**Flavonoid and Non-Flavonoid Compounds of Autumn Royal and Egnatia Grape Skin Extracts Affect Membrane PUFA's Profile and Cell Morphology in Human Colon Cancer Cell Lines**

**Valeria Tutino 1, Isabella Gigante 1, Rosa Anna Milella 2, Valentina De Nunzio 1, Riccardo Flamini 3, Mirko De Rosso 3, Maria Principia Scavo 4, Nicoletta Depalo 5, Elisabetta Fanizza 5,6, Maria Gabriella Caruso <sup>7</sup> and Maria Notarnicola 1,\***


Academic Editor: H.P. Vasantha Rupasinghe

Received: 29 June 2020; Accepted: 21 July 2020; Published: 23 July 2020

**Abstract:** Grapes contain many flavonoid and non-flavonoid compounds with anticancer effects. In this work we fully characterized the polyphenolic profile of two grape skin extracts (GSEs), Autumn Royal and Egnatia, and assessed their effects on Polyunsaturated Fatty Acid (PUFA) membrane levels of Caco2 and SW480 human colon cancer cell lines. Gene expression of 15-lipoxygenase-1 (15-LOX-1), and peroxisome proliferator-activated receptor gamma (PPAR-γ), as well as cell morphology, were evaluated. The polyphenolic composition was analyzed by Ultra-High-Performance Liquid Chromatography/Quadrupole-Time of Flight mass spectrometry (UHPLC/QTOF) analysis. PUFA levels were evaluated by gas chromatography, and gene expression levels of 15-LOX-1 and PPAR-γ were analyzed by real-time Polymerase Chain Reaction (PCR). Morphological cell changes caused by GSEs were identified by field emission scanning electron microscope (FE-SEM) and photomicrograph examination. We detected a different profile of flavonoid and non-flavonoid compounds in Autumn Royal and Egnatia GSEs. Cultured cells showed an increase of total PUFA levels mainly after treatment with Autumn Royal grape, and were richer in flavonoids when compared with the Egnatia variety. Both GSEs were able to affect 15-LOX-1 and PPAR-γ gene expression and cell morphology. Our results highlighted a new antitumor mechanism of GSEs that involves membrane PUFAs and their downstream pathways.

**Keywords:** flavonoids; non-flavonoids; membrane PUFAs profile; cell morphology; human colon cancer cells

#### **1. Introduction**

Grape (*Vitis vinifera* L.) is a fruit rich in polyphenols, bioactive compounds able to prevent the occurrence of cancer, reduce tumorigenesis, and influence important cancer-related pathways [1–3]. The anticancer effects of polyphenols are closely related to their chemical structure and concentration, as well as to the type of cancer [1,2]. Polyphenolic compounds are mainly divided into two groups: flavonoids, based on the common C6-C3-C6 skeleton which consists of two phenyl rings (A and B) linked by a heterocyclic ring (C), and non-flavonoids such as stilbenes (C6-C2-C6) and phenolic acids (C6-C1) [3,4]. The most abundant classes of flavonoids present in the grape skin and seeds include anthocyanins, flavonols, flavan-3-ols, and proanthocyanidins [5]. Flavonoids exist either as glycosides with attached sugars or as aglycones with no attached sugars, and differ in the degree of hydroxylation and substitution. These functional hydroxyl groups mediate the antioxidant effects of flavonoids and their ability to interact with biological membranes [2,6]. Stilbenes, such as resveratrol, are phytoalexins synthesized by plants in response to mechanical injury, UV irradiation, and fungal attacks [5]. Benzoic acid and cinnamic acid represent the most common phenolic acids present in the grape skin [7].

The phenolic composition of the grapes mainly depends on genotype but can be affected by environmental factors and agronomic practices [5]. Several in vivo and in vitro studies have demonstrated the antimetastatic effects of some polyphenolic compounds [8,9]. Mantena S. K. et al. evaluated the chemoprotective efficacy of grape seed proanthocyanidins in both metastatic breast cancer cells and in Balb/c mice, a mouse model of breast cancer obtained after subcutaneous implantation of the highly invasive and metastatic 4T1 mouse breast cell line [10]. There are several mechanisms of action through which grape polyphenols are able to inhibit the invasion and progression of metastasis [11–13]. These natural compounds can act on the structural components of the cytoskeleton, on cellular adhesions, and on the composition of the membrane fatty acids [11,14].

Polyphenols are able to influence cell membrane fluidity and cell motility by the stearoyl-CoA desaturase-1 (SCD1) enzyme activity, given by the oleic acid/stearic acid ratio [15,16]. The increase in the oleic acid content in cell membranes, and consequently the up-regulation of SCD1 enzyme, are known to stimulate the process of invasion and metastasis in human cancer cells [17,18]. Rearrangements in the lipidomic profile of the membrane are an important feature that distinguish cancer cells, since phospholipids are directly involved in the morphological changes occurring in tumorigenesis and tumor progression [16,19,20]. Moreover, it is now known that an imbalance in the ratio of omega-6/omega-3 (n-6/n-3) Polyunsaturated Fatty Acids (PUFAs), in favor of n-6, is associated with the development of chronic inflammatory diseases, including colon cancer [21,22]. Tumorigenesis of the colon is strongly influenced by the oxidative metabolism of PUFAs regulated by different enzymes, as 15-lipoxygenase-1 (15-LOX-1) [23,24]. 15-LOX-1 enzyme is known to exert antioxidant and antimetastatic action by activating peroxisome proliferator-activated receptor gamma (PPAR-γ) [25,26]. The preferred substrate for 15-LOX-1 is the essential fatty acid, namely linoleic acid (LA). This n-6 fatty acid with its metabolite, the 13-HODE (13-*S*-hydroxyoctadecadienoic acid), are down-expressed in human colon cancer [27].

Based on these assumptions, the main aims of the present study are: (1) to fully characterize the polyphenolic content of two table grape skin extracts (GSEs), Autumn Royal, a seedless black grape with healthy properties, and Egnatia, a new red seedless genotype obtained by breeding programs carried out by our research group; (2) to evaluate the effects of two GSEs on the membrane PUFA profile in two human colon cancer cell lines at different grade of differentiation, Caco2 and SW480; (3) to evaluate in the same treated cells the gene expression of 15-LOX-1 enzyme and its downstream factor PPAR-γ, as well as the possible changes in cell morphology.
