Predicting the physiological relevance of in vitro cancer preventive activities of phytochemicals¹

Introduction

In recent years there has been increasing interest in the use of biologically active phytochemicals in cancer prevention. In particular, many in vitro studies using a wide range of natural products have demonstrated a preferential induction of cell cycle arrest or apoptosis in tumor cell lines compared to lines derived from non-tumor tissue. On further investigation, phytochemicals have been found to modulate the expression or activity of a large number of cellular proteins which are key for cell survival and the transformed phenotype. However, there is also much concern that many of these effects^[1–3] are irrelevant in vivo, since the concentrations used are often orders of magnitude greater than appear to be achievable in the human body.

In this review, we have attempted to address this concern for a few of the most studied diet-derived compounds. For each agent, we have assimilated reported in vivo concentrations, based where possible on human data. We have then surveyed the in vitro biological effects at these or lower doses. For the indoles only, we have included data on phase I drug metabolizing activity relating to altered estrogen metabolism. Encouragingly, a significant amount of published data validates some of these changes in vivo.

For a number of reasons, some guesswork was involved in deciding on the most relevant in vitro doses for consideration. First, adding a compound directly to a cell culture may deliver a much higher local dose than occurs following ingestion in the body. Second, some if not all of the compounds may undergo metabolism in vivo to other more or less active derivatives and such metabolism may not be possible in culture. Third, higher in vivo doses than those so far reported may be achievable by administration of a pure compound rather than a dietary source, or an optimized formulation of a pure compound. Fourth, some target tissues may receive a higher (or more prolonged) dose than the reported peak levels in plasma. Fifth, some target tissues, such as skin, oral cavity, gastrointestinal tract, and bladder, may receive higher doses because they are not dependent on circulating levels. The colon, for example, can be exposed to significant amounts of (unabsorbed) excreted material and the lining of the bladder may be exposed for substantial periods of time to any compounds concentrated in urine. Finally, where diet is concerned, any one compound may be poorly bioavailable, but with dozens, even hundreds, of active molecules being ingested together, the cumulative dose of similar acting compounds may be significantly higher.

It should not be forgotten that some natural products have exhibited toxicity in vivo when given in high doses, so there is also an argument for the use of higher concentrations in vitro to indicate the full range of activity of these molecules.

Indole-3-carbinol and diindolylmethane

Indole-3-carbinol (I3C) is derived from glucobrassicin, found in cruciferous vegetables. Diindolylmethane (DIM) is an acid condensation product formed from 2 molecules of I3C. In vivo, this is thought to occur in the acid conditions of the stomach. Data for mammals only are considered here.

Bioavailability of I3C and DIM in humans Information on the bioavailability and tissue distribution of I3C or DIM in humans is very limited^[4–6]. However, there are a number of studies in which oral administration of I3C resulted in a biochemically or clinically measurable outcome, indicating that the absorption of I3C and/or its acid-condensation products does occur. Administered doses of I3C have ranged from 200 to 500 mg/d (~ 6–7 mg/kg), typically for periods of 1–6 months, although treatments up to 82 months have also been reported^[7–14]. A dose-dependent effect (placebo, 200 and 400 mg/d for 3 months) was observed in the treatment of cervical intraepithelial neoplasia^[7], and 200 or 400 mg/d were similarly effective against vulval intraepithelial neoplasia^[14]. In dose-escalation studies for breast cancer prevention, 300 mg/d (minimum) increased the urinary estrogen metabolite ratio of 2-hydroxyestrone to 16 alpha-hydroxyestrone; 800 mg/d did not provide additional benefits over 400 mg/d in adult women^[15,16]. Elevated cytochrome P450 activity was responsible for an increase in 2-hydroxylation of estrogen, increasing the ratio of 2-OH:16-OH estrone^[16], which is regarded as favorable for the prevention of breast cancer and human papilloma virus (HPV)-related neoplasias^[7–14].

Two studies detected DIM in plasma (2.5 µmol/L maximum at 2 h, gradually decreasing by 12 h) or in urine following oral administration of I3C (Table 1). I3C was not detected in plasma or serum following oral doses of 400–1200 mg^[4,5]. DIM was also detected in the urine of a patient receiving DIM^[4]. In a pilot study using a formulation of enhanced absorption DIM (BioResponse DIM, 108 mg/d for 30 d), increased 2-hydroxylation of estrogen was also reported^[6].

Table 1 Bioavailability of DIM in humans following oral administration of I3C.
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Bioavailability of I3C and DIM in animals Radio-labeled I3C was used to follow distribution and tissue content in several studies, although this method did not differentiate between I3C and related products. In rats receiving 50 mg ¹⁴C-I3C by gavage, I3C equivalents peaked at 28 µmol/L in the blood and 121 µmol/L in the liver after 30 min. The labeled product was detectable in the 100 µmol/L range from 10 min to 2 h following dosing^[17]. High maximal concentrations of the I3C equivalent, but not I3C itself, were detected in the liver (1154 µmol/L), kidney, lung (436 µmol/L), blood (320 µmol/L), and tongue of the rats given ³H-I3C in the diet for 1 week (0.88+/–0.074 mmol/kg/d)^[18]. Once a steady state had been reached, excretion in feces and urine accounted for 75% dose/d, the majority of this being present in the feces by 110 h, indicating that either the dose was not absorbed or that a major excretory route was via bile. When ¹⁴C-I3C was given to pregnant mice, it was detected in the fetal liver, stomach, kidney, intestine, and lung (100–300 µmol/L) after 8 h of maternal exposure^[19].

Anderton et al detected I3C and DIM in tissues following the dosing of mice with 250 mg/kg I3C (Table 2) using an HPLC method allowing the simultaneous identification and quantification of I3C and its derivatives^[20]. The maximum level of 28 µmol/L I3C was observed at 15 min, falling below the level of detection by 1 h after dosing. I3C was detected in the liver (170 µmol/L)>kidney (116 µmol/L)>lung and heart>plasma>brain. The levels of DIM peaked at around 2 h in the liver (16 µmol/L)>lung and kidney>heart>brain>plasma (4 μmol/L), and by 24 h, were still detectable at approximately 0.5 µg/g in the brain and liver. The presence of linear trimer, 1-(3-hydroxymethyl)-indolyl-3-indolylmethane and indolo(3,2b)carbazole, together with oxidative metabolites of I3C, was also documented. In a further study, Anderton et al compared concentrations of DIM in tissues of mice dosed with either pure DIM (250 mg/kg; Table 2) or an equivalent dose of the enhanced absorption BioResponse DIM^[21]. The tissue distribution of DIM was similar to that reported previously following the administration of I3C with maximal concentrations around 160 µmol/L in the liver^[20]. The BioRes-ponse DIM resulted in levels approximately 50% higher than those obtained with unformulated DIM.

Table 2 Bioavailability of I3C and DIM in animals following oral administration.
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Thus, following the oral administration of I3C, both I3C and DIM were detectable at µmol/L concentrations in the blood and multiple organs. I3C was rapidly absorbed and cleared from the blood and tissues within 1 h, while DIM peaked slightly later and was more persistent. The observation of I3C in the blood and tissues at these very early time points belies previous assumptions that I3C is not absorbed, but undergoes complete acid condensation in the stomach. Several studies have revealed distinct responses to I3C and DIM in animal models^[22–26]. Therefore, the in vivo activity of dietary I3C cannot be attributed completely to the production of DIM, although response due partially to DIM conversion is probable.

Physiologically relevant concentrations of I3C and DIM As no data are available for achievable levels of I3C in humans, we extrapolated from animal studies^[20,21]. The maximum plasma and tissue concentrations attained in mice for I3C were 28 and 170 µmol/L (15 min) and for DIM, 4 and 16 µmol/L (2 h), respectively (Table 2). By allometric scaling, the I3C dose given to mice would equate to a 20 mg/kg dose in humans (1200 mg/d), which yielded the maximal serum concentration 2.5 µmol/L DIM (2 h), with no I3C detectable after 1 h^[5]. Therefore, maximal detectable DIM concentrations following I3C administration are similar in mice and humans, and the discrepancy in I3C detection is likely to be caused by the sensitivity of methods and selection of time points.

The maximum levels of DIM achieved in animals following a dose of a pure compound range from 24–200 µmol/L (Table 2), with BioResponse DIM resulting in 50% higher bioavailability. The dose of BioResponse DIM used in humans was 108 mg (~1.3–1.9 mg/kg)^[6], which might be expected to give levels in the range 3–30 µmol/L.

For the purposes of this review, the effects of the physiological concentrations up to 150 µmol/L I3C and 50 µmol/L DIM in vitro have been considered.

In vitro mechanistic studies using low doses of I3C or DIM Several mechanisms are responsible for the chemo-preventive activities of I3C and DIM, as summarized in Tables 3 and 4. Both agents induce activity of phase I and II enzymes involved in the biotransformation and elimination of carcinogens and steroid hormones. While detailed molecular interactions involved have not been completely elucidated, DIM interacts with the aryl hydrocarbon receptor (AhR), resulting in its nuclear translocation and induction of the genes encoding phase1 and II enzymes^[27]. Several lines of evidence suggest that DIM exerts agonist and/or modulator activity on the AhR^[28,29]. I3C can activate the NF-E2-related factor-2 (Nrf2) transcription factor which interacts with the antioxidant response element in the promoter of many cytoprotective enzymes, as described later. The induction of cytochrome P450 (CYP450) by physiological concentrations of I3C and DIM was observed in cancer cells^[30] and confirmed by an analysis of mRNA expression profiles^[31]. Increased CYP450 activity led to increased estrogen metabolism and the degradation of estradiol (E2)^[27], which is required for the growth of estrogen receptor-alpha (ERα)-positive cancer cells. I3C (50 µmol/L) inhibited the estradiol-stimulated growth of estrogen-responsive MCF7, T47D and ZR75.1 breast and cervical cells. It inhibited receptor phosphorylation and DNA binding as well as estrogen-dependent reporter gene activity in breast tumor cells and cervical cancer cell lines^[32,33]. DIM (10 µmol/L) also inhibited the estradiol-stimulated growth of MCF7, but in the absence of 17β-estradiol it appeared to stimulate growth^[34].

Table 3 Bioactivity of I3C in vitro.
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Table 4 Bioactivity of DIM in vitro.
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DIM also exerted other estrogenic effects in these and endometrial cells^[34–36]. Conversely, earlier data indicated an inhibitory effect of DIM on E2-regulated reporter activity and ER DNA binding^[27]. Both I3C (75–100 µmol/L) and DIM (1–25 µmol/L) reduced the expression of ERα mRNA in MCF7 cells^[33,37]. DIM also interfered with androgen receptor expression, DNA binding, and signaling^[38,39].

Both compounds are growth inhibitory to a wide range of tumor cell lines, including ER-negative cancer cells. The increased loss of viability of several breast cancer cell lines when grown in a 3-D environment in the presence of I3C implied greater susceptibility in vivo than in a monolayer cell culture^[40]. Recent studies have shown that I3C decreases proliferation and induces apoptosis by reducing the expression and signaling of the genes essential for tumor cell viability, such as ER and epidermal growth factor receptor (EGFR) in breast cells of luminal A and basal-like subtypes^[33,41]. I3C (50 µmol/L) can also inhibit phosphoinositide-3-kinase (PI3K)^[42], resulting in the inhibition of protein kinase B (Akt) phosphorylation and decreased survival in cancer cells dependent on this pathway. A mechanism dependent on breast cancer-related protein (BRCA)1/2 upregulation has also been proposed^[43].

DIM, which is considerably more potent than I3C, also inhibits the growth of a range of cells (Table 4). Topoisomerase II-alpha and mitochondrial H⁺-ATP synthase were identified as direct targets of DIM. The inhibition of the latter enzyme results in increased mitochondrial reactive oxygen species (ROS) production and signaling via the p38 stress activation pathway^[44,45]. Apoptosis via the activation of the endoplasmic reticulum stress pathway has also been reported^[46,47].

Both agents have a significant effect on several other signaling pathways, such as those involving p38^[45,48] and NF-κB signaling^[49–53]. They modulate a variety of growth-, cell cycle-, and apoptotic-regulatory proteins at the mRNA or protein level, including EGFR, PI3K, transforming growth factor (TGF)-β2, fibroblast growth factor (FGF), cyclin E2, activating transcription factor (ATF), B cell lymphoma Bcl2, BclX_L, Bad, and Bax.

In vitro effects of I3C or DIM observed in vivo Both I3C and DIM clearly inhibit tumor cell growth in vivo in a range of animal models^[54]. The reduced incidence and multiplicity of mammary tumors was concurrent with increased phase I and II drug metabolizing enzymes in I3C-treated animals^[55–57]. An analysis of transgenic Nrf2^–/– mice indicated that the I3C-induced upregulation of phase II enzymes required the Nrf2 transcription factor^[58,59]. The upregulation of CYP450 activity by DIM in vivo has been proposed to occur via a mechanism involving the AhR^[30], as reported in MCF7 cells in vitro^[27]. The induction of phase I and II enzymes has been reported in liver, small intestine, and lungs of rodents receiving I3C (in the diet or by gavage)^{[30,54,57,60–63]}. DIM also induced P450 activity and flavin-containing monooxygenase 1 in the rat liver^[30,60].

Apoptosis in response to I3C was observed in vivo in initiated mammary glands with activation of caspases-8, -9, and -3^[25] and in cervical epithelium of transgenic mice (HPV16), developing cervical cancer in response to estrogen^[56]. Few studies have investigated the effect of either agent on signal transduction intermediates in vivo, but in one study, dietary I3C (0.5%) caused a significant decrease in total tyrosine phosphorylation and ornithine decarboxylase activity in the rat liver^[62]. Many of the signaling events modulated by I3C in vitro involve tyrosine phosphoryla-tion^[41], but interestingly, changes in ornithine decarboxylase activity in breast and colon cells in vitro were only observed at relatively high concentrations (>100 µmol/L)^[62,64]. The downregulation of NF-κB-regulated genes by I3C occurring in a variety of cancer cells in vitro, was also observed in mouse xenografts of MDA-MB231 cells^[52,65].

Evidence for I3C or DIM acting synergistically/ antagonistically In rats, I3C (5 mg/kg) reversed vinblastine- or vincristine-induced P-glycoprotein levels^[66]. This group also showed that a very high dose of I3C (10 mmol/L) decreased P-glycoprotein levels in vitro in the multidrug resistant cell line K562/R 10, sensitizing it to vinblastine, but had no growth-inhibitory effect on the parent K562 cell line^[67]. I3C (333 or 500 mg/kg per day) also reversed the MDR phenotype of the B16/hMDR1 (drug-resistant MDR1-expressing murine melanoma) tumor in vivo, and in combination with vinblastine, actually reduced the tumor mass^[68]. In the same study, an I3C acid-condensation mixture (12.5 µmol/L) sensitized the B16/hMDR1 cell line in vitro to vinblastine, while DIM (45 µmol/L) increased the drug content of cells by 50%^[68]. Combined treatments using I3C (50–125 µmol/L) in combination with Src or/and EGFR inhibitors reduced the viability of breast cancer cells MDA-MB468 and MCF7^[41].

DIM (25 µmol/L) plus genistein (5 µmol/L) synergistically induced growth arrest and DNA damage-inducible (GADD)34 protein levels and apoptosis, and at higher concentrations induced estrogen receptor response element (ERE)-driven reporter gene activity^[69]. I3C (50 or 100 µmol/L) has been shown to cooperate with tamoxifen (1 µmol/L) in vitro to increase cell growth inhibition and G₀/G₁ cell cycle arrest of MCF7 cells^[70]; in vivo, it reduces tumor mass and increases latency of mammary cancers^[71]. An I3C acid condensation mixture also enhanced the efficacy of vinblastine in mouse melanoma cells, while I3C itself had no effect^[68].

In vivo, dietary I3C, together with crambene (1-cyano 2-hydroxy 3-butene), another glucosinolate from vegetables, showed a greater than additive induction of glutathione-S-transferase (GST) and quinone reductase activity^[72].

Curcumin

Curcumin (diferuloylmethane) is a major constituent of the spice turmeric, derived from the roots of Curcuma longa. The major dietary source is curry, but it is also used as a food coloring and in some medicines.

Bioavailability of curcumin in humans Curcumin exhibits poor gastrointestinal absorption, with much of an oral dose passing unchanged through the gastrointestinal tract, and a further proportion undergoing conjugation, without absorption, prior to fecal loss. Absorbed curcumin undergoes sequential reduction and conjugation (glucuronidation and/or sulfation) within the gastrointestinal tract and liver, with the resultant formation of metabolites and low systemic levels of the parent compound^[73,74].

Studies in humans have demonstrated that the oral administration of curcumin furnishes very low systemic levels, mostly in the low nanomolar range (Table 5). An exception is the study by Cheng et al, which reported serum levels in the low micromolar range using the maximum tenable dose (8 g/d)^[75]. Other groups have failed to replicate this finding, with Sharma et al, for example, administering up to 3.6 g/d of curcumin to patients for up to 4 months, yet only achieving levels in the 10 nmol/L range^[76]. The discrepancy between these studies remains to be explained, but may have resulted from the use of different formulations of curcumin.

Table 5 Bioavailability of curcumin in humans following oral administration.
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Due to its poor bioavailability, curcumin levels in tissues beyond the gastrointestinal tract are also in the low nanomolar range or below. Garcea et al were unable to detect curcumin in normal liver or colorectal liver metastases in patients who had received 3.6 g/d for 1 week. In the only human study to examine colorectal tissue to date, this oral dose resulted in levels in the 10 µmol/L range^[77].

Bioavailability of curcumin in animals A number of groups have examined the bioavailability of curcumin in animals, following oral, intragastric (ig) or intraperitoneal (ip) dosing (Table 6). Studies suggest that oral dosing may give rise to significant levels of curcumin within the gastrointestinal tract. In a rat model, approximately 1.8 μmol curcumin/g of tissue was demonstrated in colonic mucosa following the dietary administration of 1200 mg/kg daily^[78]. Perkins et al reported 750 mg/kg of curcumin/d to result in ~100 nmol/g in mouse small intestine mucosa and 500 nmol/g within colonic mucosa^[79]. Following oral dosing of 400 mg per rat, liver and kidney levels were less than 20 μg per tissue^[80]. Significant levels of curcumin may also be achieved locally when administered topically to the skin or within the oral cavity, but the exact dose achieved in these scenarios remains to be confirmed.

Table 6 Bioavailability of curcumin in animals.
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It is neither practicable nor desirable to increase the oral dose of curcumin above that already investigated. Recent animal studies, however, have demonstrated that the reformulation of curcumin may enable further improvements in bioavailability. It has previously been shown that the formulation of drugs with phosphatidylcholine increases their plasma bioavailability. Such a formulation led to significantly higher levels of curcumin within plasma and the liver compared with the parent compound, although lower levels within the intestinal mucosa^[81]. Several other animal studies have also found curcumin bioavailability to be significantly increased by its administration as a phospholipid complex^[82,83]. These increases in bioavailability now require confirmation in human studies. Although the bioavailability data are lacking, in vitro and animal studies have also shown promising anticancer potential for a liposomal preparation of curcumin^[84,85]. In addition, nanoparticle-encapsulated curcumin may provide an alternative means to increase the bioavailability of this agent^[86].

Physiologically relevant concentrations of curcumin The bioavailability data suggest that in vitro studies with curcumin in the 10 µmol/L range or below might have human physiological relevance, but that its role as a chemopreventive agent may lie primarily within the gastrointestinal tract.

In vitro mechanistic studies using low doses of curcumin The anticancer effects of curcumin have been demonstrated in multiple cell types, at concentrations between 5 and 50 µmol/L^[87]. Selected studies demonstrating the anticancer activity of curcumin at or below the 10 µmol/L level achievable in the human colon in vivo are summarized in Table 7. Where available, data are presented from studies using colorectal cell lines; results from other cell types using a maximal dose of 10 µmol/L are also included. In addition to these studies, curcumin also inhibited the proliferation of squamous carcinoma SCC-25 cells^[88] and the proliferation and invasion of HBL100 breast cells^[89].

Table 7 Bioactivity of curcumin in vitro.
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In vitro effects of curcumin observed in vivo In a rat model, dietary curcumin significantly increased the apoptotic index in azoxymethane-induced colonic tumors^[90]. Rao et al demonstrated the effect of a curcumin-containing diet on azoxymethane-induced rat carcinogenesis^[91]. Curcumin significantly reduced tumor volume, as well as colonic mucosa and tumor prostaglandin (PGE)₂ expression by over 38%. Similarly, it enhanced 2-amino-1-methyl-6-phenylimidazol(4,5-b)pyridine-induced apoptosis in Min/+ mice and inhibited tumorigenesis in the proximal small intestine. Also in mice, Mahmoud et al found dietary curcumin to normalize enterocyte proliferation and restore the level of enterocyte apoptosis to that of wild-type animals^[92]. In rats, a gavage administration of curcumin (200 or 600 mg/kg) inhibited diethylnitrosamine (DEN)-induced hepatic hyperplasia and inflammation. Specifically, the increased expression of p21ras and p53 in the liver was prevented. The decreased expression of proliferating cell nuclear antigen, cyclin E, and cdc2 was also observed, along with the inhibition of DEN-induced NF-κB activation^[93].

While there are no in vitro studies for comparison, there is evidence from both animal and human studies showing that curcumin suppresses malondialdehyde-deoxyguanosine adduct (M₁dG) adduct formation in DNA^[77,78]. However, Garcea et al, while noting decreased M₁dG adduct formation in the colorectum following curcumin treatment, found no alteration in cyclooxygenase 2 (COX2) protein levels^[77].

Despite the low bioavailability of curcumin, there are examples in animal studies of its biological activity at sites distant from the locus of absorption, where levels are expected to be inefficacious based upon the results of in vitro studies. Sharma et al, for example, demonstrated increased hepatic GST expression and the attenuation of hepatotoxin-induced adduct formation following curcumin treatment^[78]. Oral curcumin also led to the complete suppression of tumor NF-κB activation in an orthotopic mouse model of pancreatic cancer^[94]. Anticancer activity has also been reported at a number of other sites distant from the gastrointestinal tract, including the breast^[95], prostate^[96], lung^[97], and liver^[98].

Evidence for curcumin acting synergistically/antagonistically The treatment of MCF7 breast cells with curcumin (10 µmol/L) and genistein (25 µmol/L) demonstrated a synergistic effect, leading to the total inhibition of proliferation induced by an endosulfane/chlordane/DDT mixture^[99]. Curcumin also synergistically potentiated the inhibitory effect of celecoxib on pancreatic carcinoma cells^[100] and additively inhibited the growth of colorectal cancer with celecoxib in the 1,2-dimethylhydrazine rat model^[101].

In an in vitro model of oral cancer, EGCG blocked cells in the G₀/G₁ phase, while curcumin blocked in the G₂/M phase of the cell cycle. The combination showed synergistic interactions in growth inhibition^[102]. While tea or curcumin individually decreased the number and volume of dimethylben-zanthracene (DMBA)-induced oral tumors in hamsters, only the combination decreased the proliferation index of squamous cell carcinoma^[103].

LNCaP prostate cancer cells are relatively insensitive to tumor necrosis factor related, apoptosis-inducing ligand (TRAIL). At low concentrations, neither TRAIL (20 ng/mL) nor curcumin (10 µmol/L) produced significant cytotoxicity, whereas cell death was markedly enhanced by the combina-tion. Both agents together induced the cleavage of procas-pases-8, -9, and -3, the truncation of Bid, the release of cytochrome c, and apoptosis^[104].

Recent studies have also demonstrated promising interactions between curcumin and established chemotherapeutic agents. In colorectal carcinoma lines, the antiproliferative and pro-apoptotic effects of curcumin and oxaliplatin increased markedly when cells were treated with both agents^[84,105]. Similarly, curcumin potentiated the pro-apoptotic effects of gemcitabine and paclitaxel in bladder cancer cell lines^[106] and the antitumor activity of gemcitabine in an orthotopic model of pancreatic cancer. Antagonistic interactions have also been demonstrated, however, with curcumin shown to inhibit chemotherapy-induced apoptosis in breast tumor lines. Camptothecin-, mechlorethamine-, and doxorubicin-induced apoptosis in MCF7, MDA-MB231 and BT474 cells was inhibited by as much as 70%, following 3 h exposure to as little as 1 μmol/L curcumin. The inhibition of both c-jun N-terminal kinase (JNK) activation and cytochrome c release occurred^[107]. The same authors, using an in vivo xenograft model, found dietary curcumin (25 g/kg) decreased the level of cyclophosphamide-induced tumor regression, again with decreased JNK activation and less apoptosis.

Epigallocatechin-3-gallate (EGCG)

Green tea and its constituent molecules, including EGCG, have been found to prevent tumor formation in a wide range of tissues in animal models. However, the possible influence of green tea on cancer in humans has been difficult to interpret due to confounding factors, such as diversity in types of tea used, preparation methods, including temperature of infusion, and frequency of tea drinking.

The relevance of in vitro studies with EGCG has been reviewed by Lambert and Yang^[108], who concluded that the effectiveness of tea consumption in cancer prevention remained unclear and required a better understanding of bioavailability and fundamental mechanisms.

Bioavailability of EGCG in humans A number of studies have reported the bioavailability of EGCG in various human body fluids (Table 8) following the administration of green tea or EGCG. Levels in plasma up to a maximum of 7.3 µmol/L (±3.6) have been reported, but more often are in the submicro-molar range. Bioavailability in two early studies found plasma levels at 0.2%–2% of the ingested amount of EGCG (up to 4 µmol/L), but higher plasma concentrations have since been reported in fasting patients compared to those who consumed catechins with food^[109–111]. The oral administration in human patients resulted in high plasma clearance levels and volume distribution, suggesting that the bioavailability of EGCG in the blood may be low, similar to the situation found in rodents^[109,112]. Dvorakova et al suggested that topical application to skin of an ointment containing 10% EGCG was likely to result in substantial intradermal uptake, but very poor systemic absorption^[113].

Table 8 Bioavailability of EGCG in humans following oral administration.
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It has been found that holding a green tea solution (1.2 g/200 mL water) in the mouth for 1 min resulted in salivary EGCG concentrations (mean) of 27 µmol/L, with values up to 48 µmol/L recorded, several fold higher than that achieved through normal drinking, and many more times greater than plasma concentrations^[114,115]. However, holding the tea in the mouth for 5 min resulted in salivary concentrations 4–5 times higher, whilst taking tea solids in capsules resulted in no detectable salivary catechin levels. Thus, drinking tea slowly may be an effective way of delivering relatively high concentrations to the oral cavity and esophagus.

EGCG can undergo metabolism through glucuronidation, sulfation, methylation, or ring fission^[108], processes which are subject to interindividual variation.

Bioavailability of EGCG in animals Surprisingly, few studies have documented the bioavailability of EGCG in animals. Most have shown a maximum plasma concentration in the nmol/L to low µmol/L range, similar to the human situation, although 1 study using a large dose of EGCG in rats reported plasma concentrations up to 20 µmol/L (Table 9). No animal studies have examined the effect of fasting on bioavailability. Fang et al, using liposomes for the local (injection) delivery of EGCG, found that a liposomal cocktail containing deoxycholic acid and ethanol greatly increased the tumor uptake of EGCG in both melanoma and colon murine tumor models^[116,117]. However, liposomal delivery was not superior following topical application.

Table 9 Bioavailability of EGCG in animals.
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Lambert and colleagues^[118] reported that piperine from black pepper enhanced the bioavailability of EGCG in mice. Small intestinal levels following EGCG administration alone resulted in a C_max of 37.5±2.5 nmol/g at 60 min, decreasing to 5.1±1.7 nmol/g at 90 min. Following cotreatment with piperine, the C_max was 31.6±15.1 nmol/g at 90 min, with levels still above 20 nmol/g at 180 min. The appearance of EGCG in the colon and feces was slower in the cotreated mice.

In rats and mice 24 h after the intragastric administration of radiolabeled EGCG, 10% of the dose was present in the blood, with around 1% in tissues, such as liver, kidney, heart, lung, and prostate^[119]. Major elimination occurred in the feces. In line with these findings, another study suggested that the transporter-mediated intestinal efflux of catechins may play a role in the systemic elimination of these compounds^[120]. Following an intravenous (iv) dose in rats, >70% was eliminated in bile and 2% in urine^[119,121]. A study in rats, in which different green tea catechins were administered by iv or ip, suggested that first-pass hepatic elimination did not play a major role in the metabolism of orally-administered epigallocatechin, epicatechin, and EGCG^[122].

Physiologically relevant concentrations of EGCG The plasma bioavailability of EGCG, whether administered as tea or a pure compound, is in the range of 0.1–7 µmol/L in humans, with concentrations over 100 µmol/L observed in saliva. No significant excretion occurred in urine (generally <0.1% of dose). Rodent data indicate levels up to 20 µmol/L may be achievable. Based on these data, we chose 20 µmol/L as the maximum concentration at which to consider in vitro findings.

In vitro mechanistic studies using low doses of EGCG Many in vitro studies show that EGCG, at concentrations ≤20 μmol/L, inhibits growth and induces cell cycle arrest or apoptosis in a variety of cell types (Table 10). A wide range of signaling molecules is affected, including growth factor receptors [EGFR, platelet derived (PDGFR), fibroblast (FGFR), vascular endothelial (VEGFR)], survival signaling pathway components [extracellular signal regulated kinase (ERK), p38, activating protein-1 (AP-1), signal transducer and activator of transcription (STAT), PI3K, Akt, and NF-κB], cell cycle regulators [cyclin D1, p21, p27, phosphorylated retinoblastoma (pRb), cyclin-dependent kinases (cdk)2/4/6], and regulators of apoptosis [Bcl2, Bax, Bad, caspases-3/7/8/9, and poly (ADP ribose) polymerase (PARP)]. One interesting effect of EGCG at the lowest doses (0.01–0.1 µmol/L) is the inhibition of VEGF-dependent phosphorylation of the VEGFR (Table 10), an anti-angiogenesis effect which also occurs in vivo, as discussed later.

Table 10 Bioactivity of EGCG in vitro.
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In vitro effects of EGCG observed in vivo There is substantial evidence that the effects of EGCG or green tea recorded in vitro have also been observed in animal models or humans. Green tea polyphenols inhibited the growth of 4T1 breast cancer cells and their metastasis to lungs in BALB/c mice. A reduction in tumor weight, increased survival time, and later tumor appearance were observed. The ratio of Bax/Bcl2 was altered in favor of apoptosis, along with a decrease in proliferating cell nuclear antigen and the activation of caspase-3^[123]. The topical application of EGCG to SKH-1 hairless mice that had been pretreated twice weekly with UVB light decreased the multiplicity of skin tumors by 44%–72% and increased the apoptotic index by 56%–92%, again measured by increased caspase-3 activity^[124]. Fang and colleagues, who demonstrated that the liposomal delivery of EGCG resulted in increased tumor uptake, also found that this delivery system led to increased antiproliferative activity in basal carcinoma cells in vitro, where the EGCG concentration in the liposomes was 21.3 μmol/L^[117].

A study investigating the effect of EGCG in murine colon 26-L5 cells found that, using in vitro assays, 1,1-diphenyl-2-picryl-hydrazyl free radicals were reduced with an ED₅₀ of 2.9 µmol/L, and cell growth was inhibited with an IC₅₀ of 41.8 µmol/L. Following the injection of these colon cells into female BALB/c mice to analyze the effect on lung metastases, they found that EGCG, administered ip, reduced the number of tumor nodules in a dose-dependent manner^[125].

When green tea extract (400–500 mg/cup, 5 cups/d) was administered for 4 weeks to 3 heavy smokers^[126], smoking-induced DNA damage was decreased, cell growth (keratino-cytes) was inhibited, and the percentage of cells in S phase was reduced, with accumulation in the G₀/G₁ phase. DNA content became less aneuploid and p53 and caspase-3 were increased. Li and colleagues found that in hamsters, 0.6% green tea inhibited DMBA-induced oral tumor number and volume, with increased apoptosis and a decreased proliferation index and microvessel density^[103]. In vitro EGCG inhibits AP-1 transcriptional activation, and this was also observed in vivo in UVB-treated transgenic mice carrying a luciferase reporter gene with an AP-1 binding sequence^[127].

EGCG (10 or 50 µg/mL) significantly decreased the proliferation of bovine capillary endothelial cells, and at 1–100 µg per disc, it also inhibited neovascularization in the chick chorioallantoic membrane assay^[128]. These authors also showed that green tea in drinking water (1.25% containing 708 µg/mL EGCG, giving plasma levels of 0.1–0.3 µmol/L) could significantly suppress VEGF-induced corneal neovascularization. Such results suggest that EGCG may be a useful in vivo inhibitor of angiogenesis.

Green tea consumption in two study groups, one in China and one in the USA, decreased oxidative DNA damage (8-hydroxy-deoxyguanosine in white blood cells and urine), lipid peroxidation (malondialdehyde in urine), and free radical generation (2,3-dihydroxy benzoic acid in urine) in smokers. Non-smokers (USA group) also exhibited a decrease in overall oxidative stress, which was correlated to decreased levels of free radicals^[129].

A recent clinical trial involving 60 volunteers with (premalignant) prostate intraepithelial neoplasia, conducted by Bettuzzi et al, showed that after 1 year, only 1 man (3%) in the group receiving 600 mg/d green tea compounds in (oral) capsule form, presented with cancer compared to 9 (30%) from the placebo group^[130]. No significant side-effects were reported. Therefore, despite the apparent poor bioavailability of green tea catechins in many studies, they appear to have great promise as chemopreventive agents.

Evidence for EGCG acting synergistically/ antagonistically There are a number of reports documenting an enhanced chemopreventive effect when EGCG or green tea is used in combination with another chemopreventive agent or a therapeutic drug.

Suganuma et al found that epicatechin significantly enhanced the uptake of labeled EGCG into human lung PC-9 cells and suggested that whole green tea was a better preventive agent than EGCG alone^[131]. In this study, the pro-apoptotic effects of EGCG were also increased by tamoxifen or sulindac. Another study using the prostate cancer cell lines PC-3, LNCaP, and CWR22Rv1 showed that while 10 μmol/L EGCG only resulted in a 12%–21% inhibition in cell viability, the addition of 10 μmol/L NS-398 (a COX2 inhibitor), resulted in a 44%–49% inhibition, greater than the additive effect of either agent alone. These results corresponded to decreases in Bcl-2, procaspases-6 and -9, phospho-p65 and peroxisome proliferator-activated receptor (PPAR)γ, and increases in Bax and PARP^[132].

EGCG at 0.1 µg/mL (equivalent to serum concentrations) markedly enhanced the growth inhibitory effects of 5-fluorouracil in head and neck squamous carcinoma cells^[133]. The IC₅₀ values for 2 different cell lines were reduced by ~4-fold (sensitive line) and 45-fold (resistant line). EGCG on its own at this concentration had no effect. The same group also found that EGCG enhanced the sensitivity of HNSCC (0.1 µg/mL) and breast (1.0 µg/mL) cells to Taxol^[134].

Min/+ mice treated with a combination of white tea (1.5%) and sulindac (80 ppm) had significantly fewer intestinal tumors than mice treated with either agent alone. While β-catenin and β-catenin/T cell factor-4-regulated genes, cyclin D1, and c-jun were detected in polyps, the expression of these proteins was markedly reduced in the normal intestine^[135]. A combination of EGCG and sulindac was also found to be efficacious in preventing azoxymethane-induced colon cancer in rat, where the combination synergistically enhanced apoptosis^[136].

Resveratrol

The phytoalexin resveratrol is found largely in grape products and peanuts, with red wine a major source of human consumption. Its potential role in disease prevention is well documented, as it exhibits vasorelaxing, anti-inflammatory, antilipidemic, anti-estrogenic, antioxidant, antifungal, and antibacterial properties^[137–139].

Whilst resveratrol appears to have great potential in vitro, the relevance to in vivo effects in both humans and animals is less clear, as its chemopreventive effect in vivo depends greatly on its absorption, metabolism, and tissue distribution.

Bioavailability of resveratrol in humans Several studies have looked at the rate of uptake in healthy human volunteers via the oral administration of either resveratrol in its pure form or when present in foodstuffs (Table 11). Estimates of the amount of resveratrol in red wine (mainly trans), vary from 0.3 to 10.6 mg/L (1.3–46 µmol/L)^[140–142]. Recent studies by Boocock et al^[143,144] found peak resveratrol plasma concentrations of 0.3–2.4 µmol/L following single oral doses of 0.5–5 g in healthy human volunteers. Observed peak plasma concentrations for resveratrol metabolites ranged between 0.92 and 4.3 µmol/L (mono-glucuronides), and 3.7–14 µmol/L (resveratrol 3-sulfate). Plasma half-lives for the parent compound and major conjugates were of a similar order (2.9–10.6 h).

Subjects receiving a lower dose of 10–25 mg pure resvera-trol/70 kg body mass were similarly found to have serum resveratrol levels between 1.83 and 2.06 µmol/L at 30 min post-dose, returning to baseline by 4 h^[145]. Wang et al^[146] found both resveratrol and resveratrol glucuronide present at up to 6 h following a dose of 1 g. It is likely that conjugated resveratrol is the main component in the circulation^[147], and plasma concentrations around 2 µmol/L appear typical (Table 11).

Several major metabolites of resveratrol are found in human subjects, including the sulfate-glucuronide, mono-glucuronides, and mono- and di-sulfates^[143,144], with sulfation thought to occur primarily via the sulfotransferase 1A1^[148] and glucuronidation via the UDP-glucuronosyltransferases^[149]. The rate of glucuronidation in the human liver ranges between 0.23 and 1.2 nmol/min/mg and the rate of sulfation is 80 pmol/min per mg^[141,150]. The presence of sulfated products in vivo may vary depending upon whether pure resveratrol is administered, as quercetin (also present in red wine) is known to inhibit its sulfation. Resveratrol glucuronide has been assumed to be pharmacologically inactive, although it has been suggested that glycosylation of polyphenols is an important step in protecting them from enzymatic oxidation, so extending their half-life and biological properties^[151]. However, it is possible that the glucuronide may be converted back to resveratrol in vivo by the action of beta-glucuronidases^[152]. The aqueous solubility of resveratrol is low, and it is thought that albumin is the main carrier in plasma, with little free resveratrol^[153]. The presence of fatty acids increases binding to serum albumin, which may have important consequences for the delivery of resveratrol to cell membranes and thus signaling events.

Bioavailability of resveratrol in animals Marier et al^[154] observed that resveratrol was absorbed within minutes following an oral dose of 50 mg/kg to rats. Resveratrol aglycone (parent compound) plasma concentrations dropped from 10 µmol/L to levels at the limit of detection by 12 h. The glucuronide, however, was present at ~100 µmol/L, falling to 3 µmol/L after 12 h, again suggesting that resveratrol is absorbed from the intestine mainly in this form. A lower dose of 20 mg/kg gave a maximal resveratrol plasma content of 3 μmol/L, falling to <0.1 µmol/L after 1 h^[140], with a 5 mg/kg dose showing maximal plasma levels of 1.5 µmol/L ^[155].

The disposition of resveratrol revealed far higher levels in tissues involved in absorption and excretion, such as the intestine, liver, and kidney, than in plasma. In mice, the highest accumulation was in intestinal mucosa^[156], brain^[157], kidneys, and liver, reaching 25–30 µmol/L following a dose of 5 mg/kg^[155]. The major tissue metabolites were resveratrol-3,4'-disulfate, -3,4',5-trisulfate and β-D-glucuronide^[158]. Following a 20 mg/kg dose, the rat lung contained 0.8 nmol/g resveratrol, the mouse liver 1.03 nmol/g, and the mouse kidney 0.17 nmol/g^[140].

Physiologically relevant concentrations of resveratrol Animal and human studies consistently indicate resveratrol levels of 1–2 µmol/L in plasma (Tables 11, 12). Concentrations 10–20 times higher than this have been achieved through ig or iv dosing in animal studies. An examination of tissue distribution revealed that concentrations in some tissues can be significantly higher than those in plasma. Therefore, in considering the relevance of in vitro studies, we have focused on those which have reported effects at concentrations of 10 µmol/L or less.

Table 11 Bioavailability of resveratrol in humans following oral administration.
Full table

Table 12 Bioavailability of resveratrol in animals.
Full table

In vitro mechanistic studies using low doses of resveratrol Cell culture studies with resveratrol indicate anticancer potential over a range of doses and in a wide variety of tissues, including breast, colon, pancreas, stomach, prostate, head and neck, ovary, liver, lung, and cervix^[137] (Table 13). At physiological concentrations of 10 µmol/L or less, resveratrol exhibits a range of activities which modulate signal transduction. These include the downregulation of growth factors (EGF and VEGF), alterations to survival signaling (ERK, JNK, AP-1, and NF-κB), cell cycle regulators (cyclinD1, cdk4/6, p21, and p53) and apoptosis regulators (PARP, ceramide, and caspases). In U937 monocytes, concentrations as low as 0.1 µmol/L effectively inhibited the production of ROS, with the inhibition of Akt phosphorylation at 10 µmol/L ^[159].

Table 13 Bioactivity of resveratrol in vitro.
Full table

In vitro effects of resveratrol observed in vivo Whether or not the IC₅₀ values in vitro are achievable in vivo depends to some extent on the target tissue. It is likely that the highest concentration of resveratrol and its metabolites following ingestion would be achieved in colorectal mucosal tissue and in the liver. Several studies administering resveratrol via ingestion of red wine have used the mean serum antioxidant capacity as a marker of efficacy. In healthy volunteers consuming 300 mL red wine over a 30 min period, blood taken up to 2 h post-dose showed significantly raised serum antioxidant capacity^[160].

MDA-MB231 xenografts in nude mice exhibited an increase in the apoptotic index and decreased angiogenesis when treated daily with 25 mg/kg resveratrol for 3 weeks, while the same cell line in the culture did not undergo apoptosis at concentrations less than 100 µmol/L^[161]. Conversely, when B16M tumor cells were inoculated into mice, 20 mg/kg resveratrol did not affect tumor growth (tumor concentration of 0.04 nmol/g), even though in the culture the cells underwent 60% apoptosis following a 5 µmol/L treatment for 24 h^[140]. The rats inoculated with the Yoshida ascites hepatoma receiving daily ip injections (1 mg/kg) of resveratrol exhibited decreased tumor growth due to the induction of apoptosis and a G₂/M cell cycle arrest. This effect was not seen in vitro using resveratrol in the range of 15–30 µmol/L over 24 h^[162]. Daily ip injections of 40 mg/kg resveratrol (estimated serum level of 25 µmol/L) reduced neuroblastoma growth in rats and increased survival by 70%. In culture, resveratrol was also cytotoxic to neuroblastoma cells in a range from 10 to 100 µmol/L ^[163]. Resveratrol at 100 mg/kg per day prolonged the survival time for rats with intracerebral tumors generated from RT-2 glioma cell xenografts. The IC₅₀ for RT-2 cells in the culture equated to 12.8 µmol/L following a 48 h treatment, with 39% cells undergoing apoptosis at the higher concentration of 25 µmol/L^[164].

A number of reports have shown that resveratrol can inhibit NF-κB activation in vitro. Banerjee et al found that in rats, 10 ppm produced striking reductions in DMBA-induced breast tumor incidence and multiplicity, while extending tumor latency^[165]. They reported that resveratrol suppressed DMBA-induced COX2 and matrix metallopro-teinase (MMP)-9 expressions through the downregulation of NF-κB activation.

Resveratrol treatment may also inhibit preneoplastic conditions. In both an experimentally induced model of colitis^[166] and the Min/+ mouse^[167], resveratrol was able to reduce damage/adenoma load and COX2 protein expression. The spontaneous development of mammary tumors in HER2/neu mice was delayed with the reduction in both size and number of tumors following resveratrol treatment^[168]. In rats, azoxymethane treatment caused the formation of aberrant crypt foci, the number of which was significantly reduced in the presence of resveratrol (200 µg/kg per day for 100 d), with decreased bax and increased p21 expression in the crypts^[169]. The treatment of dimethylhydrazine-induced aberrant crypt foci with resveratrol (8 mg/kg per day) resulted in a marked reduction in tumor incidence and degree of histological lesions^[170]. Similarly, rats fed a diet containing 15% grape extract showed a decrease in the number and area of GST-P^+ve foci^[171].

Evidence for resveratrol acting synergistically/antagonistically Resveratrol sensitized colon cancer cells to CD95 and the TRAIL-mediated induction of apoptosis, and at 10 µmol/L, sensitized HT29 cells to cisplatin-induced apopto-sis^[172]. Fulda and Debatin found that pretreatment with resveratrol cooperatively enhanced doxorubicin, cytarabine, actinomycin D, Taxol, and methotrexate-induced apoptosis and cell cycle arrest in neuroblastoma cells^[173], and enhanced TRAIL-mediated apoptosis in neuroblastoma and Jurkat T cells^[174]. Resveratrol (10 μmol/L) also enhanced the apoptotic effects of paclitaxel in A549, EBC-1, and Lu65 lung cancer cell lines^[175], and of cisplatin and doxorubicin in OVCAR-3 and Ishikawa cells, respectively^[176].

Resveratrol has been used in combination with other phytochemicals, such as betasiterol, resulting in enhanced growth inhibition due to an arrest at both the G₁ and G₂/M phases of the cell cycle in PC-3 cells^[177]. The combination of quercetin/ellagic acid with resveratrol resulted in a synergistic effect on caspase-3 activation leading to apoptosis^[178]. Lee et al^[179] found individual concentrations of resveratrol (0.5 µmol/L) or I3C (50 µmol/L) to induce the non-steroidal, anti-inflammatory, drug-activated gene (NAG)-1, a TGF-β superfamily gene associated with pro-apoptotic and anti-tumorigenic activity. However, when used in combination, the doses could be reduced to 0.025 and 1 µmol/L, respectively.

Conclusion

Plasma concentrations in humans, following normal dietary intake or administration of supplements or formula-tions, have been measured or can be estimated from animal studies for each of the agents reviewed. In general, achievable plasma concentrations were in the low micromolar range, although animal studies revealed the possibility of considerably higher concentrations in some tissues. In summarizing data from many cell culture studies, which have been carried out using the low concentrations achievable in vivo, it is apparent that all the compounds still exhibit biological activity. However, the range of activities is more limited, compared to that using a much wider dose range. While this may reflect genuine lack of activity at low doses, it is partly due to the fact that many studies have chosen to use only higher doses and shorter time points. There may therefore be many more useful preventive possibilities to be identified using lower doses.

Two very encouraging themes emerge from the data reviewed here. First, while not all in vitro findings are matched in vivo, many observations have been validated in animals or humans, giving credibility to the value of cell cultures for screening and more detailed mechanistic studies. However, some caution is required in extrapolation, as in many cases it is not known whether exactly the same signaling mechanisms are operational in vivo. There can also be significant discrepancies in the effective doses, even to the extent that where low levels are active in vivo, much higher concentrations are required to achieve the same effect in vitro.

Second, there is a growing body of evidence to suggest that even if single agents are inactive at low concentrations, combinations of 2 or more compounds might be much more efficacious. Combinations with chemotherapeutic drugs also offer the possibility of lowering the dose of the latter, with a consequent reduction of unwanted side-effects.

Thus, studies in cell culture have provided valuable insights into chemopreventive mechanisms of action, and there is now a need to pursue these at physiological doses and in novel combinations. One further aspect, which has not been tackled in any detail here, is the need to address more rigorously the question of tissue specificity and cancer subtype.

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