Kisspeptin expression by human term placenta.

<p>A) Sections of human term (TP) and first trimester (FTP) placenta were double stained with anti-Kp-54/Kp-145 and anti-CK7, a syncytiotrophoblast (STB) marker. Nuclei were counterstained with DAPI. Kisspeptin was mainly localized to apical and basal surfaces of the STBs. B) Isolated cytotrophoblasts failed to express KISS1. A contaminating positive cell, most probably a STB, has been shown by white arrow. In negative control slides, primary antibody was substituted by non-immune rabbit sera. C) Western blotting of FTP and TP (from three placentas denotes as TP1-3). Lysates were probed with anti-Kp-54/Kp-145 antibody. The antibody produced one prominent band at ~15–16 kDa, which is in good agreement with the calculated MW for Kp-145 (15.391 kDa). With purified Kp-10, anti-Kp-54/Kp-145 produced a single band at about 1.5 kDa, which is in good agreement with the theoretical MW for Kp-10-13. β-actin served as loading control. D) Western blotting of conditioned medium (CM) collected from cultured term placental explants at different time points (1–72 hr). KP was released from placental explants in as early as 1 hr after initiation of culture, peaked at 4–6 hr and then progressively decreased until 72 hr. No KISS1-specific band was observed after KP removal (CM-w/o KP) by immuno precipitation. E) RT-PCR analysis of KISS1expression in total RNA freshly isolated from TP (representative experiment). β-actin served as loading control. No amplification controls (NAC) always were shown to be negative. The presence of KPs in CM was also confirmed by capture ELISA using two fold serially-diluted CMs. The results showed a positive correlation between CM concentration and OD (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153684#pone.0153684.g001" target="_blank">Fig 1F</a>). Results are representative of 11 term and 3 first trimester placentas. KPs: Kisspeptin, CK7: Cytokeratin 7, TP: Term placenta, FTP: First trimester placenta, NC: Negative control. Scale bar: A; 100 μm, B; 50μm.</p

Effect of term placental kisspeptins on motility of MDA-MB-231 and MCF-7 breast cancer cells.

<p>MDA-MB-231 (A) and MCF-7 (B) cells were treated with supernatants of cultured term placental explants containing KP (CM), KP-free CM (CM-w/o KP), KP-10 or culture medium alone and their migration toward scratch was monitored during a period of 30 hr. Representative photographs are shown in Aa (MDA-MB-231) and Ba (MCF-7). Percent of reduction in scratched areas at each time point compared to initial time point was measured and analyzed using Image J software (Ab for MDA-MB-231 and Bb for MCF-7). Treatment with P-234 reversed the effect of CM on motility of MDA-MB-231 (Ac) and MCF-7 (Bc). Co-treatment with selective ER modulators inhibited the effect of CM on motility of MCF-7 cells (Bc). * CM <i>vs</i>. CM-w/o KP (p<0.05–0.001 for MDA-MB-231 and p<0.05–0.001), φ CM <i>vs</i>. medium (p<0.01–0.001 for MDA-MB-231 and p<0.001–0.0001 for MCF-7), ψ KP-10 <i>vs</i>. medium (p<0.05–0.01 for MDA-MB-231 and p<0.01–0.0001 for MCF-7),? CM <i>vs</i>. CM-w/o KP and CM+P-234 (p<0.05),? CM-w/o KP, CM+P-234, CM+100 nm Tam and CM+1 nm Ral <i>vs</i>. CM (p<0.05). Results are representative of 4 term placentas. Tam: Tamoxifen, Ral: Raloxifene.</p

Effect of term placental kisspeptins on cytokine production by MDA-MB-231 and MCF-7 breast cancer cells.

<p>Concentrations of IL-6 and IL-8 were measured in culture supernatants of MDA-MB-231 (A) and MCF-7 (B) cells treated with CM, CM-w/o KP, KP-10 or culture medium. To exclude overlap of placental-derived cytokines with those produced by breast cancer cells, we also measured cytokine levels in CM and CM-w/o KP. $ Cell + CM <i>vs</i>. Cell + CM-w/o KP[(IL-6: p<0.05 for MCF-7), (IL-8: p<0.01 for MDA-MB-231 and p<0.01 for MCF-7)], ε Cell + CM <i>vs</i>. CM [(IL-6: p<0.001 for both breast cancer cells), (IL-8: p<0.01 for MDA-MB-231 and p<0.0001 for MCF-7)], μ Cell + CM <i>vs</i>. Cell + medium [(IL-6: p<0.05 for MDA-MB-231), (IL-8: p<0.05 for MDA-MB-231 and p<0.01 for MCF-7)], Ω Cell + CM-w/o KP <i>vs</i>. Cell + medium [(IL-6: p<0.05 for MDA-MB-231), (IL-8: p<0.05 for MDA-MB-231 and p<0.01 for MCF-7)], δ Cell + CM-w/o KP <i>vs</i>. CM-w/o KP[(IL-6: p<0.001 for MDA-MB-231, p<0.001 for MCF-7), (IL-8: p<0.01 for MDA-MB-231 and p<0.0001 for MCF-7)], ψ Cell+KP-10 <i>vs</i>. Cell + medium [(IL-6: p<0.05 for MDA-MB-231), (IL-8: p<0.05 for MDA-MB-231 and p<0.05 for MCF-7</p

Sequence and annealing temperatures of the primers used in this study.

<p>Sequence and annealing temperatures of the primers used in this study.</p

Effect of term placental kisspeptins on proliferation of MDA-MB-231 and MCF-7 breast cancer cells.

<p>MDA-MB-231 (A) and MCF7 (B) cells were treated with different media as indicated in the figure for 24, 48 and 72 hr. All treatments were performed in six replicas. The extent of proliferation was measured by Propidium Iodide (PI) fluorometric assay. Significant differences were analyzed by Kruskal-Wallis test with a Dunnett’s posthoc test. Different dilutions from 1:2 to 1:8 were tested. CM: Term placenta conditioned medium containing KP, CM-w/o KP: KP-free CM, anti-KP: anti-Kp-54/Kp-145 antibody, Rb Ig: Rabbit immunoglobulin, Medium: Culture medium alone, KP: Kisspeptin. Results are representative of 7 term placentas. * CM <i>vs</i>. CM-w/o KP(p<0.05–0.01 for MDA-MB-231 and p<0.05–0.001 for MCF-7), φ CM <i>vs</i>. medium (p<0.01–0.001 for MDA-MB-231 and p<0.05–0.01 for MCF-7), β CM <i>vs</i>. CM+anti-KP-10 (p<0.05–0.01 for MDA-MB-231 and p<0.05 for MCF-7), Φ CM-w/o KP <i>vs</i>. medium (p<0.05–0.001 for MDA-MB-231 and p<0.05 for MCF-7), ψ KP-10 <i>vs</i>. medium (p<0.05–0.01 for MDA-MB-231 and p<0.05–0.01 for MCF-7).</p

Effect of term placental kisspeptins on adhesion of MDA-MB-231 and MCF-7 breast cancer cells.

<p>MDA-MB-231 (A) and MCF-7 (B) cells were treated with different media as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153684#pone.0153684.g003" target="_blank">Fig 3</a> for 150 min and their adhesion to fibronectin-coated plates was assessed by a colorimetric assay. Different dilutions from 1:2 to 1:8 were tested. CM: Term placenta conditioned medium containing KP, CM-w/o KP: KP-free CM, anti-KP: anti-Kp-54/Kp-145 antibody, Rb Ig: Rabbit immunoglobulin, Medium: Culture medium alone, KP: Kisspeptin. Results are representative of 11 term placentas. * CM <i>vs</i>. CM-w/o KP(p<0.05 for MDA-MB-231).</p

Effect of term placental kisspeptins on invasiveness of MDA-MB-231 and MCF-7 breast cancer cells.

<p>MDA-MB-231 (A) and MCF-7 (B) cells were treated with supernatants of cultured term placental explants containing KP (CM), CM plus 1 μM KISS1R antagonist; P-234, KP-free CM (CM-w/o KP), KP-10 or culture medium alone and their invasion toward chemo attractant (FCS) through Matrigel-coated filters was investigated. Invading cells was enumerated in 50 random fields (Ab and Bb) and expressed as percentage of corresponding controls (Aa and Ba). C) Gelatin zymographic analysis for matrix metalloproteinase (MMP) expression. MDA-MB-231 cells treated with CM released higher amounts of MMP2 and MMP9 compared to those treated with either CM-w/o KP. In parallel, cells treated with KP-10 showed slightly higher MMP9 activity in comparison to cultured in medium alone. Cells treated with Phorbol 12-myristate 13-acetate (PMA) served as positive control. CM failed to express detectable levels of MMP2 and MMP9. Results of two (denoted by numbers) out of four samples are shown. KP: Kisspeptin, MW: Molecular weight.</p