Anti-tumorigenic and Platinum-Sensitizing Effects of Apolipoprotein A1 and Apolipoprotein A1 Mimetic Peptides in Ovarian Cancer

Abstract

Objective: Apolipoprotein A1 (ApoA1) is remarkably decreased in serum and ovarian tissues of ovarian cancer patients. ApoA1 and ApoA1 mimetic peptides can sequestrate pro-inflammatory phospholipids, some of which are known to activate a variety of oncogenic pathways. Besides, more intrinsic anti-tumorigenic properties, independent from interaction with lipids, have also been described for ApoA1. We aimed to disclose the effects of ApoA1 and a mimetic peptide on the malignant phenotype of ovarian cancer cells, particularly regarding cell viability, invasiveness and platinum sensitization. Methods: Cells viability was assessed by MTS assay. Extracellular matrix invasion was assessed by transwell and spheroid invasion assays. Western blotting was performed to evaluate the effect of test compounds on intracellular pathways. Sensitization assays were performed in vitro and in the biologically relevant in ovo chorioallantoic membrane model. Results: Both ApoA1 and the mimetic peptide, at a concentration of 100 μg/mL, were able to decrease the viability of SKOV3, CAOV3, and OVCAR3 cells (p < 0.05). The peptide at this concentration was not able to affect the viability of immortalized non-neoplastic ovarian cells (p > 0.05). ApoA1 decreased SKOV3 cells invasiveness at 300 μg/mL after 72 and 96 h of exposure (p < 0.05), while the ApoA1 mimetic peptide prevented cell invasion at 50 and 100 μg/mL (p < 0.01). Treatment with 100 μg/mL of ApoA1 mimetic peptide decreased Akt phosphorylation in SKOV3 cells (p < 0.01). Accordingly, treatment with increasing concentrations of the peptide sensitized SKOV3, OVCAR3 and CAOV3 cells to cisplatin. This synergistic effect was observed both in vitro and in ovo. Conclusions: These results support the role of ApoA1 and ApoA1 mimetics as suppressors of ovarian tumorigenesis and as chemo-sensitising agents.

Keywords: ApoA1 mimetic peptides; apolipoprotein A1; invasiveness; ovarian cancer; platinum sensitization.

Figure 1

ApoA1 decreases the viability of ovarian cancer cells without affecting caspase activation. (A) SKOV3 cells were incubated with human ApoA1 up to a concentration of 100 μg/mL in serum-free conditions for 48 h. Untreated cells were exposed to vehicle only. ApoA1 treatment significantly decreased the viability of SKOV3 cells (Kruskal–Wallis test with Dunn’s multiple comparison post-test; p < 0.05). (B) SKOV3 cells were incubated with 100 μg/mL ApoA1 for 12 h (light gray bars) and 48 h (dark gray bars). Caspase 3/7 activation was quantified and normalized to the number of viable cells. No differences were observed in caspase activation between untreated cells and cells exposed to ApoA1. Differences were considered significant if p < 0.05. ^∗p < 0.05.

Figure 2

ApoA1 decreases the ability of ovarian cancer cells to invade the extracellular matrix. The 3D tumor spheroid invasion assay was performed in SKOV3 spheroids exposed to ApoA1 at a concentration of 300 μg/mL, and in unexposed spheroids (control). Invasion of extracellular matrix was assessed for up to 96 h. Exposure to ApoA1 significantly decreased the ability of SKOV3 cells to invade the extracellular matrix after 72 h (Two-way ANOVA; p < 0.001) and 96 h (p < 0.05). In (A), the relative invasion normalized to the spheroid area at time = 0 is plotted for ApoA1-treated and untreated spheroids. In (B), it is shown two spheroids, representatives of the ApoA1-treated and control conditions, at the beginning of the experiment (time = 0) and during the course of experiment (72 and 96 h). Differences were considered significant if p < 0.05. ^∗p < 0.05, ^∗∗∗p < 0.001.

Figure 3

The ApoA1 mimetic peptide decreases the viability of ovarian cancer cells without affecting non-neoplastic ovarian cells. SKOV3 (A), OVCAR3 (B), and CAOV3 (C) cells were incubated with the ApoA1 mimetic peptide 4F up to a concentration of 100 μg/mL, in serum-free conditions for 48 h. Untreated cells were exposed to vehicle only. Exposure to the highest concentration of the peptide decreased the viability of all the three cell lines (Kruskal–Wallis test with Dunn’s multiple comparison post-test; p < 0.001). Immortalized non-neoplastic human ovarian surface epithelia cells (OSEC2; D) were exposed to the highest concentration of peptide (100 μg/mL) used in the viability assay for the ovarian cancer cell lines. At this concentration the peptide did not affect the viability of OSEC2 cells. Differences were considered significant if p < 0.05. ^∗p < 0.05, ^∗∗∗p < 0.001.

Figure 4

The ApoA1 mimetic peptide hampers cancer cells invasion. A transwell migration assay was performed with SKOV3 cells exposed to the ApoA1 mimetic peptide at a concentration of 50 and 100 μg/mL, and with unexposed SKOV3 cells (control). Exposure to the ApoA1 mimetic peptide at both concentrations significantly decreased the ability of SKOV3 cells to migrate and invade, comparatively to unexposed cells (Kruskal–Wallis test with Dunn’s post-test; control vs. 50 μg/mL ApoA1 mimetic peptide: p = 0.0020; control vs. 100 μg/mL ApoA1 mimetic peptide: p = 0.0016). In (A), the number of cells per field is plotted for each condition. In (B), it is shown three images representatives of each experimental condition. Differences were considered significant if p < 0.05. ^∗∗p < 0.001.

Figure 5

The ApoA1 mimetic peptide strongly suppresses Akt signaling in ovarian cancer cells. After overnight serum starvation, SKOV3 cells were treated with the ApoA1 mimetic peptide (50 or 100 μg/mL). Untreated SKOV3 cells were used as control. Cell lysates were collected 12 or 24 h after treatment and subjected to western blot analysis. Calnexin was the loading control for these experiments. (A) Western blot for phospho Akt (Ser⁴⁷³) and phospho ERK1/2 (Thr²⁰²/Tyr²⁰⁴); (B) densitometry for Akt Ser⁴⁷³ phosphorylation; (C) densitometry for ERK1/2 Thr²⁰²/Tyr²⁰⁴ phosphorylation. The ApoA1 mimetic peptide strongly decreased phosphorylation of Akt at Ser⁴⁷³ after 12 h (two-way ANOVA; p < 0.01) and 24 h (p < 0.01) of exposure with the highest concentration of peptide (100 μg/mL). Differences were considered significant if p < 0.05. ^∗∗p < 0.001.

Figure 6

The ApoA1 mimetic peptide sensitizes ovarian cancer cells to cisplatin. SKOV3, OVCAR3, and CAOV3 cells were co-incubated with increasing concentrations of ApoA1 mimetic peptide, up to 150 μg/mL, and increasing concentrations of cisplatin, according to the sensitivity of each cell line. Exposure to the peptide and cisplatin was performed in full medium conditions. After 72 h, mitochondrial cell viability was assessed. Two-way ANOVA was performed as statistical test (A–C). ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001. Isoboles for assessing synergistic interactions between the ApoA1 mimetic peptide and cisplatin were also plotted (D–F), according to the Loewe additivity model for synergy/antagonism calculation. On the horizontal axis of the isoboles, the different concentrations of ApoA1 mimetic peptide employed in the in vitro sensitization assay are represented, while in the vertical axis it is represented the concentrations of cisplatin. The intersections represent the combined effect between the peptide and cisplatin. The color gradient ranges from dark blue (strong synergistic effect) to dark red (strong antagonistic effect). From the isoboles analysis, it is possible to identify synergies for different concentrations of ApoA1 mimetic peptide and cisplatin.

Figure 7

The ApoA1 mimetic peptide sensitizes ovarian cancer cells to cisplatin in the biologically relevant in ovo CAM model. SKOV3 xenografts were topically inoculated on the CAM, left untreated (control) or treated with the ApoA1 mimetic peptide (100 μg/mL), cisplatin (15 μM) or with the combination of both (100 μg/mL peptide plus 15 μM cisplatin). Xenograft size was calculated as the area of the fluorescent tumor relative to background. Student’s t-test was performed after testing the data normality by the Shapiro–Wilk normality test. Differences were considered significant if p < 0.05. ^∗∗p < 0.01.