Induction of endothelial cell apoptosis by rhLK8.

<p>HUVEC monolayers were incubated in EBM-2 containing 1% FBS in the presence or absence of 3 ng/ml bFGF and treated with various concentrations of rhLK8 (0.1–5 μM) for 12 or 24 h. Endothelial apoptosis was assessed by nuclear morphology after staining with Hoechst 33452. <i>(A)</i> Representative photomicrographs of control (<i>left</i>) or rhLK8 (5 μM)-treated HUVECs (<i>right</i>) in the presence of 3 ng/ml bFGF for 12 h. Apoptotic endothelial cells are indicated by arrows. <i>B</i> and <i>C</i>, the percentage of cells undergoing apoptosis was determined in cells treated with various concentrations of rhLK8 in the absence (<i>B</i>) or presence (<i>C</i>) of bFGF after 12 (<i>filled bars</i>) or 24 h (<i>open bars</i>). Each column represents the mean ± SD. (<i>D–F</i>) HUVECs were incubated with rhLK8 (5 μM) for various time periods as indicated. Cells were then collected and lysed, and whole cell proteins were separated by SDS-PAGE. (<i>D</i>) The activation of caspase-3 was determined by Western blotting using antibodies against procaspase-3 or a 20 kDa processed form of caspase-3, as indicated. (<i>E</i>) Western blotting using antibodies against procaspase-9 was performed to determine the activation of caspase-9. Actin (<i>lower panel</i>) was used as a loading control. (<i>F</i>) Cytosolic and membrane-bound proteins were prepared as described in the “Materials and Methods” and were analyzed by Western blotting using antibodies against cytochrome c to determine the release of cytochrome c into the cytosol. Protein samples loaded in <i>lane C-16</i> were prepared from cells incubated without rhLK8 for 16 h. The immunoblots shown are representative of at least three independent experiments. (Replicates of Fig. 1D, 1E, and 1F are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093794#pone.0093794.s001" target="_blank">Fig. S1</a>).</p

Suppression of liver metastasis by rhLK8.

<p>Approximately 3×10⁵ LS174T human colorectal carcinoma cells were injected into the spleen parenchyma of athymic BALB/c nude mice. Mice had daily i.p. administrations of rhLK8 (50, 10 or 2 mg/kg/day) for fourteen days. Mice were then sacrificed, and the livers were collected to analyze the metastasis of intrasplenically injected LS174T cells. (<i>A</i>) Representative photographs showing livers obtained from control (<i>left</i>) or rhLK8-treated (<i>right</i>) mice. (<i>B</i>) Number of surface tumor nodules in control and rhLK8-treated livers. *, <i>p</i><0.05; **, <i>p</i><0.01. (<i>C</i>) Sections of tumor tissues stained with H&E. <i>Arrows</i> indicate liver metastases. (<i>D</i>) The number of liver metastases per unit area (100 mm²) of randomly selected fields. *, <i>p</i><0.05; **, <i>p</i><0.01. (<i>E</i>) Immunohistochemical analyses of liver metastases of LS174T human colorectal carcinoma. Nuclei of liver specimens were stained with Hoechst 33342 (<i>a</i> and <i>c</i>), and double immunofluorescence staining was performed for CD31/PECAM-1 (red) and TUNEL (green) in control mice (<i>a</i> and <i>b</i>) or mice treated with rhLK8 (<i>c</i>, <i>d</i>, and <i>e</i>) as indicated. Apoptosis of tumor-associated endothelial cells (yellow) in the liver metastases treated with rhLK8 is indicated by arrows. Magnification, ×100. High magnification (panel <i>e</i>; ×200) of a selected region of panel (<i>d</i>), indicated by a dotted box. Bars, 100 μm. Data are representative of at least three independent experiments. (Replicates of Fig. 4A and 4B are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093794#pone.0093794.s001" target="_blank">Fig. S1</a>).</p

Inhibition of rhLK8-mediated endothelial cell apoptosis by <i>GRP78</i>-directed siRNA knockdown or anti-GRP78 antibodies.

<p>(<i>A</i>) To determine whether GRP78 may be involved in rhLK8-mediated endothelial cell apoptosis, HUVEC monolayers were treated with rhLK8 (1 or 5 μM) after pretreatment with 5 μg of GRP78 antibody for 30 min. Data are representative of three independent experiments. (<i>B</i>) HUVECs transfected with scrambled siRNA or <i>GRP78</i>-specific siRNA were treated with 5 μM of rhLK8. The subsequent induction of apoptosis was detected by antibodies against active caspase-3 or procaspase-9. The expression of GRP78 was detected by anti-GRP78 monoclonal antibody. GAPDH was used for loading control. Data are representative of two independent experiments. (C) HUVECs treated with GRP78 antibody or transfected with <i>GRP78</i>-specific siRNA were treated with rhLK8 (5 μM) and endothelial apoptosis was assessed by nuclear morphology after staining with Hoechst 33452. Representative photomicrographs are shown. Apoptotic endothelial cells are indicated by arrows. The magnifications are ×100. (Replicates of Fig. 3A and 3B are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093794#pone.0093794.s003" target="_blank">Fig. S3</a>).</p

Effects of the combination of rhLK8 and conventional chemotherapy on the suppression of colon cancer liver metastasis and host survival.

<p>Mice injected intrasplenically with LS174T human colorectal carcinoma cells were administered vehicle, 5-FU, rhLK8, or 5-FU plus rhLK8, as described in “Materials and Methods”. (<i>A</i>) Mice were sacrificed, and surface nodules were counted. *, <i>p</i><0.05 vs. control. **, <i>p</i><0.0005 vs. control. (<i>B</i>) Kaplan-Meier survival curve of control mice (•) and mice treated with 5-FU (○), rhLK8 (▾), or 5-FU plus rhLK8 (▽). Differences in survival were statistically significant, as determined by log-rank analysis: <i>p</i><0.005, <i>p</i><0.001, and <i>p</i><0.0001 vs. control mice.</p

Kaplan-Meier survival curve for mice bearing liver metastases.

<p>Athymic BALB/c nude mice were injected intrasplenically with 3×10⁵ LS174T human colorectal carcinoma cells. Mice had daily i.p. administrations of 0 (•), 10 (▾) or 50 (○) mg/kg rhLK8, and the fraction of surviving mice was monitored over time. Differences in survival were statistically significant, as determined by log-rank analysis: <i>p</i><0.005, control vs. the rhLK8-treated group (10 mg/kg); <i>p</i><0.0001, control vs. the rhLK8-treated group (50 mg/kg). Data are representative of two independent experiments.</p

Interaction of GRP78 with rhLK8 as determined by co-immunoprecipitation and flow cytometry.

<p>For immunoprecipitation (<i>IP</i>) experiments, cell extracts of (<i>A</i>) HUVECs or (<i>B</i>) HEK293 cells expressing 6×His-tagged GRP78 protein were mixed overnight at 4°C with 10 μg of HA monoclonal antibody, 2 μg of HA-tagged rhLK8, and protein G-agarose. Eluted samples were separated by SDS-PAGE. GRP78 bound to rhLK8 was detected by Western blot (<i>WB</i>) using an anti-GRP78 monoclonal antibody or anti-His antibody. To determine the binding of rhLK8 to the GRP78 protein on the surface of HUVECs, HUVECs that had been transduced with scrambled siRNA or <i>GRP78-</i>specific siRNA were harvested and stained with (<i>C</i>) FITC-labeled rhLK8 or (<i>D</i>) anti-GRP78 antibodies and analyzed by flow cytometry. Unstained cells or cells stained with FITC-labeled secondary antibody only were used as negative control. Data are representative of two independent experiments. (Replicates of Fig. 2A and 2B are available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093794#pone.0093794.s002" target="_blank">Fig. S2</a>).</p

Supplementary Table and Figures from Characterization of CD45⁻/CD31⁺/CD105⁺ Circulating Cells in the Peripheral Blood of Patients with Gynecologic Malignancies

Supplementary Table and Figures - PDF file 237K, Supplementary Table 1. Clinical characteristics of cancer patients. Supplementary Fig. 1. Isolation of CD45+/CD31- cells, CD45+/CD31bright cells, and CD45-/CD31+ cells from PBMCs using FACSAria flow cytometer. Supplementary Fig. 2. A Venn diagram showing the expression of various antigens in the endothelial cells, platelets, and monocytes</p