β₂-GPI inhibits the VEGF-induced Akt phosphorylation in HAECs.

<p>(A) A time course of Akt phosphorylation in HAECs treated with VEGF was determined by Western blot analysis. The intensity of the Akt phosphorylation band was normalized against total Akt expression and was calculated as an expression fold (relative to the control, which was set as 1). (B) The effect of β₂-GPI on VEGF-induced Akt phosphorylation was determined in HAECs treated with or without VEGF in combination with β₂-GPI at indicated concentrations. Results are presented as mean ± SEM and representative of more than three independent experiments. *<i>p</i> < 0.05 versus control; ^#<i>p</i> < 0.05 versus VEGF treatment alone.</p

β₂-GPI and deglycosylated β₂-GPI inhibits the VEGF-induced proliferation in HAECs.

<p>(A) HAECs were treated with or without VEGF in combination with β₂-GPI at indicated concentrations for 72 h and were incubated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for another 4 h. Then the effect of β₂-GPI on VEGF-induced cell proliferation was determined by MTT assay. Treatment of anti-β₂-GPI antibody and albumin was performed to confirm the specific effect of β₂-GPI on cell proliferation. (B) The effect of β₂-GPI on VEGF-induced cell proliferation was also determined by BrdU incorporation assay in HAECs with or without VEGF. Cells were cultured on a 96-well plate and were incubated with β₂-GPI in the presence of VEGF for 72 h, and then labeled with BrdU. Quantification was performed using a cell proliferation ELISA colorimetric kit. Treatment of anti-β₂-GPI antibody and albumin was used as the comparison group. (C) The purified β₂-GPI was denatured and the carbohydrate residues of β₂-GPI were removed by peptide-N-glycosidase F. Then the effect of β₂-GPI and deglycosylated β₂-GPI at 200 μg/ml on cell proliferation was compared to the cells treated with VEGF by BrdU incorporation assay. Statistics were done using one-way ANOVA and data are expressed as a percentage normalized to the control group (set as 100%). Results are expressed as mean ± SEM of at least three independent experiments. **<i>p</i> < 0.01 versus control group; ^#<i>p</i> < 0.05, ^##<i>p</i> < 0.01, ^###<i>p</i> < 0.001 versus VEGF treatment alone.</p

β₂-GPI and deglycosylated β₂-GPI inhibits the VEGF-induced tube formation in HAECs.

<p>(A) HAECs were seeded on the surface of a basement membrane extract and were treated with or without VEGF in combination with β₂-GPI at indicated concentrations. The results are representative of those observed in four separate experiments (×40). (B) The degree of tube formation in HAECs was estimated by the Metamorph tube formation module. Bar graphs represent the quantitative analysis of tube formation. (C) Images were taken in HAECs treated with or without VEGF in combination with β₂-GPI and deglycosylated β₂-GPI. The results are representative of those observed in four separate experiments. (D) The effect of β₂-GPI and deglycosylated β₂-GPI on tube formation in HAECs was estimated by the Metamorph tube formation module. Bar graphs represent the quantitative analysis of tube formation expressed as mean ± SEM and representative of more than three independent experiments. **<i>p</i> < 0.01 versus control; ^##<i>p</i> < 0.01 versus VEGF treatment alone.</p

β₂-GPI inhibits the VEGF-induced eNOS phosphorylation in HAECs.

<p>(A) A time course of eNOS phosphorylation in HAECs treated with VEGF was determined by Western blot analysis. The intensity of the eNOS phosphorylation band was normalized against total eNOS expression and was calculated as the fold of control (set as 1). (B) The effect of β₂-GPI on VEGF-induced eNOS phosphorylation was determined in HAECs treated with or without VEGF in combination with β₂-GPI at indicated concentrations. Results are expressed as mean ± SEM and representative of more than three independent experiments. *<i>p</i> < 0.05 versus control; ^#<i>p</i> < 0.05 versus VEGF treatment alone.</p

β₂-GPI inhibits the VEGF-induced angiogenesis in mice.

<p>(A) C57BL/6 mice were injected subcutaneously with 0.5 ml Matrigel containing with or without VEGF in combination with β₂-GPI at indicated concentrations (n = 6–8 per group). After 14 days, Matrigel plugs were removed and representative images were taken as shown. (B) Quantitative evaluation of angiogenesis in Matrigel plugs was determined by hemoglobin using the Drabkin’s reagent kit. Bar graphs represent the quantitative analysis of the hemoglobin content of plugs expressed as mean ± SEM. **<i>p</i> < 0.01 versus control; ^#<i>p</i> < 0.05 versus VEGF treatment alone. (C) The effect of β₂-GPI on the VEGF-induced angiogenesis was also detected using an angioreactor-based <i>in vivo</i> assay. Angioreactors with or without VEGF in combination with β2-GPI were implanted subcutaneously into the dorsal flank of C57BL/6 mice for 14 days, and vessels allowed to infiltrate. Two silicone tubes were implanted per mouse. Angioreactors are photographed using a Canon powershot G9 digital camera and the representative images were taken as shown.</p

Involvement of PI3K/AKT and STAT3 in CYT-Rx20-inhibited KYSE70 and TE8 cell viability and migration.

<p>(A) Dose effect of CYT-Rx20 on p85, AKT, and STAT3 phosphorylation was performed was performed by using immunoblotting. KYSE70 and TE8 were incubated with CYT-Rx20, and cell viability was detected by using XTT colorimetric assay. In parallel cultures, cells were pre-treated with 4 μM SC79 (B) or 0.5 μM colivelin (C) for 1 h and were further co-incubated with CYT-Rx20 for another 72 h. All values are expressed as a percentage of the control group, which is set as 100%. Results are means ± SEM (n = 3–4). *<i>P</i> < 0.05, **<i>P</i> <0.01, ***<i>P</i> <0.001 compared with the indicated group. Using a modified Boyden chamber assay, KYSE70 and TE8 were pre-treated with 4 μM SC79 (D) or 0.5 μM colivelin (E) for 1 h and further treated with CYT-Rx20 for 48 h. Representative photographs are shown with ×40 magnification. Histograms show the average area of migrated cells compared with control. Results are presented as means ± SEM (n = 3). *<i>P</i> < 0.05, **<i>P</i> <0.01, ***<i>P</i> <0.001 compared with the indicated group.</p

Effect of CYT-Rx20 on cytotoxicity and cell apoptosis in esophageal cancer cells.

<p>(A) Chemical structure of CYT-Rx20. (B) Effects of CYT-Rx20 on KYSE70 and TE8 cell viability. KYSE70 and TE8 cells were treated with various concentrations of CYT-Rx20 for 48 h and assessed by XTT colorimetric assay. The representative results are repeated at least three times and five replicate wells per CYT-Rx20 concentration in each experiment. (C) KYSE70 and TE8 cells were treated with CYT-Rx20 for 48 h, and cell death was examined with Annexin V/PI staining followed by flow cytometric analysis. (D) Effect of CYT-Rx20 on KYSE70 and TE8 cell apoptosis. TUNEL staining of cells with CYT-Rx20 was analyzed at 48 hours. One-thousand cells were counted for positivity for each stain. (E) Caspase-associated proteins were analyzed by immunoblotting after KYSE70 and TE8 cells were treated with the indicated concentrations of CYT-Rx20 for 24 h. The expression of α-tubulin was used as the internal control. The representative results are from three separate experiments. Results are means ± SEM (n = 3–5). *<i>P</i> < 0.05, **<i>P</i> <0.01, ***<i>P</i> <0.001 compared with the control group.</p

CYT-Rx20 suppressed xenograft tumor growth and decreased phospho-AKT and phospho-STAT3 expression in vivo.

<p>(A) KYSE70 cells were treated with the indicated concentrations of CYT-Rx20, followed by evaluation of anchorage-independent colony formation using soft agar assay as described in the Materials and methods section. The representative photographs are shown with ×100 magnification. (B) Female nude mice subcutaneously xenografted with KYSE70 cells were intraperitoneally treated with 0.1% DMSO in normal saline (control), 5 μg/g CYT-Rx20, or 25 μg/g CYT-Rx20 three times per week (n = 10 for each group). Tumor volumes were measured every week for each group and calculated according to the formula of width²×length/2. (C) Tumor weight was measured after sacrifice of the mice at the end of the 4-week treatment period. (D) Xenograft tumor tissues were analyzed for the expression of Ki-67, phospho-AKT, and phospho-STAT3 by IHC staining. Negative control was performed in the same procedure but without addition of Ki-67, phospho-AKT, or phospho-STAT3 antibodies. H-score was calculated as the product of percentage of stained cells and intensity of staining. The representative photographs are shown with ×200 magnification. (E) Hematoxylin and eosin (H&E) staining of tissues sections from mouse organs in control and CYT-Rx20 (25 μg/g)-administered mice. The representative photographs are shown with ×100 (Esophagus, Lung, Spleen), ×200 (Heart, Liver, Kidney) magnification. Results are means ± SEM. *<i>P</i> < 0.05, **<i>P</i> <0.01, ***<i>P</i> <0.001 compared with the control group.</p

Cytotoxicity^a of CYT-Rx20 on esophageal cancer cell lines.

<p>Cytotoxicity<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166453#t001fn001" target="_blank">^a</a> of CYT-Rx20 on esophageal cancer cell lines.</p

CYT-Rx20 suppressed orthotopic esophageal tumor growth and metastasis in vivo.

<p>(A) Luciferase-expressing KYSE70 cells were inoculated into the abdominal esophagus of female nude mice as described in the Materials and methods section. The mice were intraperitoneally injected with 0 μg/g (control) or 5 μg/g of CYT-Rx20 three times a week (n = 4–5 per group). After 4 weeks, the mice were anesthetized and intraperitoneally injected with D-luciferin (150 mg/kg) for detection of bioluminescence by IVIS Spectrum in vivo imaging system. (B) Orthotopic esophageal tumor tissues were analyzed for the expression of Ki-67 and phospho-STAT3 by IHC staining. H-score was calculated as the product of percentage of stained cells and intensity of staining. The representative photographs are shown with ×40 (H&E), ×200 (IHC) magnification. (C) Metastatic lung tumor tissues from mice with or without orthotopic esophageal tumor cell injection were analyzed for the expression of Ki-67 and phospho-STAT3. Histograms show the H-score calculated as the product of percentage of stained cells and intensity of staining. The representative photographs are shown with ×100 (H&E), ×200 (IHC) magnification. Results are means ± SEM. *<i>P</i> < 0.05, **<i>P</i> <0.01 compared with the control group.</p