Pleuromutilin Inhibits Proliferation and Migration of A2780 and Caov-3 Ovarian Carcinoma Cells and Growth of Mouse A2780 Tumor Xenografts by Down-Regulation of pFAK2

Abstract

BACKGROUND Pleuromutilin is a natural tricyclic, derived from the fungus, Pleurotus mutilus. This study aimed to investigate the effects of pleuromutilin on migration and proliferation of A2780 and Caov-3 human ovarian carcinoma cells and the growth of A2780 tumor xenografts in mice and the molecular mechanisms involved. MATERIAL AND METHODS A2780 and Caov-3 human ovarian carcinoma cells were cultured with and without 40, 160, and 200 μM of pleuromutilin. The Edu fluorescence assay, the wound-healing assay, and Matrigel were used to measure A2780 and Caov-3 cell proliferation, migration, invasion, and adhesion in vitro, respectively. Western blot measured protein levels of FAK, p-FAK, MMP-2, and MMP-9. A2780 cells were injected subcutaneously into mice to determine the effects of pleuromutilin on the growth of tumor xenografts. RESULTS Pleuromutilin significantly reduced A2780 and Caov-3 cell proliferation at 48 h in a dose-dependent manner (P<0.05), and at 200 μM, pleuromutilin reduced cell proliferation by 21.43% and 23.65%, respectively. Treatment of A2780 cells with pleuromutilin significantly reduced cell migration, invasion, and adhesion and the expression of p-FAK, MMP-2, and MMP-9 compared with untreated controls. In the mouse tumor xenograft model, treatment with pleuromutilin significantly reduced tumor size compared with the untreated group and inhibited tumor metastasis to the intestine, spleen, and peritoneal cavity. CONCLUSIONS In A2780 and Caov-3 human ovarian carcinoma cells, pleuromutilin inhibited cell proliferation, migration, invasion, and adhesion in a dose-dependent manner, and reduced tumor growth and metastases in a mouse A2780 cell tumor xenograft model.

Figure 1

The chemical structure of pleuromutilin.

Figure 2

A2780 and Caov-3 human ovarian carcinoma cell proliferation were reduced by pleuromutilin. The cells were treated with 0–200 μM concentrations of pleuromutilin. The Edu proliferation assay was used to evaluate cell proliferation and cell cytotoxicity. * P<0.05, ** P<0.02 and *** P<0.01 vs. the untreated cells.

Figure 3

The effect of pleuromutilin on A2780 and Caov-3 human ovarian carcinoma cell apoptosis. The cells after treatment with pleuromutilin at different concentrations were analyzed by flow cytometry. Cell apoptosis was quantified. * P<0.05 and ** P<0.01 vs. untreated cells.

Figure 4

The effect of pleuromutilin on A2780 and Caov-3 human ovarian carcinoma cell cycle progression. The cells were treated with increasing concentrations of pleuromutilin and then analyzed by flow cytometry.

Figure 5

The effect of pleuromutilin on A2780 and Caov-3 human ovarian carcinoma cell adhesion. (A) The cells after treatment with pleuromutilin at increasing concentrations were analyzed for adhesion. Magnification ×200. (B) The population of cell adhesion was quantified. * P<0.05, ** P<0.02 and *** P<0.01 vs. the untreated cells.

Figure 6

The effect of pleuromutilin on A2780 human ovarian carcinoma cell invasion. (A) The cells treated with pleuromutilin for 48 h were examined for invasion by the transwell assay. Magnification ×200. (B) The results were quantified. *P<0.05 and **P<0.02 vs. the untreated cells.

Figure 7

The effect of pleuromutilin on A2780 human ovarian carcinoma cell migration. (A) The cells were treated with increasing concentrations of pleuromutilin and examined by phase-contrast microscopy. Images were taken at a magnification of ×200. (B) Cell migration was quantified. * P<0.05 and ** P<0.02 vs. the untreated cells.

Figure 8

The effect of pleuromutilin on p-FAK and matrix metalloproteinases in A2780 human ovarian carcinoma cells. (A) The expression of p-FAK, MMP-2, and MMP-9 in A2780 cells at 48 h of pleuromutilin treatment assessed by Western blot. (B) Relative expression of p-FAK, MMP-2, and MMP-9 in A2780 cells. The level of β-actin used as control. * P<0.05 and ** P<0.01 vs. the untreated cells.

Figure 9

The effect of pleuromutilin on A2780 mouse xenograft tumor growth and metastasis. The mice were inoculated with A2780 cells and treated with increasing doses of pleuromutilin. The volume of tumors excised from the mice was measured on day 31 after A2780 cell inoculation. * P<0.05 and ** P<0.02 vs. the untreated mice.