Resveratrol exerts differential effects in vitro and in vivo against ovarian cancer cells.

Epithelial ovarian cancer represents the most lethal gynecological cancer, and the high mortality rate makes this malignancy a major health concern. Poor prognosis results from an inability to detect ovarian cancers at an early, curable stage, as well as from the lack of an effective therapy. Thus, effective and novel strategies for prevention and treatment with non-toxic agents merit serious consideration. Resveratrol, obtained from grapes, berries, peanuts and red wine, has been shown to have a potent growth-inhibitory effect against various human cancer cells as well as in in vivo preclinical cancer models. The objective here was to evaluate potential antitumor effects of resveratrol in both in vitro and in vivo NuTu-19 ovarian cancer models. In vitro an invasion assay was performed. After 48 h, the numbers of viable cells that invaded the extracellular matrix layer were reduced by 94% with resveratrol in comparison to control. For the in vivo anti-tumor assessment, 10 rats were injected with NuTu-19 cells into the ovarian bursa. Thereafter, half were provided with a diet mixed with a dose of 100 mg resveratrol/kg body weight/day for 28 days. Following sacrifice, anticancer effects were assessed by histological evaluation of ovarian as well as surrounding tissues, and immunohistochemical detection of cell proliferation and apoptosis, but there were no observable differences between the control and resveratrol-treated groups for any of the biological endpoints. While resveratrol is effective in suppressing the in vitro cellular invasion of NuTu-19 ovarian cancer cells, these effects do not appear to impact on in vivo NuTu-19 ovarian cancers in rats.

A phase I study of the metal ionophore clioquinol in patients with advanced hematologic malignancies.

UNLABELLED: Clioquinol is a small-molecule metal ionophore that inhibits the proteasome through a metal-dependent mechanism. Here, we report a phase I study of clioquinol in patients with refractory hematologic malignancies. Neuropathy and abdominal pain were dose-limiting toxicities. Minimal pharmacodynamic effects were observed, and there were no clinical responses. BACKGROUND: Clioquinol is a small-molecule metal ionophore that inhibits the enzymatic activity of the proteasome and displays preclinical efficacy in hematologic malignancies in vitro and in vivo. Therefore, we conducted a phase I clinical trial of clioquinol in patients with refractory hematologic malignancies to assess its safety and determine its biological activity in this patient population. METHODS: Patients with refractory hematologic malignancies were treated with increasing doses of oral clioquinol twice daily for 15 doses. Plasma and intracellular levels of clioquinol were measured. Enzymatic activity of the proteasome was measured before and after drug administration. RESULTS: Sixteen cycles of clioquinol were administered to 11 patients with 5 patients reenrolled at the next dose level as per the permitted intrapatient dose escalation. Dose-limiting neurotoxicity and abdominal pain were observed at a dose of 1600 mg twice daily. Intracellular drug levels were low. Minimal inhibition of the proteasome was observed. No clinical responses were observed. CONCLUSION: In patients with refractory hematologic malignancies, the maximal tolerated dose of clioquinol was determined. Minimal inhibition of the proteasome was observed at tolerable doses, likely due to low intracellular levels of the drug.

Chemopreventive doses of resveratrol do not produce cardiotoxicity in a rodent model of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC), one of the most lethal cancers, results in more than one million fatalities worldwide every year. In view of the limited therapeutic alternatives and poor prognosis of liver cancer, preventive control approaches, notably chemoprevention, have been considered to be the best strategy in lowering the present prevalence of the disease. Resveratrol, a naturally occurring antioxidant and antiinflammatory agent found in grapes and red wine, inhibits carcinogenesis with a pleiotropic mode of action. Recently, we have reported that dietary resveratrol significantly prevents chemically-induced liver tumorigenesis in rats. One of the mechanisms of resveratrol-mediated chemoprevention of hepatocarcinogenesis could be related to its antiinflammatory action through hepatic cyclooxygenase (COX-2) inhibition. Although several COX-2 inhibitors are known to exert chemopreventive efficacy, not all are considered ideal candidates for chemoprevention due to the risk of adverse cardiovascular events. Accordingly, the objective of the present study was to evaluate the role of resveratrol on cardiac performance during experimental hepatocarcinogenesis initiated with diethylnitrosamine and promoted by phenobarbital. Rats had free access to diet supplemented with resveratrol four weeks before the carcinogen injection and 14 weeks thereafter. The cardiotoxicity of resveratrol was assessed by monitoring the cardiac function using transthoracic echocardiography as well as Western blot analysis of cardiac tissue. Long-term dietary administration of resveratrol dose-dependently suppressed hepatic tumor multiplicity, the principal endpoint for evaluating the chemopreventive potential of a candidate agent. The chemopreventive effects of resveratrol were also reflected in histopathological assessment of hepatic tissues. Resveratrol did not exhibit any cardiotoxicity but rather improved the cardiac function in a dose-responsive fashion. Our results indicate that resveratrol-mediated chemoprevention of rat liver carcinogenesis is devoid of any adverse cardiovascular events. Resveratrol may be developed as a chemopreventive as well as therapeutic drug for human HCC.

Repeat dose study of the cancer chemopreventive agent resveratrol in healthy volunteers: safety, pharmacokinetics, and effect on the insulin-like growth factor axis.

Resveratrol, a naturally occurring polyphenol, has cancer chemopreventive properties in preclinical models. It has been shown to downregulate the levels of insulin-like growth factor-1 (IGF-I) in rodents. The purpose of the study was to assess its safety, pharmacokinetics, and effects on circulating levels of IGF-I and IGF-binding protein-3 (IGFBP-3) after repeated dosing. Forty healthy volunteers ingested resveratrol at 0.5, 1.0, 2.5, or 5.0 g daily for 29 days. Levels of resveratrol and its metabolites were measured by high performance liquid chromatography-UV in plasma obtained before and up to 24 hours after a dose between days 21 and 28. IGF-I and IGFBP-3 were measured by ELISA in plasma taken predosing and on day 29. Resveratrol was safe, but the 2.5 and 5 g doses caused mild to moderate gastrointestinal symptoms. Resveratrol-3-O-sulfate, resveratrol-4'-O-glucuronide, and resveratrol-3-O-glucuronide were major plasma metabolites. Maximal plasma levels and areas under the concentration versus time curve for the metabolites dramatically exceeded those for resveratrol, in the case of areas under the concentration versus time curve, by up to 20.3-fold. Compared with predosing values, the ingestion of resveratrol caused a decrease in circulating IGF-I and IGFBP-3 (P<0.04 for both), respectively, in all volunteers. The decrease was most marked at the 2.5 g dose level. The results suggest that repeated administration of high doses of resveratrol generates micromolar concentrations of parent and much higher levels of glucuronide and sulfate conjugates in the plasma. The observed decrease in circulating IGF-I and IGFBP-3 might contribute to cancer chemopreventive activity.

Clinical pharmacology of resveratrol and its metabolites in colorectal cancer patients.

Resveratrol is a phytochemical with chemopreventive activity in preclinical rodent models of colorectal carcinogenesis. Antiproliferation is one of the many chemopreventive modes of action it has been shown to engage in. Concentrations of resveratrol, which can be achieved in human tissues after p.o. administration, have not yet been defined. The purpose of this study was to measure concentrations of resveratrol and its metabolites in the colorectal tissue of humans who ingested resveratrol. Twenty patients with histologically confirmed colorectal cancer consumed eight daily doses of resveratrol at 0.5 or 1.0 g before surgical resection. Resveratrol was found to be well tolerated. Normal and malignant biopsy tissue samples were obtained before dosing. Parent compound plus its metabolites resveratrol-3-O-glucuronide, resveratrol-4'-O-glucuronide, resveratrol-3-O-sulfate, resveratrol-4'-O-sulfate, resveratrol sulfate glucuronide, and resveratrol disulfate were identified by high-performance liquid chromatography (HPLC) with UV or mass spectrometric detection in colorectal resection tissue. Quantitation was achieved by HPLC/UV. Cell proliferation, as reflected by Ki-67 staining, was compared in preintervention and postintervention tissue samples. Resveratrol and resveratrol-3-O-glucuronide were recovered from tissues at maximal mean concentrations of 674 and 86.0 nmol/g, respectively. Levels of resveratrol and its metabolites were consistently higher in tissues originating in the right side of the colon compared with the left. Consumption of resveratrol reduced tumor cell proliferation by 5% (P = 0.05). The results suggest that daily p.o. doses of resveratrol at 0.5 or 1.0 g produce levels in the human gastrointestinal tract of an order of magnitude sufficient to elicit anticarcinogenic effects. Resveratrol merits further clinical evaluation as a potential colorectal cancer chemopreventive agent.

Phase I dose escalation pharmacokinetic study in healthy volunteers of resveratrol, a potential cancer chemopreventive agent.

The red grape constituent resveratrol possesses cancer chemopreventive properties in rodents. The hypothesis was tested that, in healthy humans, p.o. administration of resveratrol is safe and results in measurable plasma levels of resveratrol. A phase I study of oral resveratrol (single doses of 0.5, 1, 2.5, or 5 g) was conducted in 10 healthy volunteers per dose level. Resveratrol and its metabolites were identified in plasma and urine by high-performance liquid chromatography-tandem mass spectrometry and quantitated by high-performance liquid chromatography-UV. Consumption of resveratrol did not cause serious adverse events. Resveratrol and six metabolites were recovered from plasma and urine. Peak plasma levels of resveratrol at the highest dose were 539 +/- 384 ng/mL (2.4 micromol/L, mean +/- SD; n = 10), which occurred 1.5 h post-dose. Peak levels of two monoglucuronides and resveratrol-3-sulfate were 3- to 8-fold higher. The area under the plasma concentration curve (AUC) values for resveratrol-3-sulfate and resveratrol monoglucuronides were up to 23 times greater than those of resveratrol. Urinary excretion of resveratrol and its metabolites was rapid, with 77% of all urinary agent-derived species excreted within 4 h after the lowest dose. Cancer chemopreventive effects of resveratrol in cells in vitro require levels of at least 5 micromol/L. The results presented here intimate that consumption of high-dose resveratrol might be insufficient to elicit systemic levels commensurate with cancer chemopreventive efficacy. However, the high systemic levels of resveratrol conjugate metabolites suggest that their cancer chemopreventive properties warrant investigation.

Resveratrol inhibition of Propionibacterium acnes.

OBJECTIVES: To evaluate the effects of the anti-inflammatory hydroxystilbene, resveratrol, on Propionibacterium acnes growth. METHODS: Three different strains of P. acnes were tested against resveratrol at concentrations between 0 and 200 mg/L. Piceatannol was included as a second hydroxystilbene to compare with resveratrol, and erythromycin and benzoyl peroxide were used as positive controls. RESULTS: After 24 h of treatment with resveratrol, the average 50% inhibitory concentration (IC(50)) was 73 mg/L and the average 100% inhibitory concentration (IC(100)) was 187 mg/L for the three strains of P. acnes tested. The IC(50) and IC(100) of piceatannol were 123 and 234 mg/L, respectively. The highest concentration of resveratrol tested (200 mg/L) was bactericidal, whereas lower concentrations were bacteriostatic. CONCLUSIONS: Resveratrol, an anti-inflammatory hydroxystilbene, is capable of inhibiting P. acnes growth.

Quantitation of trans-resveratrol and detection of its metabolites in human plasma and urine by high performance liquid chromatography.

We describe a reversed-phase HPLC method that uses gradient elution and UV detection (325 nm) to determine levels of resveratrol and identify six major conjugated metabolites in the plasma and urine of human volunteers after administration of a single oral dose of 1g. Waters Atlantis C18 3 microm served as the stationary phase. The gradient was formed using ammonium acetate and methanol, containing 2% propan-2-ol. Detection is linear between 5 ng/mL and 500 ng/mL in plasma (5-1000 ng/mL in urine). The coefficient of variation for intra- and inter-day variation is <10%. The average recovery of resveratrol from plasma and urine is 58+/-3%. The data presented in this report demonstrate a rapid, sensitive and accurate method for the analysis of resveratrol and its metabolites in human plasma and urine for pharmacokinetic studies.

Longitudinal evaluation of herpes simplex virus DNA load during episodes of herpes labialis.

BACKGROUND: Few studies have performed sequential evaluation of the herpes simplex virus (HSV) load using quantitative PCR during episodes of herpes labialis. OBJECTIVE: To determine HSV viral load kinetics during recurrences of herpes labialis. STUDY DESIGN: Twenty-two subjects were monitored by daily swabs during recurrences of herpes labialis. A real-time PCR detecting both HSV-1 and HSV-2 was used to quantify the viral load. RESULTS: Median duration of HSV-1 shedding was 60 h and 48 h by PCR and culture, respectively. No HSV-2 DNA was detected in that study. Peak viral DNA load (123.6 copies/cell) occurred at 48 h with no virus detected beyond 96 h of onset of symptoms. CONCLUSIONS: These viral load kinetics data could be used as surrogate markers of antiviral activity in future clinical trials.

Resveratrol suppresses nuclear factor-kappaB in herpes simplex virus infected cells.

Resveratrol inhibits herpes simplex virus (HSV) replication by an unknown mechanism. Previously it was suggested that this inhibition may be mediated through a cellular factor essential for HSV replication [Docherty, J.J., Fu, M.M., Stiffler, B.S., Limperos, R.J., Pokabla, C.M., DeLucia, A.L., 1999. Resveratrol inhibition of herpes simplex virus replication. Antivir. Res. 43, 145-155]. After examining numerous cellular factors, we report that resveratrol suppresses NF-kappaB (NF-kappaB) activation in HSV infected cells. Reports have indicated that HSV activates NF-kappaB during productive infection and this may be an essential aspect of its replication scheme [Patel, A., Hanson, J., McLean, T.I., Olgiate, J., Hilton, M., Miller, W.E., Bachenheimer, S.L., 1998. Herpes simplex type 1 induction of persistent NF-kappa B nuclear translocation increases the efficiency of virus replication. Virology 247, 212-222; Gregory, D., Hargett, D., Holmes, D., Money, E., Bachenheimer, S.L., 2004. Efficient replication by herpes simplex virus type 1 involves activation of the IkappaB kinase-IkappaB-RelA/p65 pathway. J. Virol. 78, 13582-13590]. Electromobility shift assays determined that resveratrol, in a dose dependent and reversible manner, suppressed activation of NF-kappaB in Vero cells infected with HSV-1, HSV-2 and acyclovir resistant HSV-1. Furthermore, resveratrol did not protect IkappaBalpha, a cytoplasmic NF-kappaB inhibitor, from degradation in HSV-1 infected cells. Immunohistochemical studies demonstrated that RelA/p65, a component of the dimeric NF-kappaB complex, translocated to the nucleus of HSV-1 infected cells in the presence of resveratrol. Finally, direct effects on viral transcription and DNA synthesis were evaluated. Real-time RT-PCR analysis showed that resveratrol treatment of infected cells resulted in reductions of mRNA for ICP0, ICP4, ICP8 and HSV-1 DNA polymerase by 2.1-, 3.3-, 3.8- and 3.1-fold, respectively. Plus, mRNA for glycoprotein C, an HSV late gene, was completely absent in the presence of resveratrol. Lastly, quantitative PCR showed that resveratrol significantly blocked HSV DNA synthesis. Cumulatively, these data indicate that resveratrol (i) suppresses HSV induced activation of NF-kappaB within the nucleus and (ii) impairs expression of essential immediate-early, early and late HSV genes and synthesis of viral DNA.

Resveratrol inhibition of varicella-zoster virus replication in vitro.

Resveratrol was found to inhibit varicella-zoster virus (VZV) replication in a dose-dependent and reversible manner. This decrease in virus production in the presence of resveratrol was not caused by direct inactivation of VZV or inhibition of virus attachment to MRC-5 cells. The drug effectively limited VZV replication if added during the first 30 h of infection. Western blot analysis and real-time RT-PCR studies demonstrated that protein and mRNA levels of IE62, an essential immediate early viral protein, were reduced when compared to controls. These results demonstrate that VZV replication is adversely affected by resveratrol which is negatively impacting IE62 synthesis.

Effect of resveratrol on herpes simplex virus vaginal infection in the mouse.

Resveratrol (3,5,4'-trihydroxystilbene) is a natural component of certain foods, such as grapes, that, when topically applied, has been shown to limit HSV-1 lesion formation in the skin of mice [Antiviral Res. 61:19-26, 2004]. To determine if it is active on genital HSV infection, the vagina of mice were infected with HSV-2 or HSV-1 and treated with a cream formulation of resveratrol. Mice were evaluated daily for extravaginal disease and vaginal swabs were taken regularly and assayed for infectious virus. Initial studies demonstrated that 19% resveratrol cream administered intravaginally five times a day for 5 days beginning 1h after infection significantly reduced HSV-2 replication beginning on day 1 of infection and prevented extravaginal disease when compared to animals treated with placebo. When resveratrol was tested at a concentration of 6.25% and 12.5% administered five times a day, 6.25% limited virus replication only on day 1 and delayed development of extravaginal disease by 1 day. However, 12.5% resveratrol inhibited HSV-2 replication beginning on day 1 and abolished extravaginal disease. If the number of applications per day was reduced to three for 5 days, 12.5% resveratrol inhibited HSV-2 replication only on day 1, while 19% resveratrol inhibited it throughout the 9-day assay period. When the animals with three treatments per day were examined for extravaginal disease, it was found that 12.5% resveratrol was ineffective when compared to placebo, while animals treated with 19% resveratrol did not exhibit extravaginal disease. When treatment was delayed 6h, 12.5% resveratrol did not inhibit HSV-2 replication or extravaginal lesion formation, but 19% resveratrol did. When resveratrol was used to treat vaginal HSV-1 infection, it was found that 12.5% resveratrol did not limit replication or prevent extravaginal lesion formation. In contrast, 19% resveratrol did significantly limit vaginal HSV-1 replication and reduced extravaginal lesion formation, but the latter was not significant. Mortality rates in placebo-treated animals was 37%, 6.25% resveratrol-treated animals was 40%, 12.5% resveratrol-treated animals was 24%, and 19% resveratrol-treated animals was 3%. Collectively, these results demonstrate that resveratrol cream inhibits or reduces HSV replication in the vagina of mice and limits extravaginal disease.

Resveratrol-associated renal toxicity.

Resveratrol, (3,5,4'-trihydoxystilbene) a compound found in grapes, mulberries, and peanuts, has antimycotic, antiviral, and beneficial cardiovascular and cancer preventive activities. It is being developed for several clinical indications. To evaluate the potential toxicity of resveratrol, rats were administered by gavage 0, 300, 1000, and 3000 mg trans-resveratrol per kilogram body weight per day for 4 weeks. Most of the adverse events occurred in the rats administered 3000 mg per kilogram body weight per day. These included increased clinical signs of toxicity; reduced final body weights and food consumption; elevated BUN, creatinine, alkaline phosphatase, alanine aminotransferase, total bilirubin, and albumin; reduced hemoglobin, hematocrit, and red cell counts; and increased white cell counts. Increases in kidney weights and clinically significant renal lesions, including an increased incidence and severity of nephropathy, were observed. Diffuse epithelial hyperplasia in the bladder was considered, equivocal and of limited biological significance. No histological effects on the liver were observed, despite the clinical chemistry changes and increased liver weights in the females. Effects seen in the group administered 1000 mg resveratrol per kilogram body weight per day included reduced body weight gain (females only) and elevated white blood cell count (males only). Plasma resveratrol concentrations in blood collected 1 h after dose administration during week 4 were dose related but were relatively low given the high dosage levels; conjugates were not measured. Under the conditions of this study, the no observed adverse effect level was 300 mg resveratrol per kilogram body weight per day in rats.

Effect of topically applied resveratrol on cutaneous herpes simplex virus infections in hairless mice.

Resveratrol (3,5,4'-trihydroxystilbene) is a natural component of certain foods, such as grapes, that has been shown to have anti-herpes simplex virus (HSV) activity in vitro. To determine if it is active in vivo, the abraded epidermis of SKH1 mice were infected with HSV-1 and topically treated with 12.5 or 25% resveratrol cream or cream only. Initial studies demonstrated that: (1). 25% resveratrol cream topically applied two, three, or five times a day effectively suppressed lesion development whereas 12.5% resveratrol cream effectively suppressed lesion formation when applied five times a day starting 1h after infection; (2). when treatment was begun 1, 6, or 12h after infection, both 12.5 and 25% resveratrol were effective at 1 and 6h after infection, but not if applied 12h after infection. Comparative studies between resveratrol cream, 10% docosanol cream (Abreva) and 5% acyclovir ointment (Zovirax) were also carried out. When treatment was begun 1h after infection and repeated every 3h five times a day for 5 days, 12.5 and 25% resveratrol significantly (P=0.0001) inhibited the development of HSV-1 induced skin lesions. Acyclovir was as effective (P=0.0001) as resveratrol. Animals that were topically treated with docosanol were not protected and developed lesions in a manner indistinguishable from cream only controls. These studies were repeated with an HSV-1 acyclovir-resistant virus. As before, 12.5 and 25% resveratrol cream effectively suppressed lesion formation. The skin of resveratrol-treated animals showed no apparent dermal toxicity such as erythema, scaling, crusting, lichenification, or excoriation. These studies demonstrate that topically applied resveratrol inhibits HSV lesion formation in the skin of mice.