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

<p>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. <b>A)</b> 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; (***<i>p≤0.001</i>, **<i>p≤0.01</i>, *<i>p≤0.05</i>). <b>B)</b> Western blot for VEGF expression (30 kDa). <b>C)</b> 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.</p

Generation of stable prostate cancer cell lines with LOX-1 over-expression and shRNA against <i>olr1</i>.

<p><b>A)</b> Western blot for LOX-1 (40 kDa) expression in human CaP clones with overexpression of LOX-1. <b>B)</b> Western blot for LOX-1 (40 kDa) expression in human prostate cancer cell clones with LOX-1 knockdown <b>C)</b> Real-time PCR for LOX-1 expression in three clones with overexpression of LOX-1. <b>D)</b> 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; (***<i>p≤0.001</i>, **<i>p≤0.01</i>, *<i>p≤0.05</i>).</p

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

<p>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; (***<i>p≤0.001</i>, **<i>p≤0.01</i>, *<i>p≤0.05</i>).</p

oxLDL characterization and citotoxicity assay.

<p><b>A)</b> 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>B)</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 # <i>p≤0.05</i>).</p

Analysis of <i>olr1</i> expression in prostate cancer progression using public databases arrays.

<p>The expression of <i>olr1</i> 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 <i>t</i> test (<i>*p≤0.05</i>).</p

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

<p><b>A).</b> 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>B)</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 (***<i>p≤0.001</i>, **<i>p≤0.01</i>, *<i>p≤0.05</i>).</p