Dietary phytochemicals for possible preventive and therapeutic option of uterine fibroids: Signaling pathways as target

Accepted Manuscript Title: Dietary phytochemicals for possible preventive and therapeutic option of uterine fibroids: signaling pathways as target Authors: Md. Soriful Islam James H. Segars Mario Castellucci MD, PhD Pasquapina Ciarmela Ph.D PII: DOI: Reference:

S1734-1140(16)30297-3 http://dx.doi.org/doi:10.1016/j.pharep.2016.10.013 PHAREP 585

To appear in: Received date: Revised date: Accepted date:

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Please cite this article as: Md.Soriful Islam, James H.Segars, Mario Castellucci, Pasquapina Ciarmela, Dietary phytochemicals for possible preventive and therapeutic option of uterine fibroids: signaling pathways as target, http://dx.doi.org/10.1016/j.pharep.2016.10.013 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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FULL TITLE: Dietary phytochemicals for possible preventive and therapeutic option of uterine fibroids: signaling pathways as target

SHORT TITLE: Dietary phytochemicals for uterine fibroids

Md Soriful Islam1, 2, James H. Segars3, Mario Castellucci1*, Pasquapina Ciarmela1, 4*

1

Department of Experimental and Clinical Medicine, Faculty of Medicine, Università Politecnica delle

Marche, Ancona 60020, Italy 2

Biotechnology and Microbiology Laboratory, Department of Botany, University of Rajshahi, Rajshahi

6205, Bangladesh; 3

Howard W. and Georgeanna Seegar Jones Division of Reproductive Sciences, Department of

Gynecology and Obstetrics, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA 4

Department of Information Engineering, Università Politecnica delle Marche, Ancona 60131, Italy

*Corresponding author’s: 1) Pasquapina Ciarmela, Ph.D Department of Experimental and Clinical Medicine, Faculty of Medicine, Università Politecnica delle Marche, via Tronto 10/a, 60020 Ancona, Italy Phone: +390712206270, Fax: +390712206087, E-mail: [email protected]

2) Mario Castellucci, MD, PhD Department of Experimental and Clinical Medicine, Faculty of Medicine, Università Politecnica delle Marche, via Tronto 10/a, 60020 Ancona, Italy Phone: +390712206086, Fax: +390712206087, E-mail: [email protected]

2 Abstract A growing interest has emerged on dietary phytochemicals to control diverse pathological conditions. Unfortunately, dietary phytochemical research in uterine fibroids is still under construction. Uterine fibroids/leiomyomas are benign tumors developing from the myometrium of the uterus in premenopausal women. They may occur in more than 70 % of women, and approximately 25 % of women show clinically significant symptoms. These include heavy and prolonged menstrual bleeding, pelvic pressure (urinary frequency, incontinence, and difficulty with urination), pelvic pain, pelvic mass, infertility, and reproductive dysfunction. Due to lack of medical treatments surgery has been definitive choice for fibroid management. Moreover, surgery negatively affects women’s quality of life, and its associated cost appears to be expensive. The molecular mechanism of fibroids development and growth is not fully elucidated. However, accumulated evidence shows that several signaling pathways, including Smad 2/3, PI3K/AKT/mTOR, ERK 1/2 and β-catenin are involved in the leiomyoma pathogenesis, indicating that they could serve as targets for prevention and/or treatment of this tumor. Therefore, in this review, we discuss the involvement of signaling pathways in leiomyoma development and growth, and introduce some potential dietary phytochemicals that could modulate those signaling pathways.

Keywords: Dietary phytochemicals, uterine fibroids, signaling pathways, growth factors, medical treatment

3 Introduction Uterine fibroids/leiomyomas are benign tumors developing from the myometrium of the uterus in premenopausal women [1, 2]. They may occur in more than 70 % of women [3], and among these, about 20-50% of women are reported to produce clinically significant symptoms [4]. Leiomyoma associated symptoms include: heavy and prolonged menstrual bleeding, pelvic pressure (urinary frequency, incontinence, and difficulty with urination), pelvic pain, pelvic mass, infertility, and reproductive dysfunction [5]. Surgery has been a definitive treatment of uterine fibroids. Moreover, surgery negatively affects women’s quality of life, and its associated cost appears to be expensive. For example, in United States, surgery associated cost of uterine fibroids management is approximately $5.9-34.4 billion annually [6]. Unfortunately, the current medical treatments are limited and no effective prevention strategies exist [7]. Moreover, the benefits of medical treatments are tempered by lack of efficacy or serious adverse side effects. Poor understanding of the precise molecular mechanism of uterine fibroids development and growth is may be the reason for the limitation of medical treatments. However, in recent years, significant advances to uncover the molecular mechanisms of uterine fibroid development and growth have been achieved [1, 8-11]. It is thought that uterine fibroids are monoclonal tumors, and approximately 40-50 % contain karyotypic or cytogenetic abnormalities [12]. Notably, mediator complex subunit 12 (MED12) gene mutation has been noted in 70 % of fibroids [13]. Cell signaling is the transfer of information, by which cells perceive and respond to extracellular stimuli including growth factors, neurotransmitters, and hormones. The signaling cascade starts with binding of extracellular stimuli to a cell surface receptor. The receptor then activates series of downstream signaling molecules in the cytoplasm that import signal to the nucleus for subsequent transcription of target genes. The activation of several signaling pathways, such as Smad 2/3, phosphoinositide 3-kinase (PI3K), extracellular-signal-regulated kinase 1/2 (ERK1/2), and β-catenin have been reported in leiomyoma cells [8-11]. They regulate central events (such as inflammatory response, fibrosis,

4 proliferation and angiogenesis) of leiomyoma development and growth. Therefore, signaling pathways could serve as excellent target for prevention and treatment of uterine fibroids. Dietary phytochemicals are plant based chemical compounds with disease-preventive properties, found in cereals, fruits, vegetables, legumes, herbs, spices, nuts, and beverages (such as tea, wine and beer). They are known to exert therapeutic effects on multiple pathological conditions through modulating diverse signaling pathways [14-16]. Accumulating evidences indicate that high intake of green vegetables and fruit seem to have a protective role and associated with reduced risk of uterine fibroids of US and Italian populations [17, 18]. This result supports the possible use of dietary phytochemicals for the prevention and/treatment of uterine fibroids. However, phytochemical based research in uterine fibroids is still under construction. For example, EGCG (epigallocatechin gallate), curcumin, resveratrol, isoliquiritigenin and genistein are only few dietary phytochemicals that have been partially studied in uterine leiomyoma [19-26]. They are mostly known for antiproliferative effects, however, their effects on signaling pathways have not been addressed in leiomyoma cells except curcumin [21] and genistein [27]. Hence, there is much room for future research in the area of phytochemical based studies focusing on signaling pathways as therapeutic target. Therefore, in this review, we discussed the role of signaling pathways in leiomyoma development and growth, and introduced 14 dietary phytochemicals (Fig. 1) that could modulate those signaling pathways.

Health benefits of dietary phytochemicals The father of medicine, Hippocrates, proclaimed that “Let food be thy medicine and medicine be thy food” almost 25 centuries ago. However, the relationship between diet and health is yet to explore. The human diet contains wide variety of plant-based foods that provide essential nutrients for the body. Besides, plant-based foods possess huge variety of non-nutrient components that offer beneficiary effects on health. Accumulated epidemiologic studies indicate an inverse association between diet rich in fruits and vegetables and the risk of cancers (such as colon, breast, and ovary) [28-30] and other diseases. The major

5 groups of health promoting dietary phytochemicals are phenolics [phenolic acid-gallic acid, stilbeneresveratrol, flavonoids (flavonol-quercetin, flavones-apigenin, flavanol-catechin, flavanone-naringenin, anthocyanidin-delphinidin, and isoflavonoid-genistein), lignin-matairecinol, coumarin-warfarin, and tannins)],

alkaloids-caffeine,

carotenoid-lycopene,

organosulfur

compound-sulforaphane,

and

phytosterols-sitosterol and stigmasterol [31]. For example, strawberry, a major source of anthocyanins (i.e. pelargonidin-3-glucoside), flavonols (quercetin and kaempferol), ellagitannin (ellagic acid), flavanols (catechins and procyanidins), and phenolic acid (caffeic acid), is known to exert therapeutic effects in the prevention of diabetes, obesity, cancers, cardiovascular diseases, and neurodegenerative diseases [32]. It is thought that the health benefits of fruits and vegetables are the result of additive and synergistic interactions of the phytochemicals present in whole foods [33]. Chemoprevention is characterized by the use of natural or synthetic agents to block, reverse or restrict tumorigenic steps:

initiation, promotion, and progression [16]. In recent years, dietary

phytochemicals are being considered as a cost effective, acceptable and accessible approach for cancer prevention and treatment. Therefore, dietary phytochemicals have been extensively investigated for their multiple therapeutic effects as well as their safety, and low toxicity [34].

Signaling pathways in uterine leiomyoma Accumulated evidence suggests that several signaling pathways, including Smad 2/3 (Fig.2A), PI3K (Fig.2B), ERK 1/2 (Fig.3A), and β-catenin (Fig.3B) activated by growth factors, other peptide or proteins, and hormones have been found in leiomyoma cells [8-11]. They regulate inflammatory response, fibrosis, proliferation and angiogenesis that are important phenomenon for leiomyoma pathogenesis. Since dietary phytochemicals may exert multiple therapeutic effects against diverse pathological conditions, they could regulate leiomyoma development and growth through targeting signaling pathways.

Smad 2/3 signaling

6 Smads are group of intracellular proteins that deliver extracellular signal to the nucleus induced by transforming growth factor (TGF)-β superfamily members. Smads are classified into three different groups, including receptor-regulated Smads (R-Smad), common-mediator Smad (Co-Smad), and inhibitory Smads (I-Smad). Smad1, Smad2, Smad3, Smad5 and Smad8 are known as R-Smad. R-Smads interact with Co-Smad (Smad4) to promote downstream signaling [35]. In contrast, I-Smads (Smad6 and Smad7) act as blocker of activation of R-Smads and Co-Smads [36]. In myometrial and leiomyoma cells, activin-A and TGF-β1 has been reported to induce Smad 2/3 signaling [9, 37]. They initiate signaling by binding to a type II receptor (ActRIIA or ActRIIB for activinA, and TGF-βRII for TGF-β), which recruits and phosphorylates a type I receptor [activin receptor-like kinase (ALK)-4/ActRIB for activin-A, and ALK5/TGF-βRI for TGF-β). Next, activated type-I receptor phosphorylates Smad2 and Smad3 that interact with Smad4 in the cytoplasm. This smad complex then travels to the nucleus where interacts with other transcription factors and regulates transcription of target genes [38, 39]. Uterine leiomyoma demonstrated an elevated level of Smad3, Smad4 and phosphorylated Smad3 (p-Smad3) as well as TGF-βRI and TGF-βRII expression compared to normal counterpart [40]. TGF-β1 was reported to increase p-Smad3 induction in both myometrial and leiomyoma smooth muscle cells [37]. A number of studies reported that TGF-β1 can modulate inflammatory response, fibrosis, cell growth, and apoptosis in myometrial and leiomyoma cells [41-45], which may be acquired , at least in part, through activation of Smad 2/3 signaling [37]. The involvement of Smad 2/3 signaling in leiomyoma growth was further documented by the observation that TGF-βRI kinase inhibitor, SB-525334 can block TGF-β signaling in uterine leiomyoma cells, and significantly decreased tumor (leiomyoma) size, incidence and multiplicity in Eker rat model [46]. Recently, we found that activin-A can induce phosphorylation of Smad 2/3 in both primary myometrial and leiomyoma cells [9]. The ability of activin-A to increase mRNA expression of fibronectin, collagen1A1, versican and vascular endothelial growth factor (VEGF)A in primary myometrial and/or leiomyoma cells [9, 47], demonstrating its profibrotic and angiogenic role in this cell types. Ulipristal acetate is one of the most promising therapeutic options for leiomyoma

7 was found to decrease activin-A, follistatin, ActRIIB, and ALK4 mRNA expression in leiomyoma cultured cells [47]. We also found that ulipristal acetate can block activin-A-induced mRNA expression of fibronectin and VEGF-A in myometrial and leiomyoma cultured cells [47].

PI3K signaling PI3Ks are a large family of intracellular signal transducers. Activation of PI3K signaling can occur through G protein-coupled receptors and tyrosine kinase receptors [48]. Induction of PI3K signaling by prolactin-releasing peptide (PrRP) and epidermal growth factor (EGF) has been reported in uterine leiomyoma cells [8, 49]. Upon ligand binding, receptor complex becomes activated, and phosphorylates PI3K at the cell membrane [48]. Phosphorylated PI3K converts PIP2 (phosphatidylinositol-4,5diphosphate) to PIP3 (phosphatidylinositol-3,4,5-triphosphate) [48, 50], which subsequently mediates the phosphorylation of protein kinase B (PKB/AKT) through phosphoinositide dependent protein kinase 1 (PDK1) [51]. Next, phosphorylated AKT activates/inactivates series of downstream proteins to facilitate translation of target proteins. mTOR (mammalian target of rapamycin) is a best studied downstream substrate of AKT. There are two complexes of mTOR: TORC1-Raptor complex and TORC2-Rictor complex. AKT activates mTOR through phosphorylating and inactivating TSC 1/2, which inhibits mTOR through GTP-binding protein, Rheb (Ras homolog enriched in brain). Thus, Rheb accumulates and activates the mTOR-raptor kinase complex. The activated TORC1-Raptor complex mediates phosphorylation of downstream substrates, eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and p70S6Kinase, which subsequently promote synthesis of target proteins [52]. Accumulated evidences suggest a central role of PI3K/AKT/mTOR pathway in the pathogenesis of uterine leiomyomas [8, 53-55]. Using in vivo and in vitro studies, Crabtree and co-workers confirmed the upregulation of mTOR signaling pathway in both human and rat leiomyomas/tumors [53]. They found that rapamycin analogue WAY-129327 decreased tumor size, incidence and multiplicity in Eker rats, and inhibited mTOR signaling [53]. MK-2206, an AKT Inhibitor, was reported to reduce mTOR and p70S6K phosphorylation and promote leiomyoma cell death [56]. Varghese’s group showed that PrRP promoted

8 PI3K/AKT/mTOR pathway through activation of GPR10, and increased primary leiomyoma cell proliferation [8]. EGF has been reported to increase cell proliferation, and induce phosphorylation and activation of EGFR and AKT in leiomyomal smooth muscle cells [49]. Involvement of PI3K/AKT pathway in leiomyoma growth was further evidenced by the observation that inhibition of AKT using API-59 (AKT inhibitor) decreased leiomyoma cell proliferation and cell viability, and promoted apoptosis [57]. PTEN (phosphatase and tensin homolog) is a negative regulator of PI3K pathway. Uterine leiomyoma and myometrium demonstrated an unchanged expression level of PTEN [55].

ERK 1/2 signaling ERK 1/2 is a cytoplasmic protein [58] that mediates signaling by EGF, platelet-derived growth factor (PDGF), insulin-like growth factor (IGF)-I and TGF-β in myometrial and/or leiomyoma cells [10, 44, 59]. The signaling is initiated by binding of ligands to their corresponding receptors. Upon lingand binding, receptor complex (homo- or heterodimers) become auto-and transphosphorylates and activates that leads to the association of the receptor to cytoplasmic target proteins. Grb2 (growth factor receptorbound protein 2 adapter protein), a docking protein, that binds to the activated receptors either directly or through Shc (src homology and collagen domain protein) [60]. Shc and Grb2 then recruit SOS (son of sevenless), a guanine nucleotide exchange factor, which in turn activates Ras by exchanging GDP for GTP. Ras is a small GTP-binding protein that recruits Raf and activates it. Activated Raf then phosphorylates and activates MEK 1/2, which in turn phosphorylates and activates ERK 1/2 [61]. Activated ERK 1/2 moves to the nucleus where it regulates transcription of target genes [62, 63]. EGF has been reported to stimulate proliferation of leiomyomal smooth muscle cells through activation of the EGFR-ERK1/2 pathway [49]. Stimulation of primary leiomyoma cells with EGF markedly increased intracellular reactive oxygen species (ROS) production that mediates mitogenactivated protein kinase (MAPK)3/MAPK1 (ERK 1/2) activation leading to cell proliferation [10]. The role of EGF in leiomyoma growth was also supported by the observation that AG1478 and TKS050 (selective EGFR blockers) can block leiomyoma cell proliferation by inducing cell cycle arrest and

9 apoptosis [64, 65]. PDGF has been reported to activate ERK 1/2 signaling through intracellular ROS production, and increase primary leiomyoma cell proliferation [10]. PDGF also increased collagen α1 (I) mRNA expression in both myometrial and leiomyoma cells [66]. IGF-I was reported to increase uterine leiomyoma cell proliferation by upregulating proliferating cell nuclear antigen (PCNA) expression and downregulating apoptosis by upregulation of B-cell lymphoma-2 (Bcl-2) protein expression [59, 67]. The stimulatory effect of IGF-I on leiomyoma growth was mediated, at least in part, by increasing phosphorylation of IGF-IRβ, Shc and MAPKp44/42 (ERK1/2) [59]. TGF-β1 has been shown to regulate inflammatory response, fibrosis, cell growth, and apoptosis in myometrial and leiomyoma cells [41-45], at least in part, by ERK 1/2 signaling activation [44].

β-catenin signaling β-catenin is the central component of wingless-type (WNT) signaling cascade. In the absence of WNT ligands, β-catenin is degraded by a multiprotein ‘destruction complex’ in the cytoplasm.

This

‘destruction complex’ contains APC (adenomatous polyposis coli) and AXIN, which facilitate the phosphorylation of β-catenin by CK1 (casein kinase 1) and GSK3 (glycogen synthase kinase 3) [68]. In the presence of WNT ligands, WNT binds to the Frizzled receptors and several co-receptors such as LRP5/6 (lipoprotein receptor-related protein-5/6), RYK (receptor-like tyrosine kinase) or ROR2 (receptor tyrosine kinase-like orphan receptor 2) [69] resulting in the inhibition of ‘destruction complex’ that stabilize β-catenin in the cytoplasm. Next, stabilized β-catenin moves to the nucleus and interacts with LEF (lymphoid enhancer factor)/TCF (T-cell factor) transcription factors to regulate transcription of target genes [70]. A considerable amount of evidence has suggested that WNT/β-catenin signaling is involved in the pathogenesis of uterine fibroids [11, 71-73]. Tanwar and co-investigators developed a mouse model that expresses constitutively activated β-catenin in uterine mesenchyme which gives rise to mesenchymal tumors/leiomyoma-like tumors in the uterus [71]. The constitutive activation of β-catenin induced the expression of TGF-β3 [71], which was shown to induce proliferation and extracellular matrix (ECM)

10 formation in human uterine leiomyoma cells [74]. The tumor suppressor gene, MED12, is mutated in ~ 70% of uterine leiomyomas [13], which directly binds to β-catenin and regulates WNT signaling [75]. The expression of WNT4 was found to be elevated in leiomyoma with MED12 mutations versus those without mutations [73]. An elegant experiment by Ono and co-workers uncovered a central role of the WNT/β-catenin pathway induced by estrogen and progesterone in leiomyoma growth, which was confirmed by the observation that blocking of WNT activity through inhibitor of β-Catenin and TCF4 inhibits the growth of leiomyoma-like tumors in immunodeficient mice [11]. They found that WNT11 and WNT16 were increased at mRNA levels in myometrial cells, which were remained constitutively increased (not significant) in leiomyoma cells in response to estrogen and progesterone treatment [11]. In addition, frizzled receptors, FZD1 and FZD7 were found to be significantly higher at mRNA levels in leiomyoma side-population cells than in total leiomyoma cells or leiomyoma mature population cells [11]. This group also found that LRP5 and LRP6 (co-receptors for WNT) were expressed in leiomyoma sidepopulation cells and leiomyoma mature population cells. Furthermore, estrogen and progesterone were found to selectively induce nuclear translocation of β-catenin and transcriptional activity of TCF and AXIN2 in leiomyoma side-population cells cocultured with mature myometrial cells [11].

Dietary phytochemicals that might target signaling pathways in uterine leiomyoma Here, we discuss 14 dietary phytochemicals that might target signaling pathways, including Smad 2/3 (Fig.2A), PI3K (Fig.2B), ERK 1/2 (Fig.3A), and β-catenin (Fig.3B) involved in cell proliferation, angiogenesis, inflammation and fibrosis that are linked to uterine leiomyoma development and growth. These dietary phytochemicals were chosen based on their ability to modulate signaling pathways in different pathological cell types.

Betulinic acid Betulinic acid is a pentacyclic triterpene found in leaves of rosemary, and fruits of elephant apple. Recently, Jin and co-workers reported that betulinic acid can inhibit 3-isobutyl-1-methylxanthine induced

11 melanogenesis via the downregulation of p-MEK, p-ERK and p-AKT in B16F10 cells [76]. Betulinic acid may exert antiangiogenic activity in cultured endometrial adenocarcinoma cells through decreasing expression of hypoxia-inducible factor (HIF)-1α, and VEGF [77]. In addition, betulinic acid was reported to suppress lipopolysaccharide (LPS)-induced interleukin (IL)-6 production through preventing nuclear translocation of nuclear factor-κB (NF-κB) in peripheral blood mononuclear cells [78]. The antifibrotic effect of betulinic acid was documented by the observation that betulinic acid can attenuate liver hepatic stellate cell (HSC) activation by downregulating ethanol-induced p-Smad3 expression [79].

Butein Butein is a type of chalcone derivative found in cashews. Khan and co-workers reported that butein can inhibit prostate tumor growth in vivo through inhibition of PI3K/AKT pathway [80]. Butein also suppressed breast cancer cell growth through downregulation of AKT phosphorylation [81]. Furthermore, butein was reported to inhibit cell proliferation and clonogenecity of bladder cancer cells, through reducing phosphorylation of ERK 1/2 [82]. Butein has been reported to inhibit VEGF-induced angiogenesis by downregulating the phosphorylation of AKT, mTOR, and the major downstream effectors, p70S6K, 4E-BP1, and eIF4E in endothelial progenitor cells [83]. Matrix metallopeptidase (MMP)-9 and VEGF are prominently involved in the processes of tumor cell invasion and metastasis. It was shown that butein repressed tumor necrosis factor (TNF)-α and phorbol-12-myristate-13-acetate induced expression of VEGF and MMP-9 via the suppression of NF-κB activity in human prostate cancer cells [84]. The anti-inflammatory effect of butein has been reported by several investigators [85-87]. Wang and co-workers reported that butein suppressed adipocyte inflammation in 3T3-L1 cells by downregulation of TNF-α+LPS+interferon (IFN)-γ-induced secretion and/or expression of inflammatory mediators, such as IL-6, monocyte chemoattractant protein-1 (MCP-1), inducible nitric oxide synthase (iNOS), nitric oxide (NO), NOS2 (nitric oxide synthase 2), CXCL (C-X-C motif chemokines)-1, and CXCL-10) through partly suppression of NF-κB activation and MAPKs [ERK 1/2, c-Jun N-terminal

12 protein kinase (JNK), and p38 MAPK] signaling pathways [87]. Butein also decreased TNF-α-induced monocyte cell adhesion to lung epithelial cells through inhibiting ROS generation and NF-κB activation as well as the phosphorylation of MAPKs and AKT [85]. Furthermore, butein was reported to ameliorate colitis in IL-10(-/-) mice, at least in part, by downregulation of IL-6, IL-1β, IFN-γ and MMP-9 as well as IL-6-induced activation of signal transducer and activator of transcription (STAT)3 [86]. The antifibrotic effect of butein has been documented by the observation that butein can inhibit ethanol-induced activation of liver stellate cells through inhibition of TGF-β, p38 MAPK, and JNK signaling pathways [88].

Capsaicin Capsaicin, active component of chili peppers, is known to have tumor suppressive effects. Recently, Park and co-workers reported that capsaicin can potentially inhibit the proliferation of human gastric cancer cells and induce apoptosis, through decreasing the expression of p-ERK 1/2 [89]. Capsaicin was also reported to exert anticancer effect on human colorectal cancer cells through modulating β-catenin signaling pathway [90]. This compound promoted proteosomal- degradation of β-catenin, and suppressed TCF-4 expression and disrupted the interaction between TCF-4 and β-catenin [90]. Capsaicin can induce antiangiogenic activity both in vitro and in vivo [91]. Treatment of human multiple myeloma cells with capsaicin inhibited IL-6-induced STAT3 activation, and STAT3-regulated gene products, such as cyclin D1, Bcl-2, Bcl-xL, survivin, and VEGF [92], suggesting its regulatory function in proliferation, survival and angiogenesis, and apoptosis. In addition, capsaicin was reported to increase degradation of HIF-1α, which is a key transcription factor in increasing VEGF transcription in non-small cell lung carcinoma [93] Bitencourt and co-investigators reported that capsaicin induced down-regulation of HSC activation by decreasing cyclooxygenase (COX)-2, TGF-β1 and collagen expression [94], suggesting its antifibrotic and antiinflammatory actions. Accordingly, capsaicin has been reported to suppress the

13 production of TNF-α [95], as well as prostaglandin E2 (PGE2) and NO production in macrophages via NF-κB inactivation [96].

Delphinidin Delphinidin is a polyphenolic compound found in many pigmented fruits, including cranberries, Concord grapes, and pomegranates. A recent study reported that delphinidin suppressed proliferation and migration of human ovarian clear cell carcinoma cells through blocking phosphorylation of downstream targets of PI3K (AKT and p70S6K) and MAPKs (ERK1/2 and JNK) [97]. Pal’s group demonstrated that delphinidin can inhibit growth of non-small-cell lung cancer cells through inhibition of EGFR, VEGFR2 as well as PI3K/AKT and MAPKs pathways [98]. Delphinidin also inhibited in vivo tumor growth induced by B16-F10 melanoma cell xenograft in mice, through suppression of VEGFR2-mediated endothelial cell proliferation as well as VEGFR2 signaling pathways, MAPKs (ERK1/2 and p38 MAPK) and PI3K [99]. Angiogenesis is an energetic process and VEGF-induced angiogenesis is associated with mitochondrial biogenesis. Delphinidin was reported to inhibit VEGF induced-mitochondrial biogenesis and AKT activation in endothelial cells [100]. Lamy and co-workers reported that delphinidin can inhibit smooth muscle cell migration as well as the differentiation and stabilization of endothelial cells, at least in part, by inhibiting activation of PDGFR, and PDGF-BB-induced activation of ERK-1/2 signaling pathway [101]. In athymic nude mice implanted with human prostate cancer PC3 cells, delphinidin treatment induced a significant inhibition of tumor growth, through downregulation of NF-κB expression [93]. Delphinidin also inhibited UVB-induced MMP-1 expression as well as ROS production and NOX activity in primary cultured human dermal fibroblasts partly through suppression of MAPKs phosphorylation [102]. Delphinidin has been reported to inhibit TGF-β1-induced expression of α-smooth muscle actin (α-SMA), fibronectin, and collagen as well as activation of MAPKs and NF-κB in nasal polyp-derived

14 fibroblasts [103], suggesting its role in the regulation of myofibroblast differentiation and ECM production through the MAPK/NF-κB signaling pathway.

3,3′-Diindolylmethane 3,3′-Diindolylmethane (DIM) is a polymeric compound produced after digestion of indole-3-carbinol in a low pH environment. Several cruciferous vegetables, such as Brussels sprouts, cauliflower, cabbage, broccoli, kale and turnips are known to be major sources of DIM. It has been shown that DIM can induce antiproliferative and proapoptotic effects in oral squamous cell carcinoma cells and breast cancer cells, through inactivation of NF-κB, AKT and MAPKs pathways [104, 105]. DIM also induced antiproliferative and pro-apoptotic effects in human cervical cancer cells through downregulating the expression of p-AKT, PI3K, GSK-3β, p-PDK1 as well as p-c-Raf, p-ERK1/2 and p-p38 MAPK [106]. A recent genome-wide transcriptome analysis showed that DIM can inhibit proliferation of colon cancer cells through inactivation of Wnt/β-catenin signaling pathway [107]. DIM also suppressed the growth of ovarian tumors in vitro and in vivo, at least in part, through reduction of EGFR, MEK, and ERK phosphorylation [108]. Chang and co-workers found that DIM can inhibit VEGF-induced cell proliferation in human umbilical vascular endothelial cells (HUVECs), at least in part, via downregulation of ERK1/2 and AKT [109, 110]. DIM also inhibited invasion and angiogenesis in PDGF-D-overexpressing PC3 cells through inactivation of mTOR and AKT [111]. The anti-inflammatory effect of DIM was documented by the observation that DIM can inhibit LPS-induced microglial hyperactivation and brain inflammation through attenuating DNA-binding activity of NF-κB and phosphorylation of inhibitor of κB [112]. Zhang and co-investigators reported that DIM can attenuate hepatic fibrosis by inhibiting miR-21 expression, and TGF-β induced α-SMA and COL1A1 as well as p-Smad2/3 and total Smad2/3 expression [113]. A recent study reported that DIM can attenuate TGF-β1-induced myofibroblastic transformation of

15 cardiac fibroblast through suppression of α-SMA, collagen I, collagen III, and connective tissue growth factor (CTGF) expression via downregulation of AKT/GSK-3β signaling pathways [114].

Emodin Emodin is an anthraquinone compound found in the root and rhizomes of Rhubarb. It has been shown that emodin can inhibit tumor growth in orthotopic hepatocellular carcinoma mice model, at least in part, by blocking of STAT3, JAK1/2 and AKT phosphorylation [115]. Emodin also downregulated the expression of STAT3 regulated gene products, such as cyclin D1, Bcl-2, Bcl-xL, Mcl-1, survivin and VEGF in HepG2 cells [115]. In human colorectal cancer cells, emodin downregulated the expression of β-catenin and TCF7L2, as well as several downstream proteins, including cyclin D1, c-Myc, snail, vimentin, MMP2 and MMP-9 [116]. Emodin was also reported to repress TWIST1 (Twist-related protein 1)-induced epithelial-mesenchymal transition (EMT) in head and neck squamous cell carcinoma FaDu cells through inactivation of β-catenin and AKT signaling pathways [117]. A recent study reported that emodin can inhibit the TGF-β-induced migration and invasion of human cervical cancer cells, partly through decreasing the expression of TGF-βRII, p-Smad3, Smad4, and β-catenin [118]. Kaneshiro and co-investigators reported that emodin can inhibit endothelial cell proliferation, migration, and tube formation through blocking ERK 1/2 phosphorylation [119], suggesting its antiangiogenic properties. Emodin has been reported to inhibit homocysteine-induced C-reactive protein generation in vascular smooth muscle cells, through downregulating ROS generation, and phosphorylation of ERK1/2 and p38 MAPK [120], suggesting its antiinflammatory and antiatherosclerotic effects. Yin and coinvestigators reported that emodin can protect against LPS/D-galactosamine-induced liver injury in mice through attenuating TNF-α production as well as p38 MAPK and NF-κB activation [121]. In addition, emodin was reported to ameliorate LPS-induced mastitis in mice through reducing secretion and expression of TNF-α, IL-1β and IL-6 via inactivating NF-κB and MAPKs pathways [122].

16 Emodin can exhibit antifibrotic effect on pancreatic fibrosis, at least in part, by reducing collagen and TGF-β1 expression (Wang et al., 2007a). Lee and co-workers reported that emodin suppressed TNFα-induced MMP-1 expression in human dermal fibroblast cells through inhibition of the activator protein 1 (AP-1) and MAPKs (ERK 1/2 and JNK) pathways [123]. Emodin was also reported to suppress glucose/IL-1β induced mesangial cell proliferation and ECM (fibronectin and/or collagen) production by blocking p38 MAPK pathway [124, 125].

Ferulic acid Ferulic acid is a ubiquitous polyphenolic compound in plant kingdom, found especially in artichokes, eggplants and maize bran. Ambothi and co-investigators reported that ferulic acid can inhibit UVBradiation induced photocarcinogenesis through downregulation of VEGF, iNOS, mutant p53, Bcl-2 expressions and upregulation of the Bax expression [126], suggesting its antiinflammatory, antiangiogenic and apoptosis inducing capability. A recent report indicated that ferulic acid can inhibit fibroblast growth factor (FGF)1-induced endothelial cell proliferation, migration and tube formation as well as microvessel sprouting of rat aortic rings and angiogenesis [127]. The antiangiogenic effect of ferulic acid was mediated by inactivation of FGFR1 and PI3K/PKB signaling [127]. Hou’s group reported that ferulic acid suppressed proliferation of ECV304 endothelial cells and blocked the cell cycle in G0/G1 phase, and inhibited phosphorylation of ERK 1/2 [128]. Recently, Xu and co-workers reported that ferulic acid can exhibit antifibrotic effects on hepatic stellate cells through inhibiting ERK 1/2 and Smad 2/3 signaling pathways [129]. Particularly, this compound reduced TGF-βRII, TGF-βRI and Smad4 mRNA and protein expression as well as Smad transcriptional activity, and blocked ERK 1/2 phosphorylation in HSC-T6 cells [129]. Ferulic acid also attenuated TGF-β1-induced renal cellular fibrosis in NRK-52E cells via inactivation of Smad 2/3 signaling pathway [130]. Furthermore, ferulic acid was reported to attenuate ischemia/reperfusioninduced hepatocyte apoptosis via inhibition of JNK activation [131].

17

Fisetin Fisetin is a flavonoid commonly present in apples, kiwis, strawberries, grapes, persimmons, onions and cucumbers. Khan and co-workers reported that fisetin can inhibit growth of human non-small cell lung cancer cells via suppression of PI3K/AKT/mTOR signaling [132]. In PC3 prostate cancer cells, fisetin induced autophagic cell death through inhibition of mTOR signaling pathway [133].}. Particularly, fisetin inhibited phosphorylation of mTOR kinase and downregulated the expression of Raptor, Rictor, PRAS40 and GβL as well as activated the mTOR repressor, TSC2 in this cell type [133]. Furthermore, fisetin was reported to inhibit human laryngeal carcinoma cells of TU212 cell proliferation and induce apoptosis via downregulating Raf, Ras, p-ERK1/2, PI3K, p-AKT, NF-κB and mTOR expression [134]. Fisetin has been reported to exert antiangiogenic effect by inhibiting VEGF-induced growth and survival of HUVEC as well as capillary-like tube formation on Matrigel [135]. It also inhibited the expression of eNOS (endothelial nitric oxide synthase), VEGF, iNOS, MMP-2 and MMP-9 in A549 and DU145 human cancer cells [135]. Fisetin exerts antiinflammatory effects in human mast cells by suppressing phorbol-12-myristate 13-acetate plus calcium ionophore A23187-stimulated gene expression and production of TNF-α, IL-1β, IL-4, IL-6, and IL-8 via downregulation of MAPKs and NF-κB pathways [136]. Fisetin also reduced secretion of IL-6 and TNF-α via inactivation of JNK and NF-κB in LPS-stimulated macrophage cells [137]. Furthermore, fisetin was reported to inhibit IL-1β-induced cytokines (TNF-α, IL-6) and chemokines (IL-8, MCP-1) in rheumatoid arthritic fibroblast-like synovial cells and in vivo models [138].

Kaempferol Kaempferol is a flavonoid present in green tea, broccoli, apples, strawberries and green beans. A recent study reported that kaempferol can suppress estrogen and triclosan stimulated breast cancer cell growth in cellular and xenograft breast cancer models via downregulation of p-AKT and p-MEK1/2 expression [139]. Kaempferol also inhibited proliferation of esophageal squamous cell carcinoma cells and induced

18 G0/G1 cell cycle arrest, and suppressed tumor growth in KYSE150 xenograft model via suppression of EGFR and its downstream signaling pathways, ERK 1/2 and AKT [140]. Furthermore, kaempferol was reported to suppress bladder cancer tumor growth by inhibiting cell proliferation and inducing apoptosis via downregulation of the p38 MAPK phosphorylation and c-Fos expression [141]. Kim and co-workers reported that kaempferol inhibited PDGF-BB-induced rat aortic vascular smooth muscle cell proliferation, at least in part, by downregulation of PDGFR-β phosphorylation and its downstream signal transduction pathways, ERK1/2, AKT and PLC-γ1 phosphorylation and c-fos expression [142]. The ability of kaempferol to downregulate IGF-I-induced phosphorylation of the IGF-IR and ERK 1/2 pathways in HT29 human colon cancer cells was reported by Lee and co-investigators [143]. In chorioallantoic membranes of chicken embryos, kaempferol inhibited OVCAR-3-induced angiogenesis and tumor growth, at least in part, by reducing VEGF and HIF-1α expression via downregulation of AKT phosphorylation [144]. Kaempferol also suppressed hepatocarcinoma cell survival through downregulation of HIF-1 activity and p44/42 MAPK activation [145]. Furthermore, kaempferol was reported to inhibit VEGF secretion, and in vitro angiogenesis, via downregulation of ERK phosphorylation as well as NF-κB and cMyc expression [146]. Kaempferol exhibited a protective effect on LPS-induced acute lung injury in mice via suppression of TNF-α, IL-1β and IL-6 production and MAPKs and NF-κB phosphorylation [147]. Kaempferol also attenuated myocardial ischemic injury via downregulation of TNF-α and IL-6 production as well as inhibition of MAPKs phosphorylation and NF-κB expression [148]. Yoon and coinvestigators reported that kaempferol can inhibit IL-1β-induced proliferation of rheumatoid arthritis synovial fibroblasts and the expression of COX-2, PGE2 as well as MMP-1 and MMP-3, partly through downregulation of NF-κB activation as well as MAPKs phosphorylation [149]. The antifibrotic effect of kaempferol was documented by the observation that kaempferol can suppress collagen deposition, epithelial excrescency and goblet hyperplasia in the lung of ovalbuminchallenged mice, at least in part, by inhibition of TGF-β-induced EMT by reversing E-cadherin expression and retarding the induction of N-cadherin and α-SMA [150].

19

Morin Morin is a member of flavonols found in almonds that suppressed growth and invasion of the metastatic breast cancer cell line MDA-MB 231 as well as cancer cell progression in xenograft mouse model, partly through suppression of AKT pathway [151]. Morin has been reported to attenuate ovalbumin-induced airway inflammation in mice by suppression of goblet cell hyperplasia and collagen deposition and ovalbumin-induced TNF-α, IL-4, IL13, and MMP-9 via inactivating MAPKs pathway [152]. In human bronchial epithelial cells, morin also inhibited TNF-α induced ROS generation and expression of eotaxin-1, MCP-1, and IL-8 [152]. Furthermore, morin was reported to suppress monosodium urate crystal-induced inflammation in RAW 264.7 macrophages through inhibition of expression and/or secretion of inflammatory mediators (TNF-α, IL-1β, IL-6, MCP-1, NO, PEG2, iNOS and COX-2), VEGF expression, ROS generation, and NF-κB activation [153]. Morin has been reported to ameliorate in vivo diethylnitrosamine-induced liver fibrosis in male albino Wistar rat as well as inhibit proliferation of LX-2 cells (culture-activated human HSCs), partly through inhibition of β-catenin and GSK-3β expression [154].

Naringin Naringin is a flavanone glycoside found in grapefruit and related citrus species. Li and co-workers reported that naringin can inhibit proliferation of triple-negative (ER-/PR-/HER2-) breast cancer cells (MDA-MB-231, MDA-MB-468 and BT-549), through induction of apoptosis and G1 cycle arrest [155]. They also noticed that inactivation of β-catenin signaling pathway was responsible for this anticancer effect [155]. Naringin also induced autophagy-mediated growth inhibition in AGS cancer cells by downregulating the phosphorylation of PI3K and its activated downstream targets p-AKT and p-mTOR via activation of MAPKs pathways [156].

20 Recently, Zhang and co-workers reported that naringin can prevent intestinal tumorigenesis in Apc (Min/+) mouse model through suppression of cell proliferation, induction of apoptosis and inhibition of expression and/or secretion of inflammatory mediators (Cox-2, NF-κB, TNF-α, PGE2 and IL-6) as well as β-catenin expression and GSK-3β phosphorylation [157]. Naringin also inhibited growth of HeLa cervical cancer cells and induces apoptosis, at least in part, by suppression of NF κB phosphorylation and COX 2 expression [158]. Furthermore, naringin was reported to inhibit TNF-α-induced invasion and migration of vascular smooth muscle cells as well as expression and/or secretion of MMP-9, IL-6 and IL8, at least in part, through blocking of PI3K/AKT/mTOR/p70S6K pathway via suppression of transcriptional activity of AP-1 and NF κB [159]. The protective effects of naringin against paraquat-induced acute lung injury and pulmonary fibrosis in mice have been reported, which was mediated primarily through downregulation of expression of TGF-β1 and TNF-α as well as modulation of expression and ratios of MMP-9 and TIMP-1 [160]. Naringin also inhibited renal interstitial fibrosis and collagen formation partly through inhibiting oxidative stress and NF-κB activation [161].

Pterostilbene Pterostilbene is a stilbene, biologically classified as a phytoalexin, chemically related to resveratrol. Several types of grapes and blueberries are major sources of pterostilbene. Pterostilbene has been reported to inhibit 7,12-dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate-induced mouse skin tumor formation possibly via inactivation of NF-κB and AP-1, and their upstream signaling pathways, MAPKs, PI3K and AKT [162]. Paul’s group reported that pterostilbene can inhibit the growth of cultured colon cancer HT-29 cells and induce apoptosis through downregulating TNF-α+IFN-γ+LPS-induced iNOS and COX-2 expression via suppression of p38 MAPK signaling pathway [163]. Later study reported that pterostilbene can reduce colon tumor multiplicity of non-invasive adenocarcinomas in rats by inhibiting TNF-α, IL-1β and IL-4 expression via suppression of β-catenin and NF-κB signaling pathways [164]. Chiou and co-workers reported that pterostilbene inhibited azoxymethane-induced

21 colorectal aberrant crypt foci and adenomas through upregulation of apoptosis and downregulation of iNOS, COX-2, VEGF, cyclin D1, MMP-7, MMP-26, MMP-2, and MMP-9 expression and/or activity, at least in part, via suppression of multiple signaling molecules, including p-GSK-3β, β-catenin, p-PI3K, pAKT, EGF, and EGFR [165]. Pterostilbene has been reported to suppress 12-O-tetradecanoylphorbol 13-acetate-induced invasion, migration and metastasis of HepG2 cells by downregulation of angiogenic factors, such as VEGF, EGF and EGFR expression, and their downstream signal transduction pathways, MAPKs, PI3K/AKT and PKC as well as NF-κB and AP-1 [166]. Pterostilbene has been reported to inhibit high fat-induced atherosclerosis inflammation in mice through inhibition of TNF-α, TGF-β1, IL-18, IL-6, IFN-γ, MCP-1 and IL-17 expression and/or secretion via inactivation of NF-κB [167]. Pterostilbene also reduced the expression of TNF-α, IL-1 β, IL-6, COX2, MMP-2 and MMP-9, and ROS overproduction in hyperosmotic medium exposed human corneal epithelial cells [168], suggesting its ability to protect corneal epithelial cells through antiinflammatory and antioxidative effects. It has been shown that pterostilbene inhibited dimethylnitrosamine-induced liver fibrosis in rats [169]. The antifibrotic effect of pterostilbene in the dimethylnitrosamine-treated rats was mediated by downregulation of α-SMA and MMP-2 expression as well as TGF-β1, and its signaling molecules, pSmad2 and p-Smad3 expression [169].

Silibinin Silibinin (also known as silybin) is a flavonolignan compound found artichokes. This compound can induce antiproliferative, antiinflammatory, proapoptotic and antiangiogenic effects in colorectal cancer as evidenced by decreased cell proliferation [170, 171], and TNF-α-induced NF-κB activation, and thereby NF-κB-regulated molecules, including Bcl-2, COX-2, iNOS, VEGF, HIF-1α and MMP9 [170-172]. This effect was mediated, at least in part, by downregulation of β-catenin, IGF-1Rβ, p-GSK-3β, p-ERK 1/2 and p-AKT [170, 171]. Kim’s group reported that silibinin can suppress EGF-induced phosphorylation of

22 EGFR and ERK 1/2 in SKBR3 and BT474 breast cancer cells [173]. In nude mice model, silibinin also attenuated the growth of melanoma xenograft tumors through inhibiting phosphorylation of MEK 1/2 and ERK 1/2 [174]. Antiangiogenic effect of silibinin was documented by the observation that silibinin can inhibit VEGF secretion and HIF-1α subunit accumulation in retinal pigmented epithelia cells through downregulation of p-PI3K, p-AKT, p-mTOR and p70S6K [175]. In the rat model of age-related macular degeneration, silibinin also prevented VEGF- and VEGF plus hypoxia-induced retinal oedema and neovascularization [175]. Furthermore, silibinin was reported to inhibit human cervical and hepatoma cancer cell growth by inhibiting HIF-1α protein synthesis and hypoxia-induced VEGF secretion via suppression of the p-mTOR and its effectors, p70S6K and 4E-BP1 [176]. Silibinin has been reported to suppress LPS-induced neutrophilic airway inflammation, at least in part, by downregulation of ERK phosphorylation [177]. Silibinin also inhibited ovalbumin-induced airway inflammation, and reduced the production of various cytokines (TNF-α, IL-1β, IL-4, IL-5, and IL13), partly via downregulation of NF-κB activity [178]. Furthermore, silibinin showed inhibitory effect on skin inflammation induced by 12-O-tetradecanoylphorbol-13-acetate by down-regulating IL-1β, IL-6, TNF-α and COX-2 expression via inhibiting the PI3K/AKT signaling pathway, and NF-κB activation [179]. Silibinin has been reported to attenuate cardiac hypertrophy and fibrosis by downregulating EGFR-dependent ERK1/2 and PI3K/AKT, as well as activation of NF-κB and Smad 2/3 signaling pathways [180]. Chen and colleagues reported that silibinin can inhibit myofibroblast transdifferentiation in human tenon fibroblasts and reduce fibrosis in a rabbit trabeculectomy model [181]. Particularly, silibinin inhibited TGF-β1-induced expression of α-SMA, vimentin, collagen contraction, CTGF, collagen type I in human tenon fibroblasts through downregulation of p-Smad3 [181]. A study by Cho and co-workers indicated that silibinin has the potential to prevent fibrotic skin changes by inducing the downregulation of type I collagen expression in human skin fibroblasts, partly by the inhibition of TGFβ1-induced p-Smad2 and p-Smad3 expression [182]. Silibinin exerts antiinflammatory and antifibrogenic

23 effects on human hepatic stellate cells by downregulation of human HSC cell proliferation, cell migration, and de novo synthesis of collagen type I as well as IL-1β-induced synthesis of MCP-1 and IL-8 via directly inhibiting ERK, MEK, and Raf phosphorylation [183].

Thymoquinone Thymoquinone is a bioactive component of black seed oil. It has been shown that thymoquinone can induce cell cycle arrest and apoptosis in breast cancer cells through inhibition of PI3K/AKT pathway [184, 185]. Particularly, thymoquinone reduced the phosphorylation of PTEN (inactivated form of PTEN), PDK1 and AKT that resulted in the inhibition of 4E-BP1 and p70S6K [184]. In human cholangiocarcinoma cells, thymoquinone induced inhibition of cell growth by inducing cell cycle arrest and apoptosis through downregulation of PI3K/AKT and NF-κB pathways, and their regulated gene products, including Bcl-2, COX-2, and VEGF [186]. Furthermore, thymoquinone was reported to inhibit proliferation and invasion of human nonsmall-cell lung cancer cells via downregulation of ERK pathway as confirmed by the reduced expression level of PCNA, cyclin D1, MMP-2 and MMP-9, and

p-

ERK1/2[187]. Thymoquinone has been reported to prevent tumor angiogenesis in a mouse xenograft human prostate cancer (PC3) model, and inhibited human prostate tumor growth through suppressing AKT and ERK signaling pathways [188]. Thymoquinone also exhibited antiangiogenic effects on osteosarcoma in vitro and in vivo through inactivating the NF-κB pathway as evidenced by downregulation of NF-κB DNA-binding activity, survivin and VEGF in SaOS-2 cells [189]. A recent study by Su and co-investigators reported that thymoquinone can inhibit inflammation and neoangiogenesis induced by Ovalbumin in asthma mice by reducing IL-4 and IL-5 production, as well as VEGF, p-VEGFR2, p-PI3K and p-AKT expression and tube information in HUVECs [190]. Thymoquinone also inhibited IL-1β-induced inflammation in human osteoarthritis chondrocytes as evidenced by decreased production of COX-2, iNOS, NO, and PGE2 as well as MMP-1, MMP-3, and MMP-13 expression via inhibiting NF-κB activation and IκBα degradation as well as MAPKs pathway

24 activation [191]. Furthermore, thymoquinone was reported to inhibit TNF-α-induced IL-6 and IL-8 production in rheumatoid arthritis synovial fibroblasts by inhibiting JNK and p38 MAPK signaling pathways [192]. Thymoquinone has been reported to attenuate liver fibrosis by reducing α-SMA, collagen1A1, collagen3A1, TIMP-1, L-1α, IL-1β and IL-18 levels, at least in part, via inactivating PI3K and TLR4 signaling pathways [193-195]. Thymoquinone can also block lung injury and fibrosis in rats induced by bleomycin/paraquat herbicide through downregulation of TGF-β1, α-SMA, collagen 1A1 and collagen 4A1, and inhibition of oxidative stress and NF-κB activation [196, 197].

Conclusions and future perspectives The precise molecular mechanisms of leiomyoma pathogenesis are not well understood. However, accumulating evidences suggest that several signaling pathways, such as Smad 2/3, PI3K, ERK1/2 and βcatenin are involved in regulating central events (such as inflammatory response, fibrosis, proliferation and angiogenesis) of leiomyoma development and growth. Thus, they may act as excellent target for possible prevention and treatment of uterine fibroids. Here, we introduced 14 dietary phytochemicals (betulinic acid, butein, capsaicin, delphinidin, 3,3'-diindolylmethane, emodin, ferulic acid, fisetin, kaempferol, morin, naringin, pterostilbene, silibinin and thymoquinone) that could potentially be used as therapeutic and/or preventive compounds for uterine leiomyoma (Fig. 1). These dietary phytochemicals have shown their ability to regulate major tumor initiating and promoting events such as, inflammation, fibrosis, proliferation and angiogenesis in different experimental conditions through regulating several signaling pathways, including Smad 2/3, PI3K, ERK 1/2 and β-catenin. Future research may include these dietary phytochemicals to check their ability to modulate signaling pathways in uterine fibroids. Dietary phytochemicals are not limited to these 14 compounds. Other potential dietary phytochemicals that have not yet been tested could be included for future research in uterine fibroids.

25 Funding body This work was supported by a grant from the “Fondazione Cassa di Risparmio di Fabriano e Cupramontana” (to MC and PC).

Conflict of interest None

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46 Figure legends Fig. 1. Dietary phytochemicals and their chemical structure and dietary sources.

Fig. 2A-B. Smad 2/3 (A) and PI3K (B) signaling pathways in uterine leiomyoma targeted by dietary phytochemicals.

Fig. 3A-B. ERK 2/3 (A) and β-catenin (B) signaling pathways in uterine leiomyoma targeted by dietary phytochemicals.

47 Figure 1

48 Figure 2

49 Figure 3