- 1 -

“Ιf you do not change direction you may end up where you are heading. “

Lao Tzu

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Thesis Title (in English):

“Investigation into the biological properties of Origanum onites essential oil against colon carcinoma growth”

Τίτλος Πτυχιακής Εργασίας (στα Ελληνικά):

“Διερεύνηση της βιολογικής δράσης αιθέριου ελαίου Origanum onites στην ανάπτυξη καρκινώματος παχέος εντέρου”

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Ευχαριστίες (Acknowledgments)

Με την ολοκλήρωση της παρούσας διπλωματικής ολοκληρώνεται ο κύκλος των προπτυχιακών μου σπουδών. Τα τέσσερα αυτά χρόνια αποτέλεσαν μια συναρπαστική διαδρομή, γεμάτη όμορφες αναμνήσεις και εμπειρίες, οι οποίες συνέβαλαν στην διαμόρφωση και στην ωρίμανση μου σαν άνθρωπο και σαν επιστήμονα, και για αυτό νιώθω την ανάγκη να ευχαριστήσω όσους συνέβαλαν σε μικρό ή μεγάλο βαθμό σε αυτή την πορεία.

Η παρούσα διπλωματική εργασία πραγματοποιήθηκε στο εργαστήριο Μοριακής Ανοσοβιολογίας, του Τμήματος Μοριακής Βιολογίας και Γενετικής του Δημοκρίτειου Πανεπιστημίου Θράκης, υπό την επίβλεψη της Επίκουρης καθηγήτριας κ. Αικατερίνης Χλίχλια. Θα ήθελα λοιπόν, να της απευθύνω θερμές για την εμπιστοσύνη και την πολύτιμη υποστήριξη που μου έδειξε.

Στα πλαίσια της διπλωματικής μου μου δόθηκε η ευκαιρία να συνεργαστώ με εξαιρετικούς ανθρώπους, σε ένα ευχάριστο και συνεργάσιμο περιβάλλον. Πρώτα από όλα, θα ήθελα να ευχαριστήσω την υποψήφια διδάκτωρ, Σπυριδοπούλου Κατερίνα, τον μεταπτυχιακό φοιτητή Αϊνδελή Γεώργιο και την Τιπτιρή-Κουρπέτη Αγγελική για την καθοδήγηση, την εκμάθηση, την υπομονή και την άμεση βοήθεια που μου παρείχαν. Ακόμη, αισθάνομαι ιδιαίτερη ανάγκη να ευχαριστήσω όλα τα παιδιά του εργαστηρίου, την Τομπουλίδου Ευγενία, Θεοφανίδου Θεοδώρα, Κουκουλάκη Αικατερίνη, Αραβίδου Ταμάρα, Γεωργαλλή Μαρία για την άψογη συνεργασία και την φιλική διάθεση.

Σημαντική ήταν και η συμβολή των μελών γειτονικών εργαστηρίων με τους οποίους ανέπτυξα σχέσεις και είχα την χαρά να συνεργαστώ. Επίσης, χρωστώ ένα βαθύ ευχαριστώ στην πολύ καλή φίλη Τοκαμάνη Μαρία για την συνεχή συμπαράσταση και τις συμβουλές που μου έχει δώσει.

Θα ήθελα, επίσης, να ευχαριστήσω τους συνεργάτες μας, την Παπαβασιλοπούλου Ελένη για την παροχή του ελαίου Origanum onites και την ανάλυση της σύστασης του, τον Κοτσιανίδη Ιωάννη και την Λαμπριανίδου Ελευθερία για την βοήθεια τους σχετικά με την κυτταρομετρία ροής, και τον Βασιλειάδη Σταύρο για την φροντίδα των μυών (Mus Musculus).

Τίποτα όμως από όλα αυτά δεν θα ήταν εφικτό χωρίς την ανεκτίμητη υποστήριξη των γονιών μου, Μαργαρίτα και Αθανάσιο. Σας ευχαριστώ για τον άνθρωπο που με βοηθήσατε να εξελιχθώ, για την αμέριστη υποστήριξη σας σε κάθε μου βήμα μέχρι σήμερα και τη δυνατότητα που μου παρείχατε για να ολοκληρώσω τις σπουδές μου. Ελπίζω να σας κάνω υπερήφανους.

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Περίληψη (Abstract)

Στο ερευνητικό πεδίο της φαρμακευτικής υπάρχει έντονη η τάση μελέτης

διαφόρων φυτοχημικών για τη διερεύνηση των θεραπευτικών τους

ιδιοτήτων. Το αιθέριο έλαιο που απομονώνεται από το φυτό ρίγανης

Origanum onites L., είδος που συναντάται σε μέρη της Σικελίας ως την

ανατολική Τουρκία, έχει διεγείρει το επιστημονικό ενδιαφέρον έχοντας,

μεταξύ άλλων, αντιπαρασιτική, αντιμυκητιακή και αντιβακτηριακή

δράση έναντι ποικίλων στελεχών. Παρ’ όλα αυτά, οι πιθανές

αντικαρκινικές του ιδιότητες δεν έχουν πλήρως εξεταστεί in vitro, ενώ δεν

υπάρχουν επίσημα προσβάσιμα δεδομένα σε in vivo δοκιμές.

Στη παρούσα μελέτη έγινε μια αρχική εκτίμηση της πιθανής

κυτταροτοξικής επίδρασης του ελαίου έναντι του καρκίνου του παχέος

εντέρου στις δύο κολονικές καρκινικές κυτταρικές σειρές, την CT26,

απομονωμένη από τον Mus musculus και την HT-29, ανθρώπινης

προέλευσης με τη μέθοδο SRB, καταλήγοντας στις τιμές EC50 0.0077±

0.0003 % (v/v) και 0.0106 ± 0.0002 % (v/v), αντίστοιχα. Συμπληρωματικά,

διερευνήθηκε η δυνατότητα επαγωγής της αντι-πολλαπλασιαστικής του

ιδιότητας μέσω του μηχανισμού της απόπτωσης και της αναστολής της

μετακίνησης (διήθησης) στα CT26 κύτταρα. Ακόμη, ελέγχθηκε η

χημειοπροφυλακτική του δράση με την εκ στόματος του χορήγηση με τη

μέθοδο gavage σε BALB/c ποντικούς που δέχτηκαν εκτοπική υποδόρια

ένεση συνγονικών CT26 κυττάρων. Σημειώθηκε μείωση 55,6% του όγκου

των ανεπτυγμένων απομονωμένων όγκων in vivo, ενώ παράλληλα, εξετάστηκαν τα επίπεδα κυτταροκινών σε δείγματα ορού των ζώων.

Καταληκτικά, τέθηκαν οι βάσεις για την μελέτη in vitro και in vivo αντι-

νεοπλασματικής δράσης του αιθέριου ελαίου Origanum onites σε

καρκίνωμα του κόλονος. Ωστόσο, κρίνεται αναγκαίο να συνεχιστεί η

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έρευνα στο συγκεκριμένο χώρο με σκοπό τον προσδιορισμό του

υποκείμενου μηχανισμού δράσης του, αλλά και την αναζήτηση πιθανών

τοξικών παρενεργειών με επίδραση φυσιολογικών κολονικών κυττάρων

και με μακροχρόνια χορήγηση του in vivo. Μελλοντικά, θα ήταν δυνατή η

εφαρμογή του στην αγορά ως πρόσθετο για την παραγωγή

βιολειτουργικών τροφίμων, παρέχοντας έτσι στους καταναλωτές τις

ευεργετικές του ιδιότητες.

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Abstract

Natural plant products have been used throughout human history for various purposes, such as in medicine. Recently, there is a noticeable progress on the discovery of plant-derived compounds playing a pivotal role in treating and/or preventing various ailments, including cancer. The essential oil isolated from the plant Origanum onites L. (OOEO), which is found in parts of

Greece, Turkey and Sicily, has stimulated the scientific interest as it exhibits antiparasitic, antifungal and antibacterial activity against various strains.

However, in vitro observations related to its anticancer and antitumor activity are limited and there are no published data describing in vivo experiments.

In the current work, we investigated the OOEO cytotoxic effect against two colon cancer cell lines; CT26, isolated from Mus musculus and HT-29, of human origin using the SRB assay. The EC50 values are 0.0077± 0.0003 % (v/v) and 0.0106 ± 0.0002 % (v/v), for CT26 and HT-29 cells respectively.

Additionally, the induction of apoptotic cell death as well as the inhibition of migration by OOEO were examined on CT26 cells. Moreover, its chemopreventive activity was evaluated after oral administration of oregano oil to BALB/c mice that subsequently were inoculated with syngeneic CT26 cells, resulting in a significant decrease of tumor volume (55,6%) in OOEO- treated mice. In parallel, IL—6 and IL-12 cytokines were detected in mouse serum.

To conclude, the performed preliminary experiments examined the in vitro and in vivo anti-neoplastic effect of Origanum onites essential oil on colon carcinoma. However, further experiments are needed in order to determine the underlying molecular mechanisms and potential side effects after treating normal colon cells with OOEO or long-term in vivo OOEO administration. In

- 7 - future, oregano oil could be a promising health-promoting additive for the production of functional foods.

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CONTENTS

1. INTRODUCTION ...... - 11 -

1.1 Natural Products In Treating Diseases ...... - 12 - 1.1.1 Natural Products From Plants...... - 13 - 1.1.2 The Linkage Of Natural Product Chemistry And Metabolomics For The Drug Discovery ...... - 13 - 1.1.3 Introduction to Essential Oils ...... - 14 -

1.2 Family Lamiaceae - A Large Family Of Aromatic Herbs And Shrubs ...... - 16 - 1.2.1 Introduction to the genus Origanum ...... - 16 - 1.2.1.1. General information about Origanum ...... - 17 - 1.2.1.2 Properties of its members ...... - 18 - 1.2.2 Characteristics of Origanum onites ...... - 20 - 1.2.2.1 Properties of Origanum onites essential oil (OOEO) ...... - 22 - 1.2.2.2 Studies of Origanum onites essential oil (OOEO) against cancer ...... - 23 -

1.3 Colon cancer ...... - 24 - 1.3.1. Colon anatomy ...... - 25 - 1.3.2 The stages of cancer development ...... - 25 - 1.3.3 Factors contributing to colon cancer development ...... - 26 - 1.3.4 Inflammation markers associated with colon cancer...... - 27 -

1.4 Chemoprevention ...... - 28 - 1.4.1 Identified Natural Products for contributing to treating colon cancer ...... - 29 - 1.4.2 The Role Of Essential Oils In Treating Colon Cancer ...... - 30 -

1.5 Purpose ...... - 32 -

2. MATERIAL & METHODS ...... - 33 -

2.1 Cell Cultures ...... - 34 -

2.2 Composition of Origanum onites Essential Oil ...... - 39 -

2.3 Sulforhodamine B colorimetric assay (SRB) ...... - 40 -

2.4 Flow Cytometry ...... - 42 -

2.5 Cell migration assay ...... - 45 -

2.6 In vivo experiment ...... - 48 -

2.7 ELISA (enzyme-linked immunosorbent assay) ...... - 53 -

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3. RESULTS ...... - 55 -

3.1 Antiproliferative effect of OOEO against colon cancer cell lines ...... - 56 -

3.2 Ann/Pi staining of OOEO treated CT26 cells...... - 58 -

3.3 Cell migration assay ...... - 59 -

3.4 In vivo antitumor activity of OOEO against an experimental mouse model ...... - 62 -

3.5 Serum levels of Il-6 and IL-12 in OOEO treated and tumor-bearing mice ...... - 64 -

4. DISCUSSION ...... - 67 -

5. REFERENCES...... - 71 -

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1. INTRODUCTION

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1.1 Natural Products In Treating Diseases

lthough natural products and their derivatives (also called dietary

botanicals) have been systematically investigated, as a source of A lead research for drug discovery, only a small proportion of the world’s biodiversity has been under extent study for its application in drug development (Landis-Piwowar & Iyer, 2014; Surh, 2003; Mann, 2002; Dias et al., 2002). Nowadays many novel products are investigated and tested in clinical trials for fighting diseases and more than half of the widely used drugs derive from natural sources (Molinari, 2009).

Fig. 1: The table illustrates the increasing interest for natural-product discovery between 1984

and 2003. Total natural-products patents are shown in gold. Original natural-product patents

are shown in orange (all first-time grants of patents claiming of new small-molecule

synthesis acting as pharmaceuticals).

Source: Frank E. Koehn F. E. & Carter G. T. (2005). The evolving role of natural products in drug discovery, Nature Reviews Drug Discovery, 4, 206-220.

Already in 2900 B.C. over 700 plant-based drugs were reported in the

Egyptian pharmaceutical book, Ebers Papyrus, while in Mesopotamia (2600

B.C.) there is evidence for using oils from Cupressus sempervirens and Commiphora species for cough, colds and inflammation treatment (Cragg

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& Newman, 2005). Fighting diseases using natural products was widespread also in ancient Greece as it seems from Hippocrates’s words; "let food be thy medicine and medicine be thy food." Since then there are many documents to show the human’s attempt to exploit nature for the improvement of health conditions. Among the most famous of them is the anti-inflammatory agent, acetylsalicyclic acid, alkaloids like morphine, the anti-malarial drug quinine, etc (Der Marderosian & Beutler, 2003).

1.1.1 Natural Products From Plants

The Kingdom Plantae (plant kingdom) is the most well-studied of the five

Kingdoms as the members belonging to it can produce unique, structurally diverse secondary metabolites (Dewick, 2002; Maplestone et al., 1992;

Colegate & Molyneux, 2008), as a consequence of their adaptation and reaction against the various biological factors of the environment and the weather changes (McRae et al., 2007; Fellows & Scofield, 1995). These compounds can be processed and act as therapeutic factors. Some of them are very famous, such as paclitaxel / Taxol for breast cancer [isolated from Taxus brevifolia Nutt.] (Dewick, 2002), galantamine/Razadyne for treatment of mild to moderate dementia caused by Alzheimer's disease [isolated from Narcissus pseudonarcissus or Galanthus nivalis] (Howes et al., 2003; Heinrich & Teoh,

2004)and the antiprotozoal agent Artemotil (antimalarial drug) [isolated from

Artemisia annua] (Cragg & Newman, 2005; Dewick, 2002).

1.1.2 The Linkage Of Natural Product Chemistry And Metabolomics For The Drug Discovery

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The combined approach of classical natural product chemistry and metabolomics has led to the identification of novel bioactive natural compounds. The identification of the metabolome compounds of different extracts and fractions of the plants, such as the essential oils, and taking into account the results from their biological activity assays can potentially help us match the way each compound acts (Rochfort, 2005). However, this process is very complicating due to the high chemical diversity and complexity of the organisms.

1.1.3 Introduction to Essential Oils

Essential oils have been under investigation for their several properties. It should be noticed that essential oils can be eluted only from aromatic plants

(more than 2,000) belonging to almost 60 families (e.g. Compositae,

Myrtaceae, Pinaceae and Lamiaciae) and that among those the medicinal plants are those that have one or more bioactive compounds which relieve or even treat a disease (Μαρσέλλος & Μαρσέλλος, 1981). In addition to the above, there are some plants from which we can isolate non-volatile and non- aromatic substances, such as pigments from Crocus sativus and alkaloids from

Atropa belladonna (Καρτελιά, 2003). Although those plants do not share any common characteristics they all belong to the same group. Essential oils are mixtures of volatile compounds that are responsible for the characteristic odor and are multiples of isoprene (C5H8); the monoterpenes, sesquiterpenes and diterpenes,that consist of 2, 3 and 4 linked isoprene units, respectively, forming linear chains or rings (Ρουμπελάκη- Αγγελάκη, 2003; Καραταγλής,

1994). It is recommended that they are stored in an airtight container, in a cool, dry and ventilated area away from heat sources and protected from light.

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They can be found at the glandular hairs that protrude from the surface or at the glands of the plant. They are released into the atmosphere via two possible mechanisms from one or more plant parts, such as flowers, leaves, stems, bark, wood, roots, seeds and fruits. Either by rupturing the thin hydrophobic layer which covers the outermost surface of the primary aerial tissues of land plants, cuticle, which surrounds the droplets of the plant secretions most of the times or by pores. In particular, most of these volatile compounds are eluded during the warm and sunny days, while the largest quantity of the essential oil is extracted from the dried plant materials

(Ρουμπελάκη- Αγγελάκη, 2003; Καραταγλής, 1994).

Even though the main reason for essential oil production from the plants is still not clear, there are some confirmed features, imparted by the several chemical groups linked to the terpenes referred above. According to reliable data, it seems that not only they may be toxic to herbivorous potential consumers of the plants, but also they cause the inhibition of the growth and the germination, whereas they can attract the necessary pollinators. Already since the years of the “Father of Western Medicine”, Hippocrates, it was known that they are toxic to fungi and bacteria species, too. Some of them have been used for therapeutic purposes in over the years, having special beneficial properties (Καραταγλής, 1994).

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1.2 Family Lamiaceae - A Large Family Of Aromatic Herbs And Shrubs

amiaceae, or Labiatae, is the mint family of the flowering plants. It is

the largest family of the order Lamiales as it consists of 236 genera L and more than 7,000 species. It is estimated to be the first medicinal family and the members are famous for their unique flavor and fragrance (Morteza-Semnani et al., 2006).

The members of this family are perennial, aromatic, herbaceous or subshrubs with glands and it seems that even though they diverse they also share some common features. Their stems are square or cylindrical and the simple leaves are paired and opposite, attached at the same node and usually there are no stipules. The flowers are aggregate clustered in a dense mass in the base of leaf-like bracts or higher leaves. The calyces are often tubular or funnel- shaped, with visible ribs and 4-5 calyx teeth or lobes. There are 4 stamens in two pairs didynamous, which means that the upper pair usually shorter lower, reduced to filaments with no anthers. The corolla of the individual flower is usually 2-lipped, with 2 lobes forming the upper lip and 3 lobes the lower lip. The ovaries are the most of the times on the top of the nectar disc with two carpels, while the style emerges from the base of the ovary and the stigma is often bilobed. Its fruiting part of the pistil has four lobes that remain into the calyx, each producing a nutlet with a single seed (Simpson, 2010).

1.2.1 Introduction to the genus Origanum

Already known to 7th century B.C, Hippocrates was the first who used the name for the “prince of herbs”, for Origanum, which it is believed that it is

- 16 - derived from the Greek words for mountains (oreos) and brightness/joy/beauty (ganeos). (Talbert, 2004; White, 1998) The four distinct species in Origanum history and folklore are the sweet marjoram (O. majorana), wild marjoram/oregano (O. vulgare), dittany of Crete (O. dictamnus) and Syrian oregano (O. syriacum). Origanums were thought to be good signs, associated with love, protection, purification, healing, and happiness. The genus is considered to play a significant role in ancient Greek, Roman, Egypt and pre-Judaic religions, in mythology and it is referred in the Bible, too (Talbert, 2004).

Origanum is one of the 236 genera in the Lamiaceae family and belongs to the tribe Mentheae and currently it is known that it comprises of 44 species 6 subspecies, 3 botanical varieties and 18 naturally occurring hybrids (Duman,

1998; Ietswaart, 1980).The categorization is so complex and difficult mainly due to the places where the different taxa are close and can cross and result in the appearance of many unclassified hybrids (Kintzios, 2002).

Origanums include multipurpose culinary, fragrant, medicinal and ornamental plants native to the Mediterranean and Eurasia. Despite the fact that Linnaeus (1754) had defined Origanum as a single genus, according to the

Ietswaart classification (1980) it is divided into 10 sections; Amaracus,

Anatolicon, Brevifilamentum, Longitubus, Chilocalyx, Majorana, Campanulaticalyx, Elongatispica, Prolaticorolla and Origanum.

1.2.1.1. General information about Origanum

The plant can grow in various climatic conditions, but their native habitat in which Origanum species thrive is dry, rocky, calcareous, well-drained to dry, sandy, with a neutral to alkaline pH soil and necessarily good air

- 17 - circulation/aeration. They can withstand temperatures as low as -20°C. It is rarely infected from fungi and insects. The light seems not to affect their growth despite only the scent and flower color; these features are improved when days are full of sun (Pitzer, 1996;Bown, 2001; Κανταρτζής, 1994).

Pests and diseases only rarely influence the growth of Origanum species, however there are some of these that a cultivator should be aware of. For instance, rust fungi(Pucinnia sp.) causes circular spots on the leaves and

Botrytis spp., Phytopthera and Pythium, which can cause damping off and various forms of rot. Thus, it is important to take some precautions for the proper growing conditions, including the use of sterile potting medium, keeping plants well-ventilated and avoiding excessive watering (Flint, 1990).

Lineage (full): root; cellular organisms; Eukaryota; Viridiplantae; Streptophyta; Streptophytina; Embryophyta; Tracheophyta; Euphyllophyta; Spermatophyta; Magnoliophyta; Mesangiospermae; eudicotyledons; Gunneridae; Pentapetalae; asterids; lamiids; Lamiales; Lamiaceae; Nepetoideae; Mentheae; Origanum (marjorams)

Table 1: The full lineage of Origanum genus. Source: NCBI Taxonomy Database

1.2.1.2 Properties of its members

Even though nowadays most of the species of the genus are not often used as pharmaceuticals, many of them have been widely used in the past, including

O. vulgare, O. majorana, O. dictamnus and O. syriacum, among others. They are proved to have antifungal, antimicrobial, antisecticidal, antiparasitic, antihaelminthic, antifungal, analgesic and antispasmodic properties and have been used as expectorant, for preventing diabetes complications, for

- 18 - gastrointestinal complaints and gastric ulcers, as carminative, diaphoretic, stimulant and tonic. In addition, they are widely used against colic, cough, toothaches, for the adjustment of irregular menstrual cycles, pains, the common cold, digestive aid, for rheumatism and respiratory ailments and externally for snakebite, sciatica and wounds, tonsillitis, rheumatism, amenorrhea, diabetes, kidney and liver problems, obesity, digestion and externally for wounds and headaches, to induce sweating, for urinary problems and as a diuretic, jaundice and childhood malnutrition, aphrodisiac

(Baricevic & Bartol, 2002;Baser, 2002; Skoula & Kamenopoulos, 1997).

Origanum may also affect the progesterone regulation, while it is reported it can cause uterine stimulation and abortion and this is why it is advised against its use during pregnancy (Zava at al., 1998). It is also known as a flavoring agent in perfumes and in scenting soaps, in food products and even in alcoholic beverages.

Research on Origanum species is not restricted to the aforementioned biological activites. Different groups study their effect on cancer or its implications. One of them is chronic inflammation that urges cells to divide.

Extracts of Origanum majorana L. exhibited antiulcerogenic activity (Al-

Howiriny et al., 2009), while fractions of Origanum vulgare, a well-studied species, seem to regulate the production of some cytokines on activated human THP-1 cells (Ocaña-Fuentes et al., 2010).

Several species of the genera are considered to be antioxidants, such as

Origanum vulgare L. (Koldas et al., 2015), Origanum dictamnus (Kouri et al.,

2007) and Origanum compactum (Bouhdid et al., 2008). Particularly, the essential oil of the last has been tested on bacteria and concluded to similar results.

- 19 -

Apoptosis is the programmed cell death through which the cell decides on its own to die, but in cancer this does not happen and uncontrolled cell proliferation is observed. Extracts of Origanum majorana showed not only antioxidant but also anti-proliferative activity on human lymphoblastic leukemia cell line Jurkat (Abdel-Massih et al., 2010).

Cytotoxic substances can limit tumor growth, like several extracts isolated from Origanum dictamnus against the P388 (murine leukemia) and the human bronchial epidermoid cancer NSCLC-N6 (non small cell lung cancer) cell lines

(Chinou et al., 2007) and the essential oil of Origanum vulgare L. against A549 culture cells, reducing he mitotic index and a number of chromosomal aberrations (Grondona et al., 2014). The effect of aqeous extracts of Origanum acutidens on the breast cancer cell lines MCF-7, MDAMB-468 and MDA-MB-

231, suggested their antitumor activity (Tuncer et al., 2013).

Last, Origanum minutiflorum essential oil did not react on the vascularization systems on chick embryos in a chorioallanoic membrane model, suggesting that it does not influence angiogenesis and possibly cancer metastasis (Goze et al., 2010). On the contrary, O. majorana modulated the activity and/or the expression of proteins regulating the process of cellular migration, adhesion invasion and angiogenesis and thus, being a promising chemopreventive and therapeutic product to reduce growth and metastasis (Al Dhaheri et al., 2013).

1.2.2 Characteristics of Origanum onites

Origanum onites (or Majorana onites, pot marjoram, Greek/ Turkish oregano, rhigani/rigani) is distributed from an isolated site near Siracusa in Sicily to western and southern Turkey, while it is grown in many Greek islands in the

- 20 -

Aegean Sea and it has been introduced in Tunisia, as it is shown in the Table 2.

Fig. 2: The plant Origanum onites. Source: Dipartimento di Biologia, Università di Trieste, Picture by Andrea Moro

Code Region Status Source

AE East Aegean Islands native Calculated

AE(G) Greek East Aegean Islands native Reference Reference Cr Kriti with Karpathos, Kasos & Gavdhos native

Gr Greece native Reference

Si Sicily with Malta native Reference Tn Tunisia introduced Reference

Tu Turkey native Calculated

Tu(A) Asiatic Turkey native Reference

Table 2: The distribution of Origanum onites.

Source: World Checklist of Selected Plant Families (2010), copyright © The Board of Trustees of the Royal Botanic Gardens, Kew.

- 21 -

Its flavor is hot, peppery, sharp and its odor is thyme-like. The shoot is almost 20-40 cm high, simple, erect and hairy. The light green leaves can be either vetch or hairy, there are glands, while the lower leaves are short-stalk and the upper are epiphytic, in a cardioid shape in their base. The white flowers are with spikes forming a false corymb (Gönüz & Özörgücü, 1999).

1.2.2.1 Properties of Origanum onites essential oil (OOEO)

Similar to the essential oils of other species of Origanum, the Origanum onites essential oil (OOEO) develops various activities, according to the results of modern pharmacological studies. Many surveys have proved the antimicrobial activity of the OOEO, against some pathogenic and lactic acid bacteria, e.g. Escherichia coli, Salmonella typhimurium, Lactobacillus rhamnosus,

Streptococcus thermophilus, Clavibacter michiganensis ssp. michiganensis,

Xanthomonos axonopodies, Xanthomonas campestris, Staphylococcus aureus, and

Pseudomonas aeruginosa (Aridoğan et al., 2002; Kotana et al., 2014; Ekren et al.,

2013). Additionally, its antifungal and

antiparasitic effect has led to the

application of essential oil-among other

uses- for food preservation (Koca &

Cevikbas, 2015).

Its composition analysis indicates high

amounts of components involved in the phenolic biosynthetic pathway; such as

Fig. 3: The structures of carvacrol, thymol, gamma-terpinene and p-cymene.

Source: PubChem Compound Database

- 22 - thymol and carvacrol and their precursors γ-terpinene p-cymene (Karousou

& Kokkini, 2003). Thymol and carvacrol as the main constituents of the OOEO have been investigated in many assays and their antimicrobial and antiparasitic activity against several microorganisms have been pointed out

(De Martino et al., 2009; Rozmana et al., 2007; Cicioglu Arldogan et al., 2002; Didry et al., 1994).

In addition, the above-mentioned properties, the insecticidal activity of

OOEO has also been investigated. In particular, when tested against the

Ephestia kuehniella Zeller, Plodia interpunctella Hübner, Acanthoscelides obtectus

Say and Thaumetopoea wilkinsoni Tams, OOEO showed neuroinsecticidal activity, probably due to high concentration of monoterpenoids (Ayvaz et al., 2010; Cetin et al., 2006).

1.2.2.2 Studies of Origanum onites essential oil (OOEO) against cancer

References on the anticancer properties of OOEO are restricted to in vitro studies, on hepatoma cell line HepG2 (Özkan & Erdoğan, 2011) and 5RP7, H- ras-transformed rat embryonic fibroblasts (Bostancıoğlu et al., 2012).

Furthermore, in 2011 Özkan and Erdoğan indicated its antioxidant activity on

HepG2 cells, mainly attributed to the phenolic compounds it has and/or from synergistic effect with other compounds present in the oil. A year later, in

2011, Bostancıoğlu et al. showed that OOEO inhibits cell proliferation of 5RP7 and rat adipose tissue endothelial cells (RATEC), induces apoptosis in 5RP7 and blocks in vitro formation of blood vessels and cell migration of RATEC.

Lastly, treatment of Salmonella typhimurium strains TA98 and TA100 with

OOEO resulted in antigenotoxic effects (Ipek et al., 2005).

- 23 -

1.3 Colon cancer

olorectal cancer is the fourth and third most common cancer among

men and women, respectively, while it is the second leading cause C of cancer in both sexes in Europe. Moreover, it is second in prevalence, whereas first is breast cancer and the fourth most common cause

of cancer mortality worldwide. Approximately three quarters of all cases are

sporadic and the remaining are hereditary or linked to inflammatory diseases,

while almost one third of the familial cases has been genetically determined.

Unfortunately, it is difficult to study the epidemiology because data access in

many countries is not available (Parkin et al., 2006; Haggar, & Boushey, 2009; Gellad & Provenzale, 2010).

Fig. 4: Colorectal cancer incidence and mortality rates per 100,000 people by race/ethnicity and sex are represented for the years 2006-2010 in the U.S.A. Source: Colorectal Cancer Facts & Figures 2014-2016. Published March 17, 2014. American Cancer Society, Atlanta, Ga.

Every year more than 1 million new cases of colorectal cancer are diagnosed.

It is demonstrated that the cases of colorectal cancer are increased with age

with a calculated incidence of 20 per 100,000 of 0-64 years old and rising to

- 24 -

337 per 100,000 in the over 65 group. A significant increase in the population group over 75 in the last two decades can be attributed to better health systems (Mulcahy et al., 1994).

1.3.1. Colon anatomy

The colon is a part of the digestive system, which removes and processes the nutrients from food, and finally leads to the excretion of the waste material.

The digestive system comprises of the oral cavity, throat, esophagus, stomach and the small and large intestines. Particularly, the colon is the first part of the large intestine and is about 5 feet in length. Subsequently, the rectum and anal canal form the last part of the large intestine which ends at the anus. (Irving &

Catchpole, 1992)

1.3.2 The stages of cancer development

In 1990, Fearon and Vogelstein suggested that colorectal cancer is produced by a number of successive mutations in more than one of genes related to cell proliferation or regulation or DNA damage repair. Also, the sequence of these changes is crucial to determine the progression and development of cancer.

The disease develops in several stages, whereas the first sign is a precursor benign adenomatous polyp, and then successively the formation of aberrant crypt foci, polyps, severe adenomas with an asymptomatic period of 5 years prior to the development of the next stage, the carcinoma that eventually

- 25 -

progresses to an invasive cancer. It should be noticed that invasive cancer is Fig. 5: Colon cancer develops at stages as it is illustrated in the figure. This classification system describes progressive procedure develops beginning from Stage O (Normal) and ends at Stage IV (Metastasis). Source: Green J. E. & Hudson T. (2005). The promise of genetically engineered mice for cancer prevention studies, Nature Reviews Cancer, 5, 184-198.

referred to tumor or node localized to the wall of the colon and these

represent the stages I and II, respectively. If it is not treated, then it spreads to

regional lymph nodes, which is stage III and then it metastasizes to distant

sites, the last stage. The first two stages are treated by surgical excision and a

high percentage of stage III are curable in combination with chemotherapy,

but high mortality is recorded in the group of patients at stage IV (Watson et al., 2007; Sancho et al., 2004).

1.3.3 Factors contributing to colon cancer development

Cancer is a disease attributed to environmental and genetic factors that lead

to the uncontrolled cell division. Predisposing risk factors for colorectal

cancer include the family history, endometrium or breast cancer, ulcerative

colitis or Crohn disease, polyps, weight, physical activity and diet. To begin

with, the risk rises after the age 50 and from reports it appears that most

incidents are recorded after that age. Of course, there is strong evidence that it

is inherited which means that if a person has a parent, sibling or child with

- 26 - the disease then he doubles the risk of developing colorectal cancer. It is also very important for an individual in case he had a previous colorectal cancer, high-risk adenomas, ovarian cancer and at last, inflammatory bowel disease

(Anand et al., 2008; Haggar & Boushey, 2009; Giovannucci 2002).

1.3.4 Inflammation markers associated with colon cancer

Novel technology discoveries provide the opportunity to detect potential biomarkers for early colorectal cancer diagnosis. Between them are inflammation- related serum proteins, as inflammation has been indicated to be an important mechanism by which cancer develops. Hence, some interesting biomarkers are the pro-inflammatory cytokines [e.g., interleukin

IL-6, IL-10, TNF-α, IL-5, IL-13, and IFN-γ] (Terzić et al., 2010).

IL-6 is normally produced by endothelial cells, macrophages and some activated T cells. Although it was thought to be just a pro-inflammatory cytokine, it is found in a number of pathophysiological conditions. For instance, it can act as a chemoattractant factor for leucocytes or cause the immunoglobulin production by mature B cells. In colerectal cancer, IL-6 is able to prevent Fas-mediated apoptosis through the expression of the anti- apoptotic Bcl-2 (Yuan et al., 2004) or to strongly stimulate anchorage- independent growth of some of them (Schneider et al., 2000; Mauer et al., 2015).

IL-12 is a pro-inflammatory cytokine produced mainly by phagocytic cells after immune response to antigenic stimulation. As a consequence, an inflammatory cascade is initiated and a variety of other cytokines are produced. It acts as a growth factor for activated NK and T cells (Trincheri,

1995) as it regulates the production of anti-tumoral IFN-γ from NK and T cells

- 27 - and the Th1 differentiation. IL-12 is associated with murine melanoma, mammary carcinoma, sarcoma, colon cancer and exhibits antitumor and anti- angiogenic effects, too (Coughlin et al. 1998; Li et al., 2005; Yoshimoto et al.,

1998; Yoshimoto et al. 1998).

1.4 Chemoprevention

large number of chemical compounds is under investigation for

their contribution to cancer prevention or in other words for A chemoprevention. The most common form of chemoprevention is addressed in the general population and is based on both epidemiological and experimental data (general population chemoprevention), while the other form is with regard to a limited proportion of people with increased cancer risk and is called targeted chemoprevention (Landis-Piwowar & Iyer, 2014).

There are two main lines in research for plant specific compounds inhibiting carcinogenesis. The inhibitor can be identified from raw plant material or as a synthetic component structurally-related with a compound in natural products. Moreover, another way to categorize cancer inhibitors is based on the stage of tumorigenesis in which they are efficient, namely by preventing the formation of precursors, interacting with mutagens to target sites

(blocking agents) or, not allowing a tissue exposed to a mutagen to form a neoplasm by treating it with the agent (suppressing agent) (Young-Joon, 2003;

Manson et al., 2000).

- 28 -

Both the National Cancer Institute and the American Cancer Society support that eating more fruits and vegetables daily can reduce an individual’s risk of developing cancer. It is estimated that more than 70% of cancers can be prevented (Willett, 2000) with dietary agents making a contribution of 35%

(Doll & Peto, 1981). However, the specific dietary agents still remain unidentified.

1.4.1 Identified Natural Products for contributing to treating colon cancer

The use of natural products, and especially herbs, as complementary medicine for cancer has recently increased and there is a huge range of current studies investigating natural products for the treatment of colon cancer (Reddy, 1996; Aggarwal et al. 2013). Extensive studies have revealed numerous nutrients contributing to naturally treating colon cancer. These include acetoxychavicol acetate, anacardic acid, berberine, betulinic acid, boswellic acid, butein, camptothecin, capsaicin, caraway, catechins, caffeic acid phenethyl ester, cardamonin, celastrol, chalcones, coronarin, curcumin, deguelin, diosgenin, diallyl disulfide, elephantopin, ellagic acid, emodine, embelin, escin, fisetin, flavopiridol, flavonoids, gambogic acid, garcinol, genistein, gingerol, gossypol, gossypin, guggulsterone, indole-3-carbinol, lycopene, morin, mushroom Ganoderma lucidum, naphthoquinone, nimbolide, noscapine, oleandrin, piperine, piceatannol, pinitol, plumbagin, pomegranate, resveratrol, retnoids, honokiol, quercetin, sanguinarine, sesamin, silymarin, simvastatin, sulforaphane, terpenoid, thymoquinone, tocotrienol, triptolide, ursolic acid, withanolides, xanthohumol, and zerumbone. It is suggested they mediate their effects by targeting molecular

- 29 - targets, including growth factors and their receptors, protein kinases, inflammatory biomarkers, and various transcription factors.

Fig. 6: The diagram shows the number of approved drug by FDA annually, anticancer drugs and more specifically the approved of the field of natural products.

Sources: (1) Pharmaceutical Research and Manufacturers of America PhRMA Annual Membership Survey, 1996-2014; (2) Nature Reviews Drug Discovery. 2014, 13: 85-89; (3) J Nat

Prod. 2012, 75, 311-35; 4) Front Chem 2014, 2, 20

1.4.2 The Role Of Essential Oils In Treating Colon Cancer

As previously stated, essential oils are plant-derived mixtures having a variety of properties and hence, a number of them has been tested for treating and preventing colon cancer. Owing to this, essential oils of Afrostyrax lepidophyllus, Scotonycteris zenkeri and Athanasia brownii have been demonstrated to inhibit growth of human colon carcinoma HCT 116 cells

(Fogang et al., 2014; Rasoanaivo et al., 2013), while that isolated from

- 30 -

Artemisia campestris exhibits antitumor activity against HT-29 cells(Akrout et al., 2011). Furthermore, the Melissa officinalis L. (de Sousa et al., 2004) and

Vepris macrophylla (Maggi et al., 2013) essential oils have cytotoxic effect on

Caco-2 and HCT 116 cancer cells, respectively and that from Salvia libanotica induces HCT 116 apoptosis (Itani et al., 2008).

Essential oils distilled from Origanum species are among the most well- studied oils and have been investigated for their potential anticancer effect on diverse types, but when it is colon cancer their action is partially clear and with few exceptions, the species of O. vulgare is the most examined (García-

Pérez et al., 2013; Begnini et al., 2014; Savini et al., 2009; Srihari et al., 2008).

- 31 -

1.5 Purpose

Origanum onites has been shown to exhibit anticancer properties, among others, which are associated with its composition. However, the isolated

OOEO, which is characterized by the prevalence of carvacrol, even though it has been tested in vitro, there is no evidence for its in vivo activity.

Aim of this work is to study the response of colon cancer cell lines of both human (HT-29) and murine (CT26) origin to the activity of OOEO regarding oil’s cytotoxic effect, pro-apoptotic and anti-migratory properties. Moreover, the present study extends its purpose to clarify the effect of the systematic prophylactic per os administration of the essential oil in the organism level including an in vivo experiment with BALB/c mice that were inoculated with

CT26 cancer cells. Apart from the in vivo anti-proliferative properties of

OOEO, its ability to induce anti-inflammatory response to the animals was also studied. To conclude, current work’s purpose is to investigate the anticancer activity of OOEO against colon carcinoma and to characterize its effect for the first time in vivo.

- 32 -

2. MATERIAL & METHODS

- 33 -

2.1 Cell Cultures

CT26 and HT-29 colon cancer cell lines

The implementation of cell culture provides a predictive tool for the normal physiology and biochemistry of cells, mutagenesis, carcinogenesis and the effects of drugs and toxic compounds on cell lines, conferring consistency and reproducibility of results.

Colon cancer was examined using two adherent cancer cell lines, that form a monolayer attached to the cell culture vessel and develop connections essential for proliferation. CT26 cell line (CT26.WT ATCC® CRL-2638™), is a fibroblast - derived cell line from the colon of BALB/C strain of Mus musculus. It was induced by the carcinogen, mutagen, and teratogen N- nitroso-N-methylurethane (NNMU) and Fig. 7: CT26.WT ATCC® CRL-2638™ , a expresses H-2d. CT26 cell line is widely fibroblast - derived colon cancer cell line used for testing immunotherapy from BALB/C strain of Mus musculus protocols and in studies on the host immune response. Subcutaneous inoculation in BALB/c mice leads to lethal tumor development. Moreover, when the cells are injected intravenously the animals develop pulmonary metastases.

The second cell line (HT-29,

Fig. 8: ATCC® HTB-38™, an epithelial –

derived cell line from the colon of a Caucasian 44-year old woman

- 34 -

ATCC® HTB-38™)) is epithelial –derived from the colon of Homo sapiens.

The cells were isolated from a Caucasian 44-year old woman who developed colorectal adenocarcinoma. According to ATCC these cells express a number of receptors (e.g. human adrenergic alpha2A, urokinase Receptor (u-PAR), oncogenes (myc, ras, myb, fos, sis, p53), but do not express others (abl, ros, src).

Basic Principles and equipment

When working with cell cultures you have to follow Good Laboratory

Practice (GLP) to reduce the risk of exposure to potentially infectious agents and to prevent contamination of the cell culture.

Before starting working on a laminar flow hood, the short-wave UV light should be turned on for about 10-20 minutes to sterilize the exposed surfaces of the hood. The laminar surface of the horizontal hood and the outside of any bottles must be disinfected by spraying with 70% ethanol. It the horizontal hoods the air is continuously displaced and flows directly towards the operator through a filter, which withholds particulates from the air to protect the cultures. All surfaces must be wiped out with 70% ethanol after each use, too. The hygiene compliance may prevent culture contamination, which inhibits cell growth, kills cells, and leads to inconsistent results. In these cases there is a dramatic change in the pH of the medium accompanied by a change in its color and/or cell culture death.

Cells are grown into a CO2 incubator where there is 5-10% CO2 because the growth medium contains a sodium bicarbonate/carbonic acid buffer and the pH value at 7.2-7.5 must be strictly retained. Culture flask caps are filtered or are loosened to allow the essential gas exchange. A filled pan of water is kept in the device to maintain the saturating humidity and the temperature is regulated at 37°C.

- 35 -

Bright Field inverted microscope is used for visualizing the cells with a 10X objective lens. Cultures should be examined every day for potential contamination and to determine if changing the medium or even passaging is needed depending on cell growth.

Cell culture medium differs among different cell types as each of them has highly specific growth requirements. For CT26 and HT-29 cells, Dulbecco’s modified Eagle medium (DMEM) was used. DMEM contains a mixture of amino acids, glucose, salts, vitamins, and other nutrient. FBS, Fetal bovine

Serum is added that binds and controls toxic nutrients, neutralizes trypsin and other proteases, affects cell-cell and cell-matrix interactions and contains essential nutrient and growth factors.

Dulbecco’s modified Eagle’s medium (DMEM) was supplemented with 10% fetal bovine serum, 2 mM L-glutamine and pen/strep (100 units/ml penicillin and 0.1 mg/ml streptomycin).

Cell thawing

Complete medium and the cryovial containing frozen cells are warmed in a water bath at 37°C. Next, the 1ml of cells preserved with freezing medium are resuspended in 11 ml fresh complete medium in a 15 ml microcentrifuge tube, and then transferred to the cell culture vessel. Finally, cell solution is maintained in a 12 ml flask (surface of 75 cm2).

Harvesting cells

Through trypsinization adherent cells are detached from the surface of a cell culture vessel whenever to be harvested. The medium is aspirated and discarded, while two washes with 3 ml of PBS (phosphate-buffered saline) each follow. 1.5 ml warmed 1x trypsin–EDTA solution is added and cells are incubated for about 3 min in the incubator and are observed in the

- 36 - microscope to see their status. Approximately 10 ml of complete medium are added to inactivate trypsin and pippet up and down gently.

Cells stop proliferating once they become confluent (reach a max population density) and therefore they need to be routinely passaged, which means that a fraction of the cells have to be transferred to a new cell culture vessel. Ideally, cells are harvested when they are in a semi-confluent state and are still in exponential phase. Thus, cells are harvested and the appropriate volume of the resuspended cells is transferred to a fresh cell culture vessel containing growth medium and eventually, incubated under usual conditions. HT-29 are subcultured in a ratio 1:3-1:8, while CT 26 in a ration 1:4 to 1:10 so they can grow normally. After every passage the log of each flask is changed mentioning the name of the cell line, any alterations to the standard medium, the date on which the cells were splitted and/or fed.

Counting cells using a hemocytometer

The basic method of cells’ counting is using a hemocytometer of two chambers. Each of them is ruled into 9 main squares (volume of 0.1 mm3 or 1 x 10–4 ml each). Cell concentration is determined by counting the number of cells within a defined area of known depth (volume). Using trypan blue staining it is easy to distinguish viable and nonviable cells as cells with intact membranes exclude the dye.

First, the surface of the hemocytometer is cleaned with 70% ethanol and dried with lens paper. The coverslip is also cleaned, whereas its edges are slightly wet and laid over the central area of the hemocytometer. Cells are harvested and resuspended in an appropriate volume of prewarmed full medium. It is crucial to produce a single-cell suspension after harvesting in order to find the true number of cells. The suspension is mixed well and 20ul of it are combined with 20ul of 0.4 % w/v trypan blue (0.4 gr dye diluted in 100ml

- 37 -

1xPBS). The new suspension is mixed thoroughly and is incubated for 1–2 min. 20ul of it are transferred to the edge of one side of the coverslip to fill the chamber of the hemocytometer by capillary action using a pipette and the step is repeated for the second chamber. The slide is observed to the microscope, and viewed using a 10x objective and 10x ocular. Blue-stained cells are nonviable and unstained cells are viable. The total number of viable cells is counted in the 4 corner squares, but not those that overlap the two of the four borders of squares, preventing counting cells twice. In case the cell density is really high, the cell suspension should be diluted, noting the dilution factor. The counting is repeated for the second chamber and the counted number is summarized to reduce the number of cells in 1 ml. In the end, the hemocytometer and coverslip are cleaned using 70% ethanol, then with distilled water and dried with lens paper.

Since each large square represents a total volume of 0.1 mm3 or 10-4 cm3, as referred and 1 cm3 is equivalent to approximately 1 ml, the total number of cells per ml will be determined using the following calculations:

Cells/ml = average cell count per square x dilution factor x 104

Fig. 9: Counting cells using a hemocytometer.

[A] A chamber of the hemocytometer slide under 10x objective and 10x ocular. This is divided

- 38 - into 9 major squares. [B] Detailed view of one of the 9 major squares. Only cells that overlap the two out of the four borders of squares (for instance, the top and left) should be counted. O: cells that should be counted; Ø: cells that should be ignored

2.2 Composition of Origanum onites Essential Oil

Origanum onites essential oil

composition (%) a/a compounds percentage 1 carvacrol 47.988

2 terp-1-in-4-ol 6.786 3 sabinene hydrate 6.135

4 γ-teprinene 5.206 5 p-cymene 3.847

6 a-terpineol 3.756 7 a-terpinene 2.257 8 linalool 2.048

9 borneol 1.97 10 thujanol 1.277 11 linalyl acetate 1.204

12 caryophyllene 1.167

Table 3: The Origanum onites essential oil composition (VIORYL S.A.)

- 39 -

The essential oil used in this study was provided by VIORYL S.A. Essential oil’s chemical composition was determined by GC-MS analysis, as shown in

Table 3. The proportion of thymol in this OOEO is extremely small (0.428 %) as thus, it is not shown in the table (Table 3). Essential oil composition differs depending on the plant genetics, growth conditions, the part from which it is distillated, the harvest time etc (Ozkan et al., 2010).

2.3 Sulforhodamine B colorimetric assay (SRB)

Sulforhodamine B (SRB) colorimetric assay is an in vitro method used for cell density determination of treated adherent cells for a determined period and it relies on their protein concentration measurement. In particular, SRB, is a bright-pink aminoxanthene dye with two negative charged sulfonic groups that can bind to basic amino-acid residues under mild acidic conditions of protein components of fixed cells in tissue-culture plates. The binding of the dye is stoichiometric and as a result its measured amount is directly proportional to the cell mass. The monolayer cell culture is thrypsinized and an amount of 10ul is transferred to a hemocytometer. After tests of the assay to a 96-well plate flat-bottom polystyrene tissue-culture plate for the CT26 and HT29 cell lines, it was decided that the appropriate cell number per well is 5.000 and 20.000, respectively in a total volume of 100ul. These numbers depend on the characteristics of the cell lines, such as the cell size and their growth rate in order to form a partial monolayer in the wells.

- 40 -

Fig. 10: A flat bottom 96-well plate, suitable for adherent cell lines and precise optical measurements

After seeding the plate is incubated for 24hours and the cells are attached to

the flask bottom. Then, the supernatant is removed with a p1000 pipet and the

cells are treated with a wide range of Origanum onites essential oil

concentrations in groups of wells. There is always a number of wells as

control, filled with highest studied concentration with Dimethylsulfoxide

(DMSO). DMSO is a polar oily solvent used to dilute the essential oil and

homogenize the solution in equal volume to that of oil (e.g. 0.1% of 100ul

means that 0.1 ul of DMSO are mixed with 0.1 ul of oil in 100ul of DMEM).

Until this step everything is performed in sterile conditions in the hood. The

° incubation conditions were at 37 C , in a humidified incubator with 5% CO2.

Cell monolayers are fixed with 10% (w/v) trichloroacetic acid stored in 4°C,

50ul per well, for 1hour in room temperature. Using a beaker the plate is

washed under flowing tap water and the excess of water is removed by blotting the plate on paper towels.

- 41 -

In the next step, 100ul of 0.057% (w/v) SRB solution are added to each well of the dried plate and after 30 min. The excess dye is removed by washing repeatedly with 1% (v/v) acetic acid for 3-4 times.

Finally, 200ul of 10mM Tris base solution, pH 10.5 are added in the dried wells to dissolve the protein-bound dye. The plate is left at a gyratory plate shaker for about 5 min and the OD is determined at 510nm (492nm) in the spectrophotometer Tecan SunriseTM Microplate Reader.

Each cell of the exported file corresponds to the respective well of the 96-well plate and its value is the measured absorption of the sample. The results of the measurement are analyzed with the SigmaPlot 11.0 statistical software.

The “%cell growth” is given from the equation:

%cell growth = x 100

-mean OD sample : absorbance value of test compound

-mean OD neg control : absorbance value of negative control (DMSO) and growth inhibition is

%growth inhibition=100 - %cell growth

Using the appropriate statistical data the EC50 value, namely the concentration at which the half of the cells die is found.

2.4 Flow Cytometry

- 42 -

A flow cytometer is an instrument designed to recognize and measure cellular elements in a biological liquid. Thus, it is possible not only to distinguish different cell populations in complex mixtures, but also to enumerate the diverse specific types. The instrument is divided into three parts: the fluidics, optical and electronic systems. The first has to carry the studied cells through the cytometer so they can be analyzed. A critical point in the flow cytometer is where the cells reach “the interrogation point”, as it is called, at which they intersect the laser, a single cell each time. Each cell scatters a proportion of the laser light which is collected along the axis of the laser beam (forward scatter) and at a 90° angle (side scatter) to the beam. At these points there are combinations of detectors that receive specific wavelengths of light, electrical signals are generated, amplified and sent, stored and analyzed to the computer.

According to the results from the SRB assay, OOEO seems to have a cytotoxic effect on CT26 cells. There are many ways by which a cell can finally “die”, and apoptosis is one of them. This process is genetically programmed to maintain the tissue homeostasis, under pathological conditions or in aging.

The etymology shows that the word comes from the Greek one for “falling off” of leaves from a tree and this indicates how the cell chooses to participate in its own “death”.

Apoptosis is described with some morphological features, including the loss of the cell membrane asymmetry and attachment, condensation of the cytoplasm and nucleus, and internucleosomal cleavage of DNA in succesive stages of the phenomenon.

In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is translocated from the inner to the outer side of the cell membrane, leading to the PS exposition. For this reason, in this protocol Annexin V, a Ca+2-

- 43 - dependent protein with high affinity for PS binds to it. The flow cytometer cannot detect Annexin V as a probe itself, and this is why it is conjugated to a fluorochrome, fluorescein isothiocyanate (FITC).

Next, the cells are stained with propidium iodide (PI), a compound that binds to the helix of the DNA and when this happens it fluorescents. The PS translocation causes the increase of the cell permeability and of course, the viable cells exclude the PI, while the membranes of damaged and dead are permeable to it.

Hence, the Annexin V- and PI- are viable, Annexin V+ and PI- are early apoptotic and Annexin V+ and PI+ are late apoptotic cells. But this assay cannot distinguish if the cells die due to apoptosis or necrosis in a direct way.

It is important to notice that in the case that there is necrosis, there will not be a large Annexin V+/PI- population.

The monolayer cell culture is thrypsinized and an amount of 10ul is transferred to a hemocytometer. The proper number of cells for this assay for

CT26 is 500.000 cells/well in a total volume of 3ml in a 6-well plate. The plate is incubated for 24hours, until the cells were attached to the bottom. The incubation conditions were 37°C, in a humidified incubator with 5% CO2. The next day, the supernatant is removed and the cells are treated for 48h with 3 ml of 0.007% (v/v) Origanum onites essential oil concentration. There is also a no-treated well as a negative control, filled with 3ml of cell medium (DMEM) with 0.007% (v/v) DMSO for 48h, too.

After the incubation period the supernatant is collected in a 15-ml conical centifuge tube, then the cells are washed with 500ul PBS twice and the supernatants are also collected in the respective conical centifuge tubes. 300ul of 1x thrypsin (diluted in PBS) are added in each well for almost 2 min and enzyme’s activity is deactivated with the addition of 2ml full DMEM. Up &

- 44 - down follows and finally this remaining amount collection in which the most of the viable cells are is collected, too.

In the next steps, the conical centifuge tubes must be ice-cold. They are centrifuged for 6 min (1.200 rpm), the supernatants are discarded and pellets are resuspended in 1ml PBS. This step iss repeated and cell counting is performed. Finally, cells are resuspended in Annexin binding buffer in a concentration of 2x106cells/ml. 100ul of the sample are incubated for 13 min with 5ul of Annexin V in a 5 ml round bottom polystyrene test tube. 5ul of PI and 400ul of Annexin buffer are diluted to the solution and incubated for about 5 min in the dark and then the sample are analyzed with the flow cytometer.

The results of the measurement are analyzed with the FlowJo software.

2.5 Cell migration assay

Cell migration assay is used in order to quantify the migration rate of the cells in vitro by capturing photos at the beginning and at regular interval time points of the experiment. A “Θ”-shaped silicone insert is used for the appropriate seeding of cells in two divided areas.

The inserts are introduced into the wells of a 6-well plate with sterile blunt- nosed thumb forcep. The proper number of cells for this assay for CT26 cells is 5 x 105 cells/ml. 70ul are added in each chamber of the insert and the plate is incubated for 24hours, until the cells are attached to the bottom. The next day, the insert is removed using sterile blunt-nosed thumb forcep and the cells are

- 45 - treated with 1.500ul 0.0005% (v/v) Origanum onites essential oil concentration.

This is the final concentration because although it shows cytotoxicity against the cells, a significant proportion of them do not die and are allowed to connections and migrate. A non-treated well serves as a control, filled with

1.500ul of cell medium (DMEM) with DMSO. This is a time-depending process in which photos of the wells are captured at different time points with an inverted microscope with a 30x objective lens. Photos are then compared using ImageJ to measure the rate of the gap coverage.

After we open the photo in ImageJ, we follow the successive steps:

 Process Find Edges

 Process Sharpen

 Image Type8-bit

 Image Adjust Threshold

“B & W” (Black and white) and “Default” were selected among the options, the first bar was adjusted to 0,the second to second to the value that the

resolution was optimal and pressed “Apply”. (Figure)

 Process Find Edges

 Image Lookup Tables Invert LUT

 Analyze Analyze Particles  Analyze Analyze Particles

“Circularity” was regulated to the range “0-1”, “Show outlines” was selected and “Display results” and “Summarize” were ticked. Size was the parameter adjusted in this step, so that the several gaps in the photos can be identified in an adequate degree. The value of the “% Area” in the “Summarize” table, which is equal at the percentage of the whole photo’s gap was noted.

- 46 -

Fig. 11: A photo captured with an inverted microscope with a 30x objective lens. Control, time point T=0h.

The procedure was repeated for all the photos and the “% Area” values

were analyzed using the SigmaPlot 11.0 statistical software.

Fig. 12: The “Black & White” format of the processed photo. (Fig. 11)

A graph was created following the steps:

Graph Create Graph Line Plot Multiple Straight Lines

- 47 - and “Data format” as “X many Y”. Finally, the three columns of the “Time point” and “% Open Image Area” of the two treatments were selected.

Fig. 13: The circulated areas show the areas where the brightness is higher (there are no

cells) and the noted percentages expresses the proportion that each area covers of the total image surface.

2.6 In vivo experiment

Animals are used in medical research, treatment, but their number must be limited. Any scientific study using animals should comply with the “3Rs”: tenets of replacement, reduction and refinement, based on literature and experiential data. The ultimate aim is to maximize the scientific integrity of data generated while at the same time using the minimum number of animals.

BALB/c mice are white (albino) mice with H2d

MHC Haplotype, same as the CT26 cell line. These Fig. 14: BALB/c mouse animals are used as general multipurpose model and are ideal for searching

- 48 - hybridoma development and studying monoclonal antibody production and infectious disease. Their life expectancy is 1-2 years and from the birth day until the 21st day they are regarded as infants; the next five weeks they are juveniles and after that, adults when they weight 20-40 g or 25-40 g if they are males or females, respectively. Their heart and respiratory rated per minute is between 300-800 and 100-200, while body temperature is 36.5-38 °C.

Mice are mature to breed when they reach 8-10 weeks. Estrous cycles are repeated every 4 days and last about 14 hours usually in the night. Whitten effect is a special phenomenon; even though females in the same cage may are in a different stage, synchronization of their oestrous cycles is observed when a male mouse is transported in the cage. Pregnancy lasts on average 19 days.

Mice are very sensitive animals as for their survival there must be followed certain conditions. The room temperature should be between 20-24 °C, the relative humidity 50-60 % and the light intensity less than 50 lux. Noise and the light/dark ratio (14/10) are crucial for their behavior and reproduction.

BALB/c mice of both groups were kept in separate transparent macrolon type

3 cages (900cm2 each) with a tightly fitting lid. Free of fine dust, microbes and chemicals sawdust was spread at the cage bottom as a bedding material. All the mice had the same diet in the form of pellets administrated ad libitum.

Normally, each mouse eats 3-4 g daily. Except from that, they digest microbes and consume faeces (coprophagy) and in this way can absorb certain nutrients. Fresh drinking water is available via bottles (van Zutphen, 2001).

The followed administration schedule is illustrated in Fig.16. Shortly, animals can receive 75 ml kg−1 equating to 150 ul for a 20 g mouse of the putative chemopreventive agent at innocuous doses. They are syngeneic with the implanted CT26 tumor cells to avoid rejection. Finally, volume and weight of

- 49 - tumors of the intervention group are measured and compared with animals on a relevant control diet.

Eighteen female immunocompetent BALB/c mice, approximately 6-8 weeks old were used to carry out the experiment as BALB/c mice are syngeneic to

CT26 cell line. All the animals were gavaged once daily for 15 days. They were randomized into two groups; half belonging to the control group

Fig. 15: A transparent macrolon cage with a tightly fitting lid. There is spread sawdust at the cage bottom as a bedding material, food as pellets administrated ad libitum and fresh drinking water in a bottle.

administrated with 150 ul of corn oil, whereas the others belonging to the test group were having 140 ul of corn oil mixed with 10 ul of the O. onites essential oil. On the twelfth day they received cell injection into the scruff of the neck subcutaneously (5 × 106 cells diluted in 100ul PBS per animal) and the area was observed every day for palpable tumor formation. Two days later the gavage procedure stopped until the seventh day of the experiment, when all the animals were killed.

- 50 -

Mice were killed under sterile conditions in a fume hood after anaesthesia humanely. A box was filled with cotton soaked in ether and each time a mouse was placed in the bag until it was lying on its side and slowly breathing. Then, using sterile blunt-nosed thumb forceps the eyeballs of each eye were protruded out of the socket and blood was collected on 1.5 ml microcentrifuge tubes.

Fig. 16: The performed administration schedule. Nine female BALB/c mice were administrated corn oil and other nine diluted Origanum onites essential oil for 15 days, while the 12th day all of them received an injection of syngeneic CT26 colon cancer cells in the area of the neck subcutaneously. On day 17 they were sacrificed and the removed tumors were compared between the two groups.

blunt-nosed thumb forceps. All of the samples were temporarily retained in ice. Each mice is named with a unique code, the log number (e.g. log134,

- 51 - log675) in a records book and the same number is indicated in the tubes showing to what animal each sample belongs.

Blood samples were centrifuged at 10.000 x g for 10 min at 4°C and the serum was transferred to 200ul tubes, the tumors were weighed, while five of them of each group were fixed in 10% formaldehyde in 5 ml tubes for future use.

The rest, including serums, were stored at -80°C. The volume of the tumors was calculated from caliper measurements of tumor dimensions in mm using the formula from a prolate ellipsoid:

mm3

The weight and volume of the tumors were analyzed with the SigmaPlot 11.0 statistical software in a similar way. The two used columns contained the measurement of the two groups, separately. The graphs were created following the steps:

Graph Create Graph Box Plot Vertical Box Plot and “Data format” as “X many Y”. Finally, the 3 columns of the “Group” and “Value” of either Tumor Volume or Tumor Weight were selected.

All animal experiments were performed according to international and national directives and based on the Greek law. The experiments were performed as part of a collaborative work with the University of Ioannina.

All animal experiments were complied with the requirements set by Directive

86/609/EEC and PD 160/91 and were conducted in light of 3 R’s (replacement, refinement, reduction). All mice used for the experiments were not subjected to pain or discomfort.

- 52 -

2.7 ELISA (enzyme-linked immunosorbent assay)

The ELISA (enzyme-linked immunosorbent assay) method is the most common method for quantitation of antigens in a sample, providing very sensitive and highly specific data. Typical examples of ELISA use is the measurement of cytokines, chemokines, growth factors, and other extracellular targets. The selection of Sandwich ELISA was because it performs the best, as there are involved specific, highly purified antibodies in the procedure.

In 100ul of capture antibody are added per well in 1x Coating buffer that offers the ideal conditions for the activity of the antibody in an ELISA plate.

Serum samples from both of the two mice groups were analyzed. After adding the samples/controls/blanks, the plate is sealed and incubated O/N at 4°C.

Next, the wells are washed using the Wash buffer for 3 times, 200ul/well and the non specific binding is reduced by blocking wells with 200ul/well of 1x

ELISA/ELISPOT Diluent at RT for 1h. 50 ul at RT of the samples containing unknown amount of the target cytokine of interest are added for 2h and bound to the capture antibody. After washing steps (as above) to get rid of the unbound substances, a detection antibody is added to the wells (100ul/well, sealed plate, RT, 1h), that binds to the immobilized cytokine captured during the previous incubation.

After washing (3 times, 200ul/well), 100 μL/well of Avidin-HRP are added and the plate is incubated at RT for 30 min. Following washes to remove the excess HRP conjugate, the substrate solution is added, 1x TMB, 100 μL/well,

- 53 - converted by the enzyme to a detectable colored product. The plate is incubated at RT for 15 min in darkness. The OD of this colored product is directly proportional to the concentration of antigen present in sample.

Finally, OD is measured in the spectrophotometer Tecan SunriseTM Microplate

Reader at 450 nm (492nm) and the data are analyzed using the SigmaPlot 11.0 software.

- 54 -

3. RESULTS

- 55 -

3.1 Antiproliferative effect of OOEO against colon cancer cell lines

By analyzing data from the performed SRB assay, sigmoid curves were

created and EC50 was calculated. It is apparent that the effect of OOEO is

dose-dependent for the same time point (48h). The EC50 value of the HT-29

and CT26 cell lines is approximately 0.0106% (v/v) and 0.0077% (v/v),

respectively. Thus, the murine cell line exhibited less resistance to the

essential oil activity.

A B C

A B C

Fig. 17: The figure illustrates the effect of concentrations of Origanum onites essential oil on

CT26 cells captured with a 30X objective lens.

A. control with DMEM (100% cell growth)

B. 0.006% Origanum onites. It is clear that approximately half of the cells have died (value

close to EC50, compared with the cell growth observed in control (A). C. 0.01% Origanum onites. Almost all the cells have died

- 56 -

SRB assay on HT-29 cells (48h)

120

A EC50= 0.0106 ± 0.0002 % (v/v) 100

80

60

40

% growth

20

0

control 0.0005 0.001 0.005 0.01 0.025 0.05 Essential oil concentration % v/v

SRB assay on CT26 cells (48h)

120 B EC50= 0.0077 ± 0.0003 % (v/v)

100

80

60

% growth % 40

20

0 control 0.0001 0.0005 0.001 0.005 0.01 0.05 0.1 Essential oil concentration % v/v

Fig. 18: The dose-dependent cytotoxic effect of OOEO against

EC50= 1,059e-2 ± 1,869e-4 A. HT-29 and B. CT26 cancer cell lines using the SRB assay in a 96-well format at 48h. Also, the EC50 values are shown.

- 57 -

3.2 Ann/Pi staining of OOEO treated CT26 cells

SRB assay indicated cytotoxic effect on CT26 cells, but the mechanism through which cells die under the OOEO is unknown. Flow cytometry is performed to examine necrotic vs or apoptotic cell death.

Fig.19 illustrates an apparent shift of cell profile after treatment with

Origanum onites 0.007 % (v/v) for 48 hours. Live population cells are noticeably lower, late apoptotic population is almost double, while early apoptotic cells exhibit an increase of 13%. The results of flow cytometry are shown below.

Fig. 19: Origanum onites induces apoptosis apoptotic cell death in murine CT26 colon cancer cells. Flow cytometry of CT26 cells treated with 0.007 % v/v of Origanum onites essential oil for

48 hours. Viable (annexin V- PI-, Q4), early apoptotic (annexin V+ PI-, Q3), late apoptotic

(annexin V+ PI+, Q2) and necrotic (annexin V- PI+, Q1) populations are present.

- 58 -

Origanum onites 0.007 % (v/v), 48h

100 Live Early apoptotic Late apoptic 80

60

40

% of populations populations of %

20

0

Control and Origanum onites

Fig. 20: Bar chart showing the live, early apoptotic and late apoptotic cell populations of control and Origanum onites essential oil 0.007 % (v/v)-treated samples. There is an increase of early and late apoptotic cell proportions and a decrease of live cells after treatment with Origanum onites essential oil for 48h.

3.3 Cell migration assay

Cell migration assay is a widely applied assay to investigate the migration potential of cells under the effect of compounds or substances. In this study, the effect of 0.0005% OOEO on cell migration was observed and compared to

- 59 - control (DMEM with DMSO). The initial open area in control and treatment well did not have the same dimensions and it was deemed appropriate convert the surface of the photos using ImageJ and turn it into percentage based on the total surface of each photo.

- 60 -

Fig. 21: Processed photos showing the effect of OOEO 0.0005% (v/v) on CT26 cell migration at diverse time points, until the gap at the control well is not well-distinguished. After the photo processing, the bright area is populated by cells.

During the 48 hours, and more dramatically at the first 24h, the gap under the essential oil treatment closes more slowly in contrast to control, revealing that

- 61 - the essential oil has inhibitory effect and partially inhibits CT26 cell migration and potential invasiveness.

Image Closure

120 Control Origanum onites 0.0005% (v/v) 100

80

60

40

% Image closure %Image 20

0

0h 24h 48h 72h

Time points

Fig. 22: The rate of gap closing of CT26 treated with OOEO essential oil 0.0005% (v/v) as compared to the control group.

3.4 In vivo antitumor activity of OOEO against an experimental mouse model

- 62 -

Oral administration of OOEO showed significant anti-tumor effect as tumor

weight and tumor volume of the intervention group receiving OOEO were

measured and compared with animals on a relevant control diet.

Fig. 23: Photograph of the isolated tumors. The name of each group is noted.

A

The mean value of control group is 572.2706 mm3, while for the OOEO-treated

group is 254.1115 mm3. The percentage of tumor volume decrease in treated

group lies at about 55.6%. It should be noticed that none of the mice showed

any significant weight or behavioral change, nor any other difference or signs

of discomfort after macroscopic observation of the animals during the experimental period.

Fig. 24: Chemorophylactic effect of OOEO on tumor growth in a BALB/c tumor model. Corn

oil (controls) and OOEO were administrated orally to the two groups, then mice were

inoculated with CT26 cells. Following a period of one week, euthanasia was performed and the tumors were removed and measured. A. Tumor weight in gramms and B. Tumor volume

in mm3. The decrease of tumor volume between the two groups is statistically significant (p-

value= 0.009).

- 63 -

Tumor Weight (gr) 0,8 A

0,6

0,4

Tumor weight (gr) weight Tumor 0,2

0,0

Control Origanum onites

Tumour volume (mm3) 1200 ** B

1000

)

3 800

600

400

200

Tumour volume (mm volume Tumour

0

Control Origanum onites

3.5 Serum levels of Il-6 and IL-12 in OOEO treated and tumor-bearing mice

- 64 -

After sacrifice, blood samples were taken from mice, centrifuged and serums were used for quantification of cytokines using Sandwich ELISA (enzyme- linked immunosorbent assay)-as inflammation indicators that have been associated with the disease of cancer.

IL-6 (pg/ml) IL-12 (pg/ml)

Control 51.3282 ± 8.22 23.2 ± 4.011

Origanum onites 16.011 ± 2.146 30.52 ± 6.7222

Table 4: The table shows the levels of IL-6 and IL-12 cytokines in serum samples isolated from mice receiving corn oil or Origanum onites essential oil (OOEO) according to the administration schedule mentioned above. There is a dramatic reduction of pro-inflammatory

IL-6, while IL-12 is slightly increased.

IL-6 acts as a pro-inflammatory cytokine. It is known for preventing Fas- mediated apoptosis through the expression of the anti-apoptotic Bcl-2 or strongly stimulates anchorage-independent growth of some colon cancer cells. Our results show that IL-6 is much lower in the group receiving the essential oil. On the other side, IL-12 is cytokine having antitumor effect on murine colon cancer models. The results from ELISA show a slight increase in

OOEO-treated mice, suggesting that OOEO administration may offer beneficial immune-mediated anti-tumor properties.

- 65 -

Serum IL-6 and IL-12 production

70

Control 60 Origanum onites

50

40

30

% cytokine (pg/ml) cytokine % 20

10

0

IL-6 IL-12

Fig. 25: The bar chart illustrates the different percentage of the measured cytokines in the isolated serum samples.

- 66 -

4. Discussion

- 67 -

Natural products and their derivatives have been the most efficient source of lead research, but just a small proportion of nature’s biodiversity has been studied extensively for its application in drug discovery. The major group of the initial compounds for drug development is originated from the plant kingdom. In particular, the secreted essential oils derived from almost 60 families, including Lamiaciae, exhibit many activities that are widely investigated. Origanum onites, a species of Origanum genera belonging to

Lamiaciae has been found to possess several biological activities such as antimicrobial, antifungal, antiparasitic, analgetic, insecticidal and antigenotoxic effects.

Every year there are diagnosed more than one million new cases of colorectal cancer worldwide as this type of cancer is the third most common malignancy and the fourth most common cause of cancer mortality. Several lifestyle- related factors have been associated with it and its linkage with diet is one of the strongest for any type of cancer. Plenty of nutritional elements have been found to act as chemopreventive agents. In this study Origanum onites essential oil was investigated for its potential anti-neoplastic activity on colon cancer in in vitro and in vivo experimental models.

Recently, results from previous studies have shown that OOEO inhibits cell proliferation of hepatoma HepG2 cells (Özkan & Erdoğan, 2010), 5RP7 (H-ras- transformed rat embryonic fibroblasts) and rat adipose tissue endothelial cells

(Bostancıoğlu et al., 2012) in a dose-dependent manner for 24h, while the

EC50 values are 149.12 ug/ml, 125ug/ml and 250ul/ml, respectively. This work supports these findings as it is found that 50% cell growth inhibition of OOEO was about 0.0077% (v/v) on CT26 and 0.0106% (v/v) on HT-29 colon cancer cells for 48h. OOEO may also induce apoptosis on CT26 cells, based on both our results and literature. It was shown that 5RP7 cells display characteristic features of apoptosis by DAPI staining, when treated with concentrations of

- 68 -

125, 250 and 500 ug/ml (Bostancıoğlu et al., 2010). Treatment of CT26 cells with 0.0005% v/v OOEO inhibited cell invasion. This result is similar to that found by Bonstancioglu et al. (2010) that especially 125 ug/ml of the oil can markedly decrease rat adipose tissue endothelial cell migration at 12h.

As it is reported, essential oils distilled from Origanum species have been investigated for their potential anticancer effect on colon cancer and with few exceptions, the species of O. vulgare is the most examined. In particular, isolated polyphenols from Poliomintha glabrescens Gray, a Mexican native species (García-Pérez et al., 2013), as well as Origanum vulgare exhibit cytotoxic effect against HT-29 (Begnini et al., 2014) and Caco2 (Savini et al.,

2009) cells.

In the present study, tumor volume analysis data demonstrated that tumors were decreased about 55.6% when BALB/c mice were administrated with

0.066 % (v/v) OOEO once daily in contrast to the control group. In accordance with this, 40 mg/kg Origanum vulgare is shown to modulate 1,2- dimethylhydrazine (DMH)-induced rat colon cancer orally (Srihari et al.,

2008). Examined serum samples of the animals indicated that oil administration may regulate IL-6 and IL-12 cytokine production.

Further studies are needed to examine at a molecular level how OOEO exhibits its cytotoxic activity. Besides, it would be interesting to study

OOEO’s effect against other colon cancer cell lines (e.g. Caco-2, RKO, CLT 152

[SKI 294/CLT 152]) to link potential differences with their features. Additional studies on normal colon cells are also needed to further detect possible toxic side effects. However, future studies on the oil’s major constituents can reveal biological interactions, such as synergy and competition. Therefore, further investigations on long-term administration of OOEO in vivo are needed to study its potential toxicity. Taken together, these data raise the possibility that

- 69 -

OOEO may find application in food industry and may be a promising health- promoting agent incorporated into functional foods.

- 70 -

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Βεβα ίωση

Βεβαιώνω ότι πραγματοποίησα την παρούσα εργασία αυτόνομα και δε χρησιμοποίησα άλλες πηγές και βοηθήματα εκτός από τα αναφερόμενα.

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