**2. Results and Discussion**

#### *2.1. Total Polyphenolic Content in Autumn Royal and Egnatia GSEs*

The total content of polyphenols was determined in Autumn Royal and Egnatia GSEs by a spectrophotometric assay. The results obtained confirmed that Autumn Royal is richer in total polyphenols, expressed as milligrams of gallic acid equivalents per gram of dry weight of skin (GAE/g dw), than Egnatia (53.10 ± 1.99 mg GAE/g dw versus 37.45 ± 0.73 mg GAE/g dw, *p*-value < 0.05, respectively) and that the anthocyanins were the most represented polyphenols in both varieties [28]. Polyphenolic profiles were determined by Ultra-High-Performance Liquid Chromatography/Quadrupole-Time of Flight mass spectrometry (UHPLC/QTOF) analysis. Anthocyanins were identified by positive ionization (Figure 1), the other polyphenols in negative ionization mode (Figure 2). Main MS/MS fragments and mean signal intensity of the metabolites identified in the samples are reported in the supplementary materials (Table S1). Positive and negative extract ion chromatograms (EIC), showing the signals of compounds identified, are reported in the supplementary materials (Figures S1A and S1B, respectively). More than 100 flavonoid and non-flavonoid compounds were identified in the both GSEs.

**Figure 1.** Anthocyanin composition of Autumn Royal and Egnatia table grape skin extracts expressed as the relative percentages (%) of M+• signals intensity in the positive-ion Ultra-High-Performance Liquid Chromatography/Quadrupole-Time of Flight mass spectrometry (UHPLC/QTOF) chromatogram.

The anthocyanin profiles of Autumn Royal and Egnatia GSEs were very similar and characterized by a higher percentage of anthocyanin-3-*O*-monoglucoside compounds (43% in both samples) (Figure 1). These molecules have different antitumor effects depending on the B-ring substituents [29]. Anthocyanins, whose structure contains an *o*-dihydroxy (catechol) B-ring, such as cyanidin-3-*O*-monoglucoside and delphinidin-3-*O*-monoglucoside, are characterized by tumorigenesis inhibition activity [30].

**Figure 2.** Flavonoid and non-flavonoid composition of Autumn Royal and Egnatia table grape skin extracts expressed as the relative percentages (%) of the total [M − H]<sup>−</sup> signal intensity in the negative-ion Ultra-High-Performance Liquid Chromatography/Quadrupole-Time of Flight mass spectrometry (UHPLC/QTOF) chromatogram.

Signals of phenolic acids were relevant in both varieties (22% and 35% for Autumn Royal and Egnatia, respectively), while total stilbene signals intensity was 2% in Autumn Royal and 5% in Egnatia extracts (Figure 2). Both samples showed flavonols and flavanonols as main signals, with total signal intensity 53% and 54% in Autumn Royal and Egnatia, respectively. In particular, high intensity of the B-ring trisubstituted flavonols, such as myricetin and syringetin glycosides, was found. Instead, signals of flavan-3-ols and proanthocyanidins were higher in Autumn Royal (23%) compared to Egnatia extract (6%). Several studies showed that flavan-3-ol oligomers (proanthocyanidins) are potent antioxidants and free radical scavengers also characterized by anticancer properties [31,32]. In our previous study, we found a higher antioxidant activity in Autumn Royal compared to Egnatia, and this result could also be due to the greater content of flavan-3-ols and proanthocyanidins found in Autumn Royal GSEs [28].

Within each class of flavonoids, a structural variation exists in their basic 15-carbon skeleton, leading to different physicochemical properties. An improvement in the antitumor biological activities of flavonoids is due to the 3- - and 4- -hydroxyl groups (*o*-diphenol groups), known as catechol groups, present on the B-ring [33]. Compared to the Egnatia variety, Autumn Royal presented a higher catechol percentage, calculated by summing the intensities of the signals of those metabolites whose structure includes one or more *o*-diphenol groups (100% versus 57.6%, respectively). Table 1 shows the *o*-diphenol compounds identified in both GSEs.

The number and position of hydroxyl groups also influence the interactions of flavonoids with the cell membrane lipid bilayer. The hydrophilic flavonoids, which contain more hydroxyl groups, interact with the polar head groups by hydrogen bonds, inducing membrane rigidification. On the contrary, the hydrophobic flavonoids pass through cell membranes, causing a modification of their permeability and fluidity [6,34,35]. Therefore, the contribution of each phenolic compound can be different, and their synergistic/antagonist interactions might influence their biological effects.


**Table 1.** Catechol derivatives identified in Autumn Royal and Egnatia grape skin extracts (GSEs).

#### *2.2. Membrane PUFA Profile After GSE Treatment in Human Colon Cancer Cell Lines*

To investigate the effects of Autumn Royal and Egnatia GSEs on the n-3 and n-6 PUFAs membrane composition, Caco2 and SW480 human colon cancer cell lines were treated with increasing concentrations of GSEs (20, 50 and 80 μg/mL), and the lipidomic profile was analyzed after 48 h of treatment (Table 2a,b). The choice to use these GSEs concentrations and this experimental time (48 h), was dictated by our previous study, in which we demonstrated that the greatest antiproliferative effect of GSEs in Caco2 and SW480 cell lines was observed in these experimental conditions [16].

Compared to the untreated control group (CTR), in Caco2 cells, the treatment with Autumn Royal and Egnatia GSEs caused an increase of both essential fatty acids (EFAs), linoleic acid (LA) and α-linolenic acid (ALA), already starting from the concentration of 20 μg/mL (Table 2a), and these increases did not correspond to a modification in the n-3 and n-6 fatty acids downstream pathways (Table 2a). Moreover, the increase in total PUFA levels, already at the lowest concentration (20 μg/mL) of both GSEs, was essentially due to the contribution of LA and ALA (Table 2a).

In the SW480 cell line, the treatment with Autumn Royal GSE increased LA levels at 20 μg/mL of concentration, whereas for Egnatia GSE, a higher concentration (50 μg/mL) was necessary (Table 2b). Compared to CTR, only a decrease in arachidonic acid (AA) levels was observed after exposure of both GSEs at 50 μg/mL (Table 2b). As regards the n-3 PUFAs pathway, ALA levels were induced after exposure to the two grape polyphenols, in particular after treatment with 80 μg/mL of Autumn Royal GSE and 20 μg/mL of Egnatia GSE (Table 2b). The increase in total PUFAs obtained after Autumn Royal treatment, at the concentration of 20 μg/mL, was due exclusively to the contribution of LA (Table 2b). The reduction of AA levels found at the concentration of 50 μg/mL of Autumn Royal was more pronounced than the increase of LA obtained at the same concentration of extract, thus balancing the levels of total PUFAs at 50 and 80 μg/mL (Table 2b).

Moreover, no alteration in n-6/n-3 ratio levels was found in all two cell lines and with both treatments (data not shown).

We used two human colon adenocarcinoma cell lines with different degrees of differentiation, Caco2 and SW480, in order to investigate the variations of the membrane PUFA levels induced by the quality of polyphenols contained in the two table grape varieties used. Cancer cells are in continuous proliferation and need large quantities of fatty acids and phospholipids to generate new cellular membranes [19,36]. Previously, we demonstrated the ability of Autumn Royal and Egnatia GSEs to influence membrane fluidity in Caco2 and SW480, through the inhibition of the enzyme SCD1 [16]. This enzyme, that converts saturated fatty acids (SFAs) into monounsaturated fatty acids (MUFAs), was reduced mainly in the Caco2 cell line after GSEs treatment. In this study, we demonstrated that Autumn Royal and Egnatia GSEs were also able to influence the membrane levels of total PUFAs. Different basal levels of PUFAs were found in untreated Caco2 and SW80 cells, probably due to cell type, developmental and growth stage of cells [37]. In SW480 cells with a lower degree of differentiation, the high AA levels found contribute to increasing the total PUFAs levels present in these cells, preparing the cellular pathways towards more inflammatory outcomes.


*Molecules* **2020** , *25*, 3352

concentrations

 of Autumn

**Table 2.** (**a**) Mean percentage of n-6 and n-3

Royal and Egnatia GSEs (20, 50, and 80 μg/mL) after 48 h of treatment; (**b**) mean percentage

Polyunsaturated

 Fatty Acids (PUFAs) in the Caco2 membrane cell line treated with increasing

 of n-6 and n-3

Polyunsaturated

 Fatty Acids (PUFAs) in the SW480

Both GSEs induced the levels of EFAs, LA and ALA, in all two cell lines studied. EFAs are important structural components of cell membranes that the animal cells must exclusively obtain from their environment [37]. Therefore, the increase of LA and ALA detected in treated Caco2 and SW80 cells was certainly attributable to the fatty acids contained in the grape skins.

Several studies have shown that LA, γ-linolenic acid (GLA), and dihomo-γ-linoleic acid (DGLA) have anticancer effects, unlike AA, which has been associated with the inflammation and with onset of the tumor [38–41]. Treatment with increasing concentrations of both extracts increased LA levels in SW480 cells. In addition, in these cell lines, both extracts led to a reduction in AA compared to untreated cells, exerting an anti-inflammatory effect inside cells. As regards the n-3 fatty acids pathway, no variation was observed. Hanikoglu A. et al. found differences in the reorganization of fatty acids in cell membranes of two different breast cancer cell lines, MCF-7 and MDA-MB231, after treatment with somatostatin, curcumin, and quercetin, alone or in combination [14]. Cancer cells, according to the degree of differentiation, behave differently to treatment with drugs and/or natural compounds, and this feature could explain the different response of SW480 to treatment with GSEs, with respect to Caco2 cells.

#### *2.3. Effects of GSE Treatments on the Gene Expression of 15-LOX-1 and PPAR-*γ *in Human Colon Cancer Cell Lines*

To better investigate the effects of Autumn Royal and Egnatia GSEs on membrane PUFA levels and their antitumoral and antimetastatic action, the gene expression of 15-LOX-1 and PPAR-γ, markers involved in the onset of colorectal cancer (CRC), was studied. Figure 3 shows the effects of increasing Autumn Royal and Egnatia GSE concentrations (20, 50, and 80 μg/mL) on the mRNA levels of 15-LOX-1 (Figure 3a) and PPAR-γ (Figure 3b) in Caco2 and SW480 cell lines after 48 h of treatment. Compared to CTR, Autumn Royal GSE induced a significant up-regulation of 15-LOX-1 gene expression in both cell lines studied, starting from the lowest concentration (20 μg/mL) (Figure 3a). Regarding treatment with Egnatia GSE, in Caco2 cells a higher concentration (80 μg/mL) was needed to observe a statistically significant increase in the gene expression levels of 15-LOX-1 compared to CTR (Figure 3a). For SW480 cells, the increase in the gene expression of 15-LOX-1 was already visible at 50 μg/mL of Egnatia GSE (Figure 3a). 15-LOX-1, through its product 13-S-HODE, activates PPAR-γ by inhibiting colorectal tumorigenesis. Therefore, possible changes in PPAR-γ gene expression after exposure to increasing concentrations of GSEs were investigated. Compared to CTR, Autumn Royal GSE treatment exerted an up-regulation of PPAR-γ mRNA levels, starting from 20 μg/mL in both Caco2 and SW480 cells (Figure 3b), whereas a higher concentration of Egnatia (80 μg/mL) was need to obtain the same significant increase in PPAR-γ gene expression (Figure 3b).

Carcinogenesis is known to be also caused by changes in PUFA levels of the cell membrane, including colon cancer formation [19]. 15-LOX-1 is able to oxygenate both n-3 and n-6 PUFAs. The main substrate of 15-LOX-1 is represented by LA, leading to the formation of 13(S)-HODE that activates PPAR-γ, an antimetastatic and anti-inflammatory factor in CRC [26,27].

The increase in expression of 15-LOX-1 found in the cell lines studied after GSE treatment confirms the antitumor effect exerted by these extracts, and the data obtained highlight a new mechanism of action through which GSEs inhibit colon tumorigenesis. Previously, we demonstrated the ability of GSEs to block cell migration and motility by inhibiting SCD1 and some components of the cytoskeleton [16]. The data obtained in this work show that the antiproliferative effect of GSEs also occurs through the induction of the expression of 15-LOX-1 that can be used for therapeutic purposes in CRC.

Cimen I. et al. have shown that 15-LOX-1 indirectly inhibits NF-kB through 13(S)-HODE-mediated PPAR-γ activation in HCT-116 and HT29 CRC cell lines, thus blocking cell proliferation [26]. Moreover, again in HCT-116 and HT29 cell lines, the expression of 15-LOX-1 reduced the ability of cells to adhere to fibronectin, thus inhibiting cell motility [42].

**Figure 3.** (**a**) 15-LOX-1 gene expression levels detected in Caco2 and SW480 cells treated with increasing concentrations (20, 50, 80 μg/mL) of Autumn Royal and Egnatia GSEs for 48 h of incubation; (**b**) PPAR-γ gene expression levels detected in Caco2 and SW480 cells treated with increasing concentrations (20, 50, 80 μg/mL) of Autumn Royal and Egnatia GSEs for 48 h of incubation. All data are expressed as mean ± Standard Deviation (SD) of three consecutive experiments. *p*-value was determined by ANOVA with Dunnett's posttest. \*\* *p* < 0.03 and \*\*\* *p* < 0.01 versus untreated control group (CTR).

Previous in vitro studies have shown that grape extracts can act differently on proliferation and apoptotic pathways [43,44]. These different biological effects of GSEs could depend both on the type of cancer cell and on the different polyphenolic content of grape extracts [43,45,46]. In fact, it is known that there are cell lines more sensitive to treatment with polyphenols than others in relation to cellular differentiation degree. Moreover, certain flavonoids and non-flavonoids contained in grape extracts can act together synergistically to give particular antiproliferative effects on cancer cells [46,47]. These considerations suggest that the quality of the polyphenolic content in a grape cultivar is an important factor that must be considered.

### *2.4. Autumn Royal and Egnatia GSEs Induce Cell Morphological Changes*

Figures 4 and 5 show the cell morphology of Caco2 and SW480 cell lines, respectively, treated with increasing concentrations of Autumn Royal and Egnatia GSEs (10, 20, 50, and 80 μg/mL) for 24 (T1) and 48 h (T2), analyzed by field emission scanning electron microscope (FE-SEM). To highlight the cellular morphological differences before and after each time GSEs exposure, untreated cells were used as control (CTR) at T0, T1, and T2 (Figures 4 and 5).

Caco2 CTR cells appeared firmly adherent and covered with abundant microvilli, with a visible cytoplasm and at the center a notable nucleus region without shrinkage (Figure 4). Both at T1 and T2, treatment with Autumn Royal GSE induced visible characteristic morphological changes, such as shrinkage of membrane cells, starting from low concentrations of extract (10 μg/mL), showing a typical state of cell suffering (Figure 4a). In comparison to CTR, the increase of the Autumn Royal concentration induced evident and characteristic changes in the cells, as the cytoplasmic contraction and cell membrane collapse. Moreover, at the highest concentrations of Autumn Royal GSE, it was no longer possible to distinguish cell structures (Figure 4a). As regards the treatment with Egnatia GSE, at T1, the maximum concentration (80 μg/mL) was necessary to observe clear signs of apoptosis, while at T2, already at 50 μg/mL, membrane blebbing and cell shrinkage were noted (Figure 4b).

The FE-SEM micrographs reported in Figure 5 show the untreated controls (CTR) of the SW480 cell line that appeared flat and adherent to the substrate with an evident central nucleus. The morphological changes induced by Autumn Royal GSE on SW480 cells were visible at T1 starting from the highest treatment concentrations (50 and 80 μg/mL), given that at the lowest concentrations the cells were morphologically similar to the untreated cells (Figure 5a). At T2, the proapoptotic effect of the polyphenols contained in Autumn Royal GSE was appreciated, starting from the lowest concentrations (10 μg/mL) (Figure 5a). At the concentration of 80 μg/mL of GSE, the cellular structures did not appear very detailed and appreciable, when compared to the CTR; in addition, the apparent break of the surface of the cell membrane caused cell death (Figure 5a). Both at T1 and T2, SW480 cells treated with low concentrations of Egnatia GSE were visibly adherent with a round and abundant cytoplasm (Figure 5b). Furthermore, these cells were connected with neighboring cells and extended in all directions. The higher concentrations of GSE (50 and 80 μg/mL) induced characteristic changes, as well as the reduction of cell cytoplasm and a decrease of surface microvilli, which led to cellular apoptosis (Figure 5b).

To our knowledge, this is the first study describing cell morphological changes induced by polyphenols using FE-SEM micrographs. A previous study of Wang S. et al. demonstrated the ability of Trollius chinensis flavonoids to induce apoptosis in human breast cancer MCF-7 cells using SEM analysis [48]. In these cells treated with high concentrations of flavonoids, the microvilli on the cellular surface completely disappeared and cell membranes collapsed. Other morphological changes, in particular cell shrinkage and membrane blebbing, have been found in HCT-15 colon cancer cells after treatment with diet-derived gallic acid [49]. However, our study confirms previous data obtained about antiproliferative and proapoptotic effects in human colon cancer cell lines treated with both GSEs [28] and, for the first time, demonstrates that the beneficial effects of GSE polyphenols are also due to their ability to induce morphological changes in cancer cells, preventing their growth and proliferation.

**Figure 5.** (**a**) Representative field emission scanning electron microscope (FE-SEM) micrographs (scale bar 5 μm, acquisition voltage 3 kV) of SW480 cell line treated with increasing concentrations (10, 20, 50, 80 μg/mL) of Autumn Royal GSE after 24 (T1) and 48 (T2) h of incubation; (**b**) Representative FE-SEM micrographs (scale bar 5 μm, acquisition voltage 3 kV) of SW480 cell line treated with increasing concentrations (10, 20, 50, 80 μg/mL) of Egnatia GSE after 24 (T1) and 48 (T2) h of incubation. The FE-SEM micrographs were selected as representative of a series of images collected on each sample. Untreated cells were used as control (CTR) at T0, T1, and T2. Each experiment was performed in triplicate.

### **3. Materials and Methods**

#### *3.1. Preparation of the GSEs*

Table grapes cultivar Autumn Royal, a seedless black grape variety, and Egnatia, a new red seedless genotype, were planted and grown in Apulia region at the Research Center for Viticulture and Enology of the Council for Agricultural Research and Economy (CREA-VE, Turi, BA, Italy). Grape samples were harvested at maturity in summer 2019 and berries were randomly collected and frozen at −20 ◦C. Approximately 100 frozen berries were manually peeled. To prepare the extracts, 250 mg of dry skin powder were mixed with 5 mL extraction solution of ethanol:water:hydrogen chloride 37% (70:30:1 *v*/*v*/*v*). After 24 h of complete darkness, the mixture was centrifuged, and the supernatant recovered, concentrated in a SpeedVac concentrator (Savant®SPD131DDA, Thermo Fisher Scientific, Waltham, MA, USA) for 90 min at 25 ◦C and 1.5 atmospheres of pressure and analyzed.

#### *3.2. Total Polyphenolic Content*

Total phenolic content was determined by Folin–Ciocalteu micro scale protocol with slight modification, as previously described [50]. Briefly, 1 mL of water, 0.02 mL of extract sample, 0.2 mL of the Folin reagent, and 0.8 mL of 10% sodium carbonate solution were mixed and brought to 3 mL. The absorbance was measured at 765 nm after 90 min. Results were expressed as mg GAE/g dw using calibration curves with standard gallic acid.

#### *3.3. UHPLC*/*QTOF Mass Spectrometry*

The extracts were three-fold diluted with H2O/CH3CN 95:5 (*v*/*v*) and analyzed using an Ultra-High Performance Liquid Chromatography (UHPLC) Agilent 1290 Infinity coupled to Agilent 1290 Infinity Autosampler (G4226A) and Agilent 6540 accurate-mass Quadrupole-Time of Flight (Q-TOF) Mass Spectrometer (nominal resolution 40.000) with Dual Agilent Jet Stream Ionization source (Agilent Technologies, Santa Clara, CA, USA). For each sample, two analyses in both positive and negative ionization mode by recording data in full scan acquisition mode were performed. Chromatographic separation was performed by using a Zorbax reverse-phase column (RRHD SB-C18 3 × 150 mm, 1.8 μm) (Agilent Technologies) and mobile phase composed by (A) 0.1% *v*/*v* aqueous formic acid and (B) 0.1% *v*/*v* formic acid in acetonitrile. Gradient elution program: 5% B isocratic for 8 min, from 5% to 45% B in 10 min, from 45% to 65% B in 5 min, from 65% to 90% in 4 min, 90% B isocratic for 10 min. Flow rate: 0.4 mL/min; sample injection: 10 μL; column temperature: 35 ◦C. After each sample a blank composed by the two mobile phases 1:1 *v*/*v* was analyzed to check the absence of false positives.

QTOF conditions used: sheath gas nitrogen 10 L/min at 400 ◦C; dehydration gas 8 L/min at 350 ◦C; nebulizer pressure 60 psi; nozzle voltage 0 kV (negative mode) and 1 kV (positive mode); capillary voltage ± 3.5 kV in positive and negative ion modes. Signals in the *m*/*z* 100–1700 range at acquisition rate 2 spectra/s were recorded. Mass calibrations were performed with standard mix G1969-85000 (Supelco Inc., Bellefonte, WA, USA), residual error for the expected masses between ± 0.2 ppm. Negative ionization lock masses: TFA anion at *m*/*z* 112.9856 and HP-0921 at *m*/*z* 966.0007 (ion [M + HCOO]−); positive ionization lock masses: purine at *m*/*z* 121.0509 and HP-0921 at *m*/*z* 922.0098. MS/MS fragmentation of the parent ions selected in the *m*/*z* 100–1700 range by using collision energies between 20 and 60 eV. Acquisition rate: 2 spectra/s.

Data acquisition software Agilent MassHunter version B.04.00 (B4033.2). Data analysis performed by using AgilentMassHunter Qualitative Analysis software B.05.00 (5.0.519.0). The overall identification score of compounds was calculated by the weighted average of the isotopic pattern signals (exact masses, relative abundances, *m*/*z* distances: Wmass = 100, Wabundance = 60, Wspacing = 50). Targeted identification of metabolites was performed by using the homemade database *GrapeMetabolomics* [51].

#### *3.4. Cell Culture and Treatment*

Human colon adenocarcinoma derived Caco2 cell line (ATCC: HTB 37) (well-differentiated) (G1–2) (from adenocarcinoma) and SW480 cell line (ATCC: CCL 228) (poorly-differentiated) (G3–4) (from adenocarcinoma grades III–IV) were purchased from the American Type Culture Collection (ATCC) Cell Bank (Manassas, VA, USA). Cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium, for Caco2 cells, and Dulbecco's Modified Eagle Medium (DMEM) for SW480 cells. All cell culture medium and reagents were purchased from Gibco, Life Technologies Limited, Paisley, UK. Culture medium was supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin and incubated at 37 ◦C in a humidified atmosphere containing 5% CO2 in air. Autumn Royal and Egnatia GSEs were added to the medium at increasing concentrations dissolved in 100 μL of solvent, composed by ethanol:water:hydrogen chloride 37% (70:30:1 *v*/*v*/*v*) (Sigma Aldrich, Milan, Italy), whereas the cells of control group received the same amount of the solvent. The cells were then incubated at 37 ◦C in a humidified 5% CO2 incubator for 24 and 48 h.

### *3.5. Lipids Extraction and PUFAs Analysis*

Cell membrane fatty acids were extracted after 48 h of Autumn Royal or Egnatia GSEs treatment at 20, 50, and 80 μg/mL of concentrations. Untreated cells were used as control. Lipids from cell lysate were extracted using the Folch extraction method with some modifications [52]. PUFA analysis was assessed by a gas chromatograph (ThermoFisher Scientific, Focus GC, Milan, Italy) using ChromQuest 4.1 software (Thermo Fisher Scientific, Focus GC, Milan, Italy), as previously described [16].
