Iranian Journal of Basic Medical Sciences ijbms.mums.ac.ir

Molecular targets of pomegranate (Punica granatum) in preventing cancer metastasis Naghmeh Ahmadiankia 1*

1 Cancer Prevention Research Center, Shahroud University of Medical Sciences, Shahroud, Iran

A R T I C L E I N F O A B S T R A C T Article type: Metastasis is the primary cause of mortality and morbidity among cancer patients and accounts Review article for about 90% of cancer deaths. The most common types of treatment for cancer metastasis are chemotherapy and radiotherapy. However, such therapy has many serious side effects that could Article history: Received: Sep 4, 2018 Accepted: May 14, 2019 diminishevidence showsthe quality that pomegranateof life in patients. (Punica There granatum is increased) has great appreciation potential byto inhibitthe scientific tumor growthcommunity and Keywords: that natural compounds can be potential weapons in fighting against cancer. Interestingly, much Anoikis are prerequisite for cancer metastasis. The search was performed in Google Scholar, Medline, Scopus, Cell adhesion metastasis.and PubMed In using this keywordsreview, we such discussed as metastasis, the molecular pomegranate, targets and of pomegranate,signaling pathways. specifically, Some of those the most that Chemotaxis important papers from the search results were included. Based on recent studies, some molecules, including Cytoskeleton dynamics those involved in cell-cell and cell-extracellular matrix adhesions, are affected by pomegranate. The Extracellular matrix other targets of pomegranate are modulators of cytoskeleton dynamics and regulators of cancer cell Metastasis anoikis and chemotaxis. Furthermore, the antimetastatic effect of pomegranate may be attributed to Phytochemicals Pomegranate the other targets of pomegranate regarding cancer metastasis. A wide variety of molecules can be moleculartargeted by changes pomegranate of the extracellularto suppress tumormatrix. metastasis. Pro-inflammatory A better and understanding pro-angiogenic of themolecules molecules are regulated by pomegranate is needed to provide a rational basis for its clinical application.

►Please cite this article as: Ahmadiankia N. Molecular targets of pomegranate (Punica granatum) in preventing cancer metastasis. Iran J Basic Med Sci 2019; 22:977-988. doi: 10.22038/ijbms.2019.34653.8217

Introduction adverse side effects (4). Therefore, a safe, non-toxic, Cancer is a major public health problem and one of the leading causes of death worldwide (1). It is developed. Previous studies showed that consuming estimated that metastasis is responsible for about 90% andnatural highly products effective is regimeninversely to associated fight cancer with should cancer be of cancer deaths (2). Cancer metastasis is a multistep incidence and mortality (5). Among them, pomegranate process by which tumor cells spread from the primary (Punica granatum) has been shown to inhibit the tumor site to distant organs through blood and progression of cancer in different preclinical and clinical lymphatic vessels. studies (6-8). It is an antioxidant-rich fruit containing is detachment from the primary tumor. Epithelial and different important bioactive compounds and alters endothelial cells willThe undergo first step apoptosis in cell when dissemination detached many of the cellular and molecular processes involved from the extracellular matrix (ECM), a phenomenon referred to as anoikis; however, cancer cells develop discussed the phytochemical content of pomegranate anoikis resistance that is a critical step in metastasis. inand cancer its chemotherapeutic progression (9). properties. In this review Then article, we explained we first Anoikis resistance cells leave the primary tumor site, studies on the molecular targets of pomegranate and its bioactive constituents regarding cancer metastasis. from the circulation in the distant organ where they form Understanding the mechanism of pomegranate action intravasate,a secondary circulate new tumor in vessels, (3). During and finallymultiple extravasate steps of metastasis different molecules including those involved rational basis for its clinical application. in cell–cell and cell–ECM adhesions, the modulators of and identification of its molecular targets provide a cytoskeleton dynamics, the ones that organize ECM, and Research method the signaling molecules involved in anoikis, chemotaxis, The current search was done in Google Scholar, Medline, Scopus, and PubMed through search words Nowadays, chemotherapy as a conventional treatment is such as “anoikis, cell adhesion, chemotaxis, cytoskeleton angiogenesis,available for patients and inflammatory with cancer responses metastasis. are However,involved. dynamics, extracellular matrix, phytochemicals, the current chemotherapy agents fail to selectively metastasis, and pomegranate.” The search was carried out and effectively kill cancer cells, and they usually have to include literature published as late as 1 September 2018.

*Corresponding author: Naghmeh Ahmadiankia. Cancer Prevention Research Center, Shahroud University of Medical Sciences, 7 tir square, Shahroud, Iran. Tel/Fax: +98-2332395054; +98-2332394800; Email: [email protected] Ahmadiankia Pomegranate in preventing cancer metastasis

Phytochemical content of pomegranate Pharmacokinetics of pomegranate Pomegranate is one of the oldest known fruit tree The most abundant polyphenols in pomegranate juice species, native to central Asian regions spreading are that are hydrolyzable tannins releasing from Iran to northern India, the Mediterranean area, on hydrolysis (13). The absorption of and the Middle East. It is valued for its nutritional, ellagitannins is rather low in humans (14). Interestingly, medicinal, ornamental, and industrial properties (10). gut microbiota metabolizes ellagitannins and ellagic The pomegranate plant is divided into several parts, acid leading to the formation of A, , and isourolithin A (15). The metabolites of urolithin A roots, and bark. There is a wide range of phytochemicals and urolithin B are conjugated in the liver, then excreted includingin its different fruit parts,(peel, which juice, areand summarized seeds), leaves, in Table flowers, 1. in urine (16). Due to difference in gut microbiotica,

Table 1.

Different classes of phytochemicals identified in various parts of the pomegranate tree and fruit (11, 12) Classes Phytochemicals (tissue source) Ellagitannins, Brevifolin (Leaf), Brevifolin carboxylic acid (Leaf, Flower, Heartwood), Brevifolin carboxylic acid 10-monopotassium Gallotannins, and sulphate (Leaf), (Stem bark), Casuariin (Stem bark), (Peel, Stem bark), (Peel, Leaf), derivatives Isocorilagin (Flower), Hippomanin A (Flower), Gemin D (Flower),

(Heartwood), 1,2-Di-O-galloyl-4,6-O-(S)- -DiellagicD-glucopyranoside acid rhamnosyl(1→4) (Flower), Ellagic glucopyranoside acid (Peel, Flower, Leaf), 3,3’-Di-O-methylellagic acidhexahydroxydiphenoyl (Seed), 3,3’,4’-Tri-O - βmethylellagic acid (Seed), 3-O-Methylellagic acid (Heartwood), 4,4’-Di-O-methylellagic Acid (Heartwood), 3’-O-Methyl-3,4-methylenedioxy-ellagic acid (Heartwood), Eschweilenol C (Ellagic acid 4-O- -L-rhamnopyranoside) (Heartwood), Ethyl brevifolincarboxylate (Flower),

Eucalbanin B (Aril), Eucarpanin T1 (αAril), Pomegraniin A (Aril), Pomegraniin B (Aril), (Peel), Gallic acid 3-O- -D-(6’-O-galloyl)-glucopyranoside (Flower), 6-O-Galloyl-2,3-(S)-hexahydroxydiphenoyl-D-glucose (Stem bark,

Juice)β, 5-Galloylpunicacortein D (Heartwood), 2-O-Galloylpunicalin (2-O-Galloyl-4,6-(S,S)-gallagyl-D-glucose) (Heartwood, Stem Bark), (Fruit, Leaf), (Fruit, Leaf), 2,3-(S)-Hexahydroxydiphenoyl-D-glucose (Stem bark, Juice), Lagerstannin B (Peel), Lagerstannin C (Juice), 3-O-Methylellagic acid 4-O- -L-rhamnopyranoside

(Heartwood), 3,4’-O-Dimethylellagic acid 4-O- -L-rhamnopyranoside (Heartwood), αOenothein B (Aril), I (Peel, Stem bark), Pedunculagin II α(Juice), 1,2,3,4,6-Penta-O-galloyl- -D-glucose (Leaf), 3,4,8,9,10- Pentahydroxydibenzo (b,d) pyran-6-one (Urolithin M-5) (Leaf), Phyllanthusiin E (Flower)β , Pomegranatate (Flower), Punicacortein A (Stem bark), Punicacortein B (Stem bark), Punicacortein C (Stem bark, Peel), Punicacortein D (Stem bark), Punicafolin (Leaf), A (Peel, Stem bark, Aril, Juice, Root), Punicalagin B (Peel, Stem bark, Aril, Juice, Root), (Peel, Stem bark, Aril, Juice, Heartwood), Punicatannin A (Flower), Punicatannin B (Flower), (Stem bark), (1-O-Galloyl-4,6-(S)-hexahydroxydiphenoyl-D-glucose) (Leaf), (Peel), Tercatain (1,4-Di-O-galloyl-3,6-(R)-hexahydroxydiphenoyl- -glucopyranose) (Leaf), Terminalin (Gallagyl

dilactone) (Stem bark), 1,2,4,6-Tetra-O-galloyl- -D-glucose (Leaf), 1,2,3β -Tri-O-galloyl- -glucopyranose (Leaf), 1,2,4- Tri-O-galloyl- -glucopyranose (Leaf), 1,2,6-Triβ-O-galloyl- -glucopyranose (Leaf, Flower)β , 1,3,4-Tri-O-galloyl- - glucopyranoseβ (Leaf), 1,4,6-Tri-O-galloyl- -glucopyranoseβ (Leaf), 3,4,6-Tri-O-galloyl- -glucopyranose (Flower)β, dilactone (Juice, Peel) β β

Flavonoids and anthocyanins Hovetrichoside C (Flower), Phloretin (Juice), Phlorizin (Flower), Eriodictyol-7-O- -L-arabinofuranosyl (1-6)- -D-

glucoside (Stem bark), Granatumflavanyl xyloside (Flower), Naringin (Naringeninα -7-O-rhamnoglucoside) (Peel)β , Naringenin-4 methyl ether 7-O- -L-arabinofuranosyl(1-6)- -D-glucoside (Stem bark), Pinocembrin (Juice),

Punicaflavanol′ (Flower), Apigenin α(Peel), Apigenin 4 -O- -glucopyranosideβ (Leaf), Luteolin (Peel, Flower), Luteolin 3 -O- -glucopyranoside (Leaf), Luteolin 4 -O- -glucopyranoside′ β (Leaf), Cynaroside (Luteolin 7-O-glycoside) (Peel), Luteolin′ β 3 -O- -xylopyranoside (Leaf), Tricetin′ β (Leaf), Daidzein (Seed), Genistein (Seed), Amurensin (Noricaritin 7- -D-glucopyranoside′ β ) (Juice), Kaempferol (Peel), Astragalin (Kaempferol 3-O-glucoside) (Peel), Kaempferol-3-O- rhamnoglucosideβ (Juice), Myricetin (Peel), Phellatin (Juice), Quercetin (Juice, Leaf, Seed, Peel), Hirsutrin (Quercetin- 3-O-glucoside) (Peel), Quercimeritrin (Quercetin-7-O-glucoside) (Peel), Quercetin 3-O-rhamnoside (Peel), Rutin (Quercetin-3-O-rutinoside) (Juice), Quercetin-3,4 -dimethyl ether 7-O- -L-arabinofuranosyl(1-6)- -D-glucoside

(Stem bark), Cyanidin (Juice), Chrysanthemin ′(Cyanidin-3-O-glucoside)α (Juice), Cyanin (Cyanidinβ -3,5-di-O- glucoside) (Juice), Antirrhinin (Cyanidin-3-O-rutinoside) (Juice), Catechin-cyanidin-3-hexoside (Juice), Delphinidin (Juice), Myrtillin (Delphinidin-3-O-glucoside) (Juice), Delphinidin-3,5-di-O-glucoside (Juice), Pelargonidin (Juice), Callistephin (Pelargonidin-3-O-glucoside) (Juice), Pelargonin (Pelargonidin-3,5-di-O-glucoside) (Juice), Catechin (Peel, Juice, Leaf), Epicatechin (Peel, Juice, Leaf, Seed), Epicatechin gallate (Peel), Epigallocatechin-3-O-gallate (Fruit), Gallocatechin- -catechin (Peel), Gallocatechin- -gallocatechin (Peel), Catechin- -

gallocatechin (Peel), Procyanidin(4→8) A2 (Peel), Procyanidin B1 (Peel), (4→8)Procyanidin B2 (Peel), Procyanidin B3 (Peel)(4→8)

Lignans Conidendrin (Juice), Isohydroxymatairesinol (Peel), Isolariciresinol (Juice, Peel), Matairesinol (Wood knot), Medioresinol (Juice, Wood knot, Seed), Phylligenin (Peel), Pinoresinol (Juice), Secoisolariciresinol (Peel, Juice), Syringaresinol (Juice, Wood knot, Peel, Seed), Pomegralignan (Aril, Peel), Punicatannin C (Flower)

978 Iran J Basic Med Sci, Vol. 22, No. 9, Sep 2019 Pomegranate in preventing cancer metastasis Ahmadiankia

Continued Table 1

, β , β , β

(2’,5’ N (2’ (2’

∆ ∆

, α , γ

, α , β

b b

b bapiofuranosyl(1→6) apiofuranosyl(1→6)

different urolithin metabotypes (UMs) including UM- mmol/lit, and total urolithinB of 0.01 at 6 hr (24). They A, UM-B, and UM-0, are produced in some individuals also detected ellagic acid in human plasma at a maximum (17). These circulate in the blood and reach concentration (31.9 ng/ml) after 1 hr post ingestion, many target organs where the effects of pomegranate which was rapidly eliminated within 4 hr (25). ellagitannins are noted (18, 19). Previous studies showed In another study, 1-liter pomegranate juice was that the highest production of urolithin was carried out in distal parts of the intestine in humans and pigs (20, results showed that punicalagin and ellagic acid present givenin the orallyjuice were to healthy not detected volunteers either for in five plasma days. or The in concentrations, around 24 hr after ellagic acid intake (14, urine however three microbial -derived 21).22). UrolithinMertens-Talcott appears et in al plasma. investigated and urine the at absorption significant metabolites were detected in both urine and plasma of a standardized extract from pomegranate in healthy with great interindividual variability one day after human volunteers after the acute consumption of 800 pomegranate juice consumption (18). Interestingly, mg of extract. Results indicated that ellagic acid from under simulated gastrointestinal conditions, up to 80% of the extract is bioavailable, with an observed C(max) the overall ellagic acid content was slowly released from the metabolites of urolithin A, urolithin B, hydroxyl- the slightly alkaline pHs simulating the small intestine ofurolithin 33 ng/ml A, urolithin at t(max) A-glucuronide, of 1 hr. They and dimethyl also identified ellagic fermentedenvironment pomegranate (26). It was wastes revealed over 2 hrthat incubation ellagic acid at acid-glucuronide in plasma (23). Moreover, Seeram et al. had poor absorption and rapid elimination after oral demonstrated that consuming 180 ml of pomegranate administration of pomegranate leaf extract, and part juice concentrate was associated with maximum plasma of it was absorbed from the stomach (27). In another concentrations of ellagitannins of 0.06 mmol/lit after 1 hr study, pomegranate seed oil was given to the animals and the ellagitannins metabolites, total urolithin A of 0.14 intragastrically for 40 days at concentrations of 1%, 2%,

Iran J Basic Med Sci, Vol. 22, No. 9, Sep 2019 979 Ahmadiankia Pomegranate in preventing cancer metastasis and 4%. The results have demonstrated that punicic pomegranate may protect against bladder (42), breast acid from pomegranate seed oil was metabolized and (43), cervical (44), colon (45), leukemia (46), liver (47), incorporated in the form of conjugated linoleic acid in lung (48), ovarian (49), pancreatic (50), and skin (51) different rat tissues (28). Further research is warranted cancers, as well. to determine the pharmacokinetics of pomegranate. In the next part, we bring together some of available in vitro and in vivo evidence relevant to the possible Clinical value of pomegranate molecular targets of pomegranate with respect to cancer Various chemical compounds in different parts of the metastasis (Table 2 & Figure 1). pomegranate plant have crucial roles in the treatment of many diseases. For example, the rich phytochemical Molecular targets modulated by pomegranate contents in seeds, leaves, and fruit have been attributed regarding metastasis Cell adhesion molecules and their regulators as 30). Pomegranate extract has also shown protective targets of pomegranate foreffects their against benefits acute in therenal treatment failure of(31). diabetes Moreover, (29, Adhesion (attachment) and de-adhesion pomegranate seed has been reported to regulate urine (detachment) are prerequisites for cellular motility discharge and control the burning sensation of urine and cancer metastasis (68). A group of cell adhesion (32). Punicalagin isolated from pomegranate peel molecules (CAMs) are involved in connecting cell to cell exhibited anti-viral property against the herpes virus and cell to ECM components (69). A brief description (33). Similarly, pomegranate polyphenol extract inhibits of CAMs and their regulators, which are affected by pomegranate, is presented below (Figure 2). and lowered hypertension are the other clinical values theof daily influenza pomegranate virus (34). juice Attenuation intake (11). of atherosclerosis In traditional Cadherins Cadherins are transmembrane glycoproteins that in oxidative stress-induced degenerative diseases mediate homophilic cell–cell adhesion. They constitute medicine,such as Alzheimer’s pomegranate dementia peel extract (35). could Additionally, be beneficial the the core structural component of adherence junctions. therapeutic role of peel and seed oil extract in preventing E-cadherin is a member of this superfamily that is bone loss in preclinical models of osteoporosis has been found in epithelial cells. The catenin complex, including proven (36, 37). Interestingly, pomegranate extract can accelerate the wound healing process in albino rats (38). intracellular domain of E-cadherin to the cytoskeleton α-catenin,(69). Disruption β-catenin, of γ-catenin,E-cadherin and or p120 its linkercatenin proteins link the have been proven in infertility treatment (39). Recently, is the hallmark of epithelial mesenchymal transition Moreover, the beneficial effects of pomegranate peel (EMT), which is the most important mechanism behind the initiation of cancer metastasis. During ashowed new flavoneanti-obesity glucoside, properties together (40). with Previous ellagitannins studies EMT the cellular expression of cell adhesion molecules andhave flavonesrevealed extracted that pomegranate from flowers has of pomegranateanti-cancer decreases, resulting in the formation of spindle- shaped morphology. Loss cell–cell connection allows that pomegranate extract impedes the progression of tumor cells to disseminate and eventually metastasize. effectsprostate as cancer well. (41). The mostAdditional promising studies findings indicate show that Several transcription factors have been implicated in the regulation of EMT, including the Snail family

the Twist family basic helix–loop–helix transcription zinc finger transcription factors (Snail1 and Snail2), binding homeobox proteins (ZEB1 and ZEB2). The factorsgenes encoding (Twist1 andcadherins, Twist2), claudins, and the integrins,zinc finger mucins, E-box occludin, and ZO1 are repressed via these transcription

Figure 1. Schematic description of molecular targets of pomegranate with respect to cancer metastasis. CXCR4: C-X-C chemokine receptor type 4; HAMMR: Hyaluronic acid-mediated motility receptor/ CD168; IL: Interleukin; IP-10: Induced protein-10; MARCKS: Myristoylated alanine-rich protein kinase C substrate; MCP: Monocyte

MMP: Matrix metalloproteinase; RANTES: Regulated on activation, normalchemoattractant T Cell expressed protein; MIP-1: and secreted; Macrophage SDF1: inflammatory Stromal cell-derived protein-1; Figure 2. Schematic overview of adhesion molecules and their

Vascular cell adhesion molecule-1; VEGF: Vascular endothelial growth factor;factor 1;ZO: TNC: Zonula Tenascin occludens C; TNF-α: Tumor necrosis factorα; VCAM-1: bindingregulators homeobox as targets 1; ZO: of Zonula pomegranate.NF-κB: occludens Nuclear factor-κB; VCAM-1: Vascular cell adhesion molecule-1; ZEB1: Zinc finger E-box

980 Iran J Basic Med Sci, Vol. 22, No. 9, Sep 2019 Pomegranate in preventing cancer metastasis Ahmadiankia

Table 2. Antimetastatic effects of pomegranate determined in in vitro and in vivo pre-clinical studies

In vitroin vivo 2 In vitro ( (2

2 In vitro ( (52 Decreased expression of β 2 In vitro ( (5

2, In vitro ( (5 2, In vitro (55 , , ,

2, , In vitro ( , , (5

2, In vitro, in vivo 2 (5

2 In vitro , , (5 Abolish TNFα

In vivo (

5, 2, 2 In vitro ( κB signaling2 pathway (

5 In vitro ( (5

2, 2 In vitro, in vivo , , 2 (

2 In vitro (5

, , , 5, In vitro, in vivo ( , , In vitro, in vivo κB blockade (

5, , In vitro 1α &VEGF (2

, miRNAs & inhibition of the CXCR4/SDF1α

, In vitro 2, (

5, , In vitro ( (

In vitro , , , (5

In vitro (, , 2 (

, 2 In vitro ( , (

5, 5 In vitro, in vivo ( (5

CXCR4: C-X-C chemokine receptor type 4; ECM: Extracellular matrix; EMT: Epithelial mesenchymal transition; ERK: Extracellular signal-regulated kinase; HIF-1: Hypoxia-inducible factor 1; JNK: Jun N-terminal kinases; MMP: Matrix metalloproteinase; miRNA: microRNA; mTOR: Mammalian target of rapamycin; NF-κB: Nuclear factor-κB; PLA2: Phospholipases A2; PTEN: Phosphatase and tensin homolog; SCID: Severe combined immunodeficient; SDF1: Stromal cell-derived factor 1; TNF-α: Tumor necrosis factor-α; VEGF: Vascular endothelial growth factor factors (70, 71). microarray and real-time PCR analysis revealed that In an attempt to unravel the anticancer properties of ellagic acid, luteolin, and punicic acid decrease the expression of Twist (62). Twist is a transcriptional components on the metastatic potential of prostate repressor of E-cadherin expression in breast cancer cells pomegranate,cancer cells were the effectsexamined. of specific They found pomegranate that luteolin, juice that suppresses E-cadherin through the E-box elements ellagic acid, and punicic acid as important components on its promoter (71). Moreover, it was revealed that pomegranate up- in prostate cancer cells partly through increasing regulates miR-200c and down-regulates the expression ofthe pomegranate E-cadherin juiceprotein significantly level. Moreover, decreased affymetrix invasion of ZEB1 (52, 62). The miRNA-200 family has been shown

Iran J Basic Med Sci, Vol. 22, No. 9, Sep 2019 981 Ahmadiankia Pomegranate in preventing cancer metastasis to inhibit the transcription factors of ZEB1 and ZEB2, adhesion also activate signaling pathways essential which are transcriptional repressors of the E-cadherin gene (72). It was assumed that pomegranate inhibits ZEB1 through up-regulation of miR-200c, leading to forare considered cell motile as function. biomarkers Integrins of metastasis consist (69). of α A andstudy β increased expression of E-cadherin (62). subunits.showed that The consumption integrin subunits of pomegranate α3, α5, α6, αv, peel β1, extractand β3 Moreover, previous studies suggest that miR- 9 can serve as a potential cancer biomarker. It plays an important role in cancer metastasis by activating instudies, a mice there model is notof osteoporosis, enough investigation significantly on the decreased effect of thepomegranate expression on of theintegrin expression β3 (36). of Considering integrins involved previous in targeting E- cadherin (73). Interestingly, as reported cell-ECM interactions. thepreviously, β-catenin pomegranate pathway and decreased inducing the EMT expression via directly of Endothelial CAM expression of E-cadherin. Additionally, the expression Cell adhesion molecules, such as vascular cell miR-9of miR-122 and β-catenin, was increased inhibited following EMT and pomegranate up-regulated adhesion molecule-1 (VCAM-1), intercellular adhesion treatment in prostate cancer cells (52, 62). Previous molecule-1 (ICAM-1), and endothelial leukocyte studies showed that expression of miR-122 was adhesion molecule-1 (E-selectin), are known to correlated with an increased expression of E-cadherin modulate cell-endothelium interactions. They mediate initial adhesion of cancer cells to activated endothelium, may be relevant to the antimetastatic property of andpomegranate. the linker protein of α-catenin (74). This evidence of metastatic lesions (83). It was shown previously that Interestingly, it was revealed that the adhesion theirpomegranate rolling, extravasation, juice increased and finally,the expression the establishment of miR- 126, leading to reduction of VCAM-1 expression in Azoxymethane treated rats (45). Several mRNAs such as molecule of E-cadherin is regulated by the NF-κB pathway VCAM-1 have a complementary sequence within their (75).and c-Rel) NF-κB that is a can family form of fivevarious master heterodimers transcription or factorshomodimers (NF-κB1/p105, and bind to NF-κB2/p100, consensus DNA RelA/p65, sequences RelB, at Clearly, there is a lack of studies addressing the promoter regions of responsive genes. In unstimulated 3′-untranslatedeffects of pomegranate region for on miR-126 the adhesion (84). molecules in cancer cells. cells, NF-κB dimers are inactive because of association Regulators of cytoskeleton dynamics as targets of with IκB proteins that prevent the nuclear localization pomegranate of NF-κB and DNA binding. Stimulation of cells, for The cytoskeleton is a highly dynamic network that example by cytokines, activates IκB kinase (IKK) and controls the cellular structure and movement. In this resultsproteasome-mediated in the phosphorylation degradation, of IκB which at serine results residues. in the part, the effects of pomegranate on the molecules Phosphorylated IκB is subjected to ubiquitination and involved in the modulation of cytoskeleton dynamics are discussed (Figure 3). translocationthe regulation of of NF-κB EMT genesto the nucleus,in breast where cancer it cells functions (78). Interestingly, fascin, as a key regulator of cytoskeleton as a transcription factor (76, 77). NF-κB is involved in dynamics, is down-regulated by pomegranate (62). results in down-regulation of Twist1, Snail2, and ZEB2 Fascin is an actin-crosslinking protein that is required Previous studies demonstrated that inhibition of NF-κB for the formation of actin-based cellular protrusions. Functional studies showed that fascin expression andpathway significant and consequently, increase of reversesE-cadherin the expression EMT phenotype (79). is involved in cell locomotion of both normal and Interestingly,(60, 67). Study pomegranate showed that this extract effect inhibits of pomegranate the NF-κB is neoplastic cells (85). It was revealed that fascin-1 mRNA mediated through the inhibition of IkB/IKK interaction. Flavonoids, as the main components of pomegranate, are responsible for this effect (80).

Claudins Claudins are a family of transmembrane proteins that, along with occludins, are the most important miR133-b components of the tight junctions. They are linked to the miR-205 miR-122 miR-21 zonula occludens (ZO) (81, 82). It was demonstrated that PKCε filamentouspomegranate cytoskeleton increases gene via scaffoldingexpression proteins of claudin-1 named as a member of the claudin family. Moreover, the expression of miR-122 was increased following pomegranate Fascin Profilin1 Anillin Nexilin MARCKS N-chimearin Vimentin treatment in prostate cancer cells (62). Previous studies Formation of cell protrusions, EMT &Cell motility showed that expression of miR-122 was correlated with increased expression of occludin and ZO-1 (74). Metastasis

Integrins Figure 3. Schematic overview of cytoskeletal proteins and their Integrins are the key proteins responsible for cell-ECM regulators as targets of pomegranate. EMT: Epithelial mesenchymal adhesion. They link the cytoskeleton to ECM molecules transition; MARCKS: Myristoylated alanine-rich protein kinase C providing required forces for cell migration. Cell-ECM substrate; PKC: Protein kinase C

982 Iran J Basic Med Sci, Vol. 22, No. 9, Sep 2019 Pomegranate in preventing cancer metastasis Ahmadiankia was up-regulated in accordance with miR-133b down- 2) was suppressed by pomegranate polyphenols (99). regulation (86). Interestingly, pomegranate up-regulates COX-2 inhibits anoikis by activating various pathways in the expression of miR-133b, which is accompanied by different cancer types (100, 101). More study is needed fascin down-regulation (62). to determine if pomegranate truly inhibits anoikis resistance in cancer cells. proteins, which participates in dynamic turnover andProfilin reconstruction 1 is another of the memberactin cytoskeleton of actin-binding (87). Regulators of cell chemotaxis as targets of Interestingly, decreased migration of urinary bladder pomegranate cancer cells treated with pomegranate juice coincided Chemotaxis of tumor cells in the surrounding microenvironment is essential for cancer cell Anillin and nexilin are scaffold proteins implicated dissemination during tumor progression and metastasis within the increased regulation expression of cytoskeleton of profilin structure 1 (42). and also (102). C-X-C chemokine receptor type 4 (CXCR4) and cell migration (88, 89). Interestingly, the expression of anillin and nexilin were decreased following are known as important chemotactic proteins in cancer pomegranate treatment (62). Myristoylated alanine-rich itsmetastasis ligand, stromal(103, 104). cell-derived factor 1 alpha (SDF1α) protein kinase C substrate (MARCKS) is localized to the after treatment with luteolin, ellagic acid, and punicic protein. Phosphorylation by protein kinase C (PKC) or acidThe as expression the important of CXCR4 components and SDF1α of were pomegranate decreased plasmabinding membraneto calcium-calmodulin and is an actin inhibits filament its crosslinkingassociation juice. They also inhibited prostate cancer cell migration with actin and with the plasma membrane, leading to cell migration or invasion. It has been revealed that and punicic acid together decrease the expression exogenous overexpression of MARCKS remarkably toward13 , SDF1α.PI3K, and Interestingly, p-AKT proteins luteolin, involved ellagic in acid,the promoted cell attachment (90, 91). signaling downstream of CXCR4 (105). Among different Moreover, recent studies using prostate cancer cells microRNAs,of Gα miR-1 suppresses the expression of CXCR4 showed that miR-21 promotes cell invasion by directly targeting MARCKS (92). Interestingly, pomegranate regulated by luteolin, ellagic acid, and punicic acid (62). decreased the expression of miR-21 and increased andFurthermore, its ligand SDF1α.pomegranate It was decreasedfound that themiR-1 expression was up- MARCKS in prostate cancer cells (41, 62). Reversely, of hypoxia-inducible factor-1 (HIF-1) (61). HIF-1 has pomegranate juice increased the expression of miR- been found to be a critical transcription factor for the 205 (41). It has been shown that miR-205 suppresses expression of CXCR4 in a variety of cells, including tumor cells (106, 107). More studies are necessary to N-chimearin (CHN1) (93). N-chimearin induces the better understand the effects of pomegranate on the cellformation invasion of inthe prostate actin-based cancer structures by inhibiting lamellipodia PKCε and regulators of cancer cells chemotaxis.

Structural molecules of ECM and their regulators as andof the filopodia core components (94). of the cytoskeletal network. It targets of pomegranate inducesVimentin changes is a kind in cell of intermediateshape, motility, filament and adhesion and one Since ECM is the key component of the tumor during EMT (95). In early works, the expression of microenvironment, we asked whether ECM components vimentin in cancer cells was shown to be associated are affected by pomegranate. Based on previous studies, with a high risk of metastasis and poor prognosis pomegranate can prevent metastasis by affecting (96). Bagheri et al. demonstrated that the expression structural components of ECM (Figure 4). of vimentin decreased in MDA-MB-231 cells following One of the major structural proteins in ECM is treatment with pomegranate peel extract (53). Previous studies showed that increased expression of miR-122 was correlated with reduced expression of vimentin (74). Increased expression of miR-122 following cell treatment with pomegranate components (62) may be correlated with decreased expression of vimentin in prostate cancer cells. Taken together these results provide additional evidence that the antimetastatic effect of pomegranate miR-335 miR-21 PLA2 COX2 TIMPs may at least in part be mediated via genes and proteins involved in cytoskeleton dynamics. Fibronectin TNC Collagen I MMPs HAMMR

Molecules associated with anoikis resistance as Pro-tumorigenic ECM Pro-tumorigenic targets of pomegranate signaling cascades degradation signaling cascades related to cell Cancer cells acquire anoikis resistance to survive Metastasis after detachment from the primary sites and travel through the circulatory and lymphatic systems to disseminate throughout the body (97). Researchers Figure 4. Schematic description showing the structural molecules showed that ellagic acid could induce anoikis in ovarian of the extracellular matrix (ECM) as targets of pomegranate. COX-2: Cyclooxygenase-2; HAMMR: Hyaluronic acid-mediated carcinoma cells via inducing caspase-3 mediated motility receptor/CD168; MMPs: Matrix metalloproteinases; PLA2: apoptosis by increasing the Bax/Bcl-2 ratio (98). phospholipase A2; TIMPs: Tissue inhibitors of metalloproteinases; Moreover, the expression of Cyclooxygenase-2 (COX- TNC: Tenascin C

Iran J Basic Med Sci, Vol. 22, No. 9, Sep 2019 983 Ahmadiankia Pomegranate in preventing cancer metastasis collagen. There is mounting evidence of the role partially due to inhibiting the HA signaling pathway. of collagen-dense ECM in promoting metastasis in tenascin-C (TNC) is one of the other ECM glycoproteins different cancers (108, 109). Collagen-I increases pro- that drives the progression of many types of human tumorigenic signaling cascades such as focal adhesion cancer (125). TNC is able to interact with several ECM kinase (FAK), src family kinases (SFKs), and extracellular proteins and many cell-surface receptors, which enables regulated kinase (ERK)1/2 in tumor cells (110). It was demonstrated that type I collagen, secreted by stromal that miR-335 acts as a metastasis-suppressive miRNA itin tobreast influence cancer cell by migration inhibiting (126). type There I collagen is evidence and of breast cancer cells through induction of matrix TNC (127). Interestingly, pomegranate increased the fibroblasts,metalloproteinase may augment (MMP)-9 the (111). aggressive The maincharacteristics group of expression of miR-335 and inhibited the expression of enzymes responsible for protein degradation in ECM is collagen I and TNC (62). MMPs. The degradation of ECM by different members of the MMP family removes the physical barriers for tumor Inflammatory and angiogenic molecules as targets of cells and reveals the hidden sites in the ECM, where pomegranate various cell receptors can be bound to. The ability to degrade ECM is a critical requirement for tumor invasion (112). Previous studies showed that pomegranate or its It has been increasingly recognized that inflammatory ingredients such as ellagic acid, caffeic acid, luteolin, and componentsdecreased with fire treatment up cancer of metastasis.pomegranate Interestingly, seed oil in quercetin decreased the expression of collagen 1 and/ thebreast expression cancer cells of some (54). inflammatory Moreover, pomegranate chemokines juice was or MMP-1,-2,-9, and -13(48, 49, 53, 55, 62-64, 113, 114). This evidence suggests that the anti-metastatic role of pomegranate might be attributed, at least in part, to the significantlynormal T Cell reduced expressed the leveland secreted) of secreted in IL-6,prostate IL- reduction of collagen 1 and MMPs production by cancer 12p40,cancer IL-1β,cells (128). and RANTES Previous (regulated studies onshowed activation, that cells. Moreover, the level of phospholipase A2 (PLA2) migratory and invasive properties to many tumor cell transcript was decreased by polyphenols of overexpressionlines (129). Moreover, of these pomegranate inflammatory extract cytokines suppressed confers pomegranate fermented juice, peel, and seed oil (62). PLA2 is a cytosolic enzyme responsible for prostaglandin markers, including those of inducible nitric oxide (iNOS), production. It was shown that inhibition of prostaglandin theCOX-2, expression prostaglandin of critical E2 (PEG-2), mediators and HSP90 of inflammation (99, 130). reduced the release of MMPs and inhibited invasion in Pomegranate extract has been found to down- prostate cancer cells (115). Additionally, the expression regulate the expression of angiogenic factors such as and activation of MMPs are directly correlated to the vascular endothelial growth factor (VEGF). Moreover, expression of COX-2 in tumor cells (116). Interestingly, previous studies showed that pomegranate seed oil and polyphenols in pomegranate decreased the expression fermented pomegranate juice inhibited the expression of COX-2 (117), which may lead to down-regulation of of VEGF and increased expression of migration MMPs and inhibition of metastasis. inhibitory factor (MIF) in breast cancer cells (56). It Tissue inhibitors of metalloproteinases (TIMPs) has been shown that pomegranate extract minimizes are inhibitors of active MMPs (118). Previous studies angiogenesis, tumor size, and metastasis by reducing revealed that pomegranate extract or its constituents the expression of VEGF (61). up-regulate the expression of TIMPs (58). In vitro Perspective and limitation inhibits MMP-9 and collagen 1 promoter activity and experimentsprotein expression confirmed (119). that overexpressionMoreover, recent of miR-335studies discuss the molecular targets of pomegranate with showed that miR-21 promotes invasion by targeting respectThe overallto cancer purpose metastasis. of this Previous review isstudies to briefly on MMP regulators (120, 121). Interestingly, pomegranate pomegranate were mostly focused on fruit peel and increased the level of anti-invasive miR-335 and juice. The antimetastatic activity of other tissues of decreased the expression of miR-21, which may represent antimetastatic targets of pomegranate and root should be studied in more detail. Moreover, pomegranateas yet the antimetastatic such as leaves, effect flower,of only seed,a limited heartwood, number other components of ECM decreased in breast cancer of phytochemicals in pomegranate has been studied. (62).cells treated The expression with pomegranate of fibronectin peel as extract one of(53). the Further in vitro assays and animal model studies Furthermore, hyaluronic acid (HA) is a nonsulfated should be performed to unravel the effects of other glycosaminoglycan in ECM that regulates cell adhesion phytochemicals of pomegranate on the process of and migration. One of the HA receptors is hyaluronic metastasis. Many phytochemicals in pomegranate are acid-mediated motility receptor/CD168 (HAMMR), which is a nonintegral cell surface receptor (122). HA underlying effect mechanisms of pomegranate will also binding to HAMMR stimulates downstream signaling similarpromote to our other knowledge medicinal of plants,molecular so identificationtargets of other of that activates RhoA-activated protein kinase and the functional food plants. MAPK/extracellular signal-regulated protein kinase Additionally, more comprehensive studies should be (ERK) 1/2 pathway, which results in the expression performed on the expressional changes of metastasis of genes that are required for motility and invasion related molecules following pomegranate use. A better understanding of the molecules regulated by pomegranate down-regulates HAMMR (62) suggests pomegranate will undoubtedly enable its better clinical in various cancers (123, 124). The finding that application. that the antimetastatic effect of pomegranate may be 984 Iran J Basic Med Sci, Vol. 22, No. 9, Sep 2019 Pomegranate in preventing cancer metastasis Ahmadiankia

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