Dcu.ie2

Current Pharmaceutical Design, 2004, 10, 3797-3811
Studies on Coumarins and Coumarin-Related Compounds to Determine
their Therapeutic Role in the Treatment of Cancer
Applied Biochemistry Group, School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland Abstract: The Benzopyrones are a group of compounds whose members include coumarins and flavonoids. Dietary
exposure to benzopyrones is quite significant, as these compounds are found in vegetables, fruit, seeds, nuts, coffee, tea
and wine. It is estimated that the average western diet contains approximately 1g/day of mixed benzopyrones. It is,
therefore, not difficult to see why extensive research into their pharmacological and therapeutic properties is underway
over many years. Coumarin is a natural substance that has shown anti-tumour activity in vivo, with the effect believed to
be due to its metabolites (e.g. 7-hydroxycoumarin). This review is based on recent studies of coumarins and coumarin
related compounds. Therefore, the focus will be on these relevant compounds and their therapeutic importance.
A recent study has shown that 7-hydroxycoumarin inhibits the release of Cyclin D1, which is overexpressed in manytypes of cancer. This knowledge may lead to its use in cancer therapy. Esculetin inhibits growth and cell cycle progressionby inducing arrest of the G1 phase in HL-60 leukaemia cells, resulting from the inhibition of retinoblastoma proteinphosphorylation. Recent studies investigating the potential of flavonoids as therapeutic agents have suggested they mayhave use in various therapeutic settings ranging from leukaemia treatment to the treatment of patients with HIV. Genisteinis a well-known isoflavone and is a tyrosine kinase inhibitor. Studies have indicated that genistein elicits inhibitory effectson cell growth of various carcinoma cell-lines and may be a potential candidate for cancer therapy.
In our research, we have investigated the effects of coumarins and coumarin-related compounds on a panel of cell-lines.
The most recent work involves two cell-lines, MCF-7 a breast carcinoma and A549 a lung carcinoma. Microtitre assayswere performed along with real-time analysis of cell viability using a biosensor called the Cytosensor microphysiometer.
These studies suggest that both genistein and esculetin exerted the most potent inhibitory effect on cell growth incomparison to the other two compounds.
Key Words: Coumarin, 7-hydroxycoumarin, esculetin, warfarin, genistein, benzopyrones, flavonoids, coumarin derivatives,
furanocoumarins, pyranocoumarins, pyrone-substituted coumarins, pharmacokinetics, cytochrome P450 microtitre assay, cell
proliferation, MTT, LDH, BrdU, Acid Phosphatase, Cytosensor Microphysiometer, MCF-7, A549, toxicity, signal transduction,
cancer treatment, anti-proliferation, cytostatic.
INTRODUCTION
Coumarins owe their class name to ‘Coumarou’, the vernacular name of the tonka bean (Dipteryx odorata Willd., Fabaceae), from which coumarin itself was isolated in 1820 [1]. Coumarin is classified as a member of the benzopyrone family of compounds, all of which consist of a benzene ring joined to a pyrone ring [2]. The benzopyrones can be subdivided into the benzo-α-pyrones to which the coumarins
belong and the benzo-γ-pyrones, of which the flavonoids are
principal members (Fig. 1.1).
Fig. (1.1). The chemical structures of benzopyrone subclasses, with
the basic coumarin structure (benzo-α-pyrone) [A], and flavonoid
OCCURRENCE
(benzo-γ-pyrone) structure [B].
There are four main coumarin sub-types: the simple along with their glycosides. Furanocoumarins consist of a coumarins, furanocoumarins, pyranocoumarins and the five-membered furan ring attached to the coumarin nucleus, pyrone-substituted coumarins (Table 1.1). The simple
divided into linear or angular types with substituents at one coumarins (e.g. coumarin, 7-hydroxycoumarin and 6,7- or both of the remaining benzoid positions. Pyranocoumarin dihydroxycoumarin), are the hydroxylated, alkoxylated and members are analogous to the furanocoumarins, but contain alkylated derivatives of the parent compound, coumarin, a six-membered ring. Coumarins substituted in the pyronering include 4-hydroxycoumarin [3]. The synthetic com-pound, warfarin, belongs to this coumarin subtype. By virtue *Address correspondence to this author at the Applied Biochemistry Group, of its structural simplicity coumarin has been assigned as School of Biotechnology, Dublin City University, Glasnevin, Dublin 9,Ireland; E-mail: [email protected] head of the benzo-α-pyrones, although it is generally 1381-6128/04 $45.00+.00
2004 Bentham Science Publishers Ltd.
3798 Current Pharmaceutical Design, 2004, Vol. 10, No. 30
Lacy and O’Kennedy
accepted that 7-hydroxycoumarin be regarded as the parent prises 200 species, is widely distributed in the tropical rain compound of the more complex coumarins (Table 1.1) [4].
forest where several species are used in folk medicine [10].
Genistein is an isoflavone and belongs to the benzo-γ- Although most of the natural coumarins in existence have pyrones. It is a natural component of soy and has been been isolated from the higher plants, some members have intensively investigated as a chemopreventitive agent, been discovered in microorganisms (Fig. 1.2). Some
mainly against hormonally regulated breast and prostate important coumarin members have been isolated from microbial sources e.g. novobiocin and coumermycin from Coumarins comprise a very large class of compounds Streptomyces, and aflatoxins from Aspergillus species [11, found throughout the plant kingdom [6-8]. They are found at 12]. The aflatoxins are a group of highly toxic fungal high levels in some essential oils, particularly cinnamon bark metabolites and the most commonly occurring member of oil (7,000 ppm), cassia leaf oil (up to 87,300 ppm) and the group is aflatoxin B1 [3]. Coumarin group antibiotics, lavender oil. Coumarin is also found in fruits (e.g. bilberry, such as novobiocin, coumermycin A1 and clorobiocin, are cloudberry), green tea and other foods such as chicory [9].
potent inhibitors of DNA gyrase. These antibiotics have been Most coumarins occur in higher plants, with the richest isolated from various Streptomyces species and all possess a sources being the Rutaceae and Umbelliferae. Although 3-amino-4-hydroxy-coumarin moiety and a substituted distributed throughout all parts of the plant, the coumarins deoxysugar; noviose, as their structural core that is essential occur at the highest levels in the fruits, followed by the roots, for their biological activity. Chlorobiocin differs from novo- stems and leaves. Environmental conditions and seasonal biocin in that the methyl group at the C-8 of the coumarin changes can influence the occurrence in diverse parts of the ring is replaced by a chlorine atom, and the carbamoyl at the plant [3]. Recently six new minor coumarins have been 3' of the noviose is substituted by a 5-methyl-2-pyrrolcar- isolated from the fruits and the stem bark of Calophyllum boxyl group. Coumermycin A1 contains two of the couma- dispar (Clusiaceae). The genus Calophyllum, which com- rin-noviose core joined by a 3-methyl-2,4-dicarboxyl pyrole Table 1.1. The Four Main Coumarin Subtypes. The Main Structural Features and Examples of Each Coumarin Subtype are
Illustrated in this Table
Classification
Features
Examples
SIMPLE COUMARINS
5-membered furan ring attached to benzene FURANOCOUMARINS
6-membered pyran ring attached to benzene PYRANOCOUMARINS
PYRONE-SUBSTITUTED
COUMARINS
Studies on Coumarins and Coumarin-Related Compounds
Current Pharmaceutical Design, 2004, Vol. 10, No. 30 3799
Fig. (1.2). Important coumarin members isolated from microbial sources: [A] Novobiocin, [B] Coumermycin A1, [C] Clorobiocin.
linker and has the same substituted noviose as in chloro- considered the critical value at which the distribution of a compound limits its rate of absorption. However, bothcompounds have high partition coefficients (21.5% for PHARMACOKINETICS
coumarin and 10.4% for 7-hydroxycoumarin), which isconsidered favourable for the rapid absorption of compounds The pharmacokinetics of coumarin, including the excre- once they are in aqueous solution. This coupled with the fact tion of various metabolites, were elucidated over many that coumarin is non-polar, suggests that in theory coumarin years. Coumarin is rapidly and almost completely should cross lipid bilayers easily by passive diffusion [12].
metabolised with little unchanged compound excreted [14].
Pharmacokinetic studies in humans have demonstrated Absorption and Distribution
that coumarin is completely absorbed from the GIT after oraladministration and extensively metabolised by the liver in Following oral administration, coumarin is rapidly the 1st pass with only between 2 and 6% reaching the absorbed from the gastrointestinal tract and is distributed systemic circulation intact [9]. The low bioavailability of throughout the body [9]. Coumarin and 7-hydroxycoumarin coumarin, in addition to its short half-life (1.02 hrs peroral vs are both poorly soluble in water (0.22 and 0.031 %, 0.8 hrs intravenous) has brought into question its importance respectively). These percentages indicate coumarin may have in vivo and it is now accepted that coumarin is a pro-drug, reduced bioavailability in vivo, as 0.3% solubility in water is with 7-hydroxycoumarin being the compound of main 3800 Current Pharmaceutical Design, 2004, Vol. 10, No. 30
Lacy and O’Kennedy
therapeutic relevance. At normal therapeutic plasma Coumarin is metabolised initially by specific cytochrome concentrations many drugs exist in the plasma mainly in the P-450-linked mono-oxygenase enzyme (CYP2A6) system in bound form. Ritschel [15] and colleagues have shown that liver microsomes, resulting in hydroxylation to form 7- 35% of coumarin and 47% of 7-hydroxycoumarin bind hydroxycoumarin. After 7-hydroxylation, coumarin under- plasma proteins. Availability of the compounds at their target goes a phase II conjugation reaction resulting in a tissues should not be problematic since the proportions that glucuronide conjugation associated with 7-hydroxycoumarin.
bound were well below the accepted critical value of 80% The 7-hydroxylase activity is exceptionally high in human binding. The pharmacokinetics of coumarin have been studied liver microsomes compared with its activity in the livers of in a number of species including the rat, dog, gerbil, rhesus other animal species. The activity of coumarin 3-hydroxylase monkey and in man [9]. Specific antibody recognition for its is very high in rodent microsomes but is absent in human antigen is the basis for very selective and sensitive analytical microsomes. Although coumarin may be metabolised by methods. This was exploited in numerous formats for the hydroxylation at all six possible positions (i.e. carbon atoms pharmacokinetic determination of coumarin and its deri- 3,4,5,6,7, and 8), the most common routes of hydroxylation vatives. Immunoanalytical approaches have included are at positions 7 and 3 to yield 7-hydroxycoumarin and 3- ELISA-based methods for the detection of coumarin and 7- hydroxycoumarin, respectively (Fig. 1.3). 7-hydroxylation
hydroxycoumarin in urine [16]. Antibody-based biosensors has received the most attention among the various metabolic have also been employed, with either electrochemistry, or steps, predominantly because it is the major metabolic route surface plasmon resonance (BIAcore) facilitating detection in humans and is easily analysed. Hydroxylation at carbon 3 of coumarin compounds in various matrices [17, 18].
results in further metabolism via ring opening, yielding twofurther products, o-hydroxyphenyllactic acid (o-HPLA) and Metabolism
o-hydroxyphenylacetic acid (o-HPAA) [9, 14]. Theexpression of CYP enzymes (e.g. CYP2A6) varies between Traditionally coumarin has been viewed by pharmaco- individuals due to genetic and environmental factors. These logists as the ideal model for studying the complex factors produce inter-individual variation in the metabolism metabolism of a structurally simple organic molecule, and as of drugs such as coumarin. The frequency of poor metabo- such, its metabolic fate has been extensively researched [9, lisers varies between species, races and ethnic groups. It has 19, 20]. Determining the metabolic fate of coumarin is been shown that there exists large inter-species and inter- important in order to utilise the fact that it is metabolised at individual variability in the activity of these enzymes [21].
several sites, and to access the possible dependence ofcoumarin-induced toxicity on metabolism [14].
Metabolism in Man
The superfamily of cytochromes P450 (CYPs) consists of The metabolism of coumarin has been investigated in microsomal hemoproteins that catalyse the oxidative, vivo and in vitro in a wide range of species including peroxidative and reductive metabolism of a wide variety of humans. Human metabolic studies usually involve oral endogenous and exogenous compounds. The CYP super- dosage followed by urine collection with or without timed family is divided into families and subfamilies according to fractionation [7, 22]. Analysis is by one of a number of their nucleotide sequence homology. Most biotransforma- techniques including spectrofluorimetry, HPLC and capillary tions of xenobiotics (such as drugs) are performed by electrophoresis [23-25]. Recent in vitro systems employed enzymes from the families CYP1, CYP2 and CYP3. The include tissue slices, hepatocytes, subcellular fractions, and CYP2 family have been examined using the rat, mouse and purified and cDNA-expressed enzymes [9]. In the majority rabbit model systems. This family includes seven subfami- of human subjects studied coumarin is extensively metabo- lies in mammals. In humans, the most important CYPs lised to 7-hydroxycoumarin. Some data for various coumarin regarding drug metabolism are CYP2A6, CYP2C9, dose levels and collection periods are shown in Table 1.2.
The measurement of urinary 7-hydroxycoumarin following Table 1.2. Extent of Coumarin Metabolism to 7-HC in Various Species. aCoumarin Administered Orally Unless Otherwise Stated
bShilling et al. (1963). cEgan et al. (1990)
Dose (mg/kg)a
Collection time (hr)
Urinary 7-HC (% of Dose)
Studies on Coumarins and Coumarin-Related Compounds
Current Pharmaceutical Design, 2004, Vol. 10, No. 30 3801
7-hydroxycoumarin
6-hydroxycoumarin
8-hydroxycoumarin
CYP2A6
o-hydroxyphenylpropionic acid
5-hydroxycoumarin
Coumarin
o-coumaric acid
4-hydroxycoumarin
Esculetin
3-hydroxycoumarin
3,4-epoxide
o-hydroxyphenyllactic acid
o-hydroxyphenylacetaldehyde
o-hydroxyphenylacetic acid
Fig. (1.3). Metabolism of coumarin. All biotransformations are possible, although the metabolism is species-specific.
an oral dose of coumarin has been employed as a biomarker considerable proportion of coumarin through pathways other of human hepatic CYP2A6, the cytochrome P-450 (CYP) that 7-hydroxylation such as the 3,4-epoxidation pathway to isoform which is responsible for coumarin 7 hydroxylation in human liver [14]. Some individuals can metabolise a 3802 Current Pharmaceutical Design, 2004, Vol. 10, No. 30
Lacy and O’Kennedy
In humans, there are three genes in the CYP2A sub- bile. This may result in enterohepatic circulation enhancing family, however, CYP2A6 is mainly of greater importance, the exposure of liver cells to toxic coumarin metabolites.
as the other two gene products (CYP2A7 and CYP2A13) are Species such as syrian hamster, baboon, and humans excrete either inactive or are not expressed in the liver. CYP2A6 coumarin metabolites primarily in urine. Low level exposure codes the enzyme catalysing coumarin 7-hydroxylation to coumarin from diet and from fragrances used in cosmetic (about 10% of total P450) [26]. Recently, CYP2A6 has been products would not be expected to produce any hepato- reported to be polymorphically expressed in the human liver.
toxicity even in individuals with deficient 7-hydroxylase It has been shown that CYP2A6 participates in metabolism of nicotine and its metabolite cotinine. Some drugs andchemicals, including coumarin, which is widely used as a APPLICATIONS OF COUMARIN AND COUMARIN
probe substance for CYP2A6 both in vitro and in vivo, are DERIVATIVES
also metabolised by this enzyme [27]. Substrates and The coumarins are of great interest due to their biological inhibitors currently known to be metabolised by or interfere properties. In particular, their physiological, bacteriostatic with CYP2A6 in vitro and in vivo have been summarised by and anti-tumour activity makes these compounds attractive Pelkonen [26]. Although 7-hydroxycoumarin is the main for further backbone derivatisation and screening as novel human metabolite, other hydroxylation pathways are impor- therapeutic agents. Weber [30] and co-workers have shown tant in humans and, as such, the therapeutic relevance of that coumarin and its metabolite 7-hydroxycoumarin have non-7-hydroxymetabolites should be examined rather than antitumour activity against several human tumour cell lines.
Both coumarin and coumarin derivatives have shownpromise as potential inhibitors of cellular proliferation in TOXICOLOGY
various carcinoma cell lines [12, 31, 32]. In addition it has Since 1954, coumarin has been classified as a toxic been shown that 4-hydroxycoumarin and 7-hydroxycoumarin substance by the FDA, following reports of its possible liver inhibited cell proliferation in a gastric carcinoma cell line tumour-producing properties in rats [28]. The FDA banned its use, labelling as adulterated all foods containing coumarin[12]. Due to tests performed on rodents coumarin was SIMPLE COUMARINS
referred to as a chemical carcinogen by NIOSH [National Coumarins and the benzopyrones are representative of a Institute for Occupational Safety and Health]. However, very diverse and potentially useful groups of drugs.
caution needs to be taken in extrapolating this information tohuman situations. Various tests (Ames, micronucleus) have Clinical Uses
shown that coumarin and its metabolites are non-mutagenic[6]. Preliminary results from early studies indicated that Due to its biochemical properties coumarin was proposed coumarin was a toxin, but it has been shown since, that the for use in clinical medicine. It had been evaluated for the rat is a poor model to compare with the human for this treatment of various clinical conditions, resulting in the use particular metabolism [29]. A number of studies have of a variety of dosing regimens. Recommended doses range examined the acute, chronic and carcinogenic effects of from 8mg for the treatment of venous constriction to coumarin in the rat and mouse. In studies involving the rat, 7000mg/day in anti-neoplastic therapies.
hepatic biochemical and morphological changes have beenexamined for various periods of coumarin administration (1 High Protein Oedema (HPO)
week to 2 years). Depending on dose administered, coumarin The lymph system is responsible for drainage of inters- treatment results in an increase in relative weight and titial fluid within human tissues. Oedema interferes with the changes in various hepatic biochemical parameters. Single metabolism of the tissue cells and reduces oxygen transport, oral doses of coumarin have been shown to produce liver resulting in problematic wound healing [34]. In the case of necrosis and increase plasma transaminase activities in high protein oedemas (HPO), there is an accumulation of protein in the tissue following trauma or inflammation, with In contrast, studies involving baboons, syrian hamsters resulting permeability of the capillaries causing water and certain mice strains seem to be resistant to acute leakage in the tissue spaces. Many disease states are associa- coumarin-induced hepatotoxicity. Species differences in ted with high protein oedemas, ranging from extremely coumarin-induced toxicity in vitro have been investigated in severe and chronic (e.g. lymphoedema and elephantiasis) cultured hepatocytes. These studies provide evidence for through more common and acute forms (e.g. burns, species differences in coumarin-induced toxicity in vitro.
accidental and surgical traumas). All forms have been shown The relative resistance of human and cynomolgus monkey to benefit from benzopyrone treatment [35]. Coumarin and liver slices and/or hepatocytes to coumarin toxicity correlates numerous other benzopyrones have been tested in high with coumarin 7-hydroxylation, the major pathway of protein oedema, and all have been shown to successfully coumarin metabolism in these species, being a detoxification reduce the swelling. However, according to Loprinzi [36] pathway of coumarin metabolism. However, while and colleagues, coumarin treatment alone is not effective coumarin-7-hydroxylation pathway is a detoxification therapy for women who have lymphoedema of the arm after pathway this does not appear to be the only explanation for treatment for breast cancer. It may be possible to increase the resistance of a species to coumarin-induced toxicity. In the beneficial therapeutic effect of coumarin by using it with rat coumarin-induced hepatotoxicity appears to be partially other compounds in combination treatments. The objective attributable to the excretion of coumarin metabolites in the of a recent study was to evaluate the oedema-protective Studies on Coumarins and Coumarin-Related Compounds
Current Pharmaceutical Design, 2004, Vol. 10, No. 30 3803
effect of a combination vasoactive drug, coumarin/troxerutin to the oncologist, as no satisfactory treatment for recurrent (SB-LOT) plus compression stockings in patients suffering malignant melanoma currently exists. Studies have shown from chronic venous insufficiency after decongestion of the that five years after removal of the primary lesion, the legs as recommended by the new guidelines. The study recurrence of malignant melanoma is observed in 55-80% of confirms the oedema-protective effect of SB-LOT in chronic venous insufficiency and provides a treatment option for Original work with coumarin derivatives in the treatment patients who discontinue compression after a short time [37].
of melanoma focused on the use of warfarin as a Chronic Infections
maintenance therapy. This compound was known to inhibittumour spread, and to stimulate granulocytes, lymphocytes In addition to its stimulatory effect on macrophages, and macrophages [43]. Thornes then began to assess the coumarin has been shown to activate other cells of the potential application of coumarin, the parent compound of immune system. In chronic brucellosis Brucella abortis warfarin, as an adjuvant therapy in melanoma. Like warfarin, infects macrophages, thus eluding the immune response [38].
the in vivo actions of coumarin were known to be macro- When immunostimulatory drugs such as coumarin are phage-derived. Coumarin was non-toxic and conveniently administered, the symptoms of chronic brucellosis disappear.
administered, it had no anti-coagulant activity, and a These results have encouraged the use of coumarin in other previous administration resulted in subjective improvement chronic infections such as mononucleosis, mycoplasmosis, in cancer patients [44, 45]. A more recent study by Velasco- toxoplasmosis and Q fever. A new antiplasmodial coumarin Velazquez [46] and colleagues determined the in vitro has been isolated from the roots of Toddalia asiatica. This effects of 4-hydroxycoumarin (4-HC) employing the murine finding supports the traditional use of this plant for the melanoma cell line B16-F10 and the non-malignant fibroblastic cell line B82. 4-HC disorganized the actincytoskeleton in B16-F10 cells, but not in B82 fibroblasts.
APPLICATION OF SIMPLE COUMARINS IN
Adhesion of tumour cells to extracellular matrix is required CANCER TREATMENT
during the metastatic process, therefore, 4-HC might be Anti-cancer drugs have traditionally been targeted to useful as an adjuvant therapy for melanoma.
damage the aberrantly dividing cell by interrupting the celldivision process [40]. Reagents used include DNA intercalat- Coumarin in Renal Cell Carcinoma
ing agents (e.g. adriamycin), DNA cross-linking agents (e.g. The clinical course of renal cell carcinoma (RCC) has cis-platin), topoisomerase inhibitors (e.g. campothecins), been well documented, with long-lasting stable periods and cytoskeleton-disrupting agents (e.g. vinblastin) and anti- rapid tumour growth its principal features. Surgery remains metabolites (e.g. mercaptopurine). These drugs though effec- the standard care for patients whose tumour is confined to tive, are cytotoxic, and thus exhibit severe side effects, parti- the kidney. However, many of these patients develop recur- cularly on normal proliferating tissues such as the haemato- rent or metastatic disease within months, where the lungs, poietic system. Often combination therapies, whereby liver and bones are the common sites of secondary occur- several cytotoxic agents are combined in the treatment rence [47]. The outlook for patients with metastatic RCC is regime, offer better results with fewer toxic side-effects, as poor, with a 5-year survival rate of less than 10% [48].
they are carefully regulated to allow recovery of normal, butnot malignant cells, from drug exposure [40].
Interest in the coumarin family of compounds stemmed from reports by Thornes, of the immunomodulatory activity Currently, chemotherapy, radiotherapy and surgery of coumarin and its utility in malignant melanoma [49]. The combined offer the best outcomes for cancer patients, and clinical activity of coumarin in renal cell carcinoma patients treatment combinations have been successfully applied to has been investigated by applying a treatment regime particular cancer types, for example, Hodgkin’s lymphoma, described by Thornes (coumarin at 100mg/day oral dosage, testicular cancer and various leukemias. Coumarins can be with the addition of cimetidine 4 X 300mgs/day from day used not only to treat cancer but to treat the side effects 15). This preliminary study yielded some interesting results, caused by radiotherapy. A recent study investigated the with 14 objective results among 45 patients with metastatic efficacy of coumarin/troxerutine combination therapy for the RCC, and almost no toxic side effects. Validation of this protection of salivary glands and mucosa in patients anti-tumour activity was further demonstrated by other undergoing head and neck radiotherapy. The results suggest that coumarin/troxerutine have a favourable effect in thetreatment of radiogenic sialadentis and mucositis [41]. The Following this success, it became clear that additional interest in coumarin and 7-hydroxycoumarin as anti-cancer information regarding doses and toxicities was required, and agents, arose from reports that these agents had achieved Marshall and colleagues implemented a phase I trial to objective responses in some patients with advanced mali- define the maximally tolerated dose, and dose-limiting toxicities of coumarin and cimetidine. All coumarin doseswere well tolerated, with the most common side effect of Coumarin in Malignant Melanoma
nausea attributable to the intense aroma of coumarin.
Objective responses were observed in 7 patients, all with Early diagnosis of malignant melanoma facilitates renal cell carcinoma, these responses being observed across a surgical removal of the primary lesion and achieves a good range of coumarin doses (600-5000 mgs) [52]. In vitro prognosis. However, if the lesion progresses, the risk of cytotoxic potential and mechanism of action of selected recurrence becomes serious and represents a major challenge coumarins using renal cell lines have been investigated 3804 Current Pharmaceutical Design, 2004, Vol. 10, No. 30
Lacy and O’Kennedy
recently [8]. The results obtained suggest that the coumarins hydroxycoumarin and esculetin were found to inhibit examined may have a potential therapeutic role to play in the tyrosine phosphorylation in EGF-stimulated tumour cells in a time- and dose-dependent manner. It appears that thiseffect may be achieved by reduction of the tyrosine kinase Coumarin in Prostate Cancer
Prostate cancer is the most common invasive malignancy A recent study has presented evidence that esculetin in males and is characterised by a very slow growth rate and affected phosphorylation of pRB thus inducing G1 arrest of a wide biological variability, especially with regard to human leukaemia HL-60 cells. The results demonstrated that hormonal sensitivity [53, 54]. These two traits have curbed the treatment with esculetin resulted in an accumulation of attempts at curative treatments for patients, as most effective hypophosphorylated pRB in HL-60 cells along with reduc- chemotherapeutic drugs rely on fast growth kinetics in the tions of both cyclin D1 and E. This induced the arrest of the tumour mass, and, due to differential hormonal dependencies, cell cycle at the G1 phase. Among the released proteins, the hormonal therapy (androgen deprivation) is not effective in E2F family of transcription factor has central position. Not all cases. At present, early detection, and removal of clini- only does E2F induce gene expression necessary for DNA cally significant tumours by surgery or radiation, have been synthesis, it also contributes to the regulation of the cyclin the focus of clinical strategies. However, patient survival is D1 and E genes. Esculetin treatment also induced enhanced dependent on metastases where principal metastatic sites are expression of the CDKI p27 and a reduced expression of regional lymph nodes and bone. Eventually, almost every CDK-4, thus inhibiting pRB phosphorylation [58].
prostate carcinoma that initially regressed on androgen In a separate study aiming to clarify mechanisms of deprivation will relapse into a hormonal-insensitive state and action of 7-hydroxycoumarin and coumarin, the effect on grow in the absence of androgen. Evidently, better therapeu- cell cycle progression of the human adenocarcinoma cell line tic approaches to control both metastases and hormone- A427 was investigated. These cells are pRB positive and insensitive prostate carcinomas are required.
have homozygous deletions at the gene of p16INK4A. The As coumarin had previously appeared to exert immuno- results showed that 7-hydroxycoumarin had greater cytosta- modulating effects in other cancers, a small-scale study to tic activity than coumarin. The inhibition of the cell-cycle at test the efficacy of coumarin in prostate cancer was set up transition G1/S is consistent with the cytostatic effect of 7- [55]. A phase I trial involving 40 patients with metastatic, hydroxycoumarin. Furthermore, the decrease in the percen- hormone-naïve or hormone-refractory prostatic cancer was tage of cells expressing cyclin D1 indicates that the action of conducted [56]. Participants were administered 3g of 7-hydroxycoumarin involves early events in phase G1.
coumarin daily, and evaluated for toxicity and anti-tumour Absence of changes in the level of cyclin D1 mRNA responses. 3 partial responses occurred, all in-patients with suggests a post-transcriptional effect of 7-hydroxycoumarin.
low tumour loads. One responder remained with 3 respon- A pathway which regulates post-transcriptionally the levels sive bone metastases and stable prostate specific antigen of cyclin D1 is the PI-3K/AKT pathway. If this pathway is (PSA) levels for 7 years following the trial. Myers [57] and inhibited it cannot inhibit phosphorylation of GSK-3 which co-workers examined the effects of various concentrations (0-500 µg/ml) of coumarin on the proliferation of two renalcell carcinoma cell lines (786-O and A-498) and two COUMARIN DERIVATIVES AND CANCER
malignant prostatic cell lines (DU145 and LNCaP). After 5 Furanocoumarins
days of treatment, coumarin inhibited the growth of the fourcell lines. The LNCaP prostatic cell line was most sensitive The furanocoumarins are a therapeutically important to the inhibitory effects of coumarin.
subtype as they have various clinical applications. Thefuranocoumarins consist of a 5-membered furan ring Coumarin in Leukaemia
attached to the coumarin nucleus. Two of the most importantand well known furanocoumarins are psoralen (Linear) and The effect of esculetin, coumarin and 7-hydroxycoumarin angelicin (Angular). The terms linear and angular refer to the on the cell cycle and its regulatory molecules has been orientation of the furan ring with respect to the coumarin investigated. The cytostatic and cytotoxic nature of 8-nitro- 7-hydroxycoumarin (8-NO2-7-OHC) was determined usingboth human and animal cell lines grown in vitro. This Psoralens are naturally occurring plant biosynthetic compound displayed cytotoxic properties in two human cell metabolites that have been used since ancient times in lines tested (HL-60 and K562), inducing cell death by photochemotherapy to treat a number of skin disorders apoptosis. This compound imposed a cytostatic effect on the including mycosis fungoides, psoriasis and vitiligo [60].
three other cell lines tested, exerted through a perturbation in Psoralens have recently found application in the regulation their cell cycle [31]. The effect of a number of coumarin of human cervical carcinoma cell proliferation in conjunction compounds on the growth, metabolism and cell signalling of with anti-sense technology [61]. Oligonucleotides and their human tumour cell lines was examined by Cooke [12].
analogs have been used to inhibit protein biosynthesis by Overall esculetin exhibited the strongest antiproliferative suppressing the gene expression in a sequence specific effect on the carcinoma cell lines tested [32]. Given the manner. The method is called antisense strategy and has importance of signalling anomalies in cancer cells, tests were been applied to gene therapy for incurable diseases such as performed to determine if the cellular target of 7-hydroxy- cancers and viral infections. Among various reports coumarin was a signalling pathway component. Both 7- regarding antisense technology, many have presented clear Studies on Coumarins and Coumarin-Related Compounds
Current Pharmaceutical Design, 2004, Vol. 10, No. 30 3805
evidence that the antisense mechanism participated in the metastatic tumour will ultimately form. Components of the regulation of cell growth. Some of the antiproliferative blood-clotting pathway may contribute to metastasis by effects of oligo nucleoside phosphorothioates S-oligo may be trapping cells in capillaries or by facilitating adherence of attributed to the interaction of S-oligo with certain proteins cells to capillary walls. Conceivably, anticoagulants could interfere with this step in the metastatic process [63].
Warfarin has shown particularly promising results in the Upon UVA irradiation psoralen derivatives have the treatment of SCCL (Small Cell Carcinoma Lung) a tumour ability to crosslink covalently with pyrimidine bases (e.g.
cell type that is characterised by a coagulation-associated thymine and uracil). As psoralen derivatives can inactivate pathway [64-66]. Studies by Mousa [67] have shown that gene expression via cross-linking, they have been conjugated anticoagulation with commonly used agents such as with oligonucleotides to reinforce antisense effects. During unfractionated heparin and warfarin (Coumadin) prevent in vitro experiments psoralen-conjugated S-oligos have tumour formation by limiting the ability of tumour cells to be shown resistance to nucleases and, therefore, have exhibited retained in the pulmonary microvasculature.
significant inhibitory effects upon UVA irradiation. Psoralen-conjugated S-oligos (Ps-S-oligo) were prepared and used to Recent studies suggest that anticoagulant drugs and cime- inhibit the proliferation of human cervical carcinoma cells.
tidine therapy in malignancy may improve cancer survival Upon UVA irradiation of Ps-S-oligo-treated cells, Ps-S-oligo and inhibit the metastatic process. A study investigated and complementary to the initiation codon region (Ps-P-As) of compared the effects of anticoagulant drugs (e.g. warfarin human papillomavirus (HPV)18-E6*-mRNA of human and heparin), cimetidine and a combination of cimetidine cervical carcinoma cells significantly inhibited proliferation.
with anticoagulants on adhesion of highly invasive breast The E6* protein is tightly correlated with the transformation cancer cell lines BT-549 and MDA-MB-231 in vitro. A high of human cervical cells and therefore, its suppression may anti-adhesion effect was observed with cimetidine and regulate cellular proliferation. The psoralen-conjugated warfarin. Anticoagulants such as warfarin can decrease antisense DNA has significant potential to regulate gene adhesion and tumour angiogenesis. Application of cimetidine expression, which may provide useful information to explore and anticoagulant drugs intensifies the anti-adhesion effect together with other anti-metastatic effects [68]. There is nowconsiderable evidence that the blood coagulation system Pyranocoumarins
plays an important role in the biology of malignant tumours.
This evidence has been derived from a combination of Plant materials have a long history of being successfully clinical, biochemical, histological, and pharmacological used in the treatment of cancer, both as chemotherapeutic observations that point to the possibility of favourably agents and as complementary treatments. The pyrano- affecting the course of malignant disease with agents that coumarin compound (+)-3-angeloyl-4-acetoxy-cis-khel-lac- interfere with blood coagulation pathways [67].
tone was isolated from Radix peucedani, a herb well-knownfor the treatment of respiratory diseases and pulmonary ISOFLAVONES
hypertension. Resistance of cancer cells to chemotherapeuticagents remains one of the major obstacles in achieving an For many years now, isoflavones have been investigated effective treatment for cancer. The molecular mechanism of by clinicians, pharmacologists and plant physiologists.
multidrug resistance (MDR) in cancer cells may involve Isoflavones have exceptionally interesting, multidirectional over-expression of membrane drug efflux pumps, p53 therapeutic properties and the biological activity of these mutations, and up-regulation of bcl-2, DNA repair or cellular substances is conditioned by the location of the phenyl ring detoxification enzymes. P-glycoprotein is over-expressed in near the third carbon of the benzo-γ-pyrone. Hence, these various MDR cell lines and functions as an ATP-dependent compounds, in addition to antiflammatory, antimycotic and drug efflux pump that rapidly extrudes anti-tumour drugs radical scavenging properties, also exhibit both estrogenic from target cancer cells which prevents the drugs from and anti-estrogenic effects [69]. Genistein (4,5,7-trihydroxy- exerting their cytotoxic effects. (+)-3-angeloyl-4-acetoxy- isoflavone) is a natural isoflavone phytoestrogen present in cis-khel-lactone is a P-glycoprotein inhibitor and studies soy (Fig. 1.4). In the gastrointestinal tract, the β-glucoside
were recently performed to determine its effect on MDR cell conjugates of soy are converted by the natural gut microflora lines. Work by Wu [62] and co-workers demonstrated that into free genistein and other related isoflavones, which are this pyranocoumarin causes apoptotic cell death for drug present in circulating blood, accumulate in tissue and are sensitive KB-3-1 and multidrug resistant KB-V1 cancer celllines. Strong synergistic interactions were demonstratedwhen the pyranocoumarin was combined with common anti- tumour drugs such as vincristine, doxorubicin and paclitaxel.
Hence, this pyranocoumarin may have beneficial therapeuticapplication as a MDR reversing agent. However, furtherresearch is necessary to confirm these current findings.
Warfarin
Metastasis involves several distinct steps, including one in which the tumour cell, after entry into the bloodstream, Fig. (1.4). The basic chemical structure of genistein. It belongs to
comes to rest in a capillary located at a distant site where a 3806 Current Pharmaceutical Design, 2004, Vol. 10, No. 30
Lacy and O’Kennedy
excreted in urine of people who consume high amounts of γ-irradiated K562 myeloid leukemia cells [73]. Genistein soy in their diet. Studies have revealed that individuals who structurally resembles estradiol (E2) and demonstrated weak consume a traditional diet high in soy products have a low estrogenic activity in several studies (70,71,74). In humans, incidence of certain types of cancer, such as breast, prostatic this compound appears to have both estrogenic and anti- and colon cancer [70]. Genistein has been shown to inhibit estrogenic effects, depending on the concentrations of cancer cell proliferation in vitro and this effect may be circulating endogenous estrogens and estrogen receptor attributed to the fact that it is a known tyrosine kinase (ER).The estrogen agonistic activity in the absence of inhibitor. Therefore, this compound was chosen to determine estrogen may account for the beneficial effects of genistein its effect on two cell lines in vitro. The following section against atherosclerosis, coronary artery disease, osteoporosis gives background details on this therapeutically important and post-menopausal manifestations of hot flushes etc. The protective effect of genistein against certain forms ofhormone-dependent cancers, such as prostate, may be due to GENISTEIN
Although genistein belongs to the benzopyrones is not classed as a coumarin (benzo-α-pyrone). It is a flavonoid and BENZOPYRONES AND BREAST CANCER
belongs to the benzo-γ-pyrones. Genistein is a phytoestro- While a significant amount of research regarding the gen, which belongs to the 'isoflavone' class of compounds.
clinical applications of the benzopyrones has been reported it These diphenolic compounds structurally resemble estradiol is important to emphasise that only the ‘warfarin-type’ (E2) and were shown to have some estrogenic activity [71].
anticoagulants belong to mainstream pharmacotherapy. Other Reports about environmental estrogens, or xenoestro- types of coumarin compounds are either at the experimental gens, have been widespread in the last few years. There are or early clinical trial phase. Hence, additional studies on the important distinctions between estrogenic compounds of elucidation of their mechanism of action is necessary to industrial origin and those that come from plants. Com- facilitate a greater understanding of these compounds so that pounds such as insecticide DDT and industrial PCBs have they may find future clinical application. Recent work been implicated by some researchers in causing estrogen- carried out in our laboratory involved the investigation of the dependent cancers in exposed populations. Unlike some effects of various benzopyrones on two carcinoma cell lines.
industrial xenoestrogens, which tend to bioaccumulate in The two cell lines were MCF-7, a breast carcinoma and adipose tissue and persist in the body for years, phytoestro- A549, a lung carcinoma. However, the majority of research gens are readily metabolised and spend relatively little time focused on the MCF-7 cell line especially with regard to in the body. The timing of exposure, repeated exposures and signal transduction and cell cycle regulation studies. In our levels of exposure to phytoestrogens are important. The two research the effect of benzopyrones (warfarin, 7-hydroxy- major classes of phytoestrogens that have captured the most coumarin, esculetin, and genistein) on the growth and scientific attention are isoflavones and lignans. Diets rich in metabolism of human tumour cells was examined. Signal natural anti-estrogenic substances such as isoflavones have transduction studies were also performed in order to obtain been considered as one of the main reasons for significantly information regarding the mechanism of action of warfarin, lower incidence of breast cancer in China, Japan, and South esculetin and genistein on MCF-7 cells.
East Asia. Soybeans are a particularly abundant source of Previous investigators have used cell proliferation-based isoflavones such as genistein and daidzein [72].
assays in their chemosensitivity assessment of coumarincompounds [12, 57]. Recently an in vitro proliferation assay Genistein in Cancer Research
was utilised to determine the anti-tumour activities of Genistein has been shown to inhibit cancer cell prolifera- SP500263, a novel next-generation selective estrogen tion in vitro. This effect is attributed to inhibition of several receptor modulator (SERM), tamoxifen, and raloxifene in an key enzymes, especially tyrosine kinase, which plays a in vitro and in vivo MCF-7 breast cancer model [75]. The critical role in cell proliferation and transformation. Tyrosine insensitivity of many human tumour cell lines, previously kinase is also associated with oncogene expression in breast tested to growth inhibition by coumarin, seems to confirm cancer. Breast cancer is one of the most frequently diagnosed the generally held belief that coumarin is not responsible for malignancies in women, and its incidence is increasing in the observed in vivo effects, but is a pro-drug for other active industrialised nations. Tanos [71] and co-workers have metabolites. Therefore, in our research we chose initially to investigated the effect of genistein on human dysplastic and examine three benzopyrone compounds, namely warfarin, malignant epithelial breast cell lines, expressing low and esculetin, and genistein under similar assay procedures, with high metastatic potentials. They discovered that genistein has the MCF-7 cell lines and the A549 cell lines. The cell lines a significant in vitro inhibitory effect on the growth rate of were exposed to each of the three compounds for 96 hours.
dysplastic (fibrocystic cells) and cancerous breast cells. In Proliferation assays yielded some interesting information vitro studies show that genistein also exhibited a synergistic additive effect when cancer cells were exposed to a genisteinand tamoxifen treatment. These results indicate the potential 1. The potency of growth inhibition by genistein was of genistein administration alone or in combination with greater than esculetin which, in turn was greater than tamoxifen for the treatment of breast cancer [71].
A separate study has shown that genistein treatment has a 2. MCF-7 cells tested were quite sensitive to the effects of role in triggering cell death and promotes cell cycle arrest on Studies on Coumarins and Coumarin-Related Compounds
Current Pharmaceutical Design, 2004, Vol. 10, No. 30 3807
3. A549 cells did not seem to be as sensitive to the effects (warfarin and esculetin) was monitored using this technique.
of all three compounds in comparison to MCF-7 cells.
In these experiments the basal metabolic rate of the cells was However, both genistein and esculetin displayed determined prior to drug-exposure, and this value was significant inhibitory effects on A549 cells.
normalised as 100%. This was achieved for each of the fourseparate sensor chambers in the cytosensor. Following drug The most evident trend from the results was the exposure all subsequent metabolic rates were expressed as a sensitivity of both cell lines tested to growth inhibition by percentage of the basal rate of the particular sensor chamber, esculetin (6,7-dihydroxycoumarin). This reflects well ensuring each chamber with its encased cells acted as its own previous observations by Kolodziej [76] and colleagues and Cooke & O’Kennedy [30], that a dihydroxy-function ineither an ortho- or meta-format, was an extremely potent An important result evident from the data collected was chemical structure for toxicity in human tumour cell lines.
the fact that the cytosensor microphysiometer was more Since this potency was not evident in either of the single- sensitive to the effects of the benzopyrones on cellular hydroxycoumarin compounds, the added potency may be metabolism than the MTT assay. For example, the MTT due to the existence of a double hydroxy-function on the assay detected a decrease in cellular metabolism of ~ 30% when MCF-7 cells were exposed to 20µg/ml esculetin for24hrs. According to the cytosensor results, the cellular Assessment of leakage of a wide variety of enzymes is metabolism in cells exposed to 20µg/ml esculetin decreased often used for cytotoxicity testing endpoints. Lactate by ~ 60% over this time period. In fact it would appear from dehydrogenase (LDH) is a convenient marker because of the comparing the MTT and cytosensor results for warfarin, stability of the enzyme activity in the culture system [77]. In esculetin and genistein, that the cytosensor microphysio- this study none of the four compounds tested (warfarin, 4- meter is a much more sensitive predictor of metabolic hydroxywarfarin, esculetin and 7-hydroxycoumarin) caused inhibition. The reason for this may be due to the fact that the significant membrane damage in both cell lines (MCF-7 and MTT assay relies on the activity of just one group of mitoc- A549) assessed by LDH assay. There are some drawbacks hondrial enzymes to predict adverse effects on metabolism, that should be noted when using the LDH assay. If serum and in doing so may underestimate metabolic effects. The contaminates the system with endogenous enzymes they can MTT assay has been shown to underestimate the growth mask low levels of leached enzymes, which leads to inhibitory effects of interferons (IFNs) in the past [79].
underestimation of LDH present. The main drawback of this Formazan production can also be induced by drugs that assay, however, is its reliance on the premise that membrane cause perturbations of the cell cycle [80]. Cells have also integrity and cellular viability are closely linked. This is not been observed to metabolise the tetrazolium dye when always the case, at least in the early stages of cell death; so lethally damaged and have lost the ability to exclude vital these assays may not always be true / sensitive indicators of dyes [81]. Additionally, the test compound may also react viability. Hence, other assays (MTT, AP; explained later in with other assay components, e.g. tetrazolium salts, causing text) must be performed in order to obtain a complete picture interference, which again may lead to inaccurate results [32].
of the effects of benzopyrones on cells under investigation.
Apart from the increased sensitivity of prediction, the The effects of warfarin, 7-hydroxycoumarin, esculetin, cytosensor has several other advantages over the MTT assay and genistein on cellular metabolism was examined using the for the detection of metabolic suppression. The ‘real-time’ MTT assay and the cytosensor (where only warfarin and aspect of detection with the cytosensor yields large amounts esculetin have been tested on MCF-7 cells). The MTT Assay of information on the nature of the metabolic suppression is a well-established colourimetric assay, which can be used (e.g. time of detrimental effects, etc.), per experiment. To to detect the effects of agents on cellular metabolism. The attain this information with an end-point system such as the assay is based on the cleavage of the yellow tetrazolium salt, MTT Assay would require multiple, kinetic end-point assays.
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium In addition, the exposure set-up of the cytosensor (flow- bromide), to purple formazan crystals by mitochondrial through, perfusive) mimics the in vivo drug delivery/ dehydrogenases. Therefore, cells must not only be alive, but exposure more than the static exposure set-up of the MTT also metabolically active for this reaction to occur [12]. The assay. Finally, the cytosensor microphysiometer allows for assay can be used in either a long- (96hr) or short-term reversibility studies to be performed on drug-treated cells to (24hr) drug-exposure format, to assess the effects of a drug assess their recovery, an experiment difficult, if not on cellular growth or metabolism, respectively. Previously, impossible to achieve with cells following the MTT assay.
the MTT assay has been used to assess the anti-proliferative The tested benzopyrones (warfarin, esculetin and genistein on cytosensor) all displayed a dose- and time-dependent An alternative method was used to evaluate the effect of effect on cellular metabolism, with genistein followed by coumarins on cellular metabolism. The cytosensor micro- esculetin displaying the most potent effects. For warfarin, physiometer is a biosensing instrument that determines the only the highest concentration (100µg/ml) irreversibly extracellular acidification rate of cells, which is closely suppressed the metabolism of the MCF-7 cells tested.
coupled to their basal metabolic rate. Monitoring of cellular The acid phosphatase (AP) assay is based on hydrolysis metabolism is achieved using a pH-sensitive sensor, in ‘real- of pNPP by intracellular AP in viable cells to produce p- time’, which offers distinct advantages over the end-point nitrophenol. The method shows higher sensitivity and nature of the MTT assay, when examining the effect of reproducibility in comparison to cell proliferation assays chemical agents on cellular metabolism [12]. The effect of based on reduction of tetrazolium salts (e.g. MTT assay) and 24hr exposure of MCF-7 cells to two coumarin compounds 3808 Current Pharmaceutical Design, 2004, Vol. 10, No. 30
Lacy and O’Kennedy
is especially suited to applications where a large number of inhibit both tyrosine kinase phosphorylation and its kinase samples are assayed [82, 83]. The AP assay was performed activity. Therefore, from our research it is evident that on both cells lines. Both esculetin and genistein were the esculetin alone inhibits cell proliferation, which can be most potent proliferative inhibitors. Genistein had a proli- partially attributed to its tyrosine kinase inhibition activity.
ferative effect at low concentrations only in MCF-7 cells, Esculetin is known to be a cytostatic drug and the which was expected and correlates well with previous inhibition of the cell-cycle at transition G studies [84]. Overall the AP assay was found to offer the best with the cytostatic effect of both 7-hydroxycoumarin and combination of sensitivity, linearity, flexibility, ease of esculetin. It is also evident from previous studies that performance, reproducibility and precision, of all the micro- esculetin primarily effects/inhibits events in the G the cell cycle [58, 59]. If cells are arrested in G1 phase before The investigation of how esculetin and warfarin affect the S phase progression there would be a significant decrease in signal transduction cascade and cell cycle progression in DNA synthesis expected in these arrested cells. Therefore, MCF-7 cells was performed using three different assay the results obtained indicate there is a significant decrease in formats. An ELISA for detection of tyrosine phosphorylation DNA synthesis in MCF-7 cells treated with either esculetin in whole cells was used in order to determine whether the or genistein but not warfarin. It is possible that the decrease agents tested inhibit tyrosine phosphorylation of growth in DNA synthesis in esculetin-treated MCF-7 cells is due to factor stimulated EGF-R in MCF-7 cells. The cytosensor the agent’s ability to induce G1 arrest with no progression to microphysiometer was utilised to determine if esculetin or S phase where DNA synthesis occurs. Genistein seems to warfarin inhibit tyrosine kinase activity in EGF-stimulated affect cell cycle progression differently to esculetin.
MCF-7 cells. The final assay again utilised the ELISA Genistein results show that the decline in DNA synthesis format, with the aim of determining to what extent the agents occurs at a higher concentration than esculetin and this may inhibited DNA synthesis in MCF-7 cells. Previous studies be due to the fact that genistein does not arrest cells at G1 have investigated the effects of E2 on ER stimulation and phase. Genistein has been shown to inhibit progression later signal transduction [85-87]. However, breast cancer cells can at G2/M phase transition [5, 70, 73]. This is after S phase and be stimulated to proliferate with growth factors in the therefore, some DNA synthesis may occur. However, this absence of added estrogen or progesterone. MAP kinase stop in G2/M phase usually is followed by apoptosis and not activation increases in response to stimuli such as EGF and progression into mitosis as would happen in normal cycling IGF-1. Urokinase type plasminogen activator, FGF-2 and cells. This leads to a decrease in DNA synthesis as cells die insulin activate MAP kinase in MCF-7 cells [88-90].
Therefore, it was possible in this study to successfullystimulate the EGF-R in MCF-7 cells with EGF and, in turn, Taken together the signal transduction and cell cycle activate the MAP kinase pathway in the absence of E regulation results indicate what the possible target of esculetin in MCF-7 cells is and how it affects components of The regulation of activated MAP kinase in MCF-7 cells the activated MAP kinase pathway. This inhibition affects involves inhibitory (TNF-α) as well as stimulatory (EGF) downstream molecules (e.g. Ras, ERK, Myc and Nuclear pathways. Research has shown that EGF stimulates activated ER) with important roles in MAP kinase cascade [93].
MAP kinase within 1-2 minutes in MCF-7 cells. Maximal Therefore, esculetin can inhibit the receptor tyrosine kinase stimulation occurs at 6-8 minutes with a decrease in MAP activity of growth factor receptors such as EGF-R and ER.
kinase stimulation after this time period [91]. The cytosensor This in turn will prevent growth signals reaching other results reflect this stimulation pattern as after the transient signalling intermediates such as Ras, or activation of trans- maximal stimulation of EGF-R (which will also transiently cription factors such as Myc. Another signal transduction stimulate downstream MAP kinase activation), there is a pathway involves phosphatidylinositol-3-kinase (PI3-K) decline in stimulation after a short time period of ~10 heterodimer, which is an important mediator of survival minutes. It was important to determine for this study that the factors, protecting many cell types from multiple apoptosis- EGF-R is tyrosine phosphorylated in MCF-7 cells. This has inducing stimuli. If PI3-K is inhibited (by esculetin inhibi- been demonstrated and tyrosine kinase activity was also ting phosphorylation) the signalling pathway is interrupted detected in Western blots of whole-cell protein extracts from and the cell is unable to progress into the S phase of the cell the MCF-7 cell line probed with an anti-phosphotyrosine antibody [92]. Therefore, when MCF-7 cells were pre-exposed to either esculetin or warfarin it was possible to CONCLUSIONS
determine if these agents inhibited tyrosine phosphorylationor not. The ELISA for detection of tyrosine phosphorylation Coumarin and coumarin-related compounds have proved in MCF-7 cells yielded results that indicated esculetin for many years to have significant therapeutic potential.
inhibited tyrosine phosphorylation in EGF stimulated cells They come from a wide variety of natural sources and new by up to 20% more than control cells. Warfarin is a less coumarin derivatives are being discovered or synthesised on potent inhibitor of cell proliferation and metabolic activity a regular basis. Coumarin is a simple molecule and many of than esculetin and this may be partly due to the fact that it its derivatives have been known for more than a century.
shows no significant tyrosine kinase phosphorylation However, their vital role in plant and animal biology has not inhibition in comparison to esculetin and the positive control, been fully exploited. Coumarins have multiple biological genistein. The observed results from the cytosensor micro- activities including disease prevention, growth modulation physiometer correlated well with the ELISA results as both and anti-oxidant properties. These compounds are known to sets of results demonstrate that esculetin has the ability to exert anti-tumour effects and can cause significant changes Studies on Coumarins and Coumarin-Related Compounds
Current Pharmaceutical Design, 2004, Vol. 10, No. 30 3809
in the regulation of immune responses, cell growth and Ritschel WA, Grummich KW, Kaul S, Hardt TJ.
differentiation. Research involving coumarins and their Biopharmaceutical Parameters of Coumarin and 7-hydroxycoumarin. Die Pharma Ind 1981; 43: 271-276.
antiproliferative effect on malignant melanoma, leukaemia, Egan D, O’Kennedy R. The Production and Characterisation of renal cell carcinoma, prostate and breast cancer are discussed Anti-7-Hydroxycoumarin Antibodies and Their Use in the in this review. Recent studies, including research performed Development of an Enzyme-Linked Immunosorbent Assay. J Ir in our laboratory have been presented, in order to give an overview of the various potential therapeutic applications of Dempsey E, O’Sullivan C, Smyth MR, Egan D, O’Kennedy R,Wang J. Development of an Electrochemical Biosensor for 7- coumarins. It is evident from the research described that hydroxycoumarin. Analyst 1993; 118: 411-414.
coumarin and coumarin-related compounds are a plentiful Keating G. Biosensor-based Studies on Coumarins. PhD Thesis, source of potential anti-cancer drugs deserving further study.
Dublin City University, Dublin, Ireland, 1998.
Shilling WH, Crampton RF, Longland RC. Metabolism ofCoumarin in Man, Nature, London 1969; 221: 664-665.
ACKNOWLEDGEMENTS
Moran E, O'Kennedy R, Thornes RD. Analysis of Coumarin and its The financial support of the HEA PRTLI initiative for the Urinary Metabolites by High-Performance Liquid Chromatography.
J Chromatogr 1987; 416: 165-169.
National Centre for Sensor Research at Dublin City Taavitsainen P. Cytochrome P450 Isoform-Specific In Vitro University is gratefully acknowledged.
Methods to Predict Drug Metabolism and Interactions. OuluUniversity Press, Finland, 2001.
REFERENCES
Bogan D, Deasy B, O’Kennedy R, Smyth M. Determination ofFree and Total 7-hydroxycoumarin in Urine and Serum by References 94-96 are related articles recently published in Capillary Electrophoresis. J Chromatogr B 1995; 663: 371-378.
Rautio A, Kraul H, Kojo A, Salmela E, Pelkonen O. InterindividualVariability in Coumarin 7-Hydroxylation in Healthy Individuals.
Bruneton J. Pharmacognosy, Phytochemistry, Medicinal Plants.
Second Edition, Hampshire UK, Intercept Ltd 1999; pp 263-277.
Egan D, O’Kennedy R. Spectrofluorimetric Method for the Ojala T. Biological Screening of Plant Coumarins. PhD Thesis, Quantification of 7-Hydroxycoumarin in Urine and Plasma Using University of Helsinki, Helsinki, Finland, 2001.
Both Extracted and Unextracted Samples. Analyst 1993; 118: 201- Keating G, O’Kennedy R. The Chemistry and Occurrence of Coumarins. Coumarins: Biology, Applications and Mode of Bogan D, Deasy B, O’Kennedy R, Smyth M. The Use of Capillary Action. (Eds: O’Kennedy R, Thornes RD), Chichester, John Wiley Electrophoresis for Studying Interspecies Differences in Coumarin Metabolism in Liver Microsomes. Xenobiotica 1996; 26: 437-448.
Pelkonen O, Rautio A, Raunio H, Pasanen M. CYP2A6: A Human Murray RDH, Mendez J, Brown SA. The Natural Coumarins – Coumarin 7-hydroxylase. Toxicol 2000; 144: 139-147.
Occurrence, Chemistry and Biochemistry. Chichester, John Wiley, Pelkonen O, Maenpaa J, Taavitsainen P, Rautio A, Raunio H.
Inhibition and Induction of Human Cytochrome P450 (CYP) Constantinou AI, Kamath N, Hurley JS. Genistein Inactivates Bcl- Enzymes. Xenobiotica 1998; 28: 1203-1253.
2, Delays the G2/M Phase of the Cell Cycle, and Induces Apoptosis Byrden T. The Elucidation of the Mode of Action of Coumarin and of Human Breast Adenocarcinoma MCF-7 Cells. Eur J Can 1998; Some Specified Derivatives. MSc Thesis, Dublin City University, Egan D, O’Kennedy R, Moran E, Cox D, Prosser E, Thornes RD.
Deasy B. Development of Novel Analytical Methods to Study the The Pharmacology, Metabolism, Analysis and Applications of Metabolism of Coumarin. PhD Thesis, Dublin City University, Coumarin and Coumarin-Related Compounds. Drug Metab Rev Weber US, Steffen B, Siegers CP. Antitumour-Activities of Egan D, O’Kennedy R. Rapid and Sensitive Determination of Coumarin, 7-Hydroxycoumarin and its Glucuronide in Several Coumarin and 7-Hydroxycoumarin and its Glucuronide Conjugate Human Tumour Cell Lines. Res Commun Mol Pathol Pharmacol in Urine and Plasma by High Performance Liquid Chromatography. J Chromatogr B 1992; 582: 137-143.
Egan D, James P, Cooke D, O’Kennedy R. Studies on the Finn G, Kenealy E, Creaven B, Egan D. In vitro Cytotoxic Cytostatic and Cytotoxic Effects and Mode of Action of 8-Nitro-7- Potential and Mechanism of Action of Selected Coumarins, Using Hydroxycoumarin. Cancer Letts 1997; 118: 201-211.
Human Renal Cell Lines. Cancer Letts 2002; 183: 61-68.
Cooke D, O’Kennedy R. Comparison of the Tetrazolium Salt Lake B. Coumarin Metabolism, Toxicity and Carcinogenicity: Assay for Succinate Dehydrogenase with the Cytosensor Relevance for Human Risk Assessment. Food Chem Tox 1999; 37: Microphysiometer in the Assessment of Compound Toxicities.
Guilet D, Seraphin D, Rondeau D, Richomme P, Bruneton J.
Budzisz E, Brzezinska E, Krajewska U, Rozalski M. Cytotoxic Cytotoxic Coumarins From Calophyllum Dispar. Phytochemistry Effects, Alkylating Properties and Molecular Modelling of Coumarin Derivatives and Their Phosphonic Analogues. Eur J Med Cooke D, Fitzpatrick B, O’Kennedy R, McCormack T, Egan D.
Coumarins – Multifaceted Molecules with Many Analytical and Casley-Smith JR, Casley-Smith JR. High Protein Oedema and the Other Applications. Coumarins: Biology, Applications and Mode Benzopyrones. Sydney, J.B. Lippincott 1986.
of Action. (Eds: R O’Kennedy, R.D. Thornes), Chichester, John Casley-Smith JR, Casley-Smith JR. Coumarin in the Treatment of Lymphoedema and Other High-Protein Oedemas. Coumarins: Cooke D. Studies on the Mode of Action of Coumarins (Coumarin, Biology, Applications and Mode of Action. (Eds: O’Kennedy R, 6-hydroxycoumarin, 7-hydroxycoumarin and Esculetin) at a Thornes RD), Chichester, John Wiley & Sons, 1997; pp 143-184.
Cellular Level. PhD Thesis, Dublin City University, Dublin, Loprinzi CL, Kugler JW, Sloan JA, Rooke TW, Quella SK, Novotny P, et al. Lack of Effect of Coumarin in Women With Chen H, Walsh CT. Coumarin Formation in Novobiocin Lymphedema After Treatment for Breast Cancer. N Engl J Med Biosynthesis: Beta-Hydroxylation of the Aminoacyl Enzyme Tyrosyl-S-Novh by a Cytochrome P450 NovI, Chem Biol 2001; 8: Vanscheidt W, Rabe E, Naser-Hijazi B, Ramelet AA, Partsch H, Diehm C, et al. The Efficacy and Safety of a Coumarin- Pelkonen O, Raunio H, Rautio A, Pasanen M, Lang MA. The /Troxerutin-Combination (SB-LOT) in Patients with Chronic Metabolism of Coumarin. Coumarins: Biology, Applications and Venous Insufficiency: A Double Blind Placebo-Controlled Mode of Action. (Eds: O’Kennedy R, Thornes RD), Chichester, Randomised Study. Vasa 2002; 31: 185-190.
John Wiley & Sons, 1997; pp 67-92.
Thornes RD. Acquired Immune Suppression in ChronicBrucellosis. Ir Med J 1983; 76: 225.
3810 Current Pharmaceutical Design, 2004, Vol. 10, No. 30
Lacy and O’Kennedy
Oketch-Rabah H, Mwangia J, Lisgartenb J, Mberuc E. A New Murakami A, Yamayoshi A, Iwase R, Nishida J, Yamaoka T, Antiplasmodial Coumarin from Toddalia asiatica Roots. Fitoterapia Wake N. Photodynamic Antisense Regulation of Human Cervical Carcinoma Cell Growth Using Psoralen-Conjugated Oligo Carter SK, Bakowski MT, Hellman K. Chemotherapy of Cancer.
(Nucleoside Phosphorothioate). Eur J Pharm Sci 2001; 13: 25-34.
Third Edition, New York, Wiley & Sons, 1989.
Wu JY, Fong WF, Zhang JX, Leung CH, Kwong HL, Yang MS, Grotz KA, Wustenberg P, Kohnen R, Al-Nawas B, Henneicke-von et al. Reversal of Multidrug Resistance in Cancer Cells by Zepelin HH, Bockisch A, et al. Prophylaxis Of Radiogenic Pyranocoumarins Isolated From Radix peucedani. Eur J Pharmacol Sialadenitis and Mucositis by Coumarin/Troxerutine in Patients with Head and Neck Cancer-A Prospective, Randomized, Placebo- Hejna M, Raderer M, Zielinkski C. Inhibition of Metastasis by Controlled, Double-Blind Study. Br J Oral Maxillofac Surg 2001; Anticoagulants. J Nat Can Institute 1999; 91: 22-36.
Aisner J, Goutsou M, Maurer LH, Cooper R, Chahinian P, Carey Wcislo G, Szczylik C. In process citation. Pol Merkuriusz Lek R, et al. Intensive Combination Chemotherapy, Concurrent Chest Irradiation, and Warfarin for the Treatment of Limited-Disease Thornes RD, Edlow DW, Wood S Jr. Inhibition of Locomotion in Small-Cell Lung Cancer: A Cancer and Leukaemia Group B Pilot Cancer Cells In Vivo By Anticoagulant Therapy. 1. Effect of Study. J Clin Oncol 1992; 10: 1230-1236.
Sodium Warfarin on V2 Cancer Cells, Granulocytes, Lymphocytes Maurer LH, Herndon JE, Hollis DR, Aisner J, Carey RW, Skarin and Macrophages in Rabbits. Johns Hopkins Med J 1968; 123: AT, et al. Randomized Trial of Chemotherapy and Radiation Therapy With or Without Warfarin for Limited-Stage Small-Cell Thornes RD, Daly L, Lynch G, Breslin B, Browne H, Browne HY, Lung Cancer: A Cancer And Leukaemia Group B Study. J Clin et al. Treatment With Coumarin to Prevent or Delay Recurrence of Malignant Melanoma. Cancer Res Clin Oncol 1994; 120 Suppl: Fitzpatrick B. The Production, Characterisation and Applications of Polyclonal and Monoclonal Antibodies to Warfarin. PhD Thesis, Thornes RD. Clinical and Biological Observations Associated with Dublin City University, Dublin, Ireland 2001.
Coumarins. Coumarins: Biology, Applications and Mode of Mousa S. Anticoagulants in Thrombosis and Cancer: The Missing Action. (Eds: O’Kennedy R, Thornes RD), John Wiley & Sons, Link. Expert Rev Anticancer Ther 2002; 2: 227-233.
Bobek V, Boubelik M, Kovarik J, Taltynov O. Inhibition of Velasco-Velazquez MA, Agramonte-Hevia J, Barrera D, Jimenez- Adhesion Breast Cancer Cells by Anticoagulant Drugs and Orozco A, Garcia-Mondragon MJ, Mendoza-Patino N, et al. 4- Cimetidine. Neoplasma 2003; 50: 148-151.
Hydroxycoumarin Disorganizes the Actin Cytoskeleton in B16-F10 Luczkiewicz M, Glod D. Callus Cultures of Genista Plants – In Melanoma Cells but not in B82 Fibroblasts, Decreasing their Vitro Material Producing High Amounts of Isoflavones of Adhesion to Extracellular Matrix Proteins and Motility. Cancer Phytoestrogenic Activity. Plant Sci 2003; 165: 1101-1105.
Fioravanti L, Cappelletti V, Miodini P, Ronchu E, Brivio M, Di Ebbinghaus SW, Mohler JL, Marshall ME. Renal Cell Carcinoma: Fronzo G. Genistein in the Control of Breast Cancer Cell Growth: The background, Rationale and Current Development of Coumarin Insights into the Mechanism of Action In Vitro. Cancer Letts 1998; (1,2-benzopyrone) as a Potential Therapeutic Agent. Coumarins: Biology, Applications and Mode of Action. (Eds: O’Kennedy R,Thornes RD), Chichester, John Wiley & Sons, 1997, pp 209-239.
Tanos V, Brzezinski A, Drize O, Strauss N, Peretz T. Synergistic Vuky J, Motzer RJ. Cytokine Therapy in Renal Cell Cancer. Urol Inhibitory effects of Genistein and Tamoxifen on Human Dyplastic and Malignant Epithelial Breast Cells In Vitro. Eur J Obstet Thornes RD, Lynch G, Sheehan MW. Cimetidine and Coumarin Gynecol Reprod Biol 2002; 102: 188-194.
Therapy of Melanoma. Lancet 1982; II, 328.
Barrett J. Phytoestrogens: Friends or Foes. Environ Health Perspect Dexeus FH, Logothetis CJ, Sella A, Fitz K, Amato R, Reuben JM, et al. Phase II Study of Coumarin and Cimetidine in Patients with Papazisis K, Zambouli D, Kimoundri OT, Papadakis ES, Vala V, Metastatic Renal-Cell Carcinoma. J Clin Oncol 1990; 8: 325-329.
Geromichalos GD, et al. Protein Tyrosine Kinase Inhibitor, Kokron O, Maca S, Gasser G, Schmidt PR. Cimetidine and Genistein, Enhances Apoptosis and Cell Cycle Arrest in K562 Coumarin Therapy of Renal Cell Carcinoma. Oncology 1991; 48: Cells Treated with γ-irradiation. Cancer Letts 2000; 160: 107-113.
Ratna W. Inhibition of Estrogenic Stimulation of Gene Expression Marshall ME, Butler K, Fried A. Phase I Evaluation of Coumarin by Genistein. Life Sciences 2002; 71: 865-877.
(1,2-Benzopyrone) and Cimetidine in Patients with Advanced Brady H, Desai S, Gayo-Fung LM, Khammungkhune S, McKie JA, Malignancies. Mol Biother 1991; 3: 170-178.
O'Leary E, et al . Effects of SP500263, a Novel, Potent Antiestro- Bosland MC. Prostate Cancer. Encyclopaedia of Human Biology.
gen, on Breast Cancer Cells and in Xenograft Models. Cancer Res San Diego Academic Press 1991; 6: 177-190.
Agarwal R. Cell Signalling and Regulators of Cell Cycle as Kolodziej H, Kayser O, Woerdenbag HJ, van Uden W, Pras N.
Molecular Targets for Prostate Cancer Prevention by Dietary Structure-cytotoxicity relationships of a series of natural and semi- Agents. Biochem Pharmacol 2000; 60: 1051-1059.
synthetic simple coumarins as assessed in two human tumour cell Marshall ME, Butler K, Hermansen D. Treatment of Hormone- lines. Z Naturforsch 1997; 52, 240-244.
Refractory Stage-D Carcinoma of Prostate with Coumarin (1,2- Martin A. Cytotoxicity Testing In Vitro: Investigation of 5 Benzopyrone) and Cimetidine: A Pilot Study. Prostate 1990; 17: Miniaturised, Colorimetric Assays. PhD Thesis, Dublin City Mohler JL, Gomella LG, Crawford ED, Glode LM, Zippe CD, Fair Kumi-Diaka J. Chemosensitivity of Human Prostate Cancer Cells WR, et al. Phase II Evaluation of Coumarin (1,2-Benzopyrone) in PC3 and LNCaP to Genistein Isoflavone and β-Lapachone. Biol Metastatic Prostatic Carcinoma. Prostate 1992; 20: 123-131.
Myers RB, Parker M, Grizzle WE. The Effects of Coumarin and Jabbar SAB, Twentyman PR, Watson JV. The MTT Assay Suramin on the Growth of Malignant Renal and Prostatic Cell Underestimates the Growth Inhibitory Effects of Interferons. Br J Lines. J Cancer Res Clin Oncol 1994; 120 (Suppl): S11-S13.
Wang CJ, Hsieh YJ, Chu CY, Lin YL, Tseng TH. Inhibition of CellCycle Progression in Human Leukemic HL-60 Cells by Esculetin.
Supino R. MTT Assays. Methods in Molecular Biology, vol. 43: In vitro toxicity testing protocols, (Eds: O’Hare S, Atterwill CK), Jimenez-Orozco F, Lopez-Gonzalez JS, Nieto-Rodriguez A, Totowa, USA Humana Press Inc., 1985; pp 137-149.
Velasco-Velazquez MA, Molina-Guarneros JA, Mendoza-Patino Maehara Y, Anai H, Tamada R, Sugimachi K. The ATP Assay is N, et al. Decrease of Cyclin D1 in the Human Lung Adenocarcino- More Sensitive Than the Succinate Dehydrogenase Inhibition Test ma Cell Line A-427 by 7-hydroxycoumarin. Lung Cancer 2001; for Predicting Cell Viability. Eur J Cancer & Clin Oncol 1987; 23: Diawara MM, Allison T, Kulkosky P, Williams DE. Psoralen- Yang TT, Sinai P, Kain SR. An Acid Phosphatase Assay for induced Growth Inhibition in Wistar Rats. Cancer Letts 1997; 114: Quantifying the Growth of Adherent and Nonadherent Cells. Amer Studies on Coumarins and Coumarin-Related Compounds
Current Pharmaceutical Design, 2004, Vol. 10, No. 30 3811
Si F, Shin SH, Biedermann A, Ross GM. Estimation of PC12 Cell Phosphorylation of ERK in Urokinase-Treated Cells. J Biol Chem Numbers with Acid Phosphatase Assay and Mitochondrial Dehydrogenase Assay: Dopamine Interferes with Assay Based on Vercoutter-Edouart AS, Lemoine J, Smart CE, Nurcombe V, Boilly Tetrazolium. Exp Brain Res 1999; 124: 145-150.
B, Peyrat JP, et al. The Mitogenic Signalling Pathway for Hsieh C, Santell R, Haslem S, Helferich W. Estrogenic Effects of Fibroblast Growth Factor-2 Involves the Tyrosine Phosphorylation Genistein on the Growth of Estrogen Receptor-positive Human of Cyclin D2 in MCF-7 Human Breast Cancer Cells. FEBS Letts Breast Cancer (MCF-7) Cells In vitro and In vivo. Cancer Res Flury N, Eppenberger U, Mueller H. Tumour-Necrosis Factor- Aronica SM, Kraus WL, Katzenellenbogen BS. Estrogen Action Alpha Modulates Mitogen-Activated Protein Kinase Activity of Via the cAMP Signalling Pathway: Stimulation of Adenylate Epidermal-Growth-Factor-Stimulated MCF-7 Breast Cancer Cells.
Cyclase and cAMP-regulated Gene transcription. Proc Natl Acad Mueller H, Loop P, Liu R, Wosikowski K, Kueng W, Eppenberger Migliaccio A, Di Domenico M, Castoria G, de Falco A, Bontempo U. Differential Signal Transduction of Epidermal-Growth-Factor P, Nola E, et al. Tyrosine Kinase/P21ras/MAP-Kinase Pathway Receptors in Hormone-Dependent and Hormone-Independent Activation by Estradiol-Receptor Complex in MCF-7 Cells. EMBO Human Breast Cancer Cells. Eur J Biochem 1994; 221: 631-637.
Pan SL, Huang YW, Guh JH, Chang YL, Peng CY, Teng CM.
Kelly MJ, Wagner EJ. Estrogen Modulation of G-protein-coupled Esculetin Inhibits Ras-mediated Cell Proliferation and Alleviates Receptors. Trends Endocrinol Metab 1999; 10: 369-374.
Vascular Restenosis Following Angioplasty in Rats. Biochem Alblas J, Slager-Davidov R, Steenbergh PH, Sussenbach JS, van Der BB. The Role of MAP Kinase in TPA-mediated Cell Cycle Bradshaw TD, Trapani V, Vasselin DA, Westwell AD. The aryl Arrest of Human Breast Cancer Cells. Oncogene 1998; 16: 131- hydrocarbon receptor in anticancer drug discovery: friend or foe? Curr Pharm Design 2002; 8(27): 2475-90.
Nguyen DH, Webb DJ, Catling AD, Song Q, Dhakephalkar A, Chen L, Waxman DJ. Cytochrome P450 gene-directed enzyme Weber MJ, et al. Urokinase-Type Plasminogen Activator Stimulates prodrug therapy (GDEPT) for cancer. Curr Pharm Design 2002; the Ras/Extracellular Signal-Regulated Kinase (ERK) Signalling Pathway and MCF-7 Cell Migration by a Mechanism That Patterson LH, Murray GI. Tumour cytochrome P450 and drug Requires Focal Adhesion Kinase, Src, and Shc. Rapid Dissociation activation. Curr Pharm Design 2002; 8(15): 1335-47.
of GRB2/Sps-Shc Complex is Associated with the Transient

Source: http://dcu.ie/biotechnology/abg/pdfs/rok/Studies_on_Coumarins_and_Coumarin-Related%2520Compounds%2520to%2520Determine%2520their%2520Therapeutic_Role_in_the_Treatment_of_Cancer.pdf