Phytochem Rev (2008) 7:431–443 DOI 10.1007/s11101-008-9093-5
Phytoestrogens as natural prodrugs in cancer prevention: a novel concept Randolph R. J. Arroo Æ Vasilis Androutsopoulos Æ Asma Patel Æ Somchaiya Surichan Æ Nicola Wilsher Æ Gerry A. Potter
Received: 11 October 2007 / Accepted: 28 February 2008 / Published online: 26 March 2008 Ó Springer Science+Business Media B.V. 2008
Abstract It has been generally accepted that regular consumption of fresh fruits and vegetables is linked with a relatively low incidence of cancers (e.g. breast, cervix, and colon). A number of plant-derived compounds have been identified that are considered to play a role in cancer prevention. However, at present there is no satisfactory explanation for the cancer preventative properties of the above-mentioned compound groups. The current review is an effort to develop a consistent and unambiguous model that explains how some plant-derived compounds can prevent tumour development. The model is based on recent discoveries in the fields of genomics and drugmetabolism; notably, the discovery that CYP1 genes are highly expressed in developing tumour cells but not in the surrounding tissue, and that a variety of plantderived compounds are substrates for the CYP1 enzymes. Our hypothesis is that some dietary compounds act as prodrugs, i.e. compounds with little or no biological effect as such, but become pharmaceutically effective when activated. More specifically, we state that the abovementioned prodrugs are only activated in CYP1-expressing cells—i.e. cells in the early stages of tumour development—to be converted into compounds which inhibit cell growth. Thus, the
R. R. J. Arroo (&) V. Androutsopoulos A. Patel S. Surichan N. Wilsher G. A. Potter Leicester School of Pharmacy, De Montfort University, The Gateway, Leicester LE1 9BH, UK e-mail:
[email protected]
prodrugs selectively kill precancerous cells early in tumour development. The review focuses on the identification of naturally-occurring prodrugs that are activated by the tumour-specific CYP1 enzymes and aims to assess their role in cancer prevention. Keywords CYP1 Drug metabolism Flavones Natural products Prevention
Anticancer drug discovery In the 1950s, the National Cancer Institute (NCI) started a large-scale empirical anticancer drug screening program that continued in more or less the same form until the mid-1970s. In this program, compounds were tested against a panel of mouse tumour models, mainly L1210 and P388 leukaemias (Suggit and Bibby 2005). This screening method led to the identification of a large number of compound classes that have formed the basis for most of the anticancer drugs that are currently in clinical use (Table 1). The screening program was highly compound-oriented and resulted in a golden era for phytochemistry, since natural products formed a major source of lead compounds. Indeed, a large proportion of the now conventional anticancer drugs are derived from natural sources, e.g. Vincristine and Vinblastine from Catharanthus roseus, Paclitaxel and Docetaxel from Taxus species, Doxorubicin (Adriamycin) and
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Table 1 Overview of traditional cytotoxic agents, their mode of action and side effects Class of drug
Examples
Mode of action
Side effects
Alkylating agents
Cyclophosphamide
Formation of covalent bonds with DNA, thus impeding DNA replication
Myelosuppression
Chorambucil
Antimitotic agents
Vincristine Vinblastine
GI disturbances, hair loss, Reduced fertility, increased risk of secondary malignancies
Melphalan Mitomycin Interference with microtubule assembly of cells, thereby blocking cell division.
As for alkylating agents;
Inhibition of metabolic pathways involved in DNA synthesis.
As for alkylating agents:
Neurotoxicity
Docetaxel Paclitaxel Antimetabolites
Methotrexate 5-Fluoroacil
Nephrotoxicity
6-Mercaptopurine DNA intercalators
Doxorubicin
Prevent DNA replication
Mitoxantrone Topoisomerase inhibitors
Etoposide Teniposide
As for alkylating agents; Doxorubicin can cause cardiotoxicity
Prevent unwinding of DNA strands, thereby blocking DNA replication.
As for alkylating agents
Camptothecin Irinotecan Heavy metal derivatives
Cisplatin
As metallating agents.
Carboplatin
Mitomycin from Streptomycin species, the podophyllotoxin derivatives Etoposide and Teniposide from Podophyllum species, Camptothecin and its semisynthetic derivative Irinotecan from Camptotheca acuminata. A recent estimate stated that approximately 60% of drugs in clinical trials for the multiplicity of cancers are either natural products, compounds derived from natural products, or drugs containing pharmacophores derived from natural products (Cragg and Newman 2000). The use of the mouse tumour models has led to concerns that the screening may have resulted in preferential selection of drugs that were only active against rapidly growing tumours. Indeed, most existing anticancer agents target DNA replication of dividing cells, thus inhibiting tumour cell growth but also normal cell growth. This causes many undesired side effects such as nausea and vomiting, alopecia, mucositis, myelosuppression and reproductive sterility. These side effects of chemotherapy can have a devastating impact on a patient’s quality of life. Particularly, the treatment of elderly patients (older than 70 years) presents characteristics that
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As for alkylating agents; Nephrotoxicity; Neurotoxicity and potention loss of hearing
make the choice of the correct treatment more difficult; for this reason, these patients are often undertreated (Pasetto et al. 2007).
Smart anticancer drugs The main aim of conventional cancer treatment was systemic, non-specific, high-dose chemotherapy (Abou-Jawde et al. 2003). The success of these treatments was outweighed by the systemic toxicity. However, as more was discovered about the mechanisms underlying the initiation and progression of human cancers, anticancer drug discovery moved away from the development of classic cytotoxic agents to the rational design of small molecule anticancer therapeutics. This development has prompted a transition from empirical compoundorientated preclinical screening to target-orientated drug screening (Suggit and Bibby 2005). The socalled smart drugs, which have been developed as a result of the new screening methods, are small molecules targeted at specific growth signalling
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pathways. These new drugs are expected to dominate clinical trials in the years to come either as singledrug modality or as combination treatment (van der Poel 2004). The new generation of chemotherapeutics act on specific molecular targets that are responsible for malignant phenotypes, and thus provide potentially tumour specific therapy (Table 2). Most of the compounds are synthetic molecules that are made to fit particular well-defined targets; very few of this second generation of anticancer drugs are based on natural products. Theoretically, the use of these putatively tumour specific chemotherapeutic agents, whether alone or in combination, should lead to an improved clinical response. However, the expression of the target proteins is not limited to tumour cells alone; the proteins are over-expressed in tumour cells, but also expressed at a lower frequency in normal cells. Thus, problems remained with unacceptable damage to normal cells and organs, a narrow therapeutic index, or a relatively poor selectivity for neoplastic cells. A potential strategy to overcome the limitations of chemotherapeutic agents is the use of prodrugs, i.e. compounds that need to be transformed before exhibiting their pharmacological action (Rooseboom et al. 2004). Prodrugs are often divided into two groups: 1.
prodrugs designed to increase the bioavailability of antitumour agents, e.g. when the activated drug has a low chemical stability or is rapidly broken down in vivo, more stable prodrugs can be administered.
2.
prodrugs designed to locally deliver antitumour agents (targeting)
CYP1-activated anticancer prodrugs A recent discovery in the field of oncology was that the cytochrome P450 enzyme CYP1B1 is expressed at a high frequency in a wide range of human cancers, including tumours of the breast, colon, lung, oesophagus, skin, lymph node, brain, and testis (Table 3, 4). The protein could not be detected in the surrounding normal tissues (Murray et al. 1997, 2001). Preliminary data by Stanley et al. (2001a, b) have indicated that the CYP1B1 protein is also over expressed in premalignant tissue, which suggests that CYP1B1 has a role in early tumour development. However, other authors (Gibson et al. 2003) call for more caution since they could detect CYP1B1 protein in patient samples of human colonic tissue specimens, which were excised C30 cm from the tumour, and did not show any of the characteristics of premalignant tissue. A related cytochrome P450, CYP1A1, is abundant in tumour tissue, but various xenobiotics (notably aryl hydrocarbons) can induce its expression in healthy tissue, notably in the epithelial lining of the lungs (Zhang et al. 2006). CYP1 enzymes are known to play a role in the metabolism of estrogens. Notably, CYP1B1 converts the oestrogen estradiol into 4-hydroxyestradiol. The
Table 2 Overview of targeted cancer treatments, their mode of action and side effects Class of drug
Examples
Mode of action
Side effects
Hormonal therapies
Tamoxifen
Prevent biosynthesis of oestrogen, or block the action of hormones tumour development.
Endometrial cancer, blood clots, hot flushes, fatigue, GI disturbances
Block activation of secondary messenger molecules; inhibit tyrosine kinase activity
Fluid retention, skin rashes, diarrhoea, GI disturbances
Antibodies bind to proteins that are overexpressed in tumours, e.g. CD20 antigen, or HER2 receptor.
Allergic response
Induces differentiation and death of leukaemia cells
Fever, severe respiratory distress, weight gain
Letrozole Arimidex Bicalutamide Signalling inhibitors
Imatinib Gefitinib Rituximab
Monoclonal antibodies
Trastuzumab Gemtuzumab Ozogamicin
Differentiating agents
Tretinoin
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Table 3 Expression of CYP1B1 in different types of tumours and normal tissue (after Murray et al. 1997)
Tissue
Normal (no positive/no tested)
Bladder
0/8
8/8
Brain
0/12
11/12
Breast
0/10
12/12
Colon
0/10
11/12
Connective tissue
0/9
8/9
Oesophagus
0/8
8/8
Kidney
0/11
11/11
Liver Lung
0/8 0/8
not tested 7/8
Lymph node
0/5
9/9
Ovary
0/5
7/7
Skin
0/6
6/6
Small intestine
0/5
not tested
Stomach
0/10
9/10
Testis
0/8
8/8
Uterus
0/7
7/7
Total
0/130
122/127
Table 4 Substitution patterns of selected dietary flavonols R1 3'
HO
A
OH
B
O
4' 5'
C
R2
OH OH
O
Flavonols
R1
R2
Kaempferol
H
H
Quercetin
OH
H
Myricetin
OH
OH
Isorhamnetin
OMe
H
latter compound plays a key role in the development of breast and endometrial tumours. Of particular interest was the finding that human CYP1B1 activity is regulated by estradiol via the oestrogen receptor (For a recent review see: Tsuchiya et al. 2005). Taken together the findings made CYP1B1 an ideal molecular target for the development of new anticancer drugs, where the estradiol core could be taken as a lead structure for drug design. Potter et al. designed DMU-212 (3,4,5,40 -tetramethoxy-stilbene) which showed promise in in vitro human tumour
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Tumour (no positive/no. tested)
assays, and in mouse models (Sale et al. 2004). The authors noted the close structural similarity of DMU212 with resveratrol, a naturally occurring stilbene found in red wine, which had been shown to have cancer preventative properties (Jang et al. 1997; Jang and Pezzuto 1999). Resveratrol is classified as a phytoestrogen because of its similarity to the endogenous oestrogen estradiol. It appeared that resveratrol is a substrate for the CYP1 family of enzymes (Fig. 1), and that the enzymes catalyse the conversion of resveratrol into piceatannol and a second compound, putatively identified as 3,4,5,40 -tetrahydroxystilbene (Potter et al. 2002; Piver et al. 2004). Both CYP-1 conversion products of resveratrol are known to inhibit cell proliferation (Ferrigni et al. 1984; Lu et al. 2001).
The role of diet in prevention of cancer The notion that a diet rich in fresh fruit and vegetables protects from the risk of most common epithelial cancers—including those of the digestive tract, and also several nondigestive neoplasms—is well-established (e.g. La Vecchia et al. 2001). When dietary habits of large cohorts were compared and correlated with the occurrence of cancer, a number of molecules were identified as the most
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Fig. 1 CYP1 catalysed conversions of estradiol and resveratrol
likely chemopreventive agents. At present, there is a general consensus that plant polyphenols, flavonoids, catechins, and lignans are the key constituents in cancer prevention (Kinghorn et al. 2003, 2004). The latter notion is wide-spread, also in the popular press, and a number of preparations containing the compounds mentioned above are available commercially as health-enhancing supplements. Advertisements for
the commercially available preparations often refer to scientific papers to underpin their health claims. Roughly, two models that may explain the cancer preventive properties of are cited time and time again: the antioxidant model and the phytoestrogen model. However, neither model can fully explain why certain naturally occurring dietary compounds can prevent the occurrence of cancer.
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Antioxidants and cancer prevention The antioxidant model was proposed in an influential review by Ames (1983). To date, the paper has been cited over 2,000 times. In short, reactive oxygen species, formed as a by-product of oxidative catabolism—e.g. singlet oxygen and various radicals—are possibly involved in DNA damage and tumour promotion. Antioxidants would prevent the accumulation of reactive oxygen species, and consequently any cell damage caused by these radicals. Ames lists vitamin E (tocopherol), b-carotene, and vitamin C (ascorbic acid) as dietary natural products with antioxidant activity, but he is reticent about the role of plant phenols. Nevertheless, plant phenols with antioxidant activity, e.g. stilbenes, chalcones, and flavonoids, have been a major focus of attention. Plant polyphenols have been shown to be highly effective scavengers of most types of oxidizing molecules, and the antioxidant properties of these molecules have often been cited as an explanation for their cancer preventive properties. Over 50 reviews have been written on this topic over the past five years (for a recent example, see Valko et al. 2006). However, there is a paradox here; supplements of ascorbate, vitamin E, or b-carotene—known antioxidants—do not decrease DNA damage in most studies (Halliwell 2000, 2007; Halliwell et al. 2005). In addition, in animal models antimutagens and anticarcinogens have been given in unrealistically high Fig. 2 Structural similarities between the human hormone estradiol (1), a steroid, and phytoestrogens belonging to the chemical groups of flavones (2), flavonols (3), and stilbenes (4). In an attempt to highlight the similarities, some structures are not presented in the conventional way
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doses, which do not usually reflect the human exposure situation (Knasmu¨ller et al. 2002). Intriguingly, Ames’ review also highlights the role of dietary carcinogens in the onset of cancer. In particular benzo[a]pyrene, a compound that occurs in charred meat and in cigarette smoke, has been wellinvestigated since. It is now widely accepted that benzo[a]pyrene is actually a pro-carcinogen which is activated by the human cytochrome P450 enzyme CYP1A1. It is the activated compound, benzo[a]pyrene diol epoxide, that causes cell damage (see review by Alexandrov et al. 2002).
Phytoestrogens and cancer prevention Oestrogens are hormones responsible for the female sex characteristics; they are not restricted to females since small amounts are produced in the male testes. The principal and most potent example is estradiol. The aromatic ring (Fig. 2) makes the oestrogen molecule almost planar and is essential for activity. Estradiol has been linked to breast cancer etiology, but the molecular mechanisms underlying the development of breast cancer are not completely understood (Dietel et al. 2005; Russo and Russo 2004). A variety of plant compounds have been shown to interact with human oestrogen receptors. Unsurprisingly maybe, the molecules share some structural
Phytochem Rev (2008) 7:431–443
similarity with estradiol, e.g. the almost planar configuration and the aromatic rings. Several of these so-called phytoestrogens have been linked with cancer prevention, and have been selected as candidate drugs in various studies (Usui 2006). Although to date no clear model exists that explains how phytoestrogens affect the development of hormonedependent tumours, epidemiological data suggest that the antitumour activity of phytoestrogens has little to do with their activity as endocrine disruptors. For example, genistein can stimulate estrogen receptorpositive (ER+) breast cancer growth and interfere with the antitumour activity of the endocrine disruptor tamoxifen at low levels. Thus, women who have ER+ tumours are advised not to increase their phytoestrogen intake. Several studies suggest an inhibitory effect on ER- breast cancer cell growth, and it may be reasonable for women with ER- tumours to safely consume soy and possibly other phytoestrogens (Duffy and Cyr 2003). An alternative explanation for the anticancer properties of phytoestrogens may be that they prevent the activation of pro-carcinogens like benzo[a]pyrone (Tsuji and Walle 2006) since they inhibit the activity of the enzymes CYP1A1 and CYP1B1. However, at the same time it has been observed that phytoestrogens induce CYP1A1 and CYP1B1 gene expression (Moon et al. 2006). These two observations seem to be contradictory.
Dietary prodrugs In the classical search for anticancer drugs from botanical sources, plant extracts were fractionated by various means and the fractions tested for anticancer properties in a variety of in vitro assays. However, the compounds that are present in the plant are not
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necessarily the compounds that have a pharmacological effect in the human body. This has often been cited as one of the weaknesses of in vitro testing. A few examples may demonstrate that several plantderived compounds act as prodrugs rather than drugs: 1.
2.
3.
Contrary to popular belief, the bark of the willow (Salix alba) does not contain aspirin (acetyl salicylic acid). Nevertheless, like aspirin, extract of willow bark has been effectively used to treat pains and fevers. We now know that the glucoside salicin from the bark extract is converted in the digestive tract and liver (by CYP enzymes) to form the active ingredient salicylic acid (Fig. 3). Epidemiological investigations showed a correlation between reduced risk of chronic diseases and increased concentration of the so-called ‘human’ lignans enterodiol and enterolactone enterolactone in blood plasma (Fig. 4). The ‘human’ lignans play a role in the prevention of colorectal cancer (Kuijsten et al. 2006). However, the anticancer mechanism is still unknown. In fact, the ‘human’ lignans are not formed by human metabolism at all, but by bacterial fermentation of plant-derived lignans in the colon (Rickard et al. 1996). Another example of pharmaceutically active compounds that arise as the result of fermentation by gut bacteria are equal and O-demethylangolensin (Fig. 5) which are derivatives of the dietary isoflavones formononetin and daidzein (Atkinson et al. 2005).
Based on data obtained from literature and observations in our own lab, we propose that a variety of naturally occurring compounds (notably phytoestrogens belonging to the compound class of the flavonoids) act as prodrugs with relatively little biological activity. These compounds are substrates
Fig. 3 Bioconversion of salicin
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Fig. 4 Bioactivation of dietary lignan secoisolariciresinol
for enzymes that are highly expressed in tumour cells (i.e. CYP1A1 and CYP1B1), and the conversion products inhibit cell growth (i.e. selectively inhibit growth of tumour cells). This mechanistic model that may explain the epidemiological finding that regular consumption of fruit and vegetables can prevent the development of tumours. The model also explains why flavonoid-type antioxidants, but not other antioxidants like ascorbic acid or tocopherol, seem to play a role in cancer prevention. In addition, the model explains the prominence of phytoestrogens in the lists of plant-
Fig. 5 Bioactivation of some isoflavones
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derived compounds properties.
with
possible
anticancer
Flavonoids are substrates for CYP1 enzymes In an attempt to identify whether or not dietary flavonoids interact with CYP1 enzymes, the effect of the flavonoids on CYP1-catalysed 7-ethoxyresorufin dealkylation (Burke and Mayer 1974) was monitored. Most of the flavonoids tested inhibited the dealkylation in a dose dependent manner (Table 5). All the
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Table 5 Substitution patterns of selected dietary flavones 4
R 3'
R1
7
2
6
O
A R
B
R5 4'
C
5
R3
O
Flavones
R1
R2
R3
R4
R5
Chrysin
OH
H
OH
H
H
Baicalein
OH
OH
OH
H
H
Apigenin
OH
H
OH
H
OH
Luteolin
OH
H
OH
OH
OH
Scutellarein
OH
OH
OH
H
OH
Diosmetin
OH
H
OH
OH
OMe
Eupatorin
OH
OMe
OMe
OH
OMe
Cirsiliol
OH
OMe
OMe
OH
OH
Genkwanin
OH
H
OMe
H
OH
flavonoids tested, except cirsiliol and genkwanin, inhibited CYP1B1 activity more effectively than CYP1A1 and CYP1A2. In particular the flavonols quercetin, myricetin and kaempferol were potent CYP1B1 inhibitors, whereas they seemed to be less effective inhibitors for CYP1A1 and CYP1A2.
The flavonoids which were shown to be potent inhibitors in this study, might well be potential substrates for CYP1 enzymes, e.g. resveratrol has been reported to be a substrate (Potter et al. 2002; Piver et al. 2004) and an inhibitor (Chun et al. 1999). The 7-ethoxyresorufin-O-deethylase activity assay (EROD assay) does not differentiate between the different types of inhibition, but a molecule which is a strong competitive inhibitor, would be a likely substrate, since it will fit in the active site of the enzymes. Enzyme assays, where selected flavonoids were incubated with CYP1 enzymes, confirmed that the CYP1 enzymes accept a range of flavones and flavonols as substrates. The reaction products were analysed by HPLC-DAD, or HPLC-MS. In a number of cases, the reaction products could be identified by comparison with authentic reference compounds. We can confirm that apigenin is hydroxylated to form luteolin, and that kaempferol is hydroxylated to form quercetin (Fig. 6, see also Breinholt et al. 2002; Gradolatto et al. 2004). With other flavonoids, in most cases reaction products could be observed. However, due to the absence of authentic references and the low amounts of product, a conclusive chemical identification has not been possible. Thus we can only assign putative structures to these reaction products. Control incubations, either without
Fig. 6 CYP1-catalysed conversion of the flavone apigenin (R1 = H). Analogous conversions have been found for the flavonol kaempferol (R1 = OH)
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440
CYP1 enzymes or without co-factors, did show detectable conversion of the flavonoid substrates. In addition, incubations with a range of other recombinant human cytochromes P450—CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A5—did not give detectable conversion of the flavonoids we tested. Only CYP2E1 (Piver et al. 2004) and CYP3A4 (our data) appeared to be minor catalysts. Interestingly, the isoflavonoid daidzein, a phytoestrogen from soy beans that has long been linked with prevention cancer, does not inhibit CYP1A1 or CYP1B1 activity (Chan and Leung 2003), and in our assays we could not detect any bioconversion products from daidzein.
Phytoestrogens as natural prodrugs in cancer prevention Recent developments in smart anticancer drug discovery have led to synthetic agonists of the AhR that are prodrugs activated by CYP1A1. Phortress (MacFadyen et al. 2004; Brantley et al. 2006) and aminoflavone (Kuffel et al. 2002; Pobst and Ames 2006) are both in Phase I clinical trials at the moment. Flavones have been found to induce CYP1A1 and CYP1B1 gene expression in animals, and in in vitro cultivated human cells (Durgo et al. 2007; Hodek et al. 2006), possibly through modulation of the aryl hydrocarbon receptor (AhR) (Pohl et al. 2006). Dietary flavonoids naturally occur as glycosides. During digestion of these conjugated flavonoids, the sugar moiety is split off allowing absorption of the aglycone. However, the aglycone is rapidly reconjugated and one common characteristic of flavonoids is that they occur in the bloodstream as glucuronide and sulfate conjugates (Shimoi et al. 1998; Dupont et al. 2004; Erlund 2004; Chen et al. 2007). The concentration of the conjugates of the more common dietary flavonoids in the human blood stream is in the micromolar range, and this is the concentration that reaches the tumour tissue. Around tumour tissue, significant extracellular levels of b-glucuronidase can be found, due to the liberation of this enzyme from the lysosomes of inflammatory cells (Dodds et al. 2002). Thus, tumour cells are likely to be met by flavonoid aglycones at micromolar concentrations.
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In order to screen for the presence of CYP-1 activated prodrugs, a test panel of human breast cell cultures had been designed (Potter and Butler 2004). This panel was used to test our hypothesis that phytoestrogens are selectively activated in CYP-1 expressing tumour cells. The panel consists of the human breast adenocarcinoma cell line MCF-7— which normally does not express CYP-1 enzymes but can be induced with the dioxin TCDD, the human breast adenocarcinoma cell line MDA-MB-468— which constitutively expresses CYP1s-, and the nontumorigenic epithelialbreast cell line MCF-10A— which was used a model for normal breast cells. The efficacy of a pro-drug can be expressed by determining its IC50 value (concentration needed to inhibit cell proliferation by 50%) for the different cell lines, and then calculating its activation factor or its tumour selectivity factor. When the activation factor is greater than one, cells expressing CYP1 enzymes are inhibited more by the test substances than cells not expressing CYP1 enzymes. When the tumour selectivity factor is greater than one, tumour cells are more inhibited than non-tumour cells (Tables 6, 7) shows that some common dietary flavonoids have activation factors and tumour selectivity factors ranging from 2 to 10. The activation and tumour Table 6 Inhibition of recombinant human CYP1 enzymes by dietary flavonoids. CYP1 (CYP1A1, CYP1A2, CYP1B1) microsomes (5 pmol/ml) were incubated with various concentrations of flavonoids Compound
CYP1B1
CYP1A1
CYP1A2
Quercetin Myricetin
0.7 ± 0.3 0.9 ± 0.2
7.0 ± 0.0 4.0 ± 0.0
12 ± 3.5 13 ± 0.7
Kaempferol
1.3 ± 0.2
8.0 ± 1.4
9.0 ± 2.1
2.0 ± 0.0
4.2 ± 0.0
6.0 ± 0.0
Flavonols
Flavones Apigenin Luteolin
1.8 ± 0.4
10 ± 0.7
15 ± 1.4
Diosmetin
0.5 ± 0.1
1.2 ± 0.3
18 ± 4.0
Baicalein
7.0 ± 1.1
20 ± 0.0
20 ± 0.0
Chrysin
0.5 ± 0.1
0.5 ± 0.1
2.0 ± 0.0
Eupatorin
1.1 ± 0.1
2.3 ± 0.1
25
Genkwanin
[25
[25
[25
Cirsiliol
[25
[25
[25
Results are calculated from percentage of inhibition of 7-ethoxyresorufin-O-deethylase activity compared to control, and expressed as IC50 (lM). Each value corresponds to mean ± standard deviation for n = 4 determinations
Phytochem Rev (2008) 7:431–443 Table 7 Effect of selected flavonoids on the proliferation of in vitro human cell cultures
441
Compound
MCF-7 IC50 (lM)
MCF-7 + TCDD IC50 (lM)
Activation factor
Apigenin Kaempferol
200 70
20 35
10 2
Scutellarein
30
15
2
Compound
MCF-10A IC50 (lM)
Apigenin
MDA-MB-468 IC50 (lM)
Tumour selectivity
200
50
4
Kaempferol
60
30
2
Scutellarein
40
10
4
selectivity factors presented in Table 7 indicate that the dietary flavonoids tested cannot be used in cancer therapy; the dose that stops tumour growth is too close to the dose that stops normal cell development. However, micromolar concentrations of these dietary compounds in the blood stream are sufficient to selectively inhibit the proliferation of tumorigenic cells expressing CYP1-enzymes, and thus prevent development of tumours at the very earliest stages. Thus, our hypothesis of phytoestrogens as natural prodrugs explains how micromolar amounts of dietary flavones may prevent the onset of a wide range of cancers. More research is needed to identify dietary prodrugs and characterise their activated products, and to establish the exact mechanisms by which these compounds selectively inhibit the proliferation of tumour cells. Conventional screening methods for anticancer compounds have led to a number of clinically used drugs for cancer therapy. Although, new compounds will still be discovered using conventional screening methods, it now seems to be past its peak. Most of the smart drugs, and probably none of the pro-drugs, would have been picked up by conventional screening. Many drug companies have abandoned major conventional screening programmes in favour of novel methods for smart drug development using tools that have become available the post-genomic era, i.e. specific in vitro enzyme assays or even in silico data mining.
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