Send Orders for Reprints to [email protected] Anti-Cancer Agents in Medicinal Chemistry, 2014, 14, 000-000 1 Plant : Recent Advances and Future Perspectives in Cancer Therapy

Tereza Nehybová1, Jan Šmarda1 and Petr Beneš1,2,*

1Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic and Masaryk Memorial Cancer Institute, RECAMO, Žlutý kopec 7, 656 53 Brno, Czech Republic; 2International Clinical Research Center, Center for Biological and Cellular Engineering, St. Anne's University Hospital, Brno, Czech Republic

Abstract: Natural products are often used in drug development due to their ability to form unique and diverse chemical structures. Coumestans are polycyclic aromatic plant secondary metabolites containing a moiety, which consists of a benzoxole fused to a chromen-2-one to form 1-Benzoxolo[3,2-c]chromen-6-one. These natural compounds are known for large number of biological activities. Many of their biological effects can be attributed to their action as and polyphenols. In the last decade, anticancer effects of these compounds have been described in vitro but there is only limited number of studies based on models in vivo. More information concerning their in vivo bioavailability, stability, metabolism, toxicity, estrogenicity, cellular targets and drug interactions is therefore needed to proceed further to clinical studies. This review focuses on coumestans exhibiting anticancer properties and summarizes mechanisms of their toxicity to cancer cells. Moreover, the possible role of coumestans in cancer prevention is discussed. Keywords: Cancer, cellular target, , coumestan, glycyrol, , therapy, .

INTRODUCTION also acts as antioxidant [18, 36] and prevents bone resorption by Natural products are often used in drug development due to inhibiting differentiation and function of osteoclasts and by their ability to form unique and diverse chemical structures. Natural supporting proliferation, differentiation and function of osteoblasts compounds and their derivatives are commonly used for prevention [37-42]. In addition, it inhibits adipocyte differentiation and lipogenesis and treatment of various diseases, including cancer. Interestingly, [43, 44], affects insulin sensitivity, lipid and glucose metabolism half of the small molecules approved by U.S. Food and Drug [45-48], antagonizes pregnane X receptor (PXR) [49, 50], inhibits Administration (FDA) agency in 2010 were natural products or aryl hydrocarbon receptor (AhR) activation by environmental their derivatives, including majority of the antitumor agents. This contaminants [51], and regulates metabolism by inhibiting suggests that natural products represent an important source of aromatase and 17β-hydroxysteroid dehydrogenase [52-54]. Coumestrol anticancer drugs [1, 2]. Many anticancer drugs that are currently in was also shown to be involved in epigenetic control of gene clinical use, including some of the best known as vinblastine, expression by deregulating methylation of specific genes in female vincristine, paclitaxel, camptothecin are derived from plants [1]. rat pancreas [55]. Coumestans are polycyclic aromatic plant secondary metabolites Many of the coumestrol effects mentioned above are mediated containing a coumestan moiety. These compounds were identified by receptors (ERs). Numerous studies demonstrated that and isolated from variety of plants and exhibit hepatoprotective, coumestrol is a acting as agonist of both ERα and antimyotoxic, antifibrotic, antiinflammatory, antiproteolytic, ERβ with stronger binding affinity to ERβ [19, 56, 57]. The antihemorrhagic, neuroprotective, estrogenic, antimicrobial, coumestrol binding affinity to ERα is weaker in comparison to antifungal, antihelmintic, antioxidative and immunomodulatory endogenous 17β- (E2) [19, 58]. On the other hand, binding properties [3-19]. The plant-derived coumestan family of compounds affinity of these two compounds to ERβ is similar [19, 59]. The includes coumestrol, wedelolactone, demethylwedelolactone, effects of ERα and ERβ on tumor growth, metastases and chemo- psoralidin, flemicoumestan A 1, glycyrol, erythribyssin N, aureol, sensitivity are different reflecting regulation of various genes by tephcalostan, plicadin, sophoracoumestan A, coumestoside C, D, binding to distinct regulatory elements recruiting distinct coregulators hedysarimcoumestans A, B, D, F and many others [11, 13, 20-29]. and chromatin remodelling factors. Several findings, reviewed by Moreover, other coumestan derivatives were synthesized to Leitman et al. and Burns et al., demonstrated that expression/ improve their antiviral and antimyotoxic properties [30-32]. In this activation of ERβ can modulate or even oppose pro-proliferative review, we focus on coumestrol, wedelolactone, psoralidin and effects of ERα [60, 61]. The anti-proliferative and anti-metastatic glycyrol – the coumestans with anticancer effects. Molecular effects of phytoestrogens may be therefore explained by their mechanisms mediating their effects on cancer cells are summarized higher affinity to ERβ compared to ERα [61-63]. (Fig. 1) and discussed. Both genomic and nongenomic mechanisms have been proposed to mediate phytoestrogenic effects of coumestrol (Fig. 3). COUMESTROL Coumestrol binds to nuclear ERs and induces expression of specific Coumestrol (Fig. 2) was originally isolated from alfalfa [20]. estrogen-responsive genes that regulates various cellular processes, Subsequently, this plant coumestan was found in a variety of such as growth and survival [63, 64]. A strong correlation between legumes, , brussels sprouts, clover and spinach. Coumestrol expression profiles induced by coumestrol and E2 in ERα- was demonstrated to posses a wide range of biological effects including expressing cancer cells was reported but was less obvious in the neuroprotection [33, 34] and immunomodulation [16, 17, 35]. It presence of ERβ, suggesting that activation of ERβ by coumestrol can modulate its ERα-stimulating effects in cells [63]. Coumestrol can also initiate signaling from the membrane ERs by activation of *Address correspondence to this author at the Department of Experimental 2+ Biology, Faculty of Science, Masaryk University, Kotlarska 2, 611 37 Brno, ERK1/2, JNK, PI3K, Syc and changes in Ca fluxes [65-70]. Czech Republic; Tel: +420 54949 3125; Fax: +420 54949 5533; Phytoestrogens often exert biphasic effect on the ER-responsive E-mail: [email protected] cancer cells [71]. Coumestrol in doses lower than 10-7 M enhances

1871-5206/14 $58.00+.00 © 2014 Bentham Science Publishers 2 Anti-Cancer Agents in Medicinal Chemistry, 2014, Vol. 14, No. 0 Nehybová et al.

Fig. (1). Cancer-related activities of coumestans.

Fig. (2). Chemical structures of coumestans with anticancer properties. proliferation of breast and pituitary cancer cells by stimulation of As phytoestrogens directly interacts with ERs, it has been the G1/S cell cycle transition, cyclin D1 over-expression along with suggested that they can block binding of endogenous estrogen and ERK 1/2 and JNK phosphorylation. These effects are inhibited by thus act as estrogen antagonists. This antagonism in binding to ERs the ER antagonist ICI 182,780 [69, 72-74]. At higher doses, was suggested for coumestrol as well [56, 57, 80]. However, however, coumestrol induces apoptosis/cell death of breast, coumestrol did not inhibit the E2-mediated gene transactivation pancreatic, ovarian, and prostate cancer cells. The induction of from ERE (estrogen responsive element) and the E2-induced apoptosis by coumestrol is associated with caspase activation and proliferation of breast cancer and HeLa cells [71, 80, 81]. downregulation of antiapoptotic Bcl-2 protein [35, 65, 66, 72, 75, Moreover, in some of these studies, additive effects of both 76, 77-79]. compounds were reported [82, 83]. Interestingly, coumestrol at very Coumestans in Cancer Prevention and Treatment Anti-Cancer Agents in Medicinal Chemistry, 2014, Vol. 14, No. 0 3

Fig. (3). Genomic and nongenomic effects of coumestrol in estrogen signaling. low doses attenuated the proliferative effects of E2 in rat pituitary through down-regulation of the CKII-specific Akt phosphorylation. cells [69]. These contradictory results may reflect differences in Furthermore, coumestrol-induced senescence of breast cancer doses of coumestrol and , cell and tissue specificity, the MCF-7 and colon cancer HCT116 cells was antagonized by type of reporter assays, the presence/absence/activity of ER- overexpression of CKII [91]. Inhibition of CKII by coumestrol coactivators, the ratio of ERα/ERβ or may also reflect multiple resulted in activation of NADPH oxidase, reactive oxygen species mechanisms of action of coumestrol in cells. Numerous studies (ROS) production and activation of the p53-p21 (Cip1/WAF1) reported that coumestrol acts as phytoestrogen and endocrine pathway resulting in cellular senescence [92]. disrupter in vivo by affecting several endocrine mechanisms during Coumestrol interacts with DNA [101] and posses mutagenic the estrous cycle resulting in abnormalities in the development of and clastogenic properties. It induces mutations in TA97 and male and female reproductive tracts and infertility. Pathologies, TA102 strains of Salmonella typhimurium [102] as well as DNA such as lack of ovulations, cervical and uterine lesions and strand breaks, chromosomal aberrations, micronuclei and somatic paraovarian cysts have been referred in lambs, ewes, rats, mice and mutations in Chinese hamster V79 cells, Syrian hamster embryo mares fed with a diet rich in coumestrol [84, 85]. Although cells, human lymphocytes and human lymphoblastoid cells [103- coumestrol acts as ER agonists, it does not affect activity of 106]. Although inhibition of topoisomerase II as mechanism of the androgen-, progesterone-, glucocorticoid- and thyroid receptors. coumestrol-induced clastogenicity was suggested [103, 105], this However, it does affect their expression [79, 86-88]. hypothesis has not been proved yet. Although it has been previously suggested that coumestrol Coumestrol also inhibits invasion of MDA-MB 231 breast exerts its antiproliferative effect by competitive binding to ER [89], carcinoma cells but mechanism of this effect is not known [90]. cytotoxicity of coumestrol was also observed in ER-negative cell Coumestrol is an inhibitor of α-glucosidase [107]. The α-glucosidase lines [76, 90, 91]. There are several molecular targets related to the inhibitors disrupt biosynthesis and structure of oligosacharides on coumestrol-induced cell cycle block and apoptosis. Coumestrol cell surface. The cell surface oligosacharides are important regulators inhibits activity of cassein kinase II (CKII) both in vitro and in of various steps of the cancer metastasis process and the glucosidase various cancer cells [91, 92]. CKII is Ser/Thr kinase that catalyzes inhibitors suppress the metastatic spreading of malignant tumor phosphorylation of more than 300 cytoplasmatic and nuclear cells by perturbing correct carbohydrate arrangement [108]. In proteins. CKII regulates multiple cellular processes including cell addition, coumestrol potentiates association of E-cadherin with β- proliferation, survival and RNA synthesis [93-96]. Expression of catenin, restores integrity of adherent junctions and prevents the CKII is upregulated in variety of tumor cells, thus representing a Apc-associated intestinal tumorigenesis in the mouse colorectal favorable target for cancer therapy [97]. Indeed, inhibition of CKII cancer Apc(Min/+) model. Coumestrol also significantly improves activity resulted in direct induction of apoptosis or increased rate of the enterocyte crypt-villus migration [109]. In pancreatic sensitivity to various chemotherapeutics [98-100]. Liu et al. tumor cells, coumestrol was shown to decrease expression of K-ras, demonstrated that coumestrol inhibits growth and induces apoptosis an oncogene promoting cell invasivity [66]. of three cancer cell lines (A549, Jurkat and HeLa) at least partially 4 Anti-Cancer Agents in Medicinal Chemistry, 2014, Vol. 14, No. 0 Nehybová et al.

As mentioned above, coumestrol is an antagonist of PXR that oxoETE, a metabolic product of 5-Lox [124]. In this study, authors can block the effects of PXR agonists [49]. PXR is a nuclear also reported that wedelolactone inhibited PKCε, activated JNK and receptor protein monitoring the presence of foreign toxic substances had no effect on the Akt kinase activity. The functional role of JNK and upregulating expression of proteins involved in their intracellular was subsequently confirmed, since specific JNK inhibitor transport and metabolic detoxification. Interestingly, PXR regulates suppressed the wedelolactone-induced apoptosis [124]. expression of the cytochrome P450 3A gene coding for a key There are multiple effects of wedelolactone on cell signaling enzyme of phase I drug metabolism that is involved oxidative pathways. It significantly increases phosphorylation of ERK and metabolism of significant portion of drugs and xenobiotics [110]. JNK in hepatic stellate cell line LX-2 [117], ERK but not JNK in Coumestrol also inhibits expression and/or function of xenobiotic mesenchymal stem cells [131] and decreases phosphorylation of transporters including breast cancer resistance protein (BCRP) and ERK in breast cancer MDA-MB-231 cells [119]. Wedelolactone multidrug resistance gene (mdr-1) [66, 111]. Therefore, it can be enhances the antitumor activity of IFN-γ in colon a hepatocarcinoma anticipated that coumestrol may affect the effectiveness of cell lines through inhibition of STAT1 dephosphorylation by T-cell chemotherapy of cancer. protein tyrosine phosphatase [125]. The inhibition of breast cancer With respect to the ER-targeted therapy of breast cancer, cell motility by wedelolactone was attributed to its inhibitory effect coumestrol differently affects growth of -resistant and on phosphorylation of focal adhesion kinase (FAK) [119]. tamoxifen-sensitive breast cancer cells. Moreover, it also abrogates We have recently shown that wedelolactone acts as a catalytic the growth-inhibitory effect of tamoxifen in tamoxifen-sensitive inhibitor of DNA topoisomerase IIα, the essential enzyme for cell cells [112]. Interestingly, the interaction between tamoxifen and proliferation, by blocking its binding to supercoiled plasmid DNA coumestrol in regulation of ER transcription activities depends on [132, 133]. Furthermore, we showed that the topoisomerase IIα- mutational status of ER [81]. inhibitory effect of wedelolactone is redox-dependent as it is suppressed by reducing agents. Reducing agents, such as N- WEDELOLACTONE AND DEMETHYLWEDELOLACTONE acetylcysteine (NAC) and glutathione also lower cytotoxicity of Wedelolactone (Fig. 2) is a coumestan isolated from extracts of wedelolactone to breast cancer cells. We have also documented Wedelia calandulaceae, Wedelia chinensis, Eclipta alba and Eclipta DNA damage induced by wedelolactone in vitro upon its oxidation prostrata that are commonly used in traditional medicine for by copper ions [133]. treatment of sepsis, hepatitis, viral infections and snake poisoning Wedelolactone senzitizes pancreatic cancer cells to [3-5, 21]. Extracts from these plants exhibit hepatoprotective, proapoptotic effects of TNFα by inhibiting the NFκB activity and antimyotoxic, antifibrotic, anti-phlogistic, antiproteolytic, decreasing expression of Mcl-1, a member of the Bcl-2 protein immunomodulatory and antihemorrhagic properties [113-117]. family [134]. Other members of the Bcl-2 protein family including Recently, extracts of Wedelia chinensis and a mixture of its compounds Bax and Bcl-2 were deregulated by wedelolactone in hepatic (wedelolactone, , luteolin) exhibited growth-inhibitory stellate cell line LX-2 [117]. effect on prostate cancer cells in vitro and in vivo [118]. Very Although wedelolactone is widely considered to act as recently, wedelolactone and its derivative demethylwedelolactone phytoestrogen [30, 135], to our knowledge there has not been have been shown to inhibit invasive growth and metastasis of breast published any direct evidence for this statement. Interestingly, no cancer xenografts [119]. Demethylwedelolactone (Fig. 2), which transactivation of the luciferase reporter gene from ERE occurred in has not been characterized so far, appears to be even more potent breast cancer MCF-7 cells treated with wedelolactone in media antitumor agent compared to wedelolactone itself [119]. In vitro, containing 10% FCS [131]. However, as previous studies showed wedelolactone inhibited growth and induced cell death of breast, that the weak stimulatory effect of phytoestrogens on cell growth pituitary, colon, liver, neuroblastoma, myeloma and prostate cancer and ERE transactivation in ER-responsive cells such as MCF-7 cell lines [120-125]. could be detected only in steroid-deprived conditions, the Numerous cellular molecules and signaling pathways were experiments by Lim et al. may not detect proestrogenic effects of suggested to be targeted by wedelolactone. Wedelolactone acts as wedelolactone. an inhibitor of IKK kinase, the key enzyme regulating the activity of NFκB [5]. This transcription factor, a key regulator of cancer cell PSORALIDIN survival, growth, invasion, metastasis and angiogenesis, is often Psoralidin (Fig. 2) is a coumestan isolated from seeds of constitutively active in cancers [126, 127]. Suppression of the Psoralea corylifolia (Leguminosae) [22]. This plant is used in NFκB activity by natural compounds, such as curcumin, , traditional medicine for treatment of pollakiuria, nephritis, capsaicin and wedelolactone is believed to inhibit the oncogenic osteoporosis, hypertension, asthma, leucoderma, leprosy and potential of transformed cells [119, 123, 127]. Genes coding for psoriasis. Extracts from the seeds and roots of Psoralea corylifolia matrix metalloproteinases (MMPs) were identified among the exhibits antimicrobial, antifungal, antihelmintic, antioxidative, NFκB-regulated and the wedelolactone-inhibited genes thus linking immunomodulatory, laxative, antidepressant, estrogenic and the wedelolactone-treatment to inhibition of breast cancer cell anticancer properties [6-11]. Although these extracts contain invasion and metastasis [119]. numerous bioactive compounds including various , Androgen receptor is another target of wedelolactone identified terpenes and [11], psoralidin is one of compound with in prostate cancer cells. Wedelolactone inhibited its activities and described anticancer properties. exerted stronger growth-suppressing effect in androgen-dependent Psoralidin is cytotoxic agent inducing apoptosis of prostate prostate cancer cell lines when compared to androgen-independent [136, 137], gastric [22], colon, and breast [138] cancer cells. Oral cells. Moreover, a synergistic inhibitory effect with apigenin and administration of psoralidin significantly reduces growth of prostate luteolin was observed [128]. cancer tumor xenografts in nude mice [136, 139]. Wedelolactone also inhibits activity of 5-lipoxygenase (5-Lox), Psoralidin induces production of ROS in prostate cancer cells an enzyme that catalyzes the first step in biosynthesis of but not in normal prostate epithelial cancer cell line without altering leukotrienes from arachidonic acid [129]. Previous studies have expression and activity of endogenous antioxidant defense demonstrated an essential role of 5-Lox in regulation of proliferation enzymes, such as catalase and superoxide dismutase 1 and 2 (SOD1 and survival of various types of cancer cells [130]. Sarveswaran and SOD2) [140]. Psoralidin induces apoptosis of prostate cancer et al. reported that the wedelolactone-induced caspase-dependent cell lines characterized by decreased mitochondrial membrane apoptosis of prostate cancer cells was effectively prevented by 5- potential, released cytochrome c into cytosol, activation of caspases Coumestans in Cancer Prevention and Treatment Anti-Cancer Agents in Medicinal Chemistry, 2014, Vol. 14, No. 0 5

3, 8 and 9, increased expression of Bax and decreased expression of calcineurin, NFκB and deregulation of expression of iNOS, COX-2, Bcl-2 [136, 140]. Ectopic overexpression of antioxidant defense IL-1, IL-2, IL-6 and TNF-α [13, 14]. Glycyrol induced apoptosis of enzymes or treatment with NAC reduces the psoralidin-induced human embryonic kidney 293T and Jurkat leukemia cells by ROS production and the psoralidin-induced death of prostate cancer inhibition of the NFκB activity, S-phase arrest, activation of cells. By similar approaches authors demonstrated that psoralidin caspases and enhancement of Fas but not Bcl-2 or Bax proteins inhibits cell migration, invasion and expression of epithelial- [153, 154]. mesenchymal transition (EMT) markers slug, vimentin and β– catenin in these cells [140]. COUMESTANS IN CANCER PREVENTION There are increased ROS levels in cancer cells compared to Majority of studies analyzing chemopreventive effects of normal cells resulting from their accelerated metabolism. The high coumestans were focused on coumestrol. Since it is present in ROS levels can have protumorigenic effects but at the same time legumes and soya, ingestion of these nutrients increase its they also render cancer cells more susceptible to the oxidative concentrations in serum and urine. The reported average serum stress–induced cell death. Exogenous compounds that increase concentration of coumestrol in human populations from Asia is -8 oxidative stress by disturbing balance between ROS production and approximately 10 M [155]. As stated above, coumestrol acts as scavenging may therefore cause cell damage in cancer cells more phytoestrogen within this concentration range. The effect of efficiently [141]. Interestingly, about half of the cancer phytoestrogens intake on cancer risk is still matter of intense debate chemotherapy drugs approved by the FDA until 2007 have been and was recently reviewed excellently [156, 157]. Despite the reported to cause oxidative stress [142]. numerous studies published in the last decade, there is no clear consensus regarding the cancer preventive effects of phytoestrogens Psoralidin senzitizes cancer cells to apoptosis induced by the although some meta-analyses reported protective effects with safe tumor necrosis factor α (TNF-α) ligand superfamily. Slizska et al. side-effects profiles [158-161]. We still lack an evidence that and Bronikowska et al. reported enhancement of the TRAIL- cancer incidence is affected by intake of phytoestrogens or whether induced apoptosis in prostate and cervical cancer cells [137, 143]. associations observed in previous studies just point to the role of Srinivasan et al. described similar effect of psoralidin in the TNF- phytoestrogens as markers of healthy diet [156, 157]. induced apoptosis of prostate cancer cells [136]. The mechanism of cell senzitization to the TNF ligand superfamily by psoralidin Besides its role as a phytoestrogen, coumestrol as well as other possibly include deregulation of death receptors, FADD, Bid, Bax, coumestans may be chemopreventive also by acting as polyphenols. Fas, FasL, XIAP, cIAP1/2 and Bcl-2 expression and inhibition of Polyphenols affect numerous cellular targets and signaling pathways NFκB activation/expression [136, 137]. that are related to tumorigenesis including cell proliferation, cell death, inflammation, dissemination, angiogenesis, or chemo- and Psoralidin also inhibits the LPS-induced overexpression of radioresistance [162]. These compounds can act both as inducible nitric oxide synthase (iNOS) and production of nitric antioxidants or prooxidants. Many mechanisms have been proposed oxide by macrophages by interfering with activation of the for prevention of oxidative stress and ROS generation by PI3K/Akt/NFκB signaling pathway [144, 145]. Inhibitory effect of polyphenols but radical scavenging and metal chelation are the wedelolactone and coumestrol on iNOS production in stimulated most widely published mechanisms for their antioxidant activity. immune cells was also observed [17, 146]. Whether the effect of The prooxidant activity is largely attributed to the ability of coumestans on nitric oxide production may have any relevance for polyphenolic compounds to bind and reduce Fe3+ or Cu2+ with cancer progression/treatment is unclear. NOS are produced in subsequent production of semiquinone/quinone radicals and ROS various types of cancers and nitric oxide can enhance cancer [163, 164]. Based on observation of increased concentration of progression by its promutagenic, antiapoptotic, proinvasive and cellular copper in tumors, the production of ROS by copper-initiated proangiogenic effects. Nevertheless, cytostatic and cytotoxic action redox cycling of polyphenols was suggested to be enhanced in on cancer cells has been also reported. This dual role of nitric oxide cancer cells. This may result in preferential toxicity of polyphenolic in cancer progression has been recently summarized by Choudhari compounds to cancer cells resulting in their chemotherapeutic as et al. [147]. Psoralidin was also described to inhibit the prosurvival well as chemopreventive properties [163]. PI3K/Akt/NFκB signaling pathway in prostate cancer cells [148], however, this article was subsequently retracted. The PI3K/Akt/ The radical scavenging activity of coumestans is relatively high NFκB signaling is a key regulator of cell cycle progression, compared to many other phytocompounds [9, 36]. Coumestrol cell survival, chemosensitivity, radiosensitivity and metastasis. prevents cell damage, reduces ROS production, lipid peroxidation, Numerous drugs of low molecular weight that inhibit pathway mutagenicity but increases glutathione level, SOD activities and are being developed and tested in preclinical and clinical studies survival of cells challenged with oxidative stress [165-167]. [149, 150]. Moreover, coumestans can induce expression of quinone reductase, a phase II detoxifying enzyme blocking generation of mutagenic Besides its effect on the PI3K/Akt/NFκB pathway, psoralidin quinone metabolites and carcinogens [168, 169]. also inhibits the EGFR/MAPK/ERK signaling and activates SAPK/JNK in prostate cancer cells and mouse tumor xenografts. Nevertheless, many reported effects claimed for polyphenols The functional significance of SAPK/JNK in psoralidin-induced are controversial, since correlations between their effects in vitro apoptosis was confirmed by using the JNK-specific inhibitor and in vivo are poorly established. Orally administered polyphenols SP600125 and c-Jun-specific siRNA [139]. undergo rapid and extensive conjugation in the intestinal tract. Once absorbed, polyphenols are further metabolized by the liver Psoralidin also inhibits protein tyrosine phosphatase 1B [151]. and modified by methylation, glucuronidation, and sulfation. As the role of this protein in carcinogenesis is still matter of debate Although some exceptions have been reported, most published data [152], it is not clear whether this function of psoralidin could indicate that natural polyphenols are biologically more active than contribute to its anticancer effects. their metabolites. Poor bioavailability, excessive modification and GLYCYROL limited distribution in the organism therefore complicate the use of polyphenols in clinical studies. Pharmaceutical formulations of Only few studies describing possible anticancer and cytotoxic polyphenols aiming to improve their absorption, pharmacokinetics, affects of glycyrol have been published so far. Glycyrol (Fig. 2) is a efficacy and delivery are under testing [162]. In conclusion, coumestan isolated from Glycyrrhiza uralensis with antibacterial, although there are plenty of pre-clinical and clinical studies antiinflammatory and immunosuppressive activities [12-14]. The reporting the positive role of polyphenols in cancer prevention and latter two were attributed to ability to inhibit activities of treatment, more long-term, randomized and controlled dietary 6 Anti-Cancer Agents in Medicinal Chemistry, 2014, Vol. 14, No. 0 Nehybová et al.

Table 1. Coumestrol intake and cancer risk in epidemiological studies.

Retrospective Studies Cancer Country Cases/Controls RR (95% CI) Ref Notes

Breast Mexico 141/141 ns [171] Germany 278/666 ns [172] premenopausal USA 1,326/1,657 ns [173] non-Asian origin Ovarian USA 205/390 ns [174] Testicular USA 159/136 ns [175] U-shape pattern for coumestrol (P=0.02) Thyroid USA 608/558 ns [176] Endometrial USA 424/398 ns [177] USA 500/470 ns [178] non-Asian origin Prostate USA 83/107 0.48 (0.25-0.94) [179] Sweden 1,314/782 0.57 (0.38-0.84) [182] only for carriers of ER CT/CC genotypes Gastric Mexico 257/478 0.45 (0.29-0.70) [180] Glioma USA 802/846 0.59 (0.43-0.80) [181]

Prospective Studies Cancer Country Cases/Total RR (95% CI) Ref Note Breast UK 244/25,639 ns [195] France 402/26,868 ns [183] premenopausal Sweden 1,014/49,261 0.56 (0.29-0.90) [185] only for hormone receptor negative cancer Colorectal UK 221/25,639 ns [195] Ovarian Sweden 163/47,140 0.29 (0.09-0.94) [184] only for low-malignant cancer Prostate UK 204/25,639 ns [195]

intervention studies are necessary to fully evaluate the benefit of etc. This synergism between nutrition compounds complicates polyphenol intake [170]. identification of specific factor(s) that contributes to observed Contradictory data about coumestrol and cancer risk were associations [156]. Moreover, various phytochemicals share published (data summarized in Table 1). No association between common mechanisms in regulation of cellular processes. Therefore, coumestrol intake and breast [171-173], ovarian [174], testicular combination of natural compounds may solve the problems with [175], thyroid [176] and endometrial [177, 178] cancer in bioavailability, distribution and toxicity of individual compounds. retrospective case-control studies has been reported. On the other Lowering the dose of each compound and multitarget therapy are hand, other authors reported an inverse association between additional benefits of combined treatment. Understanding the coumestrol intake and the risk of prostate, gastric and glioma synergistic mechanisms of phytocompounds may be of great cancers [179-181]. Hedelin et al. described an inverse association significance for their use in modern medicine opening broad between coumestrol intake and prostate cancer but only for carriers perspectives for “networking pharmacology“ [186]. Synergic of C allele of the -13950 ERβ promoter polymorphism in the effects between coumestans and other phytochemicals with respect retrospective case-control study [182]. In prospective studies, no to cancer treatment were documented [118, 187]. association between coumestrol intake and breast cancer was SUMMARY reported by Touillaud et al. [183]. Similarly, no association between coumestrol intake and ovarian cancer was reported in prospective, Coumestans are large family of phytocompounds with population-based study in Sweden. However, in this study a numerous biological effects. While initial studies were mostly marginally significant difference in the coumestrol intake between interested in coumestrol due to its high content in food and the patients with invasive and low-malignant ovarian cancer was observed effects on reproduction of livestock, studies performed reported [184]. Another large retrospective study of the same group in the last decade have characterized biological properties of found no association between coumestrol intake and the risk of other members of the coumestan family. These studies clearly overall breast cancer. Interestingly, an inverse association between demonstrated anticancer properties of these compounds in vitro coumestrol intake and the incidence of ER-, PR- negative breast (summarized in Table 2) but only limited number of studies on in tumors was reported in this study [185]. Inconsistency of these vivo models was published. Chemopreventive and anticancer studies may reflect differences in the study design, sample size, therapy enhancing effects of coumestans were also described. Many ethnic homogeneity, hormonal status, length of folow-up, etc. of their biological effects can be attributed to their action as Direct comparison between nutrition studies is also limited by phytoestrogens and polyphenols. However, majority of anti-cancer differences in databases of natural compounds as these often differ effects of coumestans were demonstrated in vitro using pure natural in calculation and analytical methods, references, phytoestrogen compounds and only limited data are available about absorption, content in food items, etc. [185]. metabolism and bioavailability of coumestans in human body. Therefore, data showing anticancer effects of coumestans should be Furthermore, it seems that studying only one isolated interpreted with caution. More information about their in vivo phytochemical may be inappropriate because to reduce cancer risk bioavailability, stability, metabolism, toxicity, estrogenicity, cellular the phytochemicals may act synergistically with other dietary targets and drug interactions is needed to proceed further to clinical components, such as other plant compounds, vitamins, minerals, studies. Coumestans in Cancer Prevention and Treatment Anti-Cancer Agents in Medicinal Chemistry, 2014, Vol. 14, No. 0 7

Table 2. Cancer-related biological activities and cellular targets of coumestans.

Activity Target Psoralidin Coumestrol Wedelolactone

↑? [30, 135] ↑ [192] ↑ [19, 56, 57] - [131]

Steroid signaling Androgen receptor ND ↓ [79] ↓ [128] Metabolic enzymes ND ↓ [52, 53, 54] ND PXR ND ↓ [49] ND ↓ [139] ↑ [65, 68, 69] ↑ [117, 131] ERK - [144] ↓ [38] ↓ [119] Akt ↓ [144, 190] ↓ [91] - [5, 124] ↑ [139] ↑ [69, 70] ↑ [117, 124] SAPK/JNK - [144] - [131] Cellular signaling Ca2+ influx ND ↑ [67, 68] ND FAK ND ND ↓ [119] Cassein kinase II ND ↓ [91, 92] ND K-ras ND ↓ [66] ND ROS scavenging ↑ [9] ↑ [18, 36, 166] ↑ [189] ROS production ↑ [140] ↑ [92] ↑ [133] Redox activity SODs - [140] ↑ [166, 167] ND Quinone reductase ↑ [169] ↑ [168] ND DNA damage ND ↑ [102, 103] ↑ [132, 133] DNA damage Topoisomerase IIα ND ↓? [103, 105] ↓ [132, 133] ↓ [35] NFκB ↓ [144, 148] ↓ [5, 119, 132] - [74] STAT1 ND ↓ [17] ↑ [125] Immunomodulation Cytokines ND ↓ [17, 33, 35] ↓ [193, 194] iNOS ↓ [144, 145] ↓ [17] ↓ [146] COX-2 ↓ [190] ND ↓ [191] α-glucosidase ↓ [188] ↓[107] ND Bcl-2 (Bcl-2/Bax) ↓ [136] ↓[76] ↓ [117] Others 5-lipoxygenase ↓ [190] ND ↓ [124, 129] MMPs ND ND ↓ [119]

↓ inhibition of expression/ activity; ↑ stimulation of expression/ activity; ND not determined; ? suggested effect; - effect not observed.

CONFLICT OF INTEREST [4] Wagner, H.; Geyer, B.; Kiso, Y.; Hikino, H.; Rao, G.S. Coumestans as the Main Active Principles of the Liver Drugs Eclipta alba and The author confirms that this article content has no conflict of Wedelia calendulacea. Planta Med., 1986, 52, 370-374. interest. [5] Kobori, M.; Yang, Z.; Gong, D.; Heissmeyer, V.; Zhu, H.; Jung, Y.K.; Gakidis, M.A.; Rao, A.; Sekine, T.; Ikegami, F.; Yuan, ACKNOWLEDGEMENTS C.; Yuan, J. Wedelolactone suppresses LPS-induced caspase-11 This work was funded by grants NT 13441-4/2012 (IGA, expression by directly inhibiting the IKK complex. Cell Death Differ., 2004, 11, 123-130. Ministry of Health of the Czech Republic), by European Regional [6] Zhao, L.H.; Huang, C.Y.; Shan, Z.; Xiang, B.G.; Mei, L.H. Development Fund - projects FNUSA-ICRC (CZ.1.05/1.1.00/ Fingerprint analysis of Psoralea corylifolia by HLPC and LC-MS. 02.0123), PASSEB (CZ.1.07/2.4.00/31.0155), CEB (CZ.1.07/ J. Chromatogr. B., 2005, 821, 67-74. 2.3.00/20.0183) and RECAMO (CZ.1.05/2.1.00/03.0101) via the [7] Latha, P.G.; Evans, D.A.; Panikkar, K.; Jayavardhanan, K.K. human resources project "IntegRECAMO: Intelectual Anchor" Immunomodulatory and antitumor properties of Psoralea (CZ.1.07/2.3.00/20.0097). corylifolia seeds. Fitoterapia, 2000, 71, 223-231. [8] Wang, Y.; Hong, C.; Zhou, C.; Xu, D.; Qu, H.; Cheng, Y. REFERENCES Screening antitumor compounds psoralen and isopsoralen from Psoralea corylifolia seeds. Evid. Based Complement. Alternat. [1] Newman, D.J.; Cragg, G.M. Natural products as sources of new Med., 2009, 8, 1-7. drugs over the 30 years from 1981 to 2010. J. Nat. Prod., 2012, 75, [9] Xiao, G.; Li, G.; Chen, L.; Zhang, Z.; Yin, J.J.; Wu, T.; Cheng, Z.; 311-335. Wei, X.; Wang, Z. Isolation of antioxidants from Psoralea corylifolia [2] Cragg, G.M.; Newman, D.J. Natural products: a continuing source of fruits using high-speed counter-current chromatography guided by novel drug leads. Biochim. Biophys. Acta, 2013, 1830, 3670-3695. thin layer chromatography-antioxidant autographic assay. J. [3] Mors, W.B.; do Nascimento, M.C.; Parente, J.P.; da Silva, Chromatogr. A, 2010, 1217, 5470-5476. M.H.; Melo, P.A.; Suarez-Kurtz, G. Neutralization of lethal and [10] Xu, Q.; Pan, Y.; Yi, L.T.; Li, Y.C.; Mo, S.F.; Jiang, F.X.; Qiao, myotoxic activities of South American rattlesnake venom by C.F.; Xu, H.X.; Lu, X.B.; Kong, L.D.; Kung, H.F. Antidepressant- extracts and constituents of the plant Eclipta prostrata (Asteraceae). like effects of psoralen isolated from the seeds of Psoralea Toxicon, 1989, 27, 1003-1009. 8 Anti-Cancer Agents in Medicinal Chemistry, 2014, Vol. 14, No. 0 Nehybová et al.

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Received: May 12, 2014 Revised: June 19, 2014 Accepted: July 14, 2014