producer, CAOV3, was less susceptible to cisplatin. Curcumin inhibited the production of IL‐6 in this cell suggesting that one of the mechanisms for synergy between cisplatin and curcumin was by reducing the autologous production of IL‐6. However, the synergy was also observed in the low IL‐6 producer, SKOV3, indicating that the action was most probably a result of multiple targeting. In sum, this study suggests that the compounds, curcumin and quercetin, potentially may be useful for enhancing drug sensitivity in certain cancer. J. Cell. Physiol. 194: 63–70, 2002.

Epigallocatechin-3-gallate delivers hydrogen peroxide to induce death of ovarian cancer cells and enhances their cisplatin susceptibility

The green tea polyphenol epigallocatechin‐3‐gallate (EGCG) has cancer chemopreventive properties against various types of cancers. The compound is known to attack various targets in transformed cells. In this report, we examined the action of EGCG on ovarian cancer cells. Eight ovarian cancer cell lines were tested (SKOV3, CAOV3, OVCAR3, OVCAR10, A2780, CP70, C30, and C200) and showed IC50s for EGCG at the micromolar range, including ones that are resistant to the chemotherapeutic drug cisplatin. The ovarian cancer cells were sensitive to H2O2 at similar concentrations, and EGCG treatment led to enhanced intracellular H2O2. Neutralization with pyruvate, a scavenger of H2O2, suggests that the toxicity of EGCG may be mediated by oxidative stress from the free radical. Addition of Tempol, a superoxide dismutase mimetic, demonstrates that H2O2 might be generated endogenously from superoxide. The toxicity of cisplatin and the development of cisplatin resistance are major obstacles in treatment of ovarian cancer. We found that addition of EGCG amplified the toxicity of cisplatin. EGCG increased cisplatin potency by three to six‐fold in SKOV3, CAOV3, and C200 cells, the latter being a cell line induced to have several hundred fold resistant to cisplatin above the parental line. Our findings suggest that EGCG may accentuate oxidative stress to inhibit growth of ovarian cancer cells and sensitize them to cisplatin. J. Cell. Physiol. 207: 389–396, 2006.

Curcumin inhibits the side population (SP) phenotype of the rat C6 glioma cell line: Towards targeting of cancer stem cells with phytochemicals

The phytochemical curcumin, from the Indian spice turmeric, has many biological properties, including anti-inflammatory and anti-carcinogenic activities. We have examined the effects of curcumin on the rat C6 glioma cell line. Treated and control cells were analyzed by Hoechst 33342 dye and flow cytometry. We observed a decrease in the side population (SP) of C6 cells after daily curcumin treatment of the C6 cells. Direct incubation of curcumin to C6 cells during the Hoechst assay also decreased SP. Since SP has been associated with stem cell populations, curcumin may be a dietary phytochemical with potential to target cancer stem cells.

Enhanced levels of cathepsin B mRNA in murine tumors

Relative amounts of mRNA for cathepsin B were measured in normal murine liver and three murine tumors, an invasive liver tumor (hepatoma, Hepa cl 9) and two melanoma variants (B16‐F1 and B16 amelanotic melanoma, B16a). Using a human cDNA to the cathepsin B coding region as a hybridization probe, we detected two species of cathepsin B specific RNA transcripts (2.2 and 4.1 kb) in total RNA preparation of all four tissues. The concentrations of the 2.2 and 4.1 kb species were 3.6 and 2.7‐fold greater in the highly metastatic B16a melanoma than in normal liver. The concentration of the 2.2 kb species in the invasive hepatoma wa 1.7‐fold greater than in normal liver. The increased levels of the 2.2 kb message were reflected in increases in activity of cathepsin B in both Hepa cl 9 and B16a.

A microplate assay for Leishmania amazonensis promastigotes expressing multimeric green fluorescent protein.

Abstract. Convenient and economical assays capable of screening many compounds are vital to advance the development of drug therapy. This is particularly important for many of the infections that occur mainly in the Third World. The development of such a spectrofluorometric assay for the protozoan parasite Leishmania is presented here. Using multimeric (four monomers) green fluorescent protein (GFP), Leishmania amazonensis promastigotes were generated with brightness measurable in 96-well microtiter plates. The promastigotes maintained the parental characteristics, were infective to murine macrophages and to mice, and the level of GFP fluorescence corresponded to the number of inoculated cells. The feasibility of using this assay for testing drugs kinetically and in a concentration-dependent manner, under microplate culture condition, was demonstrated with amphotericin B and the herbicide oryzalin, respectively. This assay is the first to allow a real-time analysis of antileishmanial agents with live promastigotes. The method of expressing multimeric GFP for in vitro drug screening is likely to be extendable to many species of parasitic protozoa.

Curcumin overcomes the inhibitory effect of nitric oxide on Leishmania

Upon Leishmania infection, macrophages are activated to produce nitrogen and oxygen radicals simultaneously. It is well established that the infected host cells rely on nitric oxide (NO) as the major weapon against the intracellular parasite. In India where leishmaniasis is endemic, the spice turmeric is used prolifically in food and for insect bites. Curcumin, the active principle of turmeric, is a scavenger of NO. This report shows that curcumin protects promastigotes and amastigotes of the visceral species, Leishmania donovani, and promastigotes of the cutaneous species, L. major, against the actions of S-nitroso-N-acetyl-D,L-penicillamine (SNAP) and DETANONOate, which release NO, 3-morpholino-sydnonimine hydrochloride (SIN-1), which releases NO and superoxide, and peroxynitrite, which is formed from the reaction of NO with superoxide. Thus, curcumin, as an antioxidant, is capable of blocking the action of both NO and NO congeners on the Leishmania parasite.

Structure-function analysis of antimicrotubule dinitroanilines against promastigotes of the parasitic protozoan Leishmania mexicana.

Although leishmaniasis is a major tropical disease, the currently available drugs are toxic and inadequate. We show that the antimicrotubule herbicide trifluralin has antileishmania activity. The present study aimed at deducing the relationship between the structure of the molecule and its antiprotozoan activity. Nine dinitroanilines, all of which were analogs of trifluralin, were compared. We found that pendimethalin was 2.5-fold more potent than trifluralin, and the higher efficacy may be correlated with molecular structural features that increase the accessibility to one nitro group. This association was further supported by molecular modeling. Moreover, trifluralin samples from two sources differed in their activities by more than threefold, and gas column chromatography showed that impurities were present in the more potent sample.