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Chemoprevention of Cancer and DNA Damage by Dietary Factors

35a. Protective Components of Alcoholic Beverages - Wine

Philipp Saiko* & Thomas Szekeres

Clinical Institute of Medical and Chemical Laboratory Diagnostics

Medical University of Vienna, General Hospital of Vienna

Waehringer Guertel 18-20

A-1090 Vienna, Austria

*Corresponding author:

DDr. Philipp Saiko

Phone: (43) 1 40400 ext. 54960

Fax: (43) 1 292 77 08 17

E-mail: philipp.saiko@meduniwien.ac.at

1. INTRODUCTION

1.1. General information & historical background

Wine is an alcoholic beverage made from fermentation of fruit or grape juice. Concerning grapes, a

wine may consist of a single type of grape, or may contain a blend of different grapes. The natural

chemical balance of grapes is such that they can ferment without the addition of sugars, acids,

enzymes or other nutrients. Although other fruits such as apples and berries can also be fermented,

the resultant "wines" are normally named after the fruit from which they are produced (e.g., apple

wine) and are generically known as fruit or country wine. Others, such as barley wine and rice wine

(e.g., sake) are made from starch-based materials and resemble beer more than wine, while ginger

wine is fortified with brandy. In these cases, the use of the term "wine" is a reference to the higher

alcohol content rather than to the production process. Law in many jurisdictions protects the

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commercial use of the English word „wine“ and its equivalent in other languages. Wine is produced

by fermenting crushed grapes using various types of yeast that consume the sugars being included

in the grapes and convert them into alcohol. Various varieties of grapes and strains of yeasts are

used depending on the types of wine produced.

The scientific history of wine stretches back much longer than its first written account in the Bible,

with the earliest evidence dating around 5000 BC. A pottery jar recovered in present-day Iran

provides the earliest chemical evidence so far discovered [1]. The wine was identified by the

presence of calcium salts of tartaric acid, only present in large amounts in grapes and in the resins

of terebinth trees [2]. This resin was widely used in ancient times as an additive to wine to inhibit

bacterial growth. Though wild grape trunks have been found to originate as far back as the eighth

millennium, this archaeological discovery marks the earliest scientific record of fermented wine as

part of human culture. From the fifth millennium BC wine spread from its postulated origins in the

Southern Caucasus to Palestine, Syria, Egypt and Mesopotamia and subsequently to the

Mediterranean [2], but after the fall of the Roman Empire wine making declined. During the Dark

Ages, wine making was kept alive mainly through the efforts of Christian monasteries. As the

Church extended their monasteries, they began to develop some of the finest vineyards in Europe.

In this Medieval period, wine was still considered a staple of everyday diet, because most of Europe

lacked reliable sources of drinking water. However, in the following centuries wine had to face the

rival of a clean and readily available supply of drinking water, and was no longer needed as a major

part of the daily diet.

1.2. The health effects of wine

In 1992, Renaud and de Lorgeril observed a lower mortality rate of coronary heart disease in France

in comparison to other Northern European countries and the USA, despite a similar intake of high

levels of saturated fat, identical smoking habits and lack of exercise [3]. This observation was

brought to the attention of the medical society, lay public, and became known as the “French

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Paradox”. The authors explained the paradox by the consumption of the so-called „Mediterranean

diet”, with an abundance of vegetables, fruits, olive oil, and – especially – red wine. Concerning

France, they suggested that this difference is attributed to the high consumption of (red) wines by

the French population. In the USA, a boom in red wine consumption was initiated in the 1990s by

“60 Minutes“, and other news reports on the “French paradox”. Population studies have observed a

J curve association between wine consumption and the risk of heart disease. This means that

abstainers and heavy drinkers have an elevated risk, whilst moderate drinkers have a lower risk.

They also found that moderate consumption of other alcoholic beverages might be cardioprotective,

though the association is considerably stronger for wine.

1.3. Ingredients of wine

Wine is a rich source of biologically active phytochemicals, chemicals found in plants.

Phytochemicals are divided into distinct subgroups according to their structure and function in the

plant. The polyphenols are one of the most prominent groups in disease prevention. Currently, more

than 8000 of them have been identified, which are ubiquitous in plant-borne foods. Polyphenols are

associated not only with color and with sensory properties, but also linked to the health benefits

ascribed to fruits, vegetables, and wine.

The grape polyphenols may be classified into the following three groups:

1. Nonflavonoids, derived from hydroxycinnamic acids (paracoumaric acid, caffeic acid

chlorogenic acid, and ferulic acid) and hydroxybenzoic acids (gallic acid, protecatechouric

acid, and vanillic acid),

2. Flavonoids, comprising the largest class (several thousand) of phenolic compounds

including flavonols (e.g., querecetin and myricetin), isoflavonols, flavanones, flavanals (e.g.,

catechin, epicatechin, and procyanidin), and anthocyanins (e.g., delphinidin, cyanidin, and

malvidin),

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3. Stilbenes, constituting a relatively small group of phenolic compounds that are usually

synthesized in plants in response to stress conditions. Their structures contain two benzene

rings connected by a methylene bridge. Resveratrol (3,4’,5-trihydroxy-trans-stilbene; RV) is

the most extensively studied stilbene derivative.

Most flavonoids occurring in plants are conjugated to sugar-moieties, pectins, and organic acids, or

are polymerized with other flavonoids to polymers. Products of many fruits and vegetables, such as

strawberries, blueberries, green and black tea, tomatoes, yellow onions, soy, and chocolate, contain

considerable amounts of polyphenols. Red grapes and wine of the Vitis vinifera varieties are

especially rich in these polyphenols, and more than 500 phenolic compounds have been recognized

in wine thus far [2]. Evidence from laboratory studies suggests that red wine may possess superior

health benefits including the prevention of cancer because it contains more polyphenols than white

wine, which is due to the production process (see below). Red wines obtained by traditional

maceration can have a polyphenol content of more than 3 g/L. RV is thought to be at least partly

responsible for the health benefits of red wine, since it has been shown to exert a range of both

cardioprotective as well as chemoprotective mechanisms, thus being the most prominent of these

polyphenols. Red wine contains much greater amounts of RV than white wine does, since RV is

concentrated in the grape skins and seeds, and the manufacturing procedure of red wine includes

prolonged contact of grape juice with these parts.

Plant polyphenols are recognized for their antioxidative activities, thereby protecting cells from

oxidative damage caused by free radicals. Electron acceptors such as molecular oxygen react easily

with them to become reactive oxygen species (ROS). Polyphenols scavenge free radicals, thus

breaking the free radical chain reaction of lipid peroxidation, which has been implicated in the

development of cancer. However, it is inherently difficult to evaluate the beneficial effects of

specific polyphenolic antioxidants, since a large number of individual compounds may occur in a

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single food. For example, over sixty different chemically distinct flavonoids are known to occur in a

given red wine. Numerous scientific studies have been conducted to attempt to arrive at one

consistent index for food antioxidant power. Since it has been proven that the dietary intake of

compounds exerting antioxidant activity is of great medical value, a number of chemical, biological,

and electrochemical methods have been proposed to evaluate the antioxidant potential of naturally

occurring agents such as RV.

2. PHYSICO-CHEMICAL PROPERTIES OF ACTIVE COMPOUNDS, OCCURRENCES,

AND CHEMICAL STRUCTURES

2.1. Resveratrol

2.1.1. History & sources

Resveratrol (3,4’,5,-trihydroxy-trans-stilbene; RV; Fig. 1) was first isolated in 1940 as an

ingredient of the roots of white hellebore (Veratrum grandiflorum O. Loes) and has since been

found in a wide variety of about 70 plant species, including grapes, mulberries, and peanuts [4]. RV

is a polyphenol and has been classified as a phytoalexin for being synthesized in spermatophytes in

response to injury, UV irradiation, and fungal attack. RV was identified in 1963 as the active

constituent of the dried roots of Polygonum cuspidatum, also called Ko-jo-kon in Japanese, and

used in traditional Asian medicine against suppurative dermatitis, gonorrhea, favus, and

hyperlipemia [5]. RV was first detected in grapevines (Vinis vitifera) in 1976, and then in wine in

1992 [6]. In grapes, especially when infected with Botrytis cinerea, RV is exclusively synthesized

in the leaf epidermis and in the grape skins, but not in the flesh. Fresh grape skins contain 50-

100mg RV per g, corresponding to 5-10% of their biomass [7]. Since the grape skins are not

fermented in the production process of white wines, only red wines contain considerable amounts of

RV. Its concentrations measured in a sampling of red wine varieties ranged from 2 to 40µM [7].

Figure 1. Chemical structure of resveratrol (RV).

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2.1.2. French paradox

Epidemiological studies have revealed an inverse correlation between red wine consumption and

the incidence of cardiovascular disease, a phenomenon commonly known as the “French Paradox”,

i.e. the fact that the incidence of heart infarction in France is about 40% lower than in the rest of

Europe, despite a diet being traditionally rich in saturated fat [3]. This led to the suggestion that RV

might be the active principle of red wine. Indeed, RV protects the cardiovascular system by a large

number of mechanisms including defense against ischemic-reperfusion injury, promotion of

vasorelaxation, protection and maintenance of intact endothelium, anti-atherosclerotic properties,

inhibition of low-density lipoprotein oxidation, suppression of platelet aggregation, and estrogen-

like actions [8, 9].

2.1.3. Effects of resveratrol

Besides its effects on the cardiovascular system, RV exhibits a remarkable inhibitory potential in

various stages of tumor development [9]. The antitumor activity of RV was first revealed by its

ability to reduce the incidence of carcinogen-induced development of cancers in experimental

animals [10]. Subsequently, RV has been shown to exert numerous effects that may block tumor

development at several discrete stages during the multigenic process of carcinogenesis, involving

interactions between RV and manifold targets [11]. These targets include kinases [12], steroid

hormone receptors [13], reactive oxygen species [14], ribonucleotide reductase [15], and DNA

polymerases [16]. RV causes an arrest at the S/G2 phase transition of the cell cycle [17] and is

capable of inducing differentiation and apoptosis in a multitude of human tumor cell lines. RV was

also identified as an effective inhibitor of ribonucleotide reductase (RR) [15, 18]. RR catalyzes the

rate-limiting step of de novo DNA synthesis, namely the reduction of ribonucleotides into the

corresponding deoxyribonucleoside triphosphates (dNTPs). The importance of all these targets for

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cancer development is well known and therefore RV can beneficially contribute to cancer

prevention.

As tissue inflammation provokes tumor promotion, anti-inflammatory agents are viewed as a

valuable chemopreventive modality against this mechanism of carcinogenesis [14]. RV has been

shown to exert substantial antiphlogistic activity in an in vivo rat model [19]. The key molecular

targets implicated herein are cyclooxygenases (COX-1 and COX-2). COX-1 and COX-2 are

respectively constitutive and inducible enzymes that catalyze the production of pro-inflammatory

prostaglandins from arachidonic acid [14]. Prostaglandins stimulate tumor growth by acting on cell

proliferation, angiogenesis and immunosupression. RV effects against cellular COX activity involve

its direct inhibitory action against COX-1 and COX-2 and its suppression of transcriptional COX-2

upregulation [20]. As prostaglandins not only stimulate tumor cell growth but also suppress

immune surveillance, COX enzymes are likely important targets to the cancer preventive activity of

RV [20]. Arachidonic acid is also metabolized via lipoxygenase (LOX) to produce

hydroperoxyeicosatetraenoic acids (HPETEs) or leukotrienes. Arachidonic acid metabolites derived

from LOX pathways play an important role in growth-related signal transduction, implying that

intervention through these pathways could be useful for attenuating cancer progression. RV inhibits

LOX and COX in K562 myelogenous leukemia cells [21]. LOX-derived metabolites have an

(indirect) influence on development as well as progression of human cancers [22].

Figure 2. Effects of resveratrol on arachidonic acid metabolism.

Furthermore, RV induces a multitude of effects that depend on the cell type (e.g. NF-κB modulation

in cancer cells vs. neural cells), cellular condition (normal, stressed or malignant) and concentration

(proliferative vs. growth arrest) and can have opposing activities. The final read-out depends on the

balance of these partially opposing effects. Single alterations in cell physiology, signaling and

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metabolism result often in a cascade of changes that cannot always be restored by reversion of the

single original change. RV targets whole pathways and sets of intracellular events rather than a

single enzyme and therefore offers a less specific but more gentle (fewer side effects) and possibly

more effective strategy for therapy to restore homoestasis.

Therefore, keen interest has emerged in RV due to its evident value as a cancer preventive and

cardioprotective dietary substance. RV may provide an alternative (and early) intervention approach

that could prevent/delay disease onset, emend the course of disease and/or prevent further damage.

Since the identification of RVs health benefits are largely owed to its high abundance in certain

plants and foods, the discovery of further naturally occurring stilbenes, as well as, chemically

modified analogs that are superior to RV in their cancer chemopreventive properties may be

expected.

This overview of molecular targets implicated in the antagonism of cancer by RV underscores the

complexity underlying biological responses to this drug, which is probably common to many other

molecules generated for self-defense. The efficacy of RV against distinct mechanisms of disease

development is an indicator of its potential value for the prevention of various human diseases.

Subsequently, the search and identification of more effective preventive agents among stilbene

natural products is warranted.

Table 1. Cancer-related targets, effects, and benefits of resveratrol.

2.2. Piceatannol – a naturally occurring resveratrol metabolite

In contrast to the detailed knowledge of RV activities in biological systems, little is known about

the effects of other polyhydroxylated stilbens. RV undergoes cytochrome P450 catalyzed

hydroxylation to piceatannol (3,3’,4’,5-tetrahydroxy-trans-stilbene; PCA; Fig. 3) and two other

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unidentified mono- and dihydroxy-RV analogs. This demonstrates that a natural dietary cancer

preventative agent can be converted to a compound with known chemopreventive and anticancer

activity by the enzyme CYP1B1, which is overexpressed in a wide variety of human tumors.

Importantly, these findings give insight into the functional role of the cytochrome P450 enzyme

CYP1B1 and provide evidence for the concept that CYP1B1 in tumors might be serving as a

growth suppressor enzyme [23]. Comparable with RV, PCA displays cytotoxic activity in acute

leukemia and lymphoma cells and exerts anti-proliferative activity in colon cancer cells [24].

Figure 3. Chemical structure of piceatannol (PCA).

2.3. Gallic acid

Gallic acid (3,4,5-trihydroxybenzoic acid; GA; Fig. 4) is found in gallnuts, sumac, tea leaves, oak

bark, grapes, various herbs, and in red and white wines. In particular, red wine has a high content of

this phenolic acid. GA can be present as free molecule or as part of the tannin molecule

(gallotannin). It was recently shown that GA antagonizes P-selectin mediated platelet leucocyte

interactions [25] and could be jointly responsible for the beneficial effects of red wine and the

“French paradox”. Other beneficial effects might be the antidiabetic and antiangiogenic effects of

GA containing fruit extracts [26] and the induction of Ca2+ dependent apoptosis in leukemia cells

[27]. Altogether, GA was described as an excellent free radical scavenger and as inducer of

differentiation as well as programmed cell death in numerous tumor cell lines and might play an

important role in the prevention of malignant transformation and cancer development.

Figure 4. Chemical structure of gallic acid (GA).

3. BIOAVAILABILITY AND METABOLISM OF ACTIVE COMPOUNDS

3.1. Bioavailability of resveratrol

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Several in vivo studies in animals and humans demonstrated a very low intestinal uptake of RV

leading to trace amounts in the bloodstream based on extensive metabolism in the gut and liver.

Rapid metabolism is also the main reason for the short initial half-life of the primary molecule (~8-

14 min) [28]. The bulk of an intravenous dose of RV is converted to sulfate conjugates within ~30

min in humans. A detailed analysis of plasma metabolites after oral dosing was not possible;

however, both sulfate and glucuronide conjugates were detected [29].

3.2. Metabolites of resveratrol – glucuronide and sulfate conjugates

Although modifications such as glucuronidation and sulfation typically reduce the cell permeability

of drugs and aid in their excretion, the undeniable in vivo efficacy of administered RV, despite its

low bioavailability, has led to the suggestion that its metabolites are likely to be the active principle.

However, resveratrol-3-sulfate fails to inhibit CYPs [30] and there is currently no evidence that any

metabolite is able to cross the plasma membrane.

Recently, the absorptive efficiency of three polyphenolic constituents (trans-resveratrol, +-catechin

and quercetin) was investigated after oral application to healthy human subjects in three different

media (white wine, grape juice, and vegetable juice/homogenate) [31]. All compounds were present

in serum and urine predominantly as glucuronide and sulfate conjugates, reaching peak

concentrations in the former around 30 min after consumption. The absorption of these three

polyphenols was broadly equivalent in aqueous and alcoholic matrices; however, their peak plasma

concentrations reached only 10-40nM, whereas in vitro biologic activities have been studied at 5-

100µM [31].

3.3. Bioavailability of resveratrol in grape juice compared to its pure aglycone

In grape juice, the level of free RV is rather low, and cis- and trans-Piceid (RV-3-O-β-D-glucoside;

Polydatin) are the major RV derivatives. This suggests a lower bioavailability of RV glycosides in

grape juice in comparison to its pure aglycone in wine [32]. These findings were confirmed by

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another study reporting that RV concentrations in Italian red wine ranged from 8.6 to 24.8µM,

whereas the amount in grape juice was only 1.6µM, respectively [33].

Given that in vivo concentrations of individual metabolites can be much higher than those of the

native compound, further studies are needed to determine (1) whether the metabolites represent

inactivated forms of the drug, (2) act as a pool from which free RV can be released in various

tissues, or (3) are themselves active in promoting many of the health benefits attributed to RV.

4. MECHANISMS OF PROTECTION

4.1. Results of in vitro studies

A large number of RV’s beneficial health effects, such as anticancer, antiviral, neuroprotective,

anti-aging, and anti-inflammatory effects, have been reported in vitro. Table 2 summarizes a variety

of RV’s anticancer activities.

Table 2. Results of in vitro studies.

4.2. Results of in vivo studies

As the mechanisms of RV's broad cancer chemopreventive effects are not completely understood,

continued efforts are needed, especially well designed pre-clinical studies in animal models that

closely mimic/represent human disease, to establish the usefulness of RV as cancer

chemopreventive agent. Table 3 gives an overview of such pre-clinical studies including the

observed effects.

Table 3. Results of in vivo studies.

5. RESULTS OF HUMAN STUDIES

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Overall, in vivo studies in rodents clearly show great promise for RV in the prevention and

treatment of cancers. However, only a few Phase I clinical trials are currently underway for oral RV

in humans at doses up to 7.5 g per day. A National Cancer Institute-sponsored study has been

completed recently [34], suggesting that consumption of RV does not cause serious adverse events.

RV and six metabolites were recovered from plasma and urine, among them two monoglucuronides

and RV-3-sulfate. The area under the plasma concentration curve (AUC) values for these

metabolites were up to 23 times greater than those of RV [34]. Cancer chemopreventive effects of

RV in cells in vitro require levels of at least 5 mM per litre, intimating that consumption of high-

dose RV might be insufficient to elicit systemic levels synonymous with cancer chemopreventive

efficacy. On the other hand, the high systemic levels of RV metabolites clearly suggest that they

might be the active principle of the parent drug.

6. CONCLUSIONS

Chemopreventive agents such as RV might be used not only to prevent, but also to treat cancer

since the molecular targets are similar. Due of their pharmacological safety, RV and its

metabolites/analogs may be applied in combination with other chemotherapeutic agents in order to

enhance their efficacy thus minimizing chemotherapy-induced toxicity. By inhibition of COX and

cytochrome P450 enzymes and by induction of quinone reductase, RV can simultaneously inhibit

promutagen bioactivation, stimulate carcinogen detoxification and protect the organism against the

adverse effects of diverse environmental toxins. Furthermore, RV inhibits prostaglandins, NO

formation and the generation of ROS, therefore preventing their stimulative effect on tumor

development. Besides acting as a chemopreventive agent, several in vitro, ex vivo, and in vivo

experiments have shown that RV suppresses the growth of various cancer cell lines by inhibition of

DNA polymerases, RR, and by inducing cell cycle arrest or apoptosis. Although the most exciting

in vivo data relate to its cancer chemopreventive and chemotherapeutic activity, some studies also

demonstrate beneficial effects on cardiovascular, neurological, and hepatic systems.

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RV is commonly referred to as a “dirty” molecule, meaning that it seems to interact with many

different proteins, including cyclooxygenases, ribonucleotide reductase and DNA polymerases.

Thus, its activity cannot be resumed in a unique mechanism of action but likely results from various

complementary actions of different biochemical pathways.

However, the question of whether RV itself can accumulate to bioactive levels in target organs

remains to be addressed. Opposing results and controversies involving the available data are leading

to the suggestion that RV metabolites might be the active principle, and call for additional

experiments. Long-term in vivo epidemiological studies are highly warranted to determine the

preventive and therapeutic efficacy of dietary or supplemented RV on tumor development and

progression.

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34. Boocock, D. J., Faust, G. E., Patel, K. R., Schinas, A. M., Brown, V. A., Ducharme, M. P., Booth, T. D., Crowell, J. A., Perloff, M., Gescher, A. J., Steward, W. P., and Brenner, D. E. (2007) Phase I dose escalation pharmacokinetic study in healthy volunteers of resveratrol, a potential cancer chemopreventive agent, Cancer Epidemiol Biomarkers Prev 16, 1246-1252.

17

Figure 1. Chemical structure of resveratrol (RV).

OH

OH

OH

Resveratrol (3,4',5-trihydroxy-trans-stilbene; RV)

18

Figure 2. Effects of resveratrol on arachidonic acid metabolism.

RV RVRV

RV

RV

ARACHIDONIC ACID

PHOSPHOLIPIDS

CYCLOOXYGENASES(COX-1, COX-2)

LIPOXYGENASES EPOXYGENASES(CYP450)

FREE RADICALS

ISOPROSTANESEPOXIDESHPETEsLEUKOTRIENESLIPOXINS

PROSTAGLANDINSTHROMBOXANES

PHOSPHOLIPASES

19

Table 1. Cancer-related targets and biological effects of resveratrol.

CLASS OF TARGETS MOLECULAR TARGETS BIOLOGICAL EFFECTS

Direct radical scavenging ROS DNA stability Lipidoxidation Apoptosis/cell survival

Antioxidant/phase II enzymes

SOD, catalase, GR DNA stability Lipidoxidation Apoptosis/cell survival

Heme oxidase 1 (HO-1) Radical scavenging Anti-inflammatory Anti-apoptotic

GST, NQO1, UDP-glucuronyl transferase

ARE/EpRE activation ERβ-activated

Arachidonic acid related COX 1+2, (COX 2 via NF-κB) Anti-inflammatory Anti-tumor promoting

Estrogen-related

Selective estrogen receptor modulation (SERM)

Superagonist Agonist Antagonist

CYP 1A1, CYP 1B1 Inhibition of estrogen-metabolizing phase I enzymes

Modulation of signalling kinases Raf, Src, MAPK, PKD, PKCδ

Cell growth arrest Cell death Differentiation

Modulation of global gene expression through chromatin remodelling

p300 (Acetylase), SIRT1 (Deacetylase) Cell survival Apoptosis delay Inflammatory response

Transcription factors p53/p21 Cell cycle arrest

Apoptosis IκB kinase/ NF-κB Cell survival AP1 Tumor growth promoter

Other cell cycle related

Ribonucleotide reductase, replicative DNA polymerases

DNA synthesis Cell cycle arrest

Survivin Cell survival Apoptosis

Cell death related

TRAIL/DR4+5 Apoptosis

Fas/CD95 Apoptosis Mitochondria-dependent, cytochrome c, Apaf-1 Apoptosis

PI3K/Akt, ERα-dependent Cell survival

Ceramide Apoptosis

20

Figure 3. Chemical structure of piceatannol (PCA).

OH

OHOH

OH

Piceatannol (3,3',4',5-tetrahydroxy-trans-stilbene; PCA)

21

Figure 4. Chemical structure of gallic acid (GA).

OH

OHOH

OH

O

Gallic acid (3,4,5-trihydroxybenzoic acid; GA)

22

Table 2. Results of in vitro studies. Mechanism Assay system µM1 Estrogenic/antiestrogenic and scavenging properties

MCF-7 and MVLN breast cancer cells 0.1-25

Inhibition of aryl hydrocarbon-induced cytochrome P-450 1A1 enzyme activity and CYP1A1 expression

HepG2 hepatoma cells and MCF-7 breast cancer cells

0.5-5

Modulation of the catalytic activity and mRNA expression of the procarcinogen-activating human cytochrome P450 1B1

Cultured MCF-7 human breast carcinoma cells

1-20

Inhibition of cell growth, G1-phase arrest, and induction of apoptosis

Human A431 epidermoid carcinoma cells

1-50

Inhibition of cyclooxygenase-2 transcription and activity in phorbol ester-treated human mammary epithelial cells

Human mammary 184B5/HER epithelial cells (and premalignant MSK Leuk1 oral epithelial cells)

2.5-40

Suppression of TNF-induced activation of nuclear transcription factor NF-κB, activator protein-1, and apoptosis

U937 myeloid leukemia cells, Jurkat lymphoid and HeLa and H4 epithelial cells

5

Inhibition of the binding of labeled estradiol to the estrogen receptor

MCF-7 breast cancer cells 10

Stimulation of the proliferation of estrogen-dependent breast cancer cells

T47D breast cancer cells 10

Growth inhibition, induction of apoptosis by down-regulation of Bcl-2 and up-regulation of bax

EC-9706 esophageal cancer cells 10

Modulation of ERE-Luciferase Activity and Estrogen-inducible Protein Expression

MCF-7, T47D, LY2, and S30 breast cancer cells

10-15

Inhibition of the clonal growth of tumor cells 32Dp210, HL-60, U937, and L1210 leukemia cells

10-80

Depletion of intracellular dCTP, dTTP, dATP, and dGTP pools (inhibition of ribonucleotide reductase)

HL-60 leukemia cells 12.5

Inhibition of 14C-cytidine incorporation into DNA (inhibition of ribonucleotide reductase)

HL-60 leukemia cells 12.5

Growth inhibition, accumulation of cells at the S/G2 phase transition of the cell cycle, and significant decrease of ornithine decarboxylase (ODC) activity

CaCo-2 colon cancer cells 25

Cell division cycle arrest at S/G2 phase transition

HL-60 leukemia cells 30

Increase in the content of cyclins A and B1 as well as cyclin-dependent kinases Cdk1 and Cdk2, and promotion of Cdk1 phosphorylation

SW480 colon cancer cells 30

Increase in the expression and kinase activities of positive G1/S and G2/M regulators, resulting in cell cycle blockade at the S-phase and apoptosis induction

MCF-7 breast cancer cells < 50

Inhibition of the expression and function of the androgen receptor

LNCaP prostate cancer cells 50-100

Decrease of the expression and kinase activities of positive G1/S and G2/M cell cycle regulators and inhibition of ribonucleotide reductase activity

MDA-MB-231 breast cancer cells < 200

1Lowest concentration at which reproducible changes have been observed, or IC50 or EC50, if provided.

23

Table 3. Results of in vivo studies. Species Assay system/Results Daily dose1 Male BALB/c mice Inhibition of the growth of murine hepatocellular H22

carcinoma cells implanted in mice: RV induced an S phase arrest of tumor cells, and inhibition of tumor growth was further enhanced in combination with 5-FU.

5-15mg/kg (abd.)

Female Sprague Dawley rats

Inhibition of mammary carcinogenesis induced by 7,12-dimethylbenz(a)anthracene (DMBA): RV had no effect on body weight gain and tumor volume but produced striking reductions in the incidence and multiplicity of tumors, and extended the latency period of tumor development.

10ppm (diet)

Male F344 rats Inhibition of azoxymethane (AON)-induced colon carcinogenesis: RV significantly reduced the number and multiplicity of isolated aberrant crypt foci (ACF) and abolished large ACF.

200µg/kg (d.w.)

Apc(Min+) mice Prevention of the formation of colon tumors and reduction of the formation of small intestinal tumors by 70%. RV downregulated genes that are directly involved in cell cycle progression or cell proliferation (cyclins D1 and D2, DP-1 transcription factor, and Y-box binding protein).

0.01% (d.w.)

HER-2/neu transgenic mice

Inhibition of the development of spontaneous mammary tumors: RV supplementation delayed the development of spontaneous mammary tumors, reduced the mean number and size of mammary tumors, and diminished the number of lung metastases.

0.2mg/kg (d.w.)

Female Sprague Dawley rats

Delay of N-methyl-N-nitrosourea (NMU)-induced mammary tumorigenesis: RV caused a 28-day increase in tumor latency and reduced the multiplicity of tumors from 6.0 in the control group to 3.9 in the treatment groups.

100mg/kg (gav.)

Female athymic nude mic

Inhibition of mammary carcinogenesis induced by MDA-MB-231 breast adenocarcinoma cells: RV caused significantly lower tumor growth, decreased angiogenesis, and increased the apoptotic index in ERα- ERβ+ MDA-MB-231 tumors.

25mg/kg (i.p.)

Syngeneic A/J mice Inhibition of subcutaneous neuroblastomas in mice: RV suppressed the growth rate, resulting in 70% long-term survival.

40mg/kg (i.p.)

Fischer 344 rats Lower tumor growth rate, longer animal survival time, and higher animal survival rate; immunohistochemical analyses showed that the s.c. gliomas from RV-treated rats had fewer microvessel densities than did control rats.

40mg/kg (i.p.)

Female C57BL/6 strain mice

Significant reduction of tumor volume, tumor weight, and metastasis to the lung in mice bearing highly metastatic Lewis lung carcinoma (LLC) tumors. RV dose dependently inhibited angiogenesis and had no effect on either initial or final body weight in LLC-bearing mice compared with that in normal mice.

2.5-10mg/kg (i.p.)

Female syngeneic C57BL/6N mice

Growth inhibition of B16-BL6 melanoma cells in vivo: Treatment with RV significantly delayed tumor growth, without mortality or body weight changes, but did not reduce the number of lung metastases after i.v. injection of B16-BL6 cells.

50mg/kg (i.p.)

Male Wistar rats RV administration to rats inoculated with the fast growing Yoshida AH-130 ascites hepatoma caused a 25% decrease of tumor cells, associated with an increase of cells in the G2/M phase of the cell cycle.

1mg/kg (i.p.)

Male F344 rats Inhibition of N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal tumorigenesis: RV significantly reduced number and size of NMBA-induced tumors.

1-2mg/kg (i.p. or p.o.)

Male Wistar rats Inhibition of 1,2-dimethylhydrazine (DMH) induced colon carcinogenesis: RV markedly reduced tumor incidence, the degree of histological lesions, and the size of tumors.

8mg/kg (p.o.)

24

C57Bl6/J mice Treatment with RV significantly inhibited the growth of murine T241 fibrosarcoma in mice and suppressed FGF-2- and VEGF-stimulated angiogenesis.

1mg/kg (p.o.)

Nude mice Inhibition of implanted human primary gastric carcinoma cells: RV could significantly inhibit carcinoma growth when it was injected near the carcinoma and induced implanted tumor cells to undergo apoptosis.

500-1500mg/kg (s.c.)

Female SKH-1 hairless mice

Topical application of skin with RV (both pre- and post- treatment) resulted in a highly significant inhibition in tumor incidence and in a delay in the onset of tumorigenesis.

25-50µM (top.)

1Lowest concentration at which reproducible changes have been observed, or IC50 or EC50, if provided. abd. – abdominal injection, d.w. – drinking water, gav. – gavage, i.p. – intraperitoneal, p.o. – per os, s.c. – subcutaneous injection, top. – topical application.