Lectin-like oxidized LDL receptor-1 is an enhancer of tumor angiogenesis in human prostate cancer cells

Abstract

Altered expression and function of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) has been associated with several diseases such as endothelial dysfunction, atherosclerosis and obesity. In these pathologies, oxLDL/LOX-1 activates signaling pathways that promote cell proliferation, cell motility and angiogenesis. Recent studies have indicated that olr1 mRNA is over-expressed in stage III and IV of human prostatic adenocarcinomas. However, the function of LOX-1 in prostate cancer angiogenesis remains to be determined. Our aim was to analyze the contribution of oxLDL and LOX-1 to tumor angiogenesis using C4-2 prostate cancer cells. We analyzed the expression of pro-angiogenic molecules and angiogenesis on prostate cancer tumor xenografts, using prostate cancer cell models with overexpression or knockdown of LOX-1 receptor. Our results demonstrate that the activation of LOX-1 using oxLDL increases cell proliferation, and the expression of the pro-angiogenic molecules VEGF, MMP-2, and MMP-9 in a dose-dependent manner. Noticeably, these effects were prevented in the C4-2 prostate cancer model when LOX-1 expression was knocked down. The angiogenic effect of LOX-1 activated with oxLDL was further demonstrated using the aortic ring assay and the xenograft model of tumor growth on chorioallantoic membrane of chicken embryos. Consequently, we propose that LOX-1 activation by oxLDL is an important event that enhances tumor angiogenesis in human prostate cancer cells.

Figure 1. Generation of stable prostate cancer cell lines with LOX-1 over-expression and shRNA against olr1.

A) Western blot for LOX-1 (40 kDa) expression in human CaP clones with overexpression of LOX-1. B) Western blot for LOX-1 (40 kDa) expression in human prostate cancer cell clones with LOX-1 knockdown C) Real-time PCR for LOX-1 expression in three clones with overexpression of LOX-1. D) Real-time PCR for LOX-1 expression was determined in three clones that express shRNA/LOX-1(A), and three clones that express shRNA/LOX-1(B). The data represent the means ± S.D. of three independent experiments performed in triplicate, and statistically analyzed using one-way analysis of variance and Dunnett’s post-test; (***p≤0.001, **p≤0.01, *p≤0.05).

Figure 2. oxLDL characterization and citotoxicity assay.

A) oxLDL obtained from human plasma was oxidized with 7 uM of CuSO₄, monitored by spectrophotometry at λ 243 nm and electrophoresed in 1% agarose gels with sodium borate buffer. B) Cytotoxicity assay of prostate cancer cell models treated with 25, 50, and 100 µg/mL oxLDL during 12 hours. The data represent the means ± S.D. of three independent experiments performed in triplicate and statistically analyzed using one-way ANOVA and Dunnett’s post-test (* or # p≤0.05).

Figure 3. The oxLDL ligand increases the expression of pro-angiogenic markers.

Relative quantification of LOX-1, VEGF, MMP-2, and MMP-9 expression was performed using real-time PCR in human prostate cancer cell line C4-2 incubated with increasing concentration of oxLDL (25, 50, 100 µg/mL) for 12 hours. The data represent mean ± S.D. of three independent experiments performed in triplicate, and statistically analyzed using one-way analysis of variance and Dunnet post-test; (***p≤0.001, **p≤0.01, *p≤0.05).

Figure 4. Activation of LOX-1 by oxLDL increases the expression of pro-angiogenic markers.

C4-2, C4-2/LOX-1(−), and C4-2 LOX-1(+) human prostate cancer cell models were incubated in the with or without oxLDL [100 µg/mL] for 12 hours. A) Relative quantification of VEGF, MMP-2, and MMP-9 expression was analyzed using real-time PCR. The data represent the means ± S.D. of three independent experiments performed in triplicate and statistically analyzed using one-way analysis of variance and Dunnet post-test; (***p≤0.001, **p≤0.01, *p≤0.05). B) Western blot for VEGF expression (30 kDa). C) Zymogram for MMP-2 (72 kDa pro-form; 64 kDa active-form) and MMP-9 (92 kDa pro-form; 84 kDa active-form) activities in conditioned medium.

Figure 5. The activation of LOX-1 using oxLDL promotes the generation of sprouts in mouse aortic ring assays.

A). Microphotography of mouse aortic rings and quantification of sprouts per ring incubated with conditioned medium from prostate cancer C4-2, C4-2/LOX-1(–), and C4-2 LOX-1(+) cells previously stimulated with or without oxLDL [100 µg/mL]. B) LOX-1 activation by oxLDL promotes tumor angiogenesis in CaP xenograft models on chorioallantoic membrane of chicken embryos. Microphotography and quantification of tumor blood vessels in xenografts of C4-2, C4-2/LOX-1(–), and C4-2 LOX-1(+) cells, previously incubated with or without oxLDL [100 µg/mL], in chorioallantoic membranes of 10-days-old chicken embryos. The data represent the means ± S.D. of three independent experiments performed in triplicate, and was statistically analyzed using one-way analysis of variance with Dunnett’s post-test (***p≤0.001, **p≤0.01, *p≤0.05).

Figure 6. Analysis of olr1 expression in prostate cancer progression using public databases arrays.

The expression of olr1 at different stages of prostate cancer tumor progression was determined using the public database GDS2545 at the NCBI Gene Expression Omnibus (GEO). The data represent the means ± S.E. of human normal donor tissue n = 17, primary prostate tumors n = 64 and metastatic prostate tumors n = 50, which were statistically analyzed using a t test (*p≤0.05).