Effects of ellagic acid in human neuroblastoma cells

Christina Fjæraa Alfredsson

DISSERTATION | Karlstad University Studies | 2013:48

Biomedical Sciences

Faculty of Health, Science and Technology

DISSERTATION | Karlstad University Studies | 2013:48

Effects of ellagic acid in human neuroblastoma cells

Christina Fjæraa Alfredsson

Distribution:Karlstad University Faculty of Health, Science and TechnologyDepartment of Health SciencesSE-651 88 Karlstad, Sweden+46 54 700 10 00

© The author

ISBN 978-91-7063-526-7

Print: Universitetstryckeriet, Karlstad 2013

ISSN 1403-8099

urn:nbn:se:kau:diva-29905

Karlstad University Studies | 2013:48

DISSERTATION

Christina Fjæraa Alfredsson

Effects of ellagic acid in human neuroblastoma cells

WWW.KAU.SE

Abstract

A diet rich in polyphenols has been proposed to have beneficial health effects and to reduce risk of disease. Ellagic acid, a polyphenol common in red berries and pomegranates, has potential anti-tumorigenic effects that make it interesting to further study in different cancer cell systems. Neuroblastoma is a childhood cancer that arises during development of the peripheral nervous system. Neuroblastoma, being an embryonal tumor, show loss of function of genes controlling differentiation and apoptosis. Neuroblastoma is a heterogenic tumor disease, and highly malignant neuroblastomas are difficult to treat despite different treatment modalities, identifying a need for new and combinatory treatments. A common model for human neuroblastoma is the SH-SY5Y cell line resembling immature neuroblasts that can be differentiated in vitro with several agents including the phorbol ester 12-O-tetradecanoylphorbol-13-acetate and the vitamin A-derivative all-trans retinoic acid. Here, the effect of ellagic acid on proliferation, cell detachment and apoptosis in non-differentiated and in vitro-differentiated SH-SY5Y cells were studied with the aim of identifying cellular target mechanisms and a possible therapeutic potential for ellagic acid. In non-differentiated cells, ellagic acid reduced cell number, inhibited cell cycle activity, and induced cell detachment and apoptosis. Apoptosis was partly mediated by the intrinsic pathway. 12-O-tetradecanoylphorbol-13-acetate and all-trans retinoic acid both induced morphological differentiation, while only the latter induced G0/G1-arrest. Single-cell analysis revealed that 12-O-tetradecanoylphorbol-13-acetate-treated cells continued cycling during neuritogenesis while these two read-outs were mutually exclusive in all-trans retinoic acid-treated cells. 12-O-tetradecanoylphorbol-13-acetate- and especially all-trans retinoic acid-differentiated cells showed lower sensitivity to ellagic acid-dependent cell detachment and apoptosis.

i

Papers included in the thesis

I. Fjæraa Alfredsson C., Rendel, F., Sundström, B. E. , Nånberg, E. Cell

cycle activity in differentiating SH-SY5Y human neuroblastoma cells. Manuscript

II. Fjæraa, C., Nånberg, E. Effect of ellagic acid on proliferation, cell

adhesion and apoptosis in SH-SY5Y human neuroblastoma cells. Biomed Pharmacother 2009 63(4):254-61

III. Fjæraa Alfredsson, C., Ding, M., Liang, Q., Sundström, B.E., Nånberg,

E. Ellagic acid induces a dose- and time-dependent depolarization of mitochondria and activation of caspase-9 and-3 in human neuroblastoma cells. Biomed Pharmacother 2013 10.1016/j.biopha.2013.08.010 §

IV. Fjæraa Alfredsson C., Rendel, F., Liang, Q., Sundström, B. E., Nånberg,

E. Altered sensitivity to ellagic acid in neuroblastoma cells undergoing differentiation with 12-O-tetradecanoylphorbol-13-acetate and all-trans retinoic acid. Manuscript

Additional papers, not included in this thesis:

Karlsson, S., Nanberg, E, Fjæraa, C., Wijkander, J. Ellagic acid inhibits lipopolysaccharide-induced expression of enzymes involved in the synthesis of prostaglandin E2 in human monocytes. Br J Nutr. 2010 Apr;103(8):1102-9. Reprints of Paper II and III have been made with permission from the publisher.

ii

Abbreviations

ADP adenosine diphosphate ALK anaplastic lymphoma kinase Apaf-1 apoptotic protease activating factor-1 ATP adenosine triphosphate ATRA all-trans retinoic acid Bcl-2 B-cell leukemia/lymphoma-2 BDNF brain-derived neurotrophic factor BrdU bromo-deoxy-uridine CAM cell adhesion molecule CDK cyclin-dependent kinase COX-2 cyclooxygenase-2 DAG 1,2-sn-diacylglycerol ΔΨm mitochondrial membrane potential DMSO dimethylsulfoxide DNA deoxyribonucleic acid dUTP 2`-deoxyuridine 5`triphosphate EA ellagic acid ECM extracellular matrix ERK extracellular signal-regulated kinase FACS fluorescence activated cell sorting GAP-43 growth-associated protein-43 GM-CSF granulocyte-macrophage colony-stimulating factor HDAC histone deacetylase HT hydrolysable tannins IGF insulin-like growth factor IL interleukin LDH lactate dehydrogenase MAPK mitogen-activated protein kinase MARCKS myristoylated alanine-rich C-kinase substrate MMP matrix metalloproteinase mPGE2 membrane-associated PGE synthase-2 MYC myelocytomatosis oncogene MYCN v-myc myelocytomatosis viral related oncogene neuroblastoma-derived

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NaOH natrium hydroxide (sodium hydroxide) NB neuroblastoma NF-κB nuclear factor kappa B NGF nerve growth factor NPY neuropeptide Y NSE neuron-specific enolase PARP poly ADP-ribose polymerase PCR polymerase chain reaction PDGF platelet-derived growth factor PDK phosphoinositide-dependent kinase PGE2 prostaglandine E2 PI3K phosphatidyl inositiol-3-kinase PKB protein kinase B (also known as Akt or PKB/Akt) PKC protein kinase C RA retinoic acid RAS rat sarcoma viral antigene homolouge Rho 123 rhodamine 123 ROS reactive oxygen species TH tyrosine hydroxylase Trk tropomyocin-receptor kinase TNF tumor necrosis factor TPA 12-O-tetradecanoylphorbol-13-acetate TUNEL terminal deoxynucleotidyl transferase dUTP nick end

labeling Uro urolithin VAChT vesicular acetylcholine transporter VEGF vascular endothelial growth factor VEGFR vascular endothelial growth factor-receptor VIP vasoactive intestinal peptide XTT sodium 3´[1-phenylaminocarbonyl)-3,4-tetrazolium]-bis-

(methoxy-6-nitro) benzene sulfonic acid hydrate 4-HPR 4-hydroxy(phenyl) retinamide, (also known as

fenretinide) 13-cis-RA 13-cis-retinoic acid or isotretinoin

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Table of contents

Abstract ............................................................................................................................ i Papers included in the thesis ............................................................................................ ii Abbreviations .................................................................................................................. iii Table of contents ............................................................................................................. v

Background ...................................................................................................................... 1

Polyphenols and health ................................................................................................ 1

Hydrolyzable tannins, ellagitannins and ellagic acid – a historical perspective ............... 2

Biological effects of ellagic acid .................................................................................... 4

Effects in tumor cell lines ............................................................................................... 4

Proliferation and cell cycle activity ....................................................................... 4

Apoptosis............................................................................................................. 6

Adhesion and migration ....................................................................................... 9

Ellagic acid and angiogenesis .............................................................................. 10

Effects in non-tumor cells ............................................................................................. 11

Anti-inflammatory effects .................................................................................. 11

Biological effects of ellagic acid via chelation of divalent cations ........................ 12

Coagulation mechanisms .................................................................................... 12

Data from in vivo animal models................................................................................... 13

Clinical studies .......................................................................................................... 13

Cellular uptake of ellagic acid and tissue distribution ......................................................... 14

Possible molecular target mechanisms for ellagic acid .......................................................... 15

Cancer........................................................................................................................ 17

Childhood cancer ........................................................................................................ 17

Neuroblastoma .......................................................................................................... 18

Neuroblastoma incidence, origin, characterization and staging ........................... 18

Neuroblastoma treatment .................................................................................. 20

In vitro models applying neuroblastoma cell lines ............................................................... 22

Neuroblastoma-derived cell lines........................................................................ 22

Applications were NB-derived cell lines are used ............................................... 22

The SH-SY5Y cell model ................................................................................... 22

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In vitro differentiation of SH-SY5Y cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) ..................................................................................................... 23

In vitro differentiation of SH-SY5Y cells with all-trans retinoic acid (ATRA) ..... 25

Aims of the present investigation ................................................................................... 27

Methodology .................................................................................................................. 28

Chemicals and reagents .............................................................................................. 28

TPA, ATRA, ellagic acid and staurosporine .................................................................. 28

Solvents .................................................................................................................... 28

Cell culture ................................................................................................................. 29

Methods for determination of cell number and proliferation ...................................... 29

Cell counting with Bürker chamber ................................................................................ 29

XTT ....................................................................................................................... 29

Incorporation of bromo-deoxyuridine (BrdU) ................................................................... 29

Flow cytometry (FACS) .............................................................................................. 30

Methods for determination of viability ....................................................................... 30

Trypanblue exclusion .................................................................................................. 30

LDH ...................................................................................................................... 30

Methods for determination of apoptosis .................................................................... 31

Microscopy ................................................................................................................ 31

SubG1 ..................................................................................................................... 31

TUNEL ................................................................................................................. 31

Methods for determination of apoptosis mechanisms ................................................ 32

Rhodamine 123 ......................................................................................................... 32

Apoptosis-associated protein expression, Western Blot ....................................................... 32

Methods for determination of morphological differentiation...................................... 33

Microscopy ................................................................................................................ 33

Real-time PCR ......................................................................................................... 33

Results ........................................................................................................................... 34

Effects of ellagic acid in non-differentiated SH-SY5Y ................................................ 34

Number of adherent cells ............................................................................................. 34

Proliferation and cell cycle analysis ................................................................................. 35

Apoptosis ................................................................................................................. 36

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Cell adhesion ............................................................................................................. 38

Differentiation-associated events ............................................................................... 39

Differentiation with TPA or ATRA ............................................................................ 39

Cell number .............................................................................................................. 40

Proliferation and cell cycle ............................................................................................ 41

Relationship between differentiation and proliferation ......................................................... 43

Effects of ellagic acid in differentiated SH-SY5Y ....................................................... 44

Cell number .............................................................................................................. 44

Cell adhesion ............................................................................................................. 46

Proliferation and cell cycle analysis ................................................................................. 46

Apoptosis ................................................................................................................. 47

Discussion ..................................................................................................................... 48

Summary and Conclusions ............................................................................................. 52

Future perspectives ........................................................................................................ 54

Acknowledgements ........................................................................................................ 56

Grant support ................................................................................................................ 58

References ..................................................................................................................... 58

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Background

Polyphenols and health

The use of plant material and botanical extracts in order to prevent, treat or cure disease is an ancient phenomenon. At all times in all cultures man has experimented with the use of food as medicine. Epidemiological studies have repeatedly shown a direct connection between dietary habits and human health and disease-risk. The last decades, a growing number of reports have suggested a correlation between phytochemicals, which are metabolites of plant-derived food, and reduced risk of chronic diseases such as diabetes, cardiovascular disease and cancer [1-4]. Among the phytochemicals are the polyphenols, known to have antioxidant properties [5]. In recent years, a growing number of reports have also focused on polyphenols and their anti-inflammatory, anti-proliferative and apoptosis-inducing effects, and possible roles in cancer prevention and treatment [6-9]. Some polyphenols have also been suggested to have potential effects in preventing toxic side effects from chemotherapy in cancer patients [10, 11]. However, there are discrepancies between the claimed health effects of polyphenols and available scientific evidence for biological and clinical effects [5]. Various natural products are sold in the claim of being a potential cure for different diseases without being clinically tested, and the proposed health effects of such products are sometimes a result of too far-stretched interpretation of data from basic research. Thus, there is a need for controlled studies to address and evaluate the potential therapeutic properties of compounds of interest applying scientific methods. In the project presented in this thesis, biological effects of the polyphenol ellagic acid in human neuroblastoma cells were studied.

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Hydrolyzable tannins, ellagitannins and ellagic acid – a historical perspective Polyphenols are present in virtually all plant material, and more than 8000 polyphenolic structures are known, ranging from simple molecules to highly complex polymerized compounds [12]. The polyphenols can be divided into several different groups depending on their chemical structure (Figure 1). One group is the tannins, including derived tannins and hydrolysable tannins (HT). The HTs contribute to taste, flavor, color and chemical stability in fruits and vegetables, and was the first group of plant polyphenols subjected to chemical investigations during the end of the 17th-century. These compounds were of interest because of their ability to tan leather, and oak-bark extract was the most well-known and effective tanning material available [13]. Today, HTs are of interest because of their antioxidant- and antimicrobial activities [14, 15], as well as their potential ability to lower the risk of cancer, diabetes and cardiovascular disease as suggested by an increasing number of studies [14, 16]. HTs are found in a large variety of fruits and vegetables. The largest amounts are found in vegetables of the Rosidae-family (e.g. red berries, oak, nuts, pomegranate and mango), and also in foods were tannins has been added during aging- and/or preservation (e.g. aged wine, spirits, meat or fish).

Figure 1. Classification of polyphenols. The polyphenols are divided into several different groups. Ellagic acid is a hydrolysis product of ellagitannins, polyphenols that belong to the group of hydrolysable tannins.

POLYPHENOLS

Flavonoids Stilbens Tannins Saponins Lignans Phenolic acids Other

Hydrolyzable tannins

Ellagitannins

Condensed tannins

Ellagic acid

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Amongst the HT are the ellagitannins, which are especially rich in pomegranate, blackberries, raspberries, strawberries and walnuts. The metabolism of plant polyphenols and the distribution, and bioavailability and interactions with the human body are still largely unclear. However, several in vivo-studies of orally administered ET containing foods and beverages have demonstrated that upon ingestion, ETs are hydrolyzed in the small intestine to form ellagic acid [17]. Ellagic acid is further metabolized by intestinal microorganisms to form urolithins that are readily absorbed over the intestinal mucosa into the circulation [18, 19]. (This will be further discussed below.) The first reports on ellagic acid formation were published in the early 20th century, describing a stable oxidation product of digallic acid, formed from the digallic acid backbone structure of several tannins [20]. In the 1960s, ellagic acid was studied for its effect on lowering of blood pressure [21] and on blood clotting by activation of the coagulation factor XII (also known as the Hageman factor) [22]. In the early 1980s, the first reports on ellagic acid and suspected effects on cancer were published. In 1982, Wood and co-authors reported a possible effect of ellagic acid inhibiting in vitro carcinogenesis induced by metabolites of benzo[a]pyrene in bacteria cells and in Chinese hamster V79 cells [23]. Soon after, the inhibitory effect of ellagic acid was reported to be caused by binding to and subsequent protection of DNA [24, 25]. In the last two decades, several researchers working with human cell lines derived from tumors as in vitro model systems have reported that ellagic acid has effects on proliferation and apoptosis. In more recent reports, ellagic acid has been associated with both protecting normal cells from induced oxidative damage and apoptosis [26, 27] as well as down-regulation of inflammatory-associated molecules in different cell systems [28, 29]. Ellagic acid has also been shown to protect against doxorubicin-induced cardiac injury in mice [30], methotrexate-induced small intestine damage in rats [31] and cyclophosphamide-induced renal injury, DNA-damage and genotoxicity in Swiss albino mice [32]. These findings suggest that ellagic acid may have a potential in applications to reduce adverse side effects during chemotherapy. The biological effects and suggested molecular target mechanisms will be described in more detail below.

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Figure 2. Structure of ellagic acid, (4,4′,5,5′,6,6′-Hexahydroxydiphenic acid, C14H6O8).

Biological effects of ellagic acid ETs and ellagic acid, as well as several other polyphenols have repeatedly been shown to excert effects in in vitro cultures of cell lines derived from human carcinomas [33-36]. The most commonly reported effects are on proliferation, cell cycle and apoptosis. Interactions with molecules related to cell adhesion, migration and angiogenesis have also been reported [35]. The main published findings of ellagic acid in tumor cells, descriptions of the biological effects and possible target mechanisms involved are presented below. Also, effects of ellagic acid in non-tumor cells, data from in vivo animal models and clinical studies as well as data on cellular uptake and tissue distribution of ellagic acid are described.

Effects in tumor cell lines

Proliferation and cell cycle activity The process of eukaryotic cell division is called the cell cycle. The cell cycle is a strictly controlled cycle of events, divided into four different phases, the G1, S, G2 and M. The two G phases are growth phases, where G1 is the longest and determines the division time of a cell. The S phase is when the DNA-replication and duplication occurs and M stands for mitosis, the actual cell division. Cells that are not dividing (quiescent) are said to rest in G0-phase. The cell cycle is strictly controlled by several molecules such as the cyclins (e.g. Cyclin A, B, D and E) and cyclin-dependent kinases (CDKs) (e.g. CDK 1, 4, 6) [37], and several sequential control sites [38, 39]. Cyclins bind to and activate CDKs which in turn phosphorylate target proteins (e.g. pRb) coordinating the cell cycle. This complicated system also involves small molecules called CDK-inhibitors (e.g.

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p21cip1, p27kip1, p57, p16, p15, p18 and p19) that can inactivate the cyclin/CDK-complexes and slow down or inhibit the cell cycle. Another important protein is p53, involved both in cell cycle progression and apoptosis. In response to cellular damage p53 usually either slows down the cell cycle to allow DNA-repair or initiates apoptosis by down-regulation of the anti-apoptotic protein Bcl-2 and up-regulation of the pro-apoptotic protein Bax [40]. A number of reports on ellagic acid and effect on carcinoma cell proliferation and cell cycle alterations have been published. Cell number is reduced in cultures of breast [41], colon [41-45], oral [42], bladder [46, 47], cervical carcinoma [43], ovarian [48] and prostatic cancer cells [41, 42]. Similar findings were reported in leukemia cells [49]. In several of the published reports reduced cell numbers was shown to be caused by alterations in proliferation. Cell cycle arrest in G0/G1-[43, 46, 48] or S-phase [41] have been reported as have other alterations in the cell cycle [49]. The effects have been shown to be time- and dose-dependent [45, 48]. Several molecular target mechanisms have been suggested to be involved in cell cycle activity and proliferation alterations by ellagic acid, such as elevated levels of p53 [48] and elevated levels and/or activation of p21cip1[44, 48]. Down-regulation of cyclins A and B1 [41] or cyclin D1 [48], and up-regulation [41] or decreased levels [48] of cyclin E has also been associated to ellagic acid-induced cell cycle alterations [41]. One suggested up-stream signaling pathway responsible for this is Wnt which was shown to be inhibited by ellagic acid in colonic cancer cells [50]. Ellagic acid is also a potent inhibitor of CK2, an important protein kinase involved in neoplasia and tumor cell growth activated down-stream of Wnt-signaling [51]. Down-regulation of insulin-like growth factor II (IGF-II) has been reported with ellagic acid [44], and as described below, transcription and protein levels of the kinases p38, mitogen-activated protein kinase (MAPK) and MEKK1 [47].

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Apoptosis The process of apoptosis was first described in the 1970s as a regulator of animal cell populations [52]. Apoptosis is a controlled cell death mechanism functional under both normal and pathological conditions during which cells that are damaged or redundant are eliminated within the body. This is particularly important during embryogenesis and early developmental stages when a large number of immature neuronal cells are eliminated during formation and maturation of the nervous system [53]. Apoptosis may be a consequence of several events e.g. lack of trophic support to cells, oxidative stress and DNA-damage [54]. Apoptosis is mainly mediated by two different cellular mechanisms or pathways, the extrinsic and intrinsic apoptosis pathways [55, 56]. The extrinsic pathway is initiated by activation of death receptors, while the intrinsic pathway is initiated by changes affecting the mitochondria. Both pathways involve sequential activation of a group of proteases named caspases (proteases that cleave after aspartate residues). Caspases can be divided into three groups: initiator caspases, effector caspases and other caspases (not involved in apoptosis). Caspase-8 is the initiator caspase of the extrinsic pathway, and caspase-9 is the initiator caspase of the intrinsic pathway. The effector caspase-3 is downstream of both the intrinsic and extrinsic pathways, and activates a cascade of events leading to degradation of DNA and proteins, collapse of the cells interior and exterior structures and finally cell death. The two pathways are illustrated in Figure 3. In the extrinsic pathway, upon binding of the ligand to a death receptor (e.g. Fas ligand (Fas L, also called CD95L), a member of the tumor-necrosis factor-(TNF)-receptor superfamily, recruits and binds FAS associated death domain (FADD) and procaspase-8. Procaspase-8 is cleaved to active caspase-8 that cleaves and activates caspase-3. In the intrinsic pathway, apoptosis typically start with a cellular insult causing altered structure and damage of the DNA which activates p53-dependent responses. p53 activates a pro-apoptotic member of the Bcl-2 family, Bax. The (B-cell leukaemia/lymphoma-2) Bcl-2 family consists of pro-(e.g. Bax, Bak, Bid and Bad) and anti-apoptotic (e.g. Bcl-2, Bcl-XL and Mcl-1) proteins associated with the outer mitochondrial membrane. Under normal conditions the ratio of pro- and anti-apoptotic family members is tightly controlled. When apoptosis is initiated, the ratio is skewed towards pro>anti apoptotic members

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allowing Bcl-2 and Bax to form pore channels in the outer mitochondrial membrane. Involved in this is also other mechanisms e.g. phosphorylation status of Bad, which in its phosphorylated state exists in form of a homodimer called the Bad-14-3-3-protein, but once dephosphorylated forms heterodimers with Bcl-2 and Bax thus inactivating them [57]. When pores are formed in the mitochondrial membrane, mitochondrial membrane potential (ΔΨm) is lowered, and intramembrane proteins like cytochrome c is released from the mitochondrial intermembranous compartment. Once released into the cytosol, cytochrome c is recruited to form the apoptosome complex together with Apaf-1 and procaspase-9 thus inducing proteolytic activation of caspase-9. Caspase-9 activates caspase-3. The extrinsic and intrinsic pathways also cross activate each other, since active caspase-8 also cleaves Bid, a pro-apoptotic member of the Bcl-2 family. Active caspase-3 cleaves various other proteins including cytoplasmic (e.g. actin, gelsolin, fodrin) and nuclear proteins (e.g. lamin), proteins involved in DNA metabolism and repair (e.g. poly ADP-ribose polymerase (PARP)), and various protein kinases [58]. This induces a degradation process including shrinkage of the cell and nucleus, condensation of nuclear chromatin, plasma membrane budding, and formation of apoptotic bodies. The apoptotic bodies are rapidly phagocytosed and degraded by neighbouring cells [59]. Other apoptotic pathways, independent of caspases, have been described [60, 61]. Also, non-apoptosis related functions of caspases have been reported e.g. functions in neuronal differentiation and synaptic activity [62], control of immune response-associated molecules [63], stimulation of proliferation and regeneration and possibly also resistance to chemo- and radiotherapeutic insults in apoptosis-surviving cancer cells [64]. Ellagic acid has in a large and increasing number of reports been seen to induce apoptosis in several tumor-derived cell lines, including colon [41, 42, 45], prostate [41, 42, 65], oral [42], cervix [43], breast [41], pancreas [66], ovarian [48] and bladder cells [47] and the leukemic cell line HL-60 [49]. Suggested intracellular targets of ellagic acid promoting apoptosis are decreased expression of Bcl-2 [48, 65] and Bcl-XL [45], increased expression of Bax [65], and alterations on the Bcl-2:Bax ratio [48], decrease of mitochondrial membrane potential (ΔΨm) [66],

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mitochondrial release of cytochrome c [45, 66], activation of caspases-3 [45, 47, 48, 66, 67], -6 [65] and -9 [45, 65], all involved in regulation of the mitochondrial apoptosis pathway. Also, activation of death-receptors [49] and caspase-8 [65], associated with the extrinsic apoptosis pathway, has been reported with ellagic acid. The induced apoptosis has also been associated with reduced ATP-production [41]. Suggested upstream signaling pathways responsible for this has been alterations in transcription and protein levels of the kinases p38, MAPK, and mitogen-activated protein kinase kinase kinase 1 (MEKK1) [47].

Figure 3. Apotosis pathways. The extrinsic pathway is associated with activation of death-recepotors, while the intrinsic pathway is associated with mitochondrial changes. Both pathways involves activation of caspases. The figure is adapted from Hengartner, M.O., The biochemistry of apoptosis. Nature, 2000. 407(6805): p. 770-6.

Caspase-3

FADD

Procaspase-8

Caspase-8

Caspase-9

Cytocrome c

Apaf-1

Ligand

Procaspase-3

p53Bax

Bcl-2Bcl-XL

Bid

Extrinsic pathwayIntrinsic pathwayCellular insult

Procaspase-9

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Adhesion and migration Cell adhesion molecules (CAMs) are glycoproteins expressed on the surface of cell membranes and includes integrins, cadherins (E-, N- and P-type), selectins (E-, L- and P-type), CD44 and immunoglobulin (Ig)-like CAMs (e.g. NCAM and ICAM-2) [68]. CAMs are responsible for the connection of cells to the extracellular matrix (ECM) and other cells, and for anchorage-dependent cells, functioning CAMs are necessary for survival. In epithelial cells, forming thin sheets of tissue, E-cadherin is an important adhesion molecule. During metastasis, cancer cells become less dependent on tissue-specific adhesion for their survival, often show anchorage-independent growth and become migratory [69]. Ellagic acid in combination with punicalagin and luteolin (other pomegranate-derived polyphenols), has been reported to significantly increase adhesion and decrease cell migration activity and chemotaxis in response to stromal cell-derived factor 1α (SDF1α) in cancer cells from both prostate [70] and breast [71]. The increased adhesion is accompanied by increased expression of the adhesion proteins E-cadherin, ICAM, Claudin 1 and of Myristoylated alanine-rich protein C- kinase substrate (MARCKS). Adhesion of immune cells to endothelial cells is often used as in vitro atherosclerosis models, and in these model system, ellagic acid has repeatedly been shown to decrease the expression levels of CAMs such as ICAM-1, VCAM-1 and E-selectin after induction by TNF-α or IL1-β [72, 73]. This implicates that ellagic acid also can inhibit cell adhesion in a cell specific manner. Important molecules in tumor metastasis are the matrix metalloproteases (MMPs) capable of degrading ECM-complexes [74]. Ellagic acid-induced apoptosis has been reported to be accompanied by a decrease in the levels of MMP-2 and MMP-9 [41], suggesting that ellagic acid also can inhibit early events in tumor cell metastasis. When anchorage-dependent cells detach from the ECM prior to degradation by autophagy they often undergo detachment-induced apoptosis i.e. anoikis [75]. A recent report showed that ellagic acid could induce anoikis in suspension- and agar-grown ovarian carcinoma cells characterized by anchorage-independent growth [48] thus overcoming a fundamental mechanism in metastasis.

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Ellagic acid and angiogenesis Angiogenesis, the formation of new blood vessels, is an important mechanism in solid tumors, and several growth factors are involved in this process. The family of vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs), are key factors in the control of angiogenesis. It has been reported that ellagic acid inhibits platelet-derived growth factor (PDGF) and VEGFRs involved in angiogenesis [76], and VEGFR-2 activity in breast endothelial cells [77]. A decrease of released VEGF in conditioned media has also been reported in cultures of colon, breast and prostate cancer cells treated with ellagic acid [41].

Figure 4. Suggested cellular effects of ellagic acid. Several cellular effects of ellagic acid have been imposed, and the figure presents suggested effects relevant for the study presented in this thesis. Ellagic acid can alter the expression and/or activity of protein kinases (e.g. PKB/Akt, p38 and MAPKs), transcription factors (e.g. NF-kB) and growth factors (e.g. VEGF, PDGF), illustrated by the inner ring, All these affects the cellular effects illustrated in the outer ring. Ellagic acid has been shown to alter the expression and/or activity of proteins relevant for proliferation and cell cycle (e.g. cyclins A, B, D and E, p21 cip1, p27kip1and p53), apoptosis (e.g. caspase-3, -6, -8 and -9, Bax, Bcl-2, Bcl-XL), adhesion and migration (e.g. E-cadherin, ICAM, VCAM, MMP-2 and -9, E-selectin, claudin and MARCKS, angiogenesis (e.g. VEGFR, COX-2) as well as proteins related to other cellular effects (e.g. IL-8 and IL-6).

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Effects in non-tumor cells In several of the above described reports, normal cells (non-transformed) were included to address whether ellagic acid exhibit selective effects on tumor cells. In these studies, doses up to 100 µM was tested and no apoptosis-induction were reported in normal colon [45], human umbilical vein endothelial cells (HUVEC) or normal human lung fibroblast cells [41]. Also, no effects on proliferation were seen in normal human dermal fibroblasts [78] or normal human lung fibroblasts [48].

In non-transformed cells, on the contrary, ellagic acid has been shown to have anti-apoptotic effects [79], to be protective against reactive oxygen species (ROS) and ROS-induced apoptosis, and to reduce the levels of H2O2-induced oxidative stress [47]. Also, ellagic acid has been shown to stabilize elastin fibers in human dermal fibroblasts [78].

Anti-inflammatory effects Even though much of the research on ellagic acid concern tumor cells and cancer, a rapidly growing number of reports address the role of ellagic acid and related polyphenols in inflammation. In in vitro models, ellagic acid has been shown to down-regulate expression of inflammatory-associated molecular markers. In human monocytes, stimuli-induced release of prostaglandin E2 (PGE2), and levels of membrane-associated PGE synthase-1 (mPGEs-1) protein (a PG synthesizing enzyme), cytosolic phospholipase A2 (cPLA2α) and cyclooxygenase-2 (COX-2) protein in human monocytes were reduced by ellagic acid [29]. Suggested mechanisms for this are inhibition of MAPK, extracellular signal-regulated kinase (ERK), p38 and c-Jun-terminal kinase (JNK), all downstream of NF-kB. Inhibition of COX-2 and mPGEs-1 has also been seen in human fibroblasts, and this was shown to be by inhibition of MAPK and NF-kB [28]. In other studies, the anti-inflammatory potential of ellagic acid has been investigated in animal models. Ellagic acid has been shown to inhibit phenol-induced ear edema in mice [80], suggesting a potential use in treatment of coetaneous inflammation e.g. contact dermatitis. Also, ellagic acid has been shown to protect against acid- and lipopolysaccharide (LPS)-induced acute lung injury in mice [81, 82].

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Amongst other effects related to inflammation, ellagic acid has also been suggested to inhibit eosinophilic recruitment and decrease eosinophile number in murine peritonitis and asthma models [83, 84], have anti-allergic effects [85], anti-bacterial and anti-viral effects [86] and also to have a possible role in prevention and treatment of diabetes type-2 [87].

Biological effects of ellagic acid via chelation of divalent cations Several polyphenols, including ETs are known to have metal chelating-properties, thus chelating divalent ions such as Ca2+, Cu2+, Zn2+, Co2+ and Fe2+ [88-90]. Ellagic acid is also shown to chelate nickel ions and to inhibit Ni2+-induced toxicity in rats [91]. The divalent cation Ca2+ is a very important intracellular messenger. The intracellular concentration of unbound calcium is normally around 100 nM, and by introducing a Ca2+-chelator the concentration can be made very low. That implicates that cellular signaling mediated by a rise in intracellular Ca2+ could be blocked by sequestering free Ca2+ with a chelator. One family of intracellular signal molecules that is highly dependent of Ca2+ is protein kinase C (PKC), a family of multifunctional protein kinases important for several cellular mechanisms including cell division and the cell cycle [92]. Intracellular Ca2+-signaling is also involved in activation of the protein kinase B (PKB/Akt, also known as c-Akt) pathway (see below) and induction of apoptosis in cancer cells [93].

Coagulation mechanisms Ellagic acid has been shown to shorten bleeding- and clotting-time in several animal models [94, 95]. Ellagic acid is a known activator of factor XII [22], a plasma protein involved in, but not crucial for clotting formation. Factor XII is involved in the initiation of the intrinsic- or contact-activation of blood coagulation system, and can be activated by a variety of negatively charged insoluble substances, such as the glass surface of test tubes. The ability of ellagic acid to activate factor XII has been shown to be dependent of formation of ellagic acid-metal ion complexes [88].

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Data from in vivo animal models Most of the studies on ellagic acid and inhibition of carcinogenesis and inflammation after toxic insults are performed in animal models. Ellagic acid has been shown to inhibit benzylmethylamine-induced esophageal tumorigenesis in rats [96], to inhibit rat tongue carcinogenesis induced by 4-nitroquinoline-1-oxide [97], and dose-dependently protect rats from cisplatin-induced nephrotoxicity [98]. Also much of the studies on the capacity of ellagic acid to bind to cellular protein and DNA and thereby prevent the binding of carcinogens or mutagens and possibly inhibit carcinogenesis have been performed in murine models [25, 99, 100]. In a recent study with a mouse model of pancreatic cancer, ellagic acid inhibited xenograpt growth, angiogenesis and metastasis through suppression of PKB/Akt, sonic hedgehog (Shh) and Notch signaling pathway [101]. In the same study, ellagic acid-dependent reduced proliferation and activated apoptosis were shown to involve down-regulation of proliferating cell nuclear antigen (PCNA) and Ki67, a nuclear protein important for proliferation. Also activation of caspase-3, cleavage of PARP, inhibition of Bcl-2, increased expression of Bax, inhibited expression of cyclin-D1, CDK-2 and CDK-6, were seen. All these are important molecules in direct regulation of tumor proliferation and apoptosis as described above. Ellagic acid also induced the expression of E-cadherin and inhibited the expression of Snail, MMP-2 and MMP-9 in pancreatic tumor tissues, all molecules involved in early metastasis. Also, COX-2 (related to inflammation and metastasis), was inhibited as well as VEGF, VEGF-R and HIF1alpha related to angiogenesis. Finally suppression of the pro-inflammatory cytokines interleukin (IL)-6 and IL-8 was seen [101].

Clinical studies The reports on ellagic acid from human studies and clinical trials are still very few compared with the existing in vitro data and animal studies. In one study, a combination of traditional cancer treatment (chemotherapy) together with ellagic acid was tried in patients with prostate cancer. The reported results showed that ellagic acid reduced the chemotherapy-induced toxicity, but had no direct effect on the tumors [10]. Also, ellagic acid-containing pomegranate juice (PJ) has been given to prostate cancer patients in a phase II clinical trial. In patients with rising levels of prostate specific antigen (PSA), doubling time of increase in PSA were

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drastically increased after daily intake of PJ [102], however PJ contains many components and several polyphenols and the contribution of ellagic acid to the documented effect is unclear.

Cellular uptake of ellagic acid and tissue distribution The first reports on how ellagic acid is taken up in blood and tissue were conducted in mice and rats and published in the mid-1980s. The results revealed that after oral ingestion of ellagic acid, low or no levels were detected in blood, lung or liver. After intravenous administration, ellagic acid was also very rapidly cleared, and approximately 70% of the administered dose was recovered in urine and faeces within seven hours [103]. More recent reports have stated that ellagic acid is metabolized by mircobiota in the intestinal lumen to smaller molecules called urolithins (Uro) (mainly Uro A and Uro B) [18, 19, 104]. The urolithins can be absorbed, and micromolar levels have been detected in colon [105]. This suggest that urolithins, and not ellagic acid is responsible for the reported biological effects in clinical studies and animal models [106]. Much data on cellular uptake and tissue distribution of ellagic acid (as well as the metabolites urolithins) comes from animal studies [107, 108], but in more recent studies human volunteers have been used [109-111]. Notably, the metabolism of ellagic acid to urolithins is influenced by the individual microbiotic flora, giving a high variability in the production of these metabolites between individuals [17, 18].

Gavage or peroral uptake models to investigate the safety of ellagic acid have been performed in in vivo animal models. 90 days of orally feeding of rats with different doses of ellagic acid showed no mortality or any clinical signs [101, 112]. No severe side-effects of ellagic acid were reported at the concentrations used to induce various biological effects on tumor cells in mice, suggesting it to be “safe” at the used concentrations [101]. The low bioavailability and rapid metabolism of ellagic acid after oral intake [109, 113] have imposed limited use of this substance for anti-tumor activity in vivo. In an attempt to increase the bioavailability, local delivery systems with chitosan/ellagic acid polymer films or gels are being developed [114, 115] as well as nanoparticles designed with the same aim [116].

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Possible molecular target mechanisms for ellagic acid From a perspective of the identified biological effects discussed above, two particular interesting candidate mechanisms for ellagic acid are the PI3K/Pkb/Akt pathway and the downstream NF-kB pathway [35, 117]. PKB/Akt is a central protein in intracellular signaling pathways activated by growth factors, and an important signal molecule in different processes including proliferation and apoptosis [118]. As illustrated in Figure 5, PKB/Akt is activated after up-stream events including growth factor-binding to receptors, and also by CAMs (e.g. integrins and cadherins) signaling upstream of the rat sarcoma viral antigene homolouge (Ras), PI3K, PIP3 and phosphoinositide-dependent kinase (PDK)-1. PKB/Akt can activate several important down-stream regulator molecules including NF-kB, mammalian target of rapamycin (mTOR), glycogen synthase kinase-3 (GSK-3), p21cip1, p27kip1 and caspase-9. As illustrated in Figure 5, PKB/Akt is a hypothetical target of ellagic acid and ellagic acid can possibly target up-stream signaling events, down-stream signaling events and/or PKB/Akt directly. For instance, in pancreatic stellate cells, ellagic acid inhibited PDGF-BB-induced proliferation and migration by inhibition of tyrosine phosphorylation of PDGF β-receptor and the downstream activation of ERK and PKB/Akt [119]. Also, the combinatory effect of ellagic acid and two other polyphenols (luteolin and punicalagin) in prostate cancer cells is reported to involve inhibition of PI3K and the subsequent phosphorylation of PKB/Akt [120]. In pancreatic adenocarsinoma cells, ellagic acid has been shown to induce apoptosis through inhibition of NF-kB activity [66].

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Figure 5. Possible target mechanisms for ellagic acid. PKB/Akt is a regulator of several molecules important for multiple cellular effects (e.g. apoptosis inhibition, cell cycle progression, expression of cell cycle proteins, and angiogenesis-related proteins and transcription of several other genes). PKB/Akt is a hypothetical target of ellagic acid and ellagic acid can possibly target up-stream signaling events, down-stream signaling events and/or PKB/Akt directly. In nerve cells, the binding of neurotrophins (e.g. nerve growth factor (NGF) to tropomyocin receptor kinase (Trk)-receptors (e.g. TkrA) stimulates receptor transphosphorylation, and several signaling proteins are recruited, including Sch who activates Ras which in turn activates PI3K. PI3K stimulates PDKs which activates PKB/Akt. The connection with ECM via CAMs (e.g. integrins) is important for the activity of these functions. This figure is a very simplified model for illustrating purposes only. For more details on neurotrophin signaling and the PKB/Akt pathway see [121] and [122].

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Cancer Cancer is a generic name for about 200 different diseases of malign neoplasms characterized by uncontrolled proliferation and/or no elimination of dysfunctional cells via apoptosis. Every year approximately 55.000 new cases of cancers are diagnosed in Sweden with prostate cancer and breast cancer as the most common diagnoses [123]. The onset of cancer or carcinogenesis is a gradual multistage process where several molecular and cellular alterations occur. In the review “Hallmarks of cancer: The next generation”, Hanahan and Weinberg describes six biological hallmarks for development of human tumors: “Sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis”. Cancer cells frequently have somatic mutations in genes controlling the above listed cellular functions, including activating mutations and/or overexpression of oncoproteins (e.g. Ras, Raf, PI3K and MYC) and inactivation of tumor suppressor genes (e.g. the retinoblastoma-associated (Rb-) protein, p53 and phosphatase and tensin homolog (PTEN)). Increased expression of anti-apoptotic regulators (e.g. Bcl-2 and Bcl-XL) or survival signals (e.g. IGF-I/II) and down-regulation of pro-apoptotic factors (e.g. Bax, Bim, Puma) [124] are also common events.

Childhood cancer Every year approximately 250 children (age 0-14 years) in Sweden are diagnosed with cancer. This corresponds to an annual incidence of 11/100.000 children [123]. The over-all survival figures for childhood cancer have increased significantly during the past 30 years. At present, in western European countries, the 5-year survival rate from childhood cancers is 75 % [125]. Several differences distinguishes childhood cancers from adult cancers, e.g. cancer in adults can often be linked to risk factors as smoking, dietary habits, age, UV-radiation and work-related factors, while children have not lived to be exposed to such factors unless one include pre-natal exposure and transgenerational risk-exposure that has been demonstrated in a few examples (e.g. in utero exposure to diethylstilbestrol (DES) [126]). In fetuses and small children undergoing rapid growth, a larger proportion of cells are actively dividing, therefore de-regulated differentiation and aberrant apoptosis mechanisms are more likely cellular mechanisms behind embryonal and

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early life cancer. Most childhood cancers have embryonal origin and rise in developing tissue.

Neuroblastoma

Neuroblastoma incidence, origin, characterization and staging Neuroblastoma (NB) accounts for 8-10 % of all childhood malignancies reported worldwide and is the most common extra cranial solid tumor disease in infants. In Sweden, the mean annual incidence rate for NB from 1984 to 2005 was 1.0 cases/100 000 children thus accounting for 5,6 % of all diagnosed childhood cancers. NB is a to originate from immature neural crest-derived cells that in normal development gives rise to the adrenal medulla and the sympathetic ganglia in truncus symphateticus [127]. The disease was first described in 1864 by Virchow, and the term “neuroblastoma” were coined in 1910 by J.H. Wright who first described NB tumors to be developing from “more or less undifferentiated nerve cells or neurocytes or neuroblasts” [128]. NB is a rare example of a tumor of neuronal cells and not the supporting cells in the nervous system which is the case for most brain tumors. NBs arise during the fetal or post-natal period, and are considered to be embryonal tumors. NB cells are belived to be arrested at different early stages of differentiation, and one common hypothesis is that NB develops as a result of aberrant differentiation and maturation mechanisms during differentiation from immature progenitor cells of the neural crest to mature nerve cells. Unlike most other tumor types, the clinical features of NB vary greatly between patients. Due to their symphatoadrenal origin, NB tumors may occur in the adrenal medulla or anywhere along the sympathetic ganglia in truncus symphateticus. Most primary NB tumors develop within the abdomen, other common anatomic sites are head or neck, chest or pelvis. The most common symptom of NB is an abdominal mass, often along with abdominal pain. Other symptoms are unexplained fever, weight loss and weakness. The symptoms depend on the anatomical site of the primary tumor and metastases and tumor-derived catecholamines [129, 130]. Biomarkers for NB are elevated levels of catecholamine metabolites in urine [131] and elevated

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levels of lactate dehydrogenase (LDH), neuron-specific enolase (NSE) and ferritin in serum [132-134]. A NB diagnosis is based on specific defined international criteria, defined in 1988 and revised in 1993 [135, 136]. As the clinical presentations of NB is very heterogeneous, staging systems are used to classify the tumors. Before 2009, the most common staging system was the International Neuroblastoma Staging System (INSS) defined by The International Neuroblastoma Staging System Group [135]. In 2009, a new system called the International Neuroblastoma Risk Group Staging System (INRGSS) was established. This system is based on clinical presentation and image-defined risk factors, and the tumors are divided into four groups; L1, L2, M and Ms. L1 and L2 describe localized tumors, where L2 tumors show image-defined risk factors that preclude surgical resection (i.e. due to tumor size or that it has crossed the midline of the body). M and Ms describes metastatic tumors, where Ms describes metastatic tumors in children younger than 18 months with metastases confined to skin, liver and/or bone marrow [137]. A subgroup of NB tumors, despite features of a highly malignant phenotype, has the ability of spontaneous regression or differentiation into benign ganglioneuromas. Other NB shows rapid growing tumors initially responding to treatment, but with high incidence of relapse and a low survival rate. Besides tumor histology and degree of differentiation, NB tumors vary in their cytogenetic and molecular aberrations, and different serum and tissue markers. Aneuploidi is frequently observed, with allelic loss of the short arm of chromosome 1, deletions on chromosome 11q and 14q and/or allelic gains of chromosome 17q being common aberrations [138-140]. Another important molecular marker is the NB-derived v-myc myelocytomatosis viral related (MYCN) oncogene [141]. N-myc amplification is highly correlated with advanced stages and poor prognosis [142]. Also, amplified MYCN is often associated with deletion of the short arm on chromosome 1, and increased expression of the multidrug-resistance-associated protein (MRP) [143, 144]. More recently, mutations in the tyrosine kinase receptor anaplastic lymphoma kinase (ALK) has been identified in NB [145, 146], reviewed in [147]. ALK mutations have been associated with amplification of MYCN [148], and also been showed to potentiate the oncogenic activity of MYCN in NB [149]. NB tumors do not generally express alterations in

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the p53 gene [150]. Despite large efforts and more recent large scale global sequencing projects no common genetic aberration or marker coining NB has yet been identified. The survival rate of NB varies with age at diagnosis, tumor location, stage, histology and biological features of the tumor, and presence of metastatic disease. In general, diagnosis before one year of age, extra-adrenal tumor location, low stage, absence of N-Myc amplification and chromosome 1 deletions and higher degree of differentiation are characteristics associated with better prognosis. Also, hyper diploid or near-triploid karyotypes have been associated with more favourable outcome than near diploid tumors. Markers for degree of differentiation and phenotype are expression of the neurotrophin-receptor kinases TrkA, TrkB and TrkC. Expression of TrkA and TrkC is related to low-stage NB, TrkB and MYCN amplifications have been related to advanced disease [151, 152]. In a subset of NB, lobular structures with central zones of necrosis is present. This phenomenon is suggested to be caused by hypoxia [153]. Exposure of NB cells to hypoxia in vitro and in vivo induces selection for de-differentiated cells and immature neuronal phenotype, leding to a stem cell-like phenotype expressing several genes responsive to low oxygen pressure [154, 155]. NB tumors with high expression of hypoxia-inducible trancription factors (HIFs), especially HIF-2α have been associated with poor prognosis and treatment resistance [156] . Recently, there has also been categorized an ultra-high risk group within the high-risk group of NB patients, and this group is characterized by patients with MYCN-amplification and bone metastasis. Patients with these specific alterations are examples of patients which are suitable candidates for novel treatment strategies for NB [157].

Neuroblastoma treatment The major strategies used in NB treatment are surgery, chemotherapy and radiotherapy. Also, more novel biotherapy including immunotherapy with monoclonal antibodies is used. The decision of treatment of choice is based on the factors described above, i.e. the patient age at diagnosis, location, stage, histology

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and biological features of the tumor, and eventual metastatic disease. As the main strategy is to avoid unnecessary use of chemotherapy and radiotherapy because of harmful side effects, early stage tumors are primarily removed by surgery. Chemotherapy is used in treatment of patients with advanced, refractory or metastatic NB. If NB tumors persist after surgery and chemotherapy, radiotherapy can be used [129, 130]. High-risk NB patients generally respond to initial chemotherapy, but relapse with treatment-resistant tumors. As a result of increasingly intensified treatment strategies the overall outcome for this group however has improved during the last thirty years [158, 159]. For these patients other treatment strategies are applied including bone marrow-ablative therapy, immunotherapy, differentiation therapy with vitamin A-derivatives (retinoic acids), inhibition of the MYCN oncogene, anti-angiogenesis and/or anti-histone deacetylase (HDAC)-drugs [129, 130]. Frequently, multimodal treatment modalities are applied. Currently, the standard treatment for high-risk NB consists of myeloablative therapy followed by autologous hematopoietic stem cell transplantation and maintenance with 13-cis-RA for the treatment of minimal residual disease, leading to a 3-year disease-free survival rate of about 50 % [160]. There are three vitamin-A-derivated lipophilic compounds commonly used for retinoid therapy of NB, 13-cis-retinoic acid (13-cis-RA), all-trans retinoic acid (ATRA), and N-(4-hydroxyphenyl)retinamide (4-HPR), In pediatric cancer therapy, retinoids are also used for treatment of acute promyelocytic leukemia [161]. 13-cis-RA and ATRA have similar effects on NB cells, which is morphological differentiation and cell cycle arrest [161], with 13-cis-RA having a better clinical effect due to better pharmacokinetics with longer half-life [162]. 4-HPR has another mode of action by induction of apoptosis and necrosis [160]. 13-cis-RA is also used in combination with immunotherapy and inhibitors of HDAC [160]. Immunotherapy is currently being tested for patients with highly malignant NB. One strategy is immunotherapy with the human–murine monoclonal antibody called ch14.18 against the tumor-associated disialoganglioside GD2 combined with granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-2 [163, 164].

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In vitro models applying neuroblastoma cell lines

Neuroblastoma-derived cell lines Since access to fresh NB tumor material is limited, most in vitro studies on NB are performed with cell lines derived from NB tumors. Several NB cell lines are available today, and they vary in their genotype making it possible to study e.g. NMYC-amplified and non-amplified cells. What characterizes human NB cell lines are that they are derived from highly-malignant NB tumors and can be differentiated and/or cell cycle arrested in vitro. The characteristics of existing cell lines varies with respect to for instance expression of different receptors, amplification of N-Myc, ALK mutations and atypi [165], and this mirrors the heterogeneity of NB tumors and the absence of a common genetic marker.

Applications were NB-derived cell lines are used NB cell lines are used as in vitro models for several different applications including studies of tumor biology, neuronal development, nervous tissue damage and regeneration [166, 167], neurotoxicity [168, 169], and as a tool for studies of differentiation and growth control in immature nerve cells. This makes NB cell lines model systems for discoveries of broad biological and medical significance. Frequently used cell lines for in vitro studies of human NB are LAN-2, LAN-5, IMR-32 and SH-SY5Y. In this project, SH-SY5Y was used. SH-SY5Y is a common model for NB, and also frequently used as a model system for studies of Alzheimer disease and Parkinson disease.

The SH-SY5Y cell model SH-SY5Y is a well-characterized and commonly used human NB cell line. It was originally established by Dr June Biedler and co-workers [170], and is a trice cloned (SK-N-SH -> SH-SY -> SH-SY5 -> SH-SY5Y) N-type sub-clone of the parental cell line SK-N-SH, originating from a bone marrow biopsy of a metastatic tumor from a 4-year old female patient [171]. The large majority of cells in a SH-SY5Y culture show an adherent growth pattern [172, 173], and keeping growth conditions strictly constant is an important issue when keeping cultures of SH-SY5Y cells to avoid variations in the cells phenotype.

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SH-SY5Y cells express tyrosine hydroxylase (TH), low levels of NSE [174], and chromogranin proteins and can be used as a model of undifferentiated neuroblasts. As other subclones from SK-N-SH, SH-SY5Y do not have MYCN amplification [171], but have abnormalities in chromosome 1q, 2p, 4 and 5q, 7t, 9 and 10p, 14q, 16q, 15, 17 and 22q [175] and mutated ALK (F1174L) [176]. SH-SY5Y cells exhibits normal expression of p53 [177], show no ras gene-mutations [178] or overexpression of the anti-apoptotic Bcl-2 [179]. Wild-type SH-SY5Y express low levels of neurotrophin recepors (NTRKs), but the cells can be transfected with exogenous TRKs [180, 181], and thus become responsive to NGF (Trk A) [180, 182, 183], brain-derived neurotrophic factor (BDNF) (Trk B) [184] and NT-3 (Trk C) [181]. SH-SY5Y cells can successfully be grown in 3D-cultures as spheroids [185] overcoming some of the limitations with traditional tumor cell lines grown in monolayers and more closely mimic the phenotype of untransformed cells [186]. SH-SY5Y cells can also be injected intramuscularly or subcutaneously in nude mice or rats to form human NB xenografts, and the histology and phenotype of the developing in vivo tumors are similar to the features of human NBs [187, 188]. SH-SY5Y cells can be morphologically and biochemically differentiated in vitro by several different agents including the phorbol ester 12-O-tetradecanoyl-13-phorbol-acetate (TPA) in presence of serum or defined growth factors [189], and the vitamin A derivative all-trans retinoic acid (ATRA) [190]. This will be described below, and summarised in Table I.

In vitro differentiation of SH-SY5Y cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) TPA is a phorbol ester, known to have tumor-promoting activities based on its ability to stimulate PKC by acting as a diacylglycerol (DAG) analogue. PKCs are divided into three groups of different isoforms: classical (α, βI, βII and γ), novel (δ, ε, η and θ) and atypical (ι and ζ) PKCs [191]. DAG or phorbol esters activate classical and novel isoforms of PKC. Classical isoforms also requires Ca2+ for its activation, and phorbol esters (and DAG), increases classical PKCs affinity for Ca2+. While the naturally occurring DAG will be rapidly degraded, the phorbol ester is more chemically stable, offering the possibility to study sustained activation of PKCs. Generally there is an inverse relationship between cell differentiation and

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proliferation. Activation of different isoforms of PKC by TPA can also induce differentiation and/ or inhibit cell cycle progression [192], TPA-induced differentiation is often used to study inhibition of the cell cycle machinery in several cell types [193-197]. TPA is a useful research tool for in vitro studies, but can because of its tumor promoting activity not be used for human in vivo studies. In SH-SY5Y cells, treatment with nanomolar concentrations of TPA in the presence of serum induces outgrowth of short branched processes with nodes and growth cones [189] which are gradually developed during approximately eight days [198]. TPA promotes the expression of a variety of functional neuronal marker genes and neurotransmitters, and alters the expression of different CAMs and ECM-molecules (see Table I). Taken together, this suggests differentiation towards a sympathetic adrenergic neuronal-like phenotype. Nanomolar concentration of TPA also concomitantly decrease the proliferation rate of SH-SY5Y cells [189, 198-200], reviewed in [201]. In absence of serum, SH-SY5Y cells are non-responsive to differentiation by TPA [183, 202], but serum can be replaced by defined growth factors such as insulin-like growth factor-1 (IGF-1) [202], PDGF [203], basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF) [204]. SH-SY5Y cells express protein for both classical (i.e. α βI and βII) novel (i.e. δ and ε) and atypical (i.e. ζ) isoforms of PKC [205, 206], and activation of different PKC isoforms by treatment with 16 nM TPA are shown to be important for growth, survival [205], differentiation [207] and for outgrowth [207] and maintenance of the growth cone structure [206] in SH-SY5Y. The TPA-induced effect on differentiation and growth inhibition (and also expression of noradrenalin) is highly dose-dependent, with a maximum effect around 16 nM [199]. At higher doses (e.g. 1.6 µM) TPA will result in poor differentiation [207]. This is caused by down-regulation of primarily classical PKC isoforms necessary for differentiation (i.e. an activity-dependent de-sensitization of PKCα with translocation of PKCα from cytosol to the cellular membrane) [207]. TPA also cause activation and accumulation of ERK in the cell nucleus [183], and this has been shown to be necessary for up-regulation of NPY and GAP-43 gene expression but not for neurite formation in TPA-differentiated SH-SY5Y [208]. TPA also induces PKC-dependent activation of NF-kB and up-regulation of Bcl-2 in SH-SY5Y prior to

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differentiation suggesting a requirement also for NF-kB in SH-SY5Y differentiation [209].

In vitro differentiation of SH-SY5Y cells with all-trans retinoic acid (ATRA) ATRA, or tretinoin is a vitamin A-derivative and an important factor in vertebrate development, and can be used as a differentiation tool both in vitro and in vivo. ATRA exerts its effects on gene transcription by binding to nuclear retionoic acid receptors (RARs) or retinoic x receptors (RXRs) [210]. Biologically active ATRA is found in both the central and the peripheral nervous system of mouse embryos [211], and has an important role both in early development of the nervous system and in later neuronal specification [212]. Since ATRA is a potent inducer of cellular differentiation and growth inhibition in both normal and cancer cells in vitro [213-215], it is frequently used as a research tool to study these events. In NB cells, and particularly in N-type subclones, ATRA promotes neurite outgrowth [216]. In SH-SY5Y cells, treatment with 1-10 micromolar concentrations of ATRA rapidly induces outgrowth of long, thin processes. ATRA promotes expression of a variety of functional neuronal marker genes and neurotransmitters, and alters the expression of different CAMs and ECM-molecules (see Table I). Taken together, this suggests differentiation towards a cholinergic phenotype. Micromolar concentration of ATRA also reduces cell numbers of SH-SY5Y cultures, and this is shown to be by induction of G0/G1-arrest [217]. The effect of ATRA on neurite outgrowth in NB cells is believed to be caused by regulation of the transcription of neurotrophin receptor genes [218], and ATRA has also been reported to activate kinases important for neurite outgrowth like MAPK and PI3K [219]. ATRA phosphorylates [168] and activates both PKB/Akt and ERK in SH-SY5Y [220]. The mechanism behind the actions of ATRA in SH-SY5Y regarding cell cycle arrest is unclear, but pathways dependent on ERK1/2 and PKC [35], as well as VEGF and NF-kB signaling [221] have been suggested to be involved. Increased levels of the cyclin-depent kinase inhibitor p21cip1 and p27kip1 have also been suggested [222].

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Data from in vivo animal models of NB and other tumors (e.g. leukemia) have shown potential for ATRA as a therapeutic agent. The substance has been tested in humans in both phase I and II clinical trials [223, 224]. As described above ATRA, as well as other vitamin-A-derived lipophilic compounds are used in the clinical treatment of NB [160] and another childhood tumor, acute promyelocytic leukemia [161].

Table I. Different cellular responses of SH-SY5Y to differentiation with TPA and ATRA. Abbreviations used: NSE- neuron-specific enolase, NPY- neuropeptide Y, GAP-43- growth associated protein-43, TH- tyrosine hydroxylase, MAP-2- microtubule associated protein-2, VAChT- vesicular acetylcholine transporter, VIP- vasoactive intestinal peptide. TPA ATRA Molecular structure

Optimal concentration for differentiation of NB cells

16 nM 1-10µM

Features of TPA-differentiated SH-SY5Y cells

Features of ATRA-differentiated SH-SY5Y cells

Phenotype Adrenergic Cholinergic Morphological changes Short, branched processes [189]

Growth cones [189] Long, thin processes [190]

Growth cones [190] Effects on proliferation Reduced proliferation [189]

Reduced proliferation [190] G0/G1-arrest [217]

Examples of neurotransmittors excreted

Noradrenalin [189] Adrenalin [189]

Noradrenalin [190]

Examples of general neuronal marker genes and phenotype-specific marker genes expressed

NSE [190]

NPY [208] GAP-43 [208] TH [225]

NSE [190] MAP-2 [168] Synapthophysin [168] VAChT [226] VIP [227]

Examples of CAMs and ECM proteins expressed

Integrin α2/β1 [228] ICAM-1 [228] Tenacsin [229]

Integrin α1/β1 [228] Tenascin [229]

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Aims of the present investigation

The general aim of this work has been to study the effect of ellagic acid on cell number, proliferation, apoptosis and adhesion in cultures of human neuroblastoma SH-SY5Y cells. More specifically, the work addresses

- Effect of ellagic acid in non-differentiated SH-SY5Y cells - Proliferation in differentiated SH-SY5Y cells - Effect of ellagic acid in differentiated SH-SY5Y cells

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Methodology

This is an overview of the methods used in the present investigation. Details are presented in the separate papers.

Chemicals and reagents

TPA, ATRA, ellagic acid and staurosporine The concentrations of TPA and ATRA used in this study were chosen in agreement with previous reports on TPA and ATRA and induction of differentiation and reduced proliferation and growth arrest respectively in SH-SY5Y cultures [190, 199, 201, 217, 222]. The concentrations of ellagic acid used were chosen in agreement with previous reports on ellagic acid and induction of proliferation inhibition, cell cycle arrest and apoptosis in various cell lines in vitro [41-43, 45, 46, 230]. As a positive control for apoptosis in paper II and III, 200 nM staurosporine was used.

Solvents TPA was dissolved in ethanol, ATRA and staurosporine were dissolved in dimethylsulfoxide (DMSO). Ellagic acid was dissolved in DMSO (Paper II) or 0.1 M NaOH (Paper III and IV). The change of solvent was due to an observation of increased solubility and less precipitate of high concentrations of ellagic acid (50-100 µM) with NaOH compared with DMSO. Also, DMSO had some slight effects on cellular morphology (rounding of the cells) and on cell proliferation (see Paper II, Figure 1A) and no such effects were seen with NaOH. With both solvents, ellagic acid at high doses (50, 75 and 100 µM) gave a precipitation of small grains or needles of ellagic acid in the bottom of the dishes (see Figure 8E), but the problem was reduced using NaOH as solvent. The phenomenon was protein-dependent, as no precipitate was seen in the ellagic acid solution or in ellagic acid diluted in serum-free culture media. The grains or needles were not seen directly after adding ellagic acid to the culture, and did not seem to affect any of the cellular effects induced by ellagic acid. In all experiments untreated cells and vehicle-only treated cells were included. The concentration of vehicle was standardized in all samples.

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Cell culture Human NB SH-SY5Y cells were grown in Eagle`s minimal essential medium supplemented with 10 % fetal bovine serum, L-glutamine and penicillin/streptomycin. Cells were maintained at 37 ºC in a saturated humidity atmosphere containing 95 % air and 5 % CO2. Fresh medium was added every fourth day, and the cells were sub-cultured every seventh day. For experiments, cells were allowed to plate for at least 24 hours prior to addition of reagents.

Methods for determination of cell number and proliferation

Cell counting with Bürker chamber In Paper I, II and IV, cell number was determined by counting aliquots in a Bürker chamber. Culture media containing detached cells was collected, and adherent cells were detached by trypsination before placed in a Bürker chamber and counted by bright-field microscopy.

XTT In Paper III and IV, cell number was determined by the XTT (sodium 3´[1-phenylaminocarbonyl)-3,4-tetrazolium]-bis-(methoxy-6-nitro) benzene sulfonic acid hydrate) method. Briefly, cells were incubated with a XTT reagent, which in the presence of metabolic activity was converted to a water-soluble colored formazan-product easily detected in a spectrofotometer. This method is rapid, and easy to perform and to reproduce according to standard protocols supplied by the manufacturer. As this method measures the number of metabolic active cells (i.e. functional mitochondria) the results can be confused in cases with cells that have damaged mitochondria or in cells with altered (decreased or increased) mitochondrial function or altered mitochondria number.

Incorporation of bromo-deoxyuridine (BrdU) In paper I and IV, detection of incorporated BrdU was performed. Cells were incubated with the thymidine analogue BrdU for various time periods, and the

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incorporated BrdU was detected via a coupled enzyme reaction and bright field microscopy. For this, the 5-Bromo-2`-deoxy-uridine Labeling and Detection Kit II from Roche Diagnostics was used. This method is reproducible and accurate, and easily performed according to the manufacturers protocol. Another advantage is that it is non-radioactive compared to the classical method with incorporation of the radioactively labeled [3H] thymidine. Limitations are the relatively low number (normally 300-500) of cells counted for each sample.

Flow cytometry (FACS) In Paper I and II, flow cytometry was used to study proliferation by monitoring cell cycle activity. Cells were stained with propodium iodide or Hoechst 33258 prior to analysis on a FACScan or a FACS LSR II flow cytometer respectively. This method is beneficial for analyzing a large number of cells in a short time. One of the few disadvantages is that the reagents used to stain the cells often are toxic.

Methods for determination of viability

Trypanblue exclusion In Paper II and IV, trypan blue exclusion was used to monitor cell viability. Cells with intact membranes will exclude the dye, while dead cells will stain dark blue. Relative number of trypan blue positive cells was determined by counting aliquots in a Bürker chamber. The method is rapid and cost-effective but has limitations in the relatively small number of cells per sample counted. This method does not distinguish between necrotic and apoptotic cell death. The results can be confused by living cells with somewhat affected membranes that will not fully exclude the dye and stain light blue.

LDH In Paper III and IV, cell injury was determined by the LDH method. LDH is released from cells following loss of membrane integrity resulting from both apoptosis and necrosis [231]. Briefly, culture media was withdrawn from cells and incubated with a lactate reagent, which will be converted to pyruvate in an LDH-

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catalyzed process where NAD+ is reduced to NADH/H+. A yellow tetrazolium salt is added together with a catalyst that transfers H/H+ to the tetrazolium salt converting it to a water-soluble red colored formazan-product easily detected in a spectrophotometer. This method is rapid, and easy to perform and to reproduce according to standard protocols supplied by the manufacturer. LDH can exist in the system as a contaminant of the serum in the cell culture media and it is important to always monitor the LDH-signal from blank samples (only culture media and eventual additions, no cells) and subtract this signal from the samples before analyzing the results. Methods for determination of apoptosis Microscopy In Paper III, morphological signs of apoptosis was evaluated using bright field and phase contrast microscopy. The cells were examined in a Leica DMI 6000 B inverted microscope (Leica Microsystems) and photographed with a digital camera. Morphological signs of apoptosis were defined as cell shrinkage, membrane blebbing, and formation of apoptotic bodies and detachment of cells from the culture dishes. This method is rapid and cost-effective, but has limitations in the relatively small number of cells per sample counted per sample.

SubG1 In Paper III and IV, apoptosis was determined by SubG1-analysis. Cells were stained with propodium iodide prior to analysis on a FACScan flow cytometer. The method for this is identical to the method for monitoring cell cycle activity with FACS. The subG1 fraction of cells is determined as cells with less DNA than cells in the G0/G1-phase of the cell cycle. The method is beneficial for analyzing a large number of cells in a short time.

TUNEL In Paper II and IV, apoptosis was monitored by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-staining prior to analysis on a FACScan flow cytometer. This method is specific and highly sensitive since the

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TUNEL-stain with a FITC-coupled specific antibody will only detect cells with DNA strand breaks. As well as the general advantages and disadvantages for flow cytometry described above, a challenge with this method is that two fluorescent dyes, FITC and propidium iodide are used at the same time and these may interact with each other confusing the data unless the wavelength settings of the flow cytometer are correct. Methods for determination of apoptosis mechanisms Rhodamine 123 In Paper III, determination of mitochondrial membrane potential (ΔΨm) was performed in cells stained with rhodamine 123 and analyzed on a FACscan flow cytometer. In this method, live cells are stained and analyzed. Since the rhodamine 123 is highly light sensitive, this procedure has to be performed in a dark room. This method also has the general advantages and disadvantages for flow cytometry as described above.

Apoptosis-associated protein expression, Western Blot In Paper III and IV, expression and cleavage of apoptosis-related proteins were measured with western blot. Cells were harvested and lysed, and protein concentration determined. Lysates were separated on pre-cast polyacrylamide gels, and proteins were electrotransferred to polyvinylidene fluoride membranes. The membranes were blocked with bovine serum albumin (BSA) or dry milk before incubation with appropriate primary antibodies, washing and incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies prior to detection with enhanced chemiluminiscence (ECL) and capturing of the signals on X-ray film. Between incubations with different primary antibodies, membranes were stripped. Western blot is a semi-quantitative method for detection of protein levels, and the results rely on the specificity of the antibodies used.

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Methods for determination of morphological differentiation Microscopy In Paper I and IV, morphological signs of differentiation were evaluated using bright field and phase contrast microscopy. The cells were examined in a Leica DMI 6000 B inverted microscope (Leica Microsystems) and photographed with a digital camera. Morphological signs of differentiation were defined as cells bearing one or more neurite processes longer than two cell body lengths. For quantification of morphologically differentiated cells, 300 ± 30 cells or more per dish were counted. The method has the general advantages and disadvantages of microscopy as described above.

Real-time PCR In Paper I, real-time polymerase chain reaction (PCR) was used to monitor several different markers of differentiation. Total RNA was isolated, first strand cDNA was synthesized and quantitative PCR was run on a Step One plus Real time PCR system using SYBR green as a stain. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a stably expressed gene, was included with to normalize input DNA. The method has high precision for quantitative detection of changes in mRNA expression, however changes in mRNA levels do not always reflect changes in protein levels.

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Results

Effects of ellagic acid in non-differentiated SH-SY5Y

Number of adherent cells Ellagic acid decreased the number of adherent cells in cultures of SH-SY5Y, and the effect was dose- and time-dependent. Cells were treated with different doses ranging from 1-100 µM ellagic acid (dissolved in DMSO or NaOH) for varying time-periods from 24-96 hours and counted in a Bürker chamber or measured with XTT. At the low doses (1-10 µM ellagic acid), cell numbers were only slightly decreased. A concentration-range was identified between 30-75 µM ellagic acid, where adherent cell numbers were significantly decreased after 24 hours (Paper II and III), and further decreased at 36 (Paper IV), 48, 72 and 96 hours (data not shown). With 100 µM ellagic acid the decrease in cell number was very pronounced and full effect was seen already after 24-48 hours (no or very few adherent cells left) (Paper II and III). This is illustrated below in Figure 7A. The effect of ellagic acid lowering the number of adherent cells was highly dose-dependent with overlapping concentration ranges, despite different time for treatment of the cultures, methods for estimation of cell number and the vehicle used. When cultures were treated with ellagic acid for eight days a reduction in cell numbers compared with control was apparent already after two days, and further pronounced compared with control cultures after four, six and eight days (Paper II). This is illustrated in Figure 7B. The total cell number (adherent + detached cells) was also slightly lowered with ellagic acid (Paper II, III and IV), however this effect was marginal compared with the decrease in number of adherent cells. This will be further discussed below in the section on adhesion.

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Figure 7. Ellagic acid-induced effects on number of adherent cells. Ellagic acid reduced the number of cells in cultures of SH-SY5Y and the effect was (A) dose- and (B) time-dependent. (A): A number of 105 cells were seeded in 60 mm culture dishes and were treated with nothing (Control), vehicle (DMSO) or different doses of ellagic acid (1-100 µM) for 96 hours before adherent cells were harvested and counted in a Bürker chamber. (B): Cells were seeded as in (A) and treated with nothing (Control, open circles), vehicle (DMSO, filled circles) or 30 µM ellagic acid (filled squares) added each fourth day as indicated by the arrows. Every second day, adherent cells were harvested and counted as in (A). Data are mean (± SEM) values from three experiments with triplicate samples (A) or two experiments with duplicate samples (B). * indicates statistical significance (p < 0.05). The figure is adopted from Paper II, Figure 1.

Proliferation and cell cycle analysis The decrease in cell number induced by ellagic acid was showed to be partly due to decreased proliferative activity. When cells were treated with 30 µM ellagic acid for 96 hours and analyzed by FACS, the cell cycle distribution was revealed to be altered with decreased percentage of cells in S-phase, and increased percentages of cells in G0/G1 and in G2/M phases (Paper II). Only the increase in number of G0/G1-cells was statistically significant. This is illustrated below in Table II. In Paper III, the same method of analysis based on DNA-content was used for detection of SubG1-events in cells treated with 50 µM ellagic acid for 24 hours (data not shown), however no ellagic acid-induced shift in cell cycle distribution was seen during the initial 24 hours. In Paper IV, data from a 24 hour long BrdU-chase on cells incubated with ellagic acid for 24 hours showed a dose-dependent

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decrease of BrdU-positive cells with 30 or 50 µM ellagic acid compared with control cells. Table II. Ellagic acid-induced effects on cell cycle after 96 hours. A number of 3x106 cells were seeded in 100 mm culture dishes and treated with nothing (Control),vehicle (DMSO) or 30 µM ellagic acid for 96 hours before harvested, fixated and stained with propidium iodide. The distribution of cells in G0/G1-, S- and G2/M-phase was determined by FACS-analysis. Data are mean values from three to four experiments with duplicate samples. Data are adapted from Paper II, Figure 2. G0/G1 S G2/M

Control 66 % 31 % 3 % DMSO (vehicle) 65 % 32 % 3 % Ellagic acid, 30 µM 69 % 27 % 4 %

Apoptosis The decrease in cell number induced by ellagic acid was shown to be partly mediated by induction of apoptosis. In cells incubated with 30 µM ellagic acid for 96 hours, DNA-degradation was detected by TUNEL-staining (Paper II). In Paper III, dose-dependency was investigated in cells treated with doses ranging from 1-100 µM ellagic acid. The cell morphology was extensively analyzed by microscopy during the initial 30 hours of treatment (Paper III) and then further examined after 36 (Paper IV), 48, 72 and 96 hours (data not shown). The results showed a time course of events with an initial rounded morphology, plasma membrane blebbing, formation of apoptotic bodies, pycnotic irregular cell morphology and finally detachment and fragmentation of the cells (Paper III, Figure 1B and Table I). The effect was dose-dependent, and results were in accordance with the data presented on reduced number of adherent cells presented above. No or limited effects were seen with 1-10 µM ellagic acid but with increased doses, gradually increasing visual signs of apoptosis occurred. This is illustrated in Figure 8. The effects were also time-dependent as all visual signs of apoptosis became more pronounced with increasing time (48, 72 or 96 hours, data not shown) for all doses of ellagic acid except for 100 µM where the effect again was very pronounced with most of the cells detached and degraded already after 24-48 hours. In agreement with an induction of cell death, ellagic acid also caused a dose-dependent increase of LDH-release from the cells, indicative of cell damage (by apoptosis and/or necrosis) (Paper III). Steadily higher LDH-release was also monitored after 48, 72 and 96

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hours, confirming a time-dependency of the ellagic acid-induced cell damage (not shown). SubG1-analysis showed a dose-dependent ellagic acid-induced DNA-degradation (Paper III). The 50 µM concentration of ellagic acid, close to an EC50-dose with respect to effect on apoptosis (this was supported by results from Paper IV showing ellagic acid-induced LDH-release and DNA-degradation) was chosen for further analysis of apoptotic pathways.

Figure 8. Ellagic acid-induced effects on apoptosis. Ellagic acid induced morphological changes associated with apoptosis. Cells were incubated with A: nothing (Control), B: Vehicle (NaOH), C: 1 µM ellagic acid, D: 10 µM ellagic acid, D: 50 µM ellagic acid, or E: 100 µM ellagic acid for 24 ± 2 hours. The cells were examined in a Leica DMI 6000B inverted microscope and photographed. Rounded cells are indicated by white arrows and plasma membrane blebbing is indicated by black arrows. Data are representative pictures from one of three experiments with duplicate samples. The figure is adapted from Paper III, Figure 1 and Table I. The mechanism of ellagic acid-dependent apoptosis was shown to be partly mediated by the intrinsic, mitochondrial pathway (Paper III). This was evidenced by decrase of mitochondrial membrane potential (ΔΨm) and cleavage and activation of caspase-3 and -9. The ellagic acid-induced decrease of ΔΨm was shown to be dose- and time-dependent. Again, no alterations were seen with 1 µM ellagic acid, while 50 and 100 µM strongly increased the relative amount of cells with low ΔΨm and concomitantly decreased the relative amount of cells with intact ΔΨm. The proportion of cells with low ΔΨm was significantly increased already after 6 hours, and further increased after 24 and 48 hours of 50 µM ellagic acid-treatment. Similarly, activating cleavage of caspase-3 and -9 were dose-dependent. No effects on caspases were seen after treatment with 1 µM ellagic acid for up to 12 hours, but with 50 and 100 µM, both capase-3 and -9 were proteolytically cleaved to form their catalytically active forms (Paper III, Figure 4). The specific caspase-9 inhibitor z-LEDH-fmk almost completely blocked cleavage of caspase-3 but only partly inhibited the ellagic acid-induced DNA-degradation suggesting that other pathways are also involved in ellagic acid-induced apoptosis in SH-SY5Y cells. Antibodies against intact and cleaved capase-8 were tested, but protein

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expression could not be detected (data not shown). Data from Paper IV showed that the cells expressed the anti-apoptotic protein Bcl-2, and the pro-apoptotic protein Bax, but that the protein levels were not changed (Bcl-2) or only slightly decreased (Bax) after treatment with ellagic acid. The time-course and sequence of the observed events of ellagic acid-induced apoptosis in SH-SY5Y cells is illustrated in Figure 9.

Figure 9. Time-course and sequence of observed events during ellagic acid-induced apoptosis. Data are adapted from Paper III, Figure 1, 2, 3 and 4.

Cell adhesion The observed decrease in proliferative activity and induction of apoptosis discussed above could not alone explain the strong negative effect ellagic acid had on the number of recovered adherent cells, and further analyses reveled that cell detachment was an important effect of ellagic acid-dependent decrease of cell numbers. This was originally indicated by data in paper II (illustrated below in Figure 10) and analyzed in greater detail in Paper IV. When cells were treated 50 µM ellagic acid for 36 hours before detached and adherent cells were counted, 43 % of the cells were in the detached fraction compared with 17 % in control cultures (Paper IV). The viability of the detached cells, judged by trypan blue staining, was low, and the viability of the remaining adherent cells were also shown to be decreased compared with cells treated with vehicle only. Notably, after 36 hours with 50 µM ellagic acid, the relative number of trypan-blue positive cells in the detached fraction was lower (77 %) compared with control cultures (84%). This suggested that different cell death mechanisms were activated i.e. cell death prior to detachment, and possibly also anoikis. A significant increase in number of detached cells was also seen already after 24 hours, and was further seen at 36, 48 and 96 hours (Papers II and IV).

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Figure 10. Ellagic acid-induced effects on cell detachment (A) and viability (B). A number of 105 cells were seeded in 60 mm culture dishes and were treated with nothing (Control), vehicle (DMSO) or 30 µM ellagic acid for 96 hours before detached and adherent cells were harvested, stained with trypan blue and counted in a Bürker chamber. Panel (A) shows relative cell number, related to total cell number in Control (set to 100 %). Panel (B) shows cell viability determined by trypan blue exclusion. Data are mean (± SEM) values from two experiments with duplicate samples. * indicates statistical significance (p< 0.05). The figure is adapted from Paper II, Figure 3.

Differentiation-associated events

Differentiation with TPA or ATRA Treatment with TPA and ATRA induces morphological changes of the SH-SY5Y cells, i.e. outgrowth of long, thin processes, so called neurites, and altered shape of the cell bodies. Here, morphological differentiation was defined as outgrowth of neurites longer than two cell body lengths. Differentiation with TPA induced formation of relatively short, multi-branched neurites with varicosities and extensive growth cones, while differentiation with ATRA induced outgrowth of longer, thinner processes with less extended growth cones. This is illustrated in Figure 11. The effect of TPA on morphology was gradual and increased over four to eight days while the effect of ATRA was more rapid. (Paper I). Similar morphological changes were seen in Paper II and IV when cells were differentiated with TPA and ATRA for seven days prior to treatment with ellagic acid. A concurrent increase in expression of mRNA for different neuronal marker genes was seen. In accordance with previously published reports, the catecholaminergic

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markers TH and NPY and cholinergic markers VAChT and VIP were increased with TPA and ATRA respectively [226, 227, 232], (Paper I).

Figure 11. Morphological differentiation of SH-SY5Y cells with TPA or ATRA. TPA and ATRA induced morphological changes associated with differentiation. A number of 105 cells were seeded in 60 mm culture dishes and were incubated with nothing (Non-differentiated, left panel), 16 nM TPA (TPA-differentiated, middle panel) or 1 µM ATRA (ATRA-differentiated, right panel) for eight days. The cells were examined in a Leica DMI 6000B inverted microscope and photographed at 20 and 100 x magnification. Data are representative pictures from multiple (> 5) experiments with duplicate samples. The figure is adapted from data presented in Paper I and IV.

Cell number The cell number was significantly decreased in both TPA- and ATRA-differentiated cultures, and ATRA had a more pronounced effect compared with TPA. This was evidenced when cells were counted every day during eight days of treatment (Paper 1), and is illustrated in Figure 12. This was further confirmed in Paper II and IV, where cells were differentiated with TPA or ATRA before treatment with ellagic acid. Cell loss by detachment from the culture dishes was taken into consideration, but results were repeatedly negative for this for both TPA and ATRA (not shown). On the contrary, cell detachment seemed to be less frequent in cultures treated with TPA and ATRA, suggesting increased adhesion in differentiated cells. The relatively few detached cells in these cultures were to a

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lesser extent stained with trypan blue compared with non-differentiated cells (Paper II and IV). This was supported by data from TUNEL- and SubG1-analysis where TPA- and ATRA-differentiated cells showed lower relative number of both TUNEL- and SubG1-events compared with non-differentiated cells (Paper IV). This could possibly be explained by an increased expression of the anti-apoptotic protein Bcl-2 and no changes in the pro-apoptotic protein Bax seen with TPA and ATRA (Paper IV), and will be further discussed below.

Figure 12. Differentiation-induced effects on cell number. TPA and ATRA decreases the number of cells in cultures of SH-SY5Y. A number of 3 x 105 cells were seeded in 100 mm culture dishes as were treated with nothing (open circles), 16 nM TPA (closed circles) or 1 µM ATRA (open squares) added every fourth day as indicated by the arrows. Every day during eight days, adherent cells were harvested and counted in a Bürker chamber. Data are mean (± SEM) values from two experiments with duplicate samples. The figure is presented in Paper I (Paper I, Fig 1 B).

Proliferation and cell cycle The decrease in cell number seen with TPA or ATRA was associated with reduced proliferative activity or cell cycle arrest respectively. This is illustrated below in Figure 13. When cell cycle activity was analyzed in cultures differentiated for eight days it became apparent that with TPA, no alterations in BrdU-incorporation were seen after a short-time BrdU-chase (15 minutes), and no alterations in cell cycle distribution was seen compared with control cultures. When analyzed by BrdU using longer time chases (2, 24 and 48 hours) it was evident that the relative number of BrdU-positive cells was decreased in TPA-treated cultures, suggesting reducd proliferative activity in this cultures rather than a cell cycle arrest (Paper I).

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In the case of ATRA, decreased BrdU-incorporation was seen with both short (15 minutes) and longer (2, 24 or 48 hours) BrdU-chases and cell cycle analysis with FACS showed an arrest in G0/G1 (Paper I).

Figure 13. Differentiation-induced effects on proliferation and cell cycle. Effect of TPA and ATRA

on cell cycle activity. (A) A number of 105 cells were seeded in 60 mm culture dishes and were treated with nothing, 16 nM TPA or 1 µM ATRA for eight days. The cultures were incubated with 1 µg/ml BrdU-labeling reagent for 15 minutes prior to fixation and immunocytochemical staining. The relative number of

BrdU-positive cells was determined by bright field microscopy. (B) A number of 3 x105 cells were seeded in 100 mm culture dishes and were treated with nothing, 16 nM TPA or 1 µM ATRA for eight days. Adherent cells were harvested, stained with Hoechst 33258 and the cell cycle distribution of cells was determined by flow cytometric analysis. Data are mean (± SEM) values from two experiments with triplicate samples (A) or three experiments with duplicate samples (B). * indicates significance (p < 0.05). The figure is presented in Paper I (Paper I, Figure 4).

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Relationship between differentiation and proliferation Single-cell analysis revealed that in TPA-treated cultures, morphological differentiation (shown as neurite outgrowth and increased expression of mRNA for neuronal marker genes TH and NPY) was distinct from proliferation (Paper I). This was evident since a relatively large number of morphologically differentiated cells were positive for BrdU-incorporation, hence retained their proliferative activity. In contrast, in the ATRA-treated cultures there was a clear correlation between the observed morphological changes and increased expression of neuronal markers VAChT and VIP and growth arrest, and with a large majority of cells morphologically differentiated and negative for BrdU-incorporation as reveled by the single-cell analyses. This finding further supports previous data on that TPA and ATRA differentiates SH-SY5Y cells into morphologically and functionally different phenotypes, and also suggest that in this cells, morphological differentiation (i.e. outgrowth of neurites) does not always correlate with attenuated proliferation. This is illustrated in Figure 14.

Figure 14. Relationship between differentiation and proliferation. A number of 105 cells were seeded in 60 mm culture dishes respectively and were treated with nothing (Control), 16 nM TPA or 1 µM ATRA for eight days. The cultures were incubated with 1 µg/ml BrdU-labeling reagent for 2 hours prior to fixation and immunocytochemical staining. The relative number of neurite bearing cells versus BrdU-positive cells was determined by bright field microscopy. Data are mean (± SEM) values from two experiments with at least duplicate samples. The figure is presented in Paper I (Paper I, Figure 7).

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Effects of ellagic acid in differentiated SH-SY5Y As described above, ellagic acid induced reduced cell numbers, inhibited cell cycle activity, and induced cell detachment and apoptosis in SH-SY5Y. To further explore this, and to investigate whether differentiation alters the sensitivity to ellagic acid with respect to the above described cellular effects, the outcome of ellagic acid-treatment was analyzed in TPA- and ATRA-differentiated cultures.

Cell number Differentiation with TPA or ATRA lowered the sensitivity to ellagic acid-induced decrease of cell number (Paper IV). This is illustrated in Figure 15. As described above, ellagic acid strongly decreased the number of adherent cells, increased the number of detached cells and slightly lowered the total cell number in cultures of non-differentiated SH-SY5Y (Papers II, III and IV). When cells were differentiated with TPA for seven days before treatment with 50 µM ellagic acid for 36 hours, the number of adherent cells as well as the total cell number was significantly decreased, but no large increase was seen in the number of detached cells. In ATRA-differentiated cultures, neither the number of adherent cells, detached cells, nor total cell number was significantly changed after treatment with ellagic acid. In non-differentiated and TPA- and ATRA-differentiated cells the XTT-signal was decreased with ellagic acid, and LDH-release was concomitantly increased in non-differentiated and TPA-differentiated cells indicating cell death. When cells were differentiated with ATRA and treated with ellagic acid for 48 hours, the number of adherent cells, detached cells and total cell numbers were all slightly reduced (Paper II).

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Figure 15. Ellagic acid-induced effects on cell number and adhesion in differentiated cells. Cells were differentiated with 16 nM TPA and 1 µM ATRA or cultured in regular medium for seven days prior to incubation with vehicle (-) or 50 µM ellagic acid (+) for 36 hours. Detached and adherent cells were collected and stained with trypan blue and counted in a Bürker chamber. Grey bars depict data on adherent cells, open bars depict data on detached cells. The numerical data presented below the bars represents the fractional (number of adherent and detached out of total) distribution of the cells (%), and the relative number of trypan blue positive cells (%). Data are mean (± SEM) values from four experiments with duplicate samples (ATRA-data for detached cells are from three experiments with duplicate samples). Symbols a-d indicates significance, p < 0.05, of the following: (a) effect of differentiation on total cell number, effect of ellagic acid on(b) total cell number of each phenotype, or (c) number of detached cells, or (d) number of adherent cells. n.s. = non significant. The figure is presented in Paper IV (Paper IV, Figure 1)

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Cell adhesion Differentiation with TPA and ATRA lowered the sensitivity to ellagic acid-induced cell detachment (Paper IV). As presented above, in non-differentiated cells, ellagic acid strongly increased the number of detached cells. This was evident in Paper II, III and IV, and is illustrated in Figure 10 and Figure 15 above. In ellagic acid-treated cultures, the relative number of trypan blue-positive cells was increased compared with vehicle-only in the adherent fraction of cells, and decreased in the detached fraction. When cells were differentiated with TPA or ATRA for seven days before treatment with 50 µM ellagic acid for 24 hours, only low levels of cell detachment were seen, thus treatment with TPA or ATRA lowered the cells sensitivity to ellagic acid-induced cell detachment. In TPA-treated cultures, the total cell number was decreased after treatment with ellagic acid, suggesting a possibility that cell detachment was present, but that the cells had been degraded before counting occurred. This however was unlikely, since the relative number of trypan blue-positive cells was low in TPA- and ATRA-treated cells compared with non-differentiated cells indicating resistance against cell death after treatment with TPA or ATRA. The decreased sensitivity to ellagic acid-induced detachment in at least ATRA-differentiated cells could possibly be explained by increased adhesion to ECM-components [233].

Proliferation and cell cycle analysis The effect of ellagic acid on cell cycle activity in differentiated cells was tested only once (data not shown). More experiments are needed for any conclusions on this. It has been discussed if ellagic acid and other polyphenols are more active against proliferating cells, and in Paper IV, the effect of ellagic acid in SH-SY5Y cells growth deprived by reduced serum levels was analyzed. The results showed that the growth-deprived cells were less sensitive to ellagic acid-induced cell detachment. This observation suggested that ellagic acid is possibly targeting proliferative active cells, and that reduced proliferation and/or growth arrest could contribute to the lower sensitivity to ellagic acid-induced effects seen in differentiated cells.

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Apoptosis Differentiation with TPA or ATRA also decreased the cells sensitivity to ellagic acid-induced apoptosis (Paper IV). Both TPA- and ATRA-differentiated cells showed lower degree of ellagic acid-induced DNA-degradation demonstrated by TUNEL- and SubG1-analysis, compared with non-differentiated cells. This is illustrated below in Figure 16. The expression of the anti-apoptotic protein Bcl-2 was increased 2-fold with both TPA and ATRA, and treatment with ellagic acid did not alter the expression of Bcl-2 in these cells. This suggested that Bcl-2 possibly is involved in the decreased sensitivity to ellagic acid-induced apoptosis in differentiated cells. The expression of the pro-apoptoic protein Bax was not changed with TPA- or ATRA-differentiation and also did not change after treatment with ellagic acid for 24 hours (Paper IV).

Figure 16. Ellagic acid-induced effects on apoptosis in differentiated cells. A number of 3 x 105 cells were seeded in 100 mm culture dishes and incubated with medium (control), 16 nM TPA or 1 µM ATRA for 7 days prior to incubation with vehicle (-) or 50 µM ellagic acid (+) for 24 hours. The cell culture media were discarded and adherent cells harvested and subjected to TdT labeling (TUNEL), propidium iodide staining (SubG1) and flow cytometric analysis on a FACScan flow cytometer. For each sample, a total number of 20.000 events were monitored. Panel A shows TUNEL-data, panel B shows SubG1-data. Data are from one experiment with duplicate samples. Symbols a and b indicates significance, p < 0.05 for (a) effect of differentiation, or (b) effect of ellagic acid on each phenotype. The figure is presented in Paper IV (Paper IV, Figure 3).

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Discussion

The aim of this study was to investigate the effect of the polyphenol ellagic acid in non-differentiated and TPA- and ATRA-differentiated human NB cells. The data show that in non-differentiated SH-SY5Y cells, ellagic acid reduced the cell number, inhibited cell cycle activity, and induced cell detachment and apoptosis. The apoptosis was shown to be partly mediated by the intrinsic apoptosis pathway. TPA and ATRA both induced morphological differentiation, while only the latter induced a more pronounced G0/G1-arrest. The results also show that TPA-treated cells remained actively cell cycling despite undergoing neuritogenesis while these two read-outs were mutually exclusive in ATRA-differentiated cells. Both TPA- and especially ATRA-differentiated cells showed lower sensitivity to ellagic acid-dependent cell detachment and apoptosis. The results from cell counting and analysis of proliferation and cell cycle with BrdU and FACS show that ellagic acid reduced the cell numbers and inhibited cell cycle activity in SH-SY5Y. Thus, our herein reported data on cells of neuronal origin agree with the observations made by others describing similar effects on cell number, proliferation and cell cycle in in vitro cultures of cells of epithelial origin [43, 45, 46], Our findings thus suggests that the reported effects of ellagic acid are more general and not cell type-specific. Our studies of ellagic acid-related cell morphology events and analysis of DNA-degradation by FACS show that the observed reduction in number of adherent cells was to some extent explained by induction of apoptosis. Analysis of mitochondrial membrane potential and expression of apoptosis-regulating proteins (i.e. caspases) reveal that the ellagic acid-induced apoptosis is partly mediated by activation of the intrinsic pathway mechanism including mitochondrial events and cleavage and activation of capase-9 and -3. The statues of intact and cleaved capase-8 was not analysed, but published data from others show that neither undifferentiated nor RA/BDNF-differentiated SH-SY5Y cells express caspase-8 protein [234, 235]. Again, our reported data agree with observations on carcinoma cells [41-43, 45, 46], suggesting similar responses to ellagic acid in cells of neuronal and epithelial origin.

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Of the tested doses, significant effects on cell number, proliferation, cell cycle and apoptosis were seen after treatment with concentrations between 30 and 100 µM. These results are consistent with two previous reports where the IC50 for cytotoxicity of ellagic acid in in vitro cultures of tumor cells was calculated to be 78.5 µM and 33.7 µM respectively [47, 236]. A significant part of the in our studies observed reduction in numbers of adherent cell after treatment with ellagic acid is explained by increased cell detachment. Data on trypan blue-exclusion showed that in non-differentiated (and ATRA-differentiated) ellagic acid-treated cultures, the relative amount of the detached cells that was viable (excluding trypan blue) even after 24 hours, remained almost the same or increased. These data suggests that the cells that detach from the culture dishes do not undergo immediate detachment-induced apoptosis (anoikis). Reduced adhesion and adhesion-dependence are important cellular features during tumor progression, where cells able to avoid anoikis have a potential to migrate and initiate metastasis. If these kinds of alterations in cellular characteristics are promoted by treatment with ellagic acid, the use of this compound and similar substances in cancer treatment could possibly be fatal. Studies on ellagic acid-treatment during extended periods of time and preferably also in in vivo animal-models are needed to further investigate this. None of the previously published reports from cells of epithelial origin specifically addresses this issue or discusses cell detachment as a possible mechanism behind the reduction in cell numbers seen with ellagic acid. Thus from available data we cannot draw conclusions on whether decreased cell adhesion is a more general cellular effect induced by ellagic acid. Neuroblastoma tumors consist of transformed neuronal cells with different degrees of differentiation and maturity and stromal components. Our model system SH-SY5Y cells can be successfully differentiated in vitro by several agents including TPA and ATRA, and during the differentiation a number of cellular characteristics are altered, e.g. expression of differentiation maker genes, the proliferation rate and expression of CAMs. In vitro differentiation of SH-SY5Y cells has been reported to alter the cells sensitivity to different chemical and physical insults including chemotherapeutically used agents [230, 237-239], γ-radiation [239] the cytokine TNF-α and hypoxia [240]. Shortly after the study

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presented in paper II had been published, Das et al. [221] reported that SH-SY5Y cells in vitro differentiated with ATRA (or one of two other tested retinoids) for seven days were more susceptible to apoptosis induced by the polyphenols (-)epigallocatechin-3-gallate (EGCG) and genistein (GST). Taken together, the available data suggested further investigations analysing the effect of ellagic acid in TPA- and ATRA-differentiated SH-SY5Y cells compared with non-differentiated cells. The optimal conditions with respect to dose and time to induce morphological differentiation and reduced proliferation in SH-SY5Y cells in response to TPA and ATRA has been thoroughly investigated by others, hence the experimental conditions were chosen in agreement with previous reports [53-54, 65-66, 119, 116-117]. Morphological examination, measurements of neurite length and analyses of mRNA for neuronal marker genes showed that again both TPA and ATRA induce morphological differentiation in SH-SY5Y. Analysis of cell numbers, proliferation and cell cycle by cell counting, BrdU and FACS reveiled that while both TPA and ATRA reduces cell numbers and proliferation, only ATRA induced a more complete G0/G1-arrest. Furthermore, single-cell analyses of BrdU-stained cells showed that TPA-treated cells remained cell cycling during neuritogenesis while these two read-outs were mutually exclusive in ATRA-differentiated cultures. Thus while almost all the ATRA-treated cells that incorporated BrdU showed no signs of differentiation, the majority of the non-proliferating cells were morphologically differentiated, suggesting a possibly more mature neuronal phenotype induced by ATRA compared with TPA in SH-SY5Y. Interestingly, a fraction of the ATRA-treated cells (about 20 %) were BrdU-positive. Also, data from FACS-analyses indicated that a fraction of the cells did not respond to ATRA by either entering G0/G1 arrest or by any signs of morphological differentiation, suggesting a sub-

population of ATRA-refractile cells. Our here presented results showed that TPA- and ATRA-differentiated cells seemed to have an altered sensitivity to ellagic acid-induced detachment, DNA-degradation and cell death. A protective effect of ATRA-treatment was more pronounced compared with TPA-related effects. In the ATRA-differentiated cultures both cell loss, cell detachment and decrease in cell viability was less pronounced compared with the ellagic acid-induced effects seen in non-

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differentiated cultures. In contrast, in the TPA-differentiated cultures we identified a significant ellagic acid-dependent decrease in cell number and the data also suggests that the detached cells are more vulnerable to undergo ellagic acid-induced apoptosis or other cell destructing mechanisms compared with non-differentiated cultures. During treatment with TPA the SH-SY5Y cells show an altered shape and substrate adhesion of the cell bodies compared with control cultures and that is especially visible just after addition of TPA and media changes. This is previously reported by others [198]. Measurement of viability by trypan blue staining however indicates that the cells that detach from the dishes in TPA-treated cultures (and ATRA) are less susceptible to cell death compared with detached cells from control dishes. TPA and ATRA induce increased protein expression of anti-apoptotic Bcl-2, and this can possibly explain the observed increase in cell survival and is in agreement with previous reports from others [179, 241]. Ellagic acid does not alter the expression of the pro-apoptotic Bax or the expression of Bcl-2 protein in non-differentiated cells. Published scientific reports on cellular effects of ellagic acid may suggest a higher sensitivity to cytostatic and cytotoxic effects of ellagic acid in proliferating cancer cells compared with non-transformed cells and that this may be related to a more selective effect on proliferating cells. In cultures of SH-SY5Y that were cultured under low-serum conditions and thereby proliferation-inhibited [242], we saw a decrease in ellagic acid-induced cell detachment, a finding supporting this theory. Furthermore, in experiments with BrdU-incorporation, ellagic acid reduced not only the number of cells remaining attached to the dishes (not shown), but at the same time the relative number of BrdU-positive cells amongst the remaining cells after a 24h treatment. This was not explained by an ellagic acid-dependent cell cycle arrest at this early time-point but rather the data strongly suggest that ellagic acid selectively hits on and affects actively proliferating cells. So, can ellagic acid possibly be used in combinatory or complementary treatment of NB? To answer this question, several other questions have to be answered first. One very important issue raised by our data is if ellagic acid induces cell detachment in NB cells, and that a fraction of these non-adhering cells are viable even though they have lost their adherence to surface. Are these cells resistant to

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anoikis, and capable to migrate and re-attach to ECM? Or will they subsequently undergo apoptosis and be eliminated? If the detached cells survive and are able to grow independent of substrate adherence, ellagic acid is probably not suited for NB treatment. Another important issue raised by these studies is if there is a fraction of cells unresponsive to ellagic acid? When cells were treated with ellagic for 96 hours and then cultured in normal media, proliferation was relatively quickly (about 3-4 days, data not showed) back to normal rates and this indicates that some cells also will survive ellagic acid treatment and retain their ability to proliferate. These are important questions that have to be answered in the context of evaluating ellagic acid as a potential therapeutic compound.

Summary and Conclusions

The main findings of this study is that in non-differentiated SH-SY5Y cells, ellagic acid induces reduced cell number and cell cycle activity and cell detachment and apoptosis. The apoptosis was shown to be partly mediated by the intrinsic apoptosis pathway. SH-SY5Y cells become morphologically differentiated by both TPA- and ATRA-treatment, and the morphological event are accompanied by decreased cell numbers, decreased proliferative activity, and in the case of ATRA cell cycle arrest in G0/G1. While TPA-differentiated cells retain their proliferative capacity during differentiation, ATRA-treated cells demonstrates a close correlation between morphological differentiation and reduced proliferation at single cell level. TPA- and ATRA-differentiated cells were shown to be less sensitive to ellagic acid-induced cell detachment and apoptosis. This is summarized in Figure 17.

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Figure 17. Summary of the data presented in this thesis. Differentiation of SH-SY5Y cells with TPA and ATRA induces formation of neurites, expression of mRNA of differentiation marker genes, reduced cell number and reduced proliferative activity. The effect of ATRA is more pronounced than the effects of TPA (Panel A). Treatment of non-differentiated cultures of SH-SY5Y with ellagic acid gives reduced cell numbers, reduced cell cycle activity, induction of apoptosis and loss of adhesion. Differentiation with TPA or ATRA alters the sensitivity to ellagic acid (Panel B).

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Future perspectives

When exploration of the possibility to use ellagic acid and other polyphenols as inhibitors of cancer cells in vitro was started, ellagic acid looked very promising as a future alternative for combinatory treatment for cancer. With increasing amount of available data on low bioavailability of polyphenols and rapid degradation of the bioactive metabolites being identified, the idea of peroral administration of polyphenols for cancer treatment has become less attractive. Currently, local delivery systems with chitosan/ellagic acid polymer films or gels for potential delivery of ellagic acid directly at tumor site are being developed as well as nanoparticles designed with the same aim [114-116]. In this study, effects of ellagic acid were investigated in SH-SY5Y cells cultured in a monolayer. Often, studies of different chemicals with a potential activity in vitro have revealed different or lower effects in in vivo tumor models and more complex in vivo systems [5]. SH-SY5Y cells can be cultured as spheroids, a vitro 3-D cell system resembling NB tumor models. In this kind of system cell-cell adhesion and exchange between cells more resembles the situation in a native NB tumor [185]. One interesting aspect would be to further study effects of ellagic acid on cell-cell adhesion and viability in SH-SY5Y cells cultured as spheroids, as well as expression and functionality of CAMs in SH-SY5Y in both monolayers and 3D-models. Hopefully, this would give us a better understanding of the cellular and molecular target mechanisms of ellagic acid-dependent cell detachment and apoptosis and hence better insight in the potential role of ellagic acid in anti-tumorigenic treatment. More detailed molecular explanations for the ellagic-induced effects in NB are not yet addressed. The general knowledge of the mechanisms behind the biological effect of ellagic acid and related polyphenols are however rapidly growing. The PKB/Akt signaling pathway has been mentioned in recent reports [77, 243]. Neuroblastoma cells are dependent of functioning PI3K and PKB/Akt-activity for cell cycle progression, differentiation and survival and these signaling molecules are dependent of functioning cell adherence as well as functioning receptor signaling. Studies of the protein expression and phosphorylation levels of PKB/Akt (e.g. by western blot) after ellagic acid treatment of non-differentiated and TPA- and ATRA-differentiated SH-SY5Y could possibly add valuable data to the discussion

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of the herein described data. NB cells containing MYCN amplification have been shown to be more sensitive to growth inhibition by inhibitors of PKB/Akt compared with cell lines containing no MYCN amplification [244], hence the effect of ellagic acid in MYCN-amplified cells (e.g. IMR-32 or LAN-5) could also add valuable data to the discussion. The intracellular concentration of Ca2+ is important for cellular signaling. As polyphenols are known chelators of divalent cations, the effect of ellagic acid on intracellular Ca2+ would be interesting to investigate. Here, differentiation with TPA and ATRA was seen to protect the cells from ellagic acid-induced detachment and apoptosis, however total cell number was slightly decreased in both non-differentiated, TPA- or ATRA-differentiated cultures after treatment with ellagic acid. As TPA- and ATRA-differentiated cultures includes cells of various degree of morphologically and functionally differentiated cells, an interesting question is which sub fraction of the cells that are protected and not protected against ellagic acid. In both TPA- and ATRA-treated cultures there are still some non-differentiated cells after seven days, is it this population of cells that are responsive to ellagic acid? This could be further explored on single-cell level with microscopy and/or flow cytometry. Several different research fields are and will in the future be interested in ellagic acid and other polyphenols. Some important questions are how crops can be modified to contain more polyphenols, how extraction and purification of these compounds can be standardized and improved, how bioavailability and uptake of the substances can best be monitored, and how can it be increased. Another important question is whether valuable polyphenols can be extracted from materials and substances today seen as waste products (e.g. food waste and waste products from the forest industry). According to information found on the internet, ellagic acid can cure or prevent any health problem from “dull smokers’ skin” to “the development of life-threatening cells”. The ambition is that the findings presented in thesis add scientifically sound and evidence-based knowledge about ellagic acid.

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Acknowledgements

I would like to express my deepest gratitude to the following persons, who in one way or another have contributed to this work. My supervisor Professor Eewa Nånberg for that you have given me the opportunity to participate in this project. Thank you for sharing your knowledge and experience, for all your support and for never losing faith in me. My co-supervisor Associate Professor Birg itta Sundström for all your support, for new ideas and for sometimes seeing things from a different angle. Special thanks for support with the FACS, and in the writing-process. My co-author Filip Rendel for the work your contribution to Paper I and IV. I wish you all luck with your future work! My co-authors, former project workers Menglei Ding and Quili Liang. It was nice to work with you! All other former project workers who in one way or another participated in the project. Special thanks to Maria Lidh och Mikaela Hoffman who participated at the very beginninig and Evelin Sjöö who contributed in the startup of what became Paper III. Lars Renman for help with statistics, especially the ANOVA-calculations. Trond Stokke og Kirsti Solberg Landsverk at Department of Radiation Biology at Radiumhospitalet in Oslo for kindly giving me permission to use your LSR II flow cytometer. All former and present PhD-students that I have worked with during my 6 (± 2) years as a PhD-student, especially Sofia Karlsson, Cecilia Roos, Rozbeh ”Robban” Jafari, Ola Olsson and Alexander Engström for sharing good (and worse) times in the lab. After all these years, there are a too many of you to mention. I thank you all for the fun we have had. (Maybe we are all just salmons in Lake Vänern). An extra thanks to Ola and “Robban” for handling toxic chemicals when I was pregnant and Filip for counting an endless numbers of cells this and last fall. All former and present colleges at the (now former) department of Chemistry and Biomedical Sciences for creating a nice atmosphere. Special thanks to engineers Elisabeth Björk and Micke Andersén for technical help.

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Det finns även några personer utanför akademin som förtjänar ett tack. Stina Andersson och Hannah Heidkamp för god mat och goda samtal. Eli-Anne og Karianne Marthinsen a.k.a. to tredjedeler av «Tresset» som alltid er der uansett om det er en uke eller ett år siden sist. ”Tonsenhagen Hotell” (som rakk å flytte underveis) for kost og logi ved besök i Oslo. Der det er hjerterom er det husrom! Ann Mari, du har alltid vært et forbilde, og har lært meg noe viktig: om man virkelig ønsker seg noe så får man det. Mamma og pappa for at dere alltid støtter og stiller opp, uansett. Ekstra takk for at dere har kommet og passet barna i flere omganger den siste intensive tiden. Benny och Gittan och övriga Alfredssons, för att ni alltid ställer upp och finns där. Ni är guld värda! Jimmy, för att du alltid lyssnat och stöttat mig i alla perioder av denna resa. (Att den här avhandlingen nu är klar är nog skönt för dig med.) Elliot och Lova, ni kommer alltid vara nummer ett. ♥

THANK YOU, TACK, TAKK!

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Grant support

This work was financially supported by The County Council of Värmland, The Swedish Childrens Cancer Society and The Royal Swedish Academy of Sciences.

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Effects of ellagic acid in human neuroblastoma cells

A diet rich in polyphenols has been proposed to have beneficial health effects and to reduce risk of disease. Ellagic acid, a polyphenol common in red berries and pomegranates, has potential anti-tumorigenic effects that make it interesting to further study in different cancer cell systems.

Neuroblastoma is a childhood cancer that arises during development of the peripheral nervous system. Neuroblastoma is a heterogenic tumor disease, and highly malignant neuroblastomas are difficult to treat despite different treatment modalities, identifying a need for new and combinatory treatments.

Here, the effect of ellagic acid in non-differentiated and in vitro-differentiated human neuroblastoma SH-SY5Y cells were studied with the aim of identifying cellular target mechanisms and a possible therapeutic potential for ellagic acid. In non-differentiated cells, ellagic acid reduced cell number, inhibited cell cycle activity, and induced cell detachment and apoptosis. Cells differentiated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate or the vitamin A- derivative all-trans retinoic acid showed lower sensitivity to ellagic acid-dependent cellular effects, a finding with interesting future implications.

DISSERTATION | Karlstad University Studies | 2013:48

ISSN 1403-8099

ISBN 978-91-7063-526-7

urn:nbn:se:kau:diva-29905