Auto-antibodies against apolipoprotein A-1 block cancer cells proliferation and induce apoptosis

Abstract

Auto-antibodies against apoA-1 (anti-apoA-1 IgGs) have been identified as important actors of atherosclerosis development through pro-inflammatory and pro-atherogenic properties and to also induce apoptosis in tumoral neuronal and lymphocyte derived cell lines through unknown mechanisms. The purpose of this study was to explore the cellular pathways involved in tumoral cell survival modulated by anti-apoA-1 antibodies. We observed that anti-apoA-1 antibodies induce growth arrest (in G2/M phase) and cell apoptosis through caspase 3 activation, accompanied by a selective p53 phosphorylation on serine 15. RNA sequencing indicated that anti-apoA-1 IgGs affect the expression of more than 950 genes belonging to five major groups of genes and respectively involved in i) cell proliferation inhibition, ii) p53 stabilisation and regulation, iii) apoptosis regulation, iv) inflammation regulation, and v) oxidative stress. In conclusion, anti-apoA-1 antibodies seem to have a role in blocking tumoral cell proliferation and survival, by activating a major tumor suppressor protein and by modulating the inflammatory and oxidative stress response. Further investigations are needed to explore a possible anti-cancer therapeutic approach of these antibodies in very specific and circumscribed conditions.

Keywords: anti-apoA-1 antibodies; apoptose; cell proliferation; oxidative stress.

Figure 1. Morphology change of cells cultured in presence of anti-apoA-1 IgG or control isotype IgG.

Pictures of U251 (A), SUPT1 (B) or HEK293 cells (C) in culture were taken at objective ×10 using a Axiovert 25 (Zeiss) microscope equipped with a CETI SI-3000 High-Definition Digital camera. Cells were incubated with goat polyclonal anti-apoA-1 IgG (aAPO-A1) or isotype CTL IgG (CTL IgG) at 150 μg/ml for 48 or 72 h.

Figure 2. Apoptosis and necrosis quantification after incubation of Hela, HEK293 and macrophages with anti-apoA-1 IgGs.

Data are expressed as % of apoptotic (A, C, E) (activated caspase 3 positive cells) or necrotic (B, D, F) events quantified by flow cytometry after 24, 48 or 72 h of cell incubation with polyclonal goat anti-apoA-1 or polyclonal goat control antibodies (150 μg/ml). Data are presented in Box and Whisker graph with median, IQR and range. Significant difference between anti-apoA-1 and control IgGs was assessed by Mann Whitney test on 5 to 8 independent experiments (^* p < 0.05; ^**** p < 0.001), only 2 experiments were performed with macrophages.

Figure 3

Anti-apoA-1 IgGs induced Caspase 3 and PARP cleavage in U251 (A) and SUPT1 (B). Dot plot analysis of cleaved caspase 3 according to cell treatment for 48 h or 72 h. The percentage of cleaved caspase 3 enriched cells is indicated in the Low-right panel. Western blot analysis of PARP cleavage after 48 and 72 h of U251 (A) and SUPT1 (B) cells treated with anti-apoA-1 IgGs or CTL IgGs at 150 μg/ml. Graphs present the mean+/–SD ratio of cleaved PARP over PARP, normalized to β-actin from 2 experiments.

Figure 4. Effect of anti-ApoA-1 IgGs on cell proliferation.

Cell proliferation and viability were quantified by MTT assay over 96 hours in tumoral cell lines (A) and non tumoral cell line and primary cell (B). Apoptosis inductor staurosporine was used at 1 μM and polyclonal goat anti-apoA-1 and polyclonal goat CTL IgGs at 150 μg/ml. Data are expressed as means + SD for 2 to 4 independent experiments. Significant differences between anti-apoA-1 and control IgGs, and untreated cells were assessed by Mann Whitney test (^* p < 0.05).

Figure 5. Modification of cell cycle phases according to cell treatment.

Cell cycle profiles were determined by flow cytometry and percentage of cells in G1, S and G2 phases were calculated using Watson model. Data are expressed as mean of % of cells from 8 experiments for U251, 4 experiments for SUPT1 and Hela, 2 experiments for HEK293. Significant differences between anti-apoA-1 and control IgG, and untreated cells were assessed by Mann Whitney test (^* p < 0.05).

Figure 6. Anti-apoA-1 IgGs induced p53 phosphorylation on Serine 15.

Western blot analysis of p53 total expression and phosphorylation on Ser 15 after 24, 48 and 72 h of U251 (A) and SUPT1 (B) cells treated with anti-apoA-1 IgG or CTL IgG at 150 μg/ml. Graphs present the expression of the p53 or phosphoSer15-p53 normalized to b-actin in Box and Whisker graph with median, IQR and range. Significant differences were assessed by Mann Whitney test on 6 independent experiments (^* p < 0.05).

Figure 7. Anti-apo-A1 IgGs induced a down-expression of cyclin B1 in U251.

Western blot analysis of cyclin B1 expression, CDK1 phosphorylation on Tyrosine 15 and Rb phosphorylation on Ser 807/811 in U251 after treatment with anti-apoA-1 IgGs or CTL IgGs at 150 μg/ml. Graphs represent the quantification of the target proteins normalized to b-actin from 2 (cyclin B1), 8 (CDK1-P-tyr15) or 6 (Rb-P-ser81) experiments in Box and Whisker graph with median, IQR and range.

Figure 8. Summary of potential anti-apoA-1 antibody induced signalings involved in cancer cell growth arrest and apoptosis.