Figure 1.
Binding affinity of Ec-LDP-Hr fusion protein to esophageal cancer cells.
EC9706 cells (A), KYSE150 cells (C) or NIH 3T3 cells (E) were incubated with FITC-labeled Ec-LDP-Hr protein at indicated concentrations, and the mean fluorescence intensities (MFIs) were analyzed by flow cytometer. Increased concentrations of FITC-labeled Ec-LDP-Hr proteins were incubated with EC9706 cells (B) or KYSE150 cells (D). Following FACS, the MFIs were plotted versus protein concentrations.
Figure 2.
Cytotoxicity of Ec-LDP-Hr-AE on esophageal cancer cells.
(A) Expression of EGFR and HER2 on different esophageal cancer cells and NIH 3T3 cells analyzed by Western blot. (B) The cell killing effects of Ec-LDP-Hr-AE on esophageal cancer cell lines KYSE150, EC9706, ECa109 and EC-1and mouse fibroblast cell line NIH 3T3 were tested by MTT assays. Cells were exposed to various concentrations of Ec-LDP-Hr-AE for 48 h and the results were obtained from three independent experiments. (C) The IC50 values of lidamycin (LDM), Ec-LDP-Hr-AE, Ec-LDP-AE, and LDP-Hr-AE against 4 kinds of esophageal cancer cells and NIH 3T3 cells were measured by MTT assay. Columns, mean of triplicate experiments, bars, SD.
Figure 3.
Cell cycle distribution of esophageal cancer cells after treatment with Ec-LDP-Hr-AE.
EC9706 cells or KYSE150 cells were exposed to Ec-LDP-Hr-AE for 48 h at the indicated concentrations and cell cycle distribution was determined by flow cytometry after PI staining. Columns, mean of triplicate experiments, bars, SD.
Figure 4.
The effects of apoptosis-induction by Ec-LDP-Hr-AE treatment.
(A) KYSE150 cells were treated by Ec-LDP-Hr-AE at indicated concentrations for 48 h, and then stained by Hoechst 33342. The images were observed under a fluorescence microscope at ×200. EC9706 cells (B), KYSE150 cells (C) or NIH 3T3 cells (D) were exposed to the indicated concentrations of Ec-LDP-Hr-AE for 48 h. Cells were harvested and stained with a combination of FITC-Annexin V and PI. The lower left quadrants (FITC−/PI−) indicated the viable cells, and the lower right quadrants (FITC+/PI−) indicated the early apoptotic cells. The upper right quadrants (FITC+/PI+) indicated the late apoptotic cells, and the upper left quadrants (FITC−/PI+) indicated the dead cells.
Figure 5.
Effects of Ec-LDP-Hr-AE on the EGFR/HER2 signaling.
The expression of apoptosis related molecules (e.g. Caspase 3, Caspase 7 and PARP) and key molecules in the EGFR/HER2 signaling pathways (e.g. phosphorylated-EGFR, -HER2, -ERK, -p38MAPK, -JNK, -AKT) were determined by Western blot analysis. β-actin was served as a loading control.
Figure 6.
In vivo inhibitory effects of lidamycin (LDM) and Ec-LDP-Hr-AE.
Nude mice (n = 7) bearing human esophageal carcinoma KYSE150 xenografts were treated with LDM or Ec-LDP-Hr-AE on day 11 and day 21 after tumor inoculation by tail vein injection. The mean tumor volumes (A) and the mean body weights of mice (B) in each group are shown. Ec-LDP-Hr-AE at the dose of 0.3 mg/kg inhibited the growth of KYSE150 xenografts by 88%, which showed statistically significant differences compared with LDM treated group at the maximum tolerated dose (0.05 mg/kg) (P<0.05).