In Vitro Anti-Proliferative and Apoptotic Effects of Hydroxytyrosyl Oleate on SH-SY5Y Human Neuroblastoma Cells

Abstract

The antitumor activity of polyphenols derived from extra virgin olive oil and, in particular the biological activity of HTyr, has been studied extensively. However, the use of HTyr as a therapeutic agent for clinical applications is limited by its low bioavailability and rapid excretion in humans. To overcome these limitations, several synthetic strategies have been optimized to prepare lipophenols and new compounds derived from HTyr to increase lipophilicity and bioavailability. One very promising ester is hydroxytyrosyl oleate (HTyr-OL) because the chemical structure of HTyr, which is responsible for several biological activities, is linked to the monounsaturated chain of oleic acid (OA), giving the compound high lipophilicity and thus bioavailability in the cellular environment. In this study, the in vitro cytotoxic, anti-proliferative, and apoptotic induction activities of HTyr-OL were evaluated against SH-SY5Y human neuroblastoma cells, and the effects were compared with those of HTyr and OA. The results showed that the biological activity of HTyr was maintained in HTyr-OL treatments at lower dosages. In addition, the shotgun proteomic approach was used to study HTyr-OL-treated and untreated neuroblastoma cells, revealing that the antioxidant, anti-proliferative and anti-inflammatory activities of HTyr-OL were observed in the unique proteins of the two groups of samples.

Keywords: SH-SY5Y human neuroblastoma cells; anti-proliferative activity; apoptosis induction activity; cytotoxicity; hydroxytyrosol; hydroxytyrosol oleate; plant-derived compounds.

Figure 1

Chemical structure of hydroxytyrosol (HTyr).

Figure 2

Chemical structure of hydroxytyrosyl oleate (HTyr-OL) and oleic acid (OA).

Scheme 1

Synthesis of HTyr.

Scheme 2

Synthesis of HTyr-OL.

Figure 3

(a) Graph bar of cytotoxic activity. The cell viability percentages of SH-SY5Y are plotted against treatment concentrations; (b) Line graphs of the anti-proliferative effects of HTyr-OL, (c) HTyr, and (d) OA on SH-SY5Y neuroblastoma cells at 24, 48, and 72 h. Results are expressed as mean of cell viability against concentration (µM). The values are expressed as % ± SD. *, p-value < 0.05 significant differences compared with ctrl group; a, p-value < 0.05 significant differences compared with 48 and 72 h; b, p-value < 0.05 significant differences compared with 24 and 72 h; c, p-value < 0.05 significant differences compared with 24 and 48 h.

Figure 4

Apoptosis detection by flow cytometry. Flow cytometric analysis of the HTyr-OL, HTyr and OA treatments on SH-SY5Y cells for apoptosis determination using AnnexinV-FITC/PI staining. L: live cells; EA: early apoptotic cells; LA: late apoptotic cells; N: necrotic cells.

Figure 5

SEM micrographs of SH-SY5Y cells at 24 h of EC₅₀ treatments, with: (a) HTyr-OL; (b) HTyr; (c) OA; (d) Ctrl or untreated cells; (e) VBL. Arrowheads for membrane blebbing of apoptotic cells. Scale bars 5 µm.

Figure 6

Western blot analysis for expression of cleaved caspase-3, studied using a cleaved caspase-3 monoclonal antibody. (a) In the lower panel, the representative image of detection of cleaved caspase-3 in SH-SY5Y cells treated with HTyr-OL, HTyr, OA; Ctrl and VBL cells were negative and positive controls, respectively (one of the three experiments). In the upper panel, detection of β-actin as the internal control; (b) Graph bar showing differences in intensity of caspase-3 obtained by pictures in (a), quantitatively evaluated using the ImageJ program.

Figure 6

Western blot analysis for expression of cleaved caspase-3, studied using a cleaved caspase-3 monoclonal antibody. (a) In the lower panel, the representative image of detection of cleaved caspase-3 in SH-SY5Y cells treated with HTyr-OL, HTyr, OA; Ctrl and VBL cells were negative and positive controls, respectively (one of the three experiments). In the upper panel, detection of β-actin as the internal control; (b) Graph bar showing differences in intensity of caspase-3 obtained by pictures in (a), quantitatively evaluated using the ImageJ program.

Figure 7

Identified proteins and Gene Ontology analysis. (a) Venn diagram representing the number of reproducibly quantified proteins for Ctrl (3911 total proteins) and HTyr-OL (3400 total proteins) samples. (b) Molecular functions comparison between Ctrl and HTyr-OL groups. (c) Cellular components comparison between Ctrl and HTyr-OL groups. In (b,c), Ctrl is on the outer chart and HTyr-OL is on the inner chart.

Figure 8

Volcano plot showing proteins differentially expressed in HTyr-OL and Ctrl datasets. Proteins with statistically significant differential expression (S0 = 2, p-value > 0.05) are in the top, right and left, quadrants. Upregulated proteins in HTyr-OL are represented in blue; downregulated proteins in HTyr-OL are in orange.

Figure 9

Protein–protein interaction (PPI) network of DEPs identified in HTyr-OL that describes the node proteins, wherein nodes in the same cluster are shown in the same color. The lines connecting the nodes indicate the associations between the proteins. The first cluster “Histone fold” is in red, and the second cluster “Apoptosis and response to oxidative stress” is in green. Proteins in blue are not included in a cluster.