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

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

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

Effect of CYT-Rx20 on KYSE70 and TE8 cell migration and invasion.

<p>(A) The effect of CYT-Rx20 on cell migration was detected by using a modified Boyden chamber assay, as described in the Materials and methods section. The representative photographs are shown with ×40 magnification. Histograms represent quantification of cell migration and are expressed as the average area of the migrated cells in four fields of the group. (B) Effect of CYT-Rx20 on KYSE70 and TE8 cell invasion. Invasion of cells through transwell inserts containing Matrigel-coated membranes was assessed 48 h after CYT-Rx20 treatment. (C) Epithelial to mesenchymal transition (EMT) proteins were analyzed by immunoblotting after KYSE70 and TE8 cells were treated with the indicated concentrations of CYT-Rx20 for 24 h. The intensity of the protein band normalized to the internal control β-actin was calculated as the fold of controls (set as 1) and then depicted as histograms.</p