Endostar inhibits ascites formation and prolongs survival in mouse models of malignant ascites

Abstract

Endostar, a modified recombinant human endostatin, inhibits the growth of a variety of tumors by suppressing neovascularization. Vascular endothelial growth factor (VEGF) has an important role in malignant ascites formation. In order to determine whether Endostar can suppress the formation of ascites and prolong survival times, mouse models of malignant ascites were established using S180 and H22 tumor cells. The experimental mice were randomly divided into four groups: The three treatment groups received different doses of Endostar (4, 8 and 16 mg/kg), and the control group received 0.9% w/v NaCl. The volume of ascites, and the tumor cell, red blood cell (RBC), VEGF protein and mRNA content of the ascites was measured alongside the peritoneal permeability and the mouse survival time. In vitro analysis of cultured Endostar-treated S180 and H22 cells was also performed in order to examine cellular proliferation and the level of VEGF secreted protein and mRNA. The results revealed that Endostar suppressed the ascites volume, decreased the level of tumor cells, RBCs and VEGF in the ascites fluid, and lowered the permeability of the peritoneum. The tumor cells collected from the ascites in the Endostar-treated mice demonstrated a decrease in the expression of VEGF mRNA. The survival rates of the 8 and 16 mg/kg Endostar-treated mice were longer than those of the controls. The in vitro experiments revealed a significant inhibition of VEGF protein secretion and VEGF mRNA by Endostar, but no effect on cellular proliferation. In conclusion, Endostar lowers ascites production by downregulating VEGF expression, and may therefore be effective for the treatment of malignant ascites.

Keywords: Endostar; antiangiogenesis; malignant ascites model; peritoneum permeability; vascular endothelial growth factor.

Figure 1.

Body-weight growth curves of the experimental mice inoculated with (A) S180 and (B) H22 tumor cells. Three groups of mice received different doses of Endostar (4, 8 and 16 mg/kg) by intraperitoneal injection for 10 consecutive days. The control group received 0.9% normal saline. The results indicate that the 8 and 16 mg/kg Endostar-treated mice gained body weight more slowly than the control group.

Figure 2.

(A) Results of the peritoneal permeability assay in the S180 and H22 mouse models. Endostar (4, 8 and 16 mg/kg) significantly decreased the permeability of the peritoneum (all P<0.05 vs. control group). (B) Results of the ELISA for VEGF levels in ascites. The cell-free ascites fluid was collected from the peritoneal cavity of the experimental mice. The 8 and 16 mg/kg Endostar-treated groups exhibited marked differences in the level of VEGF (P<0.05 vs. control group). (C) Results of the ELISA for in vitro VEGF secretion in S180 and H22 cells. In total, 1×10⁴ cells/well were treated with various concentrations of Endostar (0, 2.5, 10 and 40 µg/ml) for 48 h. The media from the wells was then individually collected and analyzed. Endostar reduced VEGF secretion (all P<0.05 vs. control group). (D) Results of the in vitro MTT proliferation assay for S180 and H22 cells. The S180 and H22 cells were treated with Endostar at various doses (0.1, 0.5, 2.5, 12.5 and 62.5 µg/ml). Endostar did not reduce the proliferation of S180 and H22 cells (all P>0.05 vs. control group). The data are expressed as the mean ± standard deviation. VEGF, vascular endothelial growth factor; OD, optical density.

Figure 3.

Survival analysis curves for the Endostar-treated mouse models. (A) S180 mice and (B) H22 mice. In total, 18 mice from each of the four groups received 0, 4, 8 and 16 mg/kg Endostar or 0.9% w/v NaCl solution for 10 consecutive days. The mice were then monitored until they succumbed to the disease.

Figure 4.

Agarose gel of the reverse transcription polymerase chain reaction revealing the levels of VEGF mRNA from tumor cells collected from the control- and Endostar-treated (4, 8 and 16 mg/kg) mice. (A) S180 and (B) H22 mice. The β-actin gene was used as an internal control. The results indicate a decrease in the level of VEGF mRNA in the Endostar-treated (8 and 16 mg/kg) mice. VEGF, vascular endothelial growth factor.

Figure 5.

Agarose gel of the reverse transcription polymerase chain reaction of VEGF mRNA from control- and Endostar-treated (2.5, 10 and 40 mg/kg) cultured tumor cells. The β-actin gene was used as an internal control. The results indicate a decrease in the level of VEGF mRNA in Endostar-treated (2.5, 10 and 40 mg/kg) cells.