Joseph M. Wu

What Is New for an Old Molecule? Systematic Review and Recommendations on the Use of Resveratrol

Background Resveratrol is a natural compound suggested to have beneficial health effects. However, people are consuming resveratrol for this reason without having the adequate scientific evidence for its effects in humans. Therefore, scientific valid recommendations concerning the human intake of resveratrol based on available published scientific data are necessary. Such recommendations were formulated after the Resveratrol 2010 conference, held in September 2010 in Helsingør, Denmark. Methodology Literature search in databases as PubMed and ISI Web of Science in combination with manual search was used to answer the following five questions: 1Can resveratrol be recommended in the prevention or treatment of human diseases?; 2Are there observed “side effects” caused by the intake of resveratrol in humans?; 3What is the relevant dose of resveratrol?; 4What valid data are available regarding an effect in various species of experimental animals?; 5Which relevant (overall) mechanisms of action of resveratrol have been documented? Conclusions/Significance The overall conclusion is that the published evidence is not sufficiently strong to justify a recommendation for the administration of resveratrol to humans, beyond the dose which can be obtained from dietary sources. On the other hand, animal data are promising in prevention of various cancer types, coronary heart diseases and diabetes which strongly indicate the need for human clinical trials. Finally, we suggest directions for future research in resveratrol regarding its mechanism of action and its safety and toxicology in human subjects.

Resveratrol: a cardioprotective substance.

Coronary heart disease (CHD) is a major and preventable cause of morbidity and death in the United States. Recently, significant research efforts have been directed at an epidemiological phenomenon known as the “French paradox.” This observation refers to the coexistence of high risk factors with unanticipated low incidence of CHD, and is postulated to be associated with low‐to‐moderate consumption of red wine. In vivo studies have shown that red wine intake is more CHD‐preventative in comparison to other alcoholic drinks; enhanced cardioprotection may be attributed to grape‐derived polyphenols, e.g., resveratrol, in red wine. This review summarizes results of in vitro and animal studies showing that resveratrol exerts multifaceted cardioprotective activities, as well as evidence demonstrating the presence of proteins specifically targeted by resveratrol, as exemplified by N‐ribosyldihydronicotinamide:quinone oxidoreductase, NQO2. A mechanism encompassing nongenomic and genomic effects and a research roadmap is proposed as a framework for uncovering further insights on cardioprotection by resveratrol.

Detection of caspases activation by fluorochrome‐labeled inhibitors: Multiparameter analysis by laser scanning cytometry

BACKGROUND The fluorochrome-labeled inhibitors of caspases (FLICA) were recently used as markers of activation of these enzymes in live cells during apoptosis (Bedner et al.: Exp Cell Res 259:308-313, 2000). The aims of this study were to (a) explore if FLICA can be used to study intracellular localization of caspases; (b) combine the detection of caspase activation with analysis of the changes with cell morphology detected by microscopy and laser scanning cytometry (LSC); and (c) adapt the assay to fixed cells that would enable correlation, by multiparameter analysis, of caspase activation with the cell attributes that require cell permeabilization in order to be measured. METHODS Apoptosis of human MCF-7, U-937, or HL-60 cells was induced by camptothecin (CPT) or tumor necrosis factor-alpha (TNF-alpha) combined with cycloheximide (CHX). Binding of FLICA to apoptotic versus nonapoptotic cells was studied in live cells as well as following their fixation and counterstaining of DNA. Intensity of cell labeling with FLICA and DNA-specific fluorochromes was measured by LSC. RESULTS Exposure of live cells to FLICA led to selective labeling of cells that had morphological changes characteristic of apoptosis. The FLICA labeling withstood cell fixation and permeabilization, which made it possible to stain DNA and measure its content for identification of the cell cycle position of labeled cells. When fixed cells were treated with FLICA, both apoptotic and nonapoptotic cells became strongly labeled and the labeling pattern was consistent with the localization of caspases as reported in the literature. A translocation of the FLICA binding targets from mitochondria to cytosol was seen in the MCF-7 cells treated with CPT. FLICA binding was largely (> 90%) prevented by the substrates of the caspases or by the unlabeled caspase inhibitors having the same peptide moiety as the respective FLICA. CONCLUSIONS The detection of caspase activation combined with cell permeabilization requires exposure of live cells to FLICA followed by their fixation. Cell reactivity with the respective FLICA, under these conditions, identifies the activated caspases and makes it possible to correlate their activation with the cell cycle position and other cell attributes that can be measured only after cell fixation/permeabilization. FLICA can also be used to study intracellular localization of caspases, including their translocation.

Mechanism of fenretinide (4-HPR)-induced cell death

Abstract4-HPR (fenretinide) is a synthetic analog of retinoic acid (RA) whose potential as a chemopreventative agent has gained support from in vitro and animal experiments and in limited clinical trials. Comparative analyses of cellular, biochemical, and molecular properties of fenretinide with RA using various tissue culture cells reveal that a key distinction between these two retinoids lies in the ability of fenretinide to induce programmed cell death, also known as apoptosis. Here we review the composite evidence for induction of apoptosis in fenretinide-treated cells. Assays used to validate apoptosis in various cell types are also summarized. Apoptosis in response to fenretinide primarily occurs by a receptor-independent mechanism, which is accompanied by increases in signaling molecules, e.g., ceramide, and cysteine-dependent aspartate-directed proteases, termed caspases, including execution caspase-3. Both caspase-3 inhibitor DEVD-CHO and ceramide synthase inhibitor fumonisin B1 (FB1) block fenretinide-induced apoptosis. Increase in caspase-3 appears to result from fenretinide-elicited stabilization of procaspase-3 zymogen. We also review apoptotic regulatory proteins such as inhibitor of apoptosis (IAPs) and second mitochondria-derived activator of caspase (SMACs) that participate in the coordinate control of caspase activities. The existence of a large number of proteins capable of modulating apoptosis via activation or inhibition of caspases, coupled with the fact that both the initiation and execution phases of apoptosis utilize pre-existing zymogens, which, once set in motion, culminates in an irreversible apoptotic cascade, raise the possibility that the on/off switch of apoptosis is linked to an intricate intracellular regulatory network, capable of responding to external stimuli such as fenretinide. This network functions to provide checks/balances of the need for apoptosis as well as to minimize and prevent untimely errors in apoptosis. We suggest that dynamic and coordinated regulation of apoptosis by such a hypothetical network in vivo may involve co-localization of pro- and anti-apoptotic proteins and their respective activators/inhibitors in a macromolecular modular unit which we propose to be named caspasomes. Fenretinide also induces apoptosis by elevating reactive oxygen species (ROS), unrelated to changes in ceramide-caspases. Thus multiple, distinct pathways contribute to the induction of apoptosis by fenretinide.

Effect of resveratrol on intimal hyperplasia after endothelial denudation in an experimental rabbit model.

The ability of resveratrol to inhibit vascular intimal thickening was tested in an experimental model in which endothelial denudation was performed in the normal rabbit iliac artery. Resveratrol (2 approximately 4mg/ kg/d) was administered intragastrically for 5 weeks beginning 1 week before denudation. At the higher concentration of resveratrol, the intimal hyperplasia of injured vascular wall was effectively inhibited; the intimal proliferation index also was significantly less than that in the untreated control group (0.28 +/- 0.07 vs 0.41 +/- 0.13, respectively, p<0.01); the relative luminal area increased from 0.38 +/- 0.06 in the untreated control group to 0.53 +/- 0.10 in the resveratrol treatment group (p < 0.001); and the count of smooth muscle cells in the thickened intima was statistically significantly reduced in the high dose resveratrol treatment group than that in the untreated group (1,115 +/- 510 vs 1,796 +/- 963, respectively, p < 0.05). Resveratrol added to the culture media of cultured rabbit vascular smooth muscle cells inhibited DNA synthesis in a dose-dependent manner. These results showing that resveratrol is capable of inhibiting intimal hyperplasia of injured artery raise the possibility that this polyphenol might have clinical potential in prevention and treatment of restenosis after angioplasty.

Regulation of androgen receptor (AR) and prostate specific antigen (PSA) expression in the androgen‐responsive human prostate LNCaP cells by ethanolic extracts of the Chinese herbal preparation, PC‐SPES

As part of the study on the potential use of natural product‐based combination therapy for treating prostate cancer, we have investigated the effects of a “HPLC standardized” herbal preparation, PC‐SPES, on the prostate LNCaP cell line. Proliferation of the LNCaP cells was inhibited by a 4‐6 day incubation with ethanolic extracts of PC‐SPES. Decrease of cell growth was accompanied by a 60‐70 % down‐regulation of the proliferating cell nuclear antigen (PCNA) and level of secreted PSA. A smaller and more variable decrease (20‐40%) in the level of intracellular PSA was also observed. The PC‐SPES‐modulated PSA changes occurred concurrently with the decrease of AR expression, based on Western blot analysis and binding to the radioactive ligand [3H]R1881. A 60% decrease in R1881 binding occurred after a 24 h incubation with PC‐SPES. These results suggest that PC‐SPES negatively affects cell growth in part through its ability to modulate changes in PCNA, and may decrease PSA levels indirectly by suppressing AR expression.

Regulation of G1/S transition and induction of apoptosis in HL‐60 leukemia cells by fenretinide (4HPR)

We previously reported that all‐trans retinoic acid (RA) and fenretinide (4HPR) suppress HL‐60 leukemia cell growth and cause partial cell arrest in the G1‐to‐S phase. Moreover, 4HPR but not RA induces apoptosis in HL‐60 cells. To investigate further the observed biological effects, cyclin D1 and cdk4 expression and the level of phosphorylation of the retinoblastoma protein Rb were assessed. Cyclin D1 and cdk4 expression and Rb phosphorylation were significantly reduced, by 40–75%, after 24 hr of treatment with RA or 4HPR; these decreases were either transient, e.g., only at 24 hr for cdk4, or sustained for 72 hr. In general, more pronounced decreases were seen in the 4HPR‐treated cells. Evidence for 4HPR‐induced apoptosis comes from (1) cleavage of the enzyme poly(ADP‐ribose) polymerase (PARP) to an 89‐kDa truncated product, (2) appearance of DNA ladders on agarose gel electrophoresis, and (3) higher incorporation in situ of digoxigenin nucleotides into the free 3′‐ends of DNA. Overnight pretreatment with 0.5–5.0 μM of the CPP32 inhibitor DEVD, but not the ICE inhibitor YVAD, significantly reduced the specific processing of PARP, suggesting that CPP32 is involved in the mechanism of action of 4HPR. Analysis of 2 lipid‐derived second messengers, ceramide and diacylglycerol (DAG), as a function of time of treatment with RA or 4HPR, showed ceramide but not DAG to be significantly albeit transiently increased 2‐fold at 3 hr, by 4HPR. To test further whether ceramide may be involved in the signaling cascade that culminates in the induction of apoptosis in 4HPR‐treated HL‐60 cells, the effects of fumonisin B1, an inhibitor of ceramide synthase, were studied. Simultaneous treatment of cells with 4HPR and 25–100 μM fumonisin B1 resulted in a dose‐dependent reduction in the elevation in ceramide, the extent of PARP cleavage, and induction of apoptosis. Pretreatment with DEVD or YVAD, on the other hand, had no effect on the 4HPR‐induced increase in ceramide. Int. J. Cancer 78:53–61, 1998.© 1998 Wiley‐Liss, Inc.

Induced cytoskeletal changes in bovine pulmonary artery endothelial cells by resveratrol and the accompanying modified responses to arterial shear stress

BackgroundAtherosclerosis and coronary heart disease (CHD) are significant contributors to morbidity and mortality in developed countries. A noted exception is the low mortality of CHD in France, particularly the southwest region. This phenomenon, commonly referred to as the French paradox, may be associated with high consumption of red wine. We investigate whether the cardioprotective activity of red wine may involve the grape skin-derived polyphenol, resveratrol. We further test the possibility that resveratrol acts by modulating structural and functional changes in endothelial cells lining the blood vessel wall.ResultsBovine pulmonary artery endothelial cells (BPAEC) were incubated with resveratrol, with and without concurrent exposure to simulated arterial shear stress. Resveratrol significantly affected proliferation and shape of BPAEC; growth was suppressed and cells became elongated, based on morphologic analysis of rhodamine-conjugated phalloidin stained F-actin by confocal microscopy. Using selective signaling inhibitors, we showed that the resveratrol-induced cellular phenotype was dependent on intracellular calcium and tyrosine kinase activities, and assembly of actin microfilaments and microtubules, but was unrelated to PKC activity. Exposure to simulated arterial flow revealed that, whereas controls cells easily detached from the culture support in a time-dependent manner, resulting in total cell loss after a 5 min challenge with simulated arterial flow conditions, a significant percentage of the treated cells remained attached to the cultured plastic coverslips under identical experimental conditions, suggesting that they adhered more strongly to the surface. Western blot analysis shows that whereas cells treated with 25 μM and 100 μM resveratrol had no change in total ERK1/2, treatment did result in an increase in phosphorylated ERK1/2, which probably involved stabilization of the active enzyme. An increase in nitric oxide synthase expression was detected as early as 6 h and persisted for up to 4 days of treatment.ConclusionsResults of our studies show that resveratrol interacts with endothelial cells in vitro to elicit morphological and structural changes; the observed changes support the interpretation that resveratrol acts as a cardioprotective agent.

Effects of fenretinide (4-HPR) on prostate LNCaP cell growth, apoptosis, and prostate-specific gene expression

Although fenretinide (4‐HPR) is currently being evaluated in a phase II clinical study for the chemoprevention of prostate cancer [Greenwald et al.: CA 45:31–49, 1995], the mechanism underlying its antineoplastic activity has not been elucidated.

Antimicrobial susceptibility of Ehrlichia phagocytophila.

ABSTRACT Human granulocytic ehrlichiosis is a recently described disease caused by an obligate intracellular gram-negative organism recently named Ehrlichia phagocytophila. To expand our knowledge of the susceptibility of E. phagocytophila, we tested six New York State isolates for susceptibility to 12 antimicrobials using an HL-60 cell culture system. All of the isolates were susceptible to doxycycline (MIC, ≤0.125 μg/ml; minimum bactericidal concentration [MBC], 0.125 to 0.5 μg/ml), rifampin (MIC, ≤0.125 μg/ml; MBC, ≤0.125 μg/ml), ofloxacin (MIC, ≤2 μg/ml; MBC, ≤2 μg/ml), levofloxacin (MIC, ≤1 μg/ml; MBC, ≤1 μg/ml), and trovafloxacin (MIC, ≤0.032 μg/ml; MBC, ≤0.032 μg/ml). Isolates were uniformly resistant to amoxicillin, ceftriaxone, erythromycin, azithromycin, clarithromycin, and amikacin. For one strain, the MBC of chloramphenicol was ≤8 μg/ml. These data suggest that quinolone antibiotics and rifampin may be alternative agents for patients with intolerance to tetracyclines.