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, Isabella Gigante, Rosa Anna Milella, Valentina De Nunzio, Riccardo Flamini, Mirko De Rosso, Maria Principia Scavo, Nicoletta Depalo, Elisabetta Fanizza, Maria Gabriella Caruso, Maria Notarnicola, Valeria Tutino, Isabella Gigante, Rosa Anna Milella, Valentina De Nunzio, Riccardo Flamini, Mirko De Rosso, Maria Principia Scavo, Nicoletta Depalo, Elisabetta Fanizza, Maria Gabriella Caruso, Maria Notarnicola

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

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
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.
Figure 2
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]− signal intensity in the negative-ion Ultra-High-Performance Liquid Chromatography/Quadrupole-Time of Flight mass spectrometry (UHPLC/QTOF) chromatogram.
Figure 3
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).
Figure 4
Figure 4
(a) Representative field emission scanning electron microscope (FE-SEM) micrographs (scale bar 5 µm, acquisition voltage 3 kV) of Caco2 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 Caco2 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.
Figure 5
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.

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