Marion M. Chan

Antimicrobial effect of resveratrol on dermatophytes and bacterial pathogens of the skin.

The phytoalexin resveratrol is commonly found in food and drinks, including red wine, grapes, and peanuts. Many studies have shown that this compound has anti-inflammatory properties, and it has been ascribed as having health benefits that help to prevent cancer and coronary heart disease. A treatment that combines antimicrobial and anti-inflammatory actions may be desirable for alleviating many skin conditions that range in severity. Therefore, we evaluated the antimicrobial activity of resveratrol against bacteria and dermatophytes that are major etiologic agents of human skin infections. Using the broth microdilution protocol of the National Committee for Clinical Laboratory Standards (NCCLS) M7-A5, growth of the bacterial species Staphylococcus aureus, Enterococcus faecalis, and Pseudomonas aeruginosa was inhibited at 171-342 microg/mL of resveratrol in the solvent dimethyl sulfoxide. Using the NCCLS protocol M38-P, activity against the fungal species Trichophyton mentagrophytes, Trichophyton tonsurans, Trichophyton rubrum, Epidermophyton floccosum, and Microsporum gypseum was also tested. The growth of dermatophytes was inhibited at 25-50 microg/mL of resveratrol. Thus, this study indicates a novel application for resveratrol, a molecule of plant defense, to combat human fungal pathogens. Resveratrol and its analogs may have wide application to skin conditions that afflict a significant portion of our population, and may also have promising clinical potentials in diabetic wounds.

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.

Resolution of Inflammation in Murine Autoimmune Arthritis Is Disrupted by Cyclooxygenase-2 Inhibition and Restored by Prostaglandin E2-Mediated Lipoxin A4 Production

Acute inflammation follows defined phases of induction, inflammation and resolution, and resolution occurs by an active process that requires cyclooxygenase-2 (COX-2) activity. This study aims to address whether this paradigm extends to recognized model of chronic inflammation. We demonstrated that murine collagen-induced arthritis follows a similar sequential course. Interestingly, COX-2 and its metabolite, the presumably proinflammatory PGE2, are present in the joints during resolution, and blocking COX-2 activity and PGE2 production within this period perpetuated, instead of attenuated, inflammation. Repletion with PGE2 analogs restored homeostasis, and this function is mediated by the proresolving lipoxygenase metabolite, lipoxin A4, a potent stop signal. Thus, the study provided in vivo evidence for a natural, endogenous link between the cyclooxygenase–lipoxygenase pathways and showed that PGE2 serves as a feedback inhibitor essential for limiting chronic inflammation in autoimmune arthritis. These findings may explain the enigma regarding why COX-2 inhibitors are palliative rather than curative in humans, because blocking resolution may mitigate the benefit of preventing induction.

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.

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.

Long-term use of an antiinflammatory, curcumin, suppressed type 1 immunity and exacerbated visceral leishmaniasis in a chronic experimental model.

Inflammation is considered the underlying cause of numerous disorders, and the practice of taking antiinflammatories as diet supplements has become increasingly prevalent. This study addresses the bioavailablity of a well-established dietary antiinflammatory, curcumin, and examines its effect on adaptive immunity. Visceral leishmaniasis is a major parasitic disease which protection relies on cell-mediated immunity and production of nitric oxide. We found that long-term, low-dose, oral consumption of curcumin activates peroxisome proliferator-activated receptor-γ, deactivates type 1 response, inhibits inducible nitric oxide synthase, and interferes with adaptive immunity to exacerbate the pathogenesis of Leishmania donovani infection in vivo. These in vivo effects can be correlated to activities on infected residential macrophages in vitro. Therefore, when reactive radicals generated from inflammation play the dominant role in elimination of pathogens, excessive use of the antioxidative supplements may compromise microbial defense. Nonetheless, it should be noted with equal importance that our finding, conversely, also strengthens the prospect that curcumin may alleviate type 1 response disorders.

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.

Peroxisome proliferator-activated receptor (PPAR): balance for survival in parasitic infections.

Parasitic infections induce a magnitude of host responses. At the opposite ends of the spectrum are those that ensure the hosts needs to eliminate the invaders and to minimize damage to its own tissues. This review analyzes how parasites would manipulate immunity by activating the immunosuppressive nuclear factor, peroxisome proliferator-activated receptors (PPARs) with type 2 cytokines and free fatty acids from arachidonic acid metabolism. PPARs limit the action of type 1 immunity, in which classically activated macrophages act through the production of proinflammatory signals, to spare the parasites. They also favor the development of alternately activated macrophages which control inflammation so the host would not be destroyed. Possibly, the nuclear factors hold a pivotal role in the establishment of chronic infection by delicately balancing the pro- and anti-inflammatory signaling mechanisms and their ligands may be used as combination therapeutics to limit host pathology.