Inhibition of mammalian target of rapamycin signaling by CCI-779 (temsirolimus) induces growth inhibition and cell cycle arrest in Cashmere goat fetal fibroblasts (Capra hircus)

Abstract

The mammalian target of rapamycin (mTOR) is a Ser/Thr kinase. It plays an evolutionarily conserved role in regulating cell growth, proliferation, survival, and metabolism via different cellular processes. The purpose of this study was to explore the inhibitory effects of CCI-779 (temsirolimus), a specific mTOR inhibitor, on mTOR signaling, and examine the mechanism of cell growth suppression by CCI-779 in Cashmere goat fetal fibroblasts (GFb cells). GFb cells were sensitive to CCI-779 and the survival rate of cells treated with >3.0 μM of CCI-779 was significantly reduced compared with the control (p<0.01). CCI-779 inhibited the phosphorylation of mTOR (at Ser2448) and S6 (at Ser240/244), and the expression of mTOR, p70S6K, and S6. Thus, CCI-779 was toxic to GFb cells, and it induced a dose-dependent decrease in cell proliferation and caused G1/S cell cycle arrest. Taken together, these data show that CCI-779 can inhibit mTOR signaling and proliferation in GFb cells in vitro. Therefore, mTOR is an important regulator for GFb cell growth and proliferation.

FIG. 1.

Effect of CCI-779 and vehicle (0.5% ethanol; v/v) on cell proliferation of GFb cells examined by trypan blue exclusion assay. Survival rate of cells treated with >3.0 μM of CCI-779 was significantly reduced compared with the vehicle only and untreated controls. GFb, goat fetal fibroblasts. *p<0.05.

FIG. 2.

CCI-779 suppresses the proliferation of GFb cells. CCI-779 treatments were started at day 1 and cell numbers were assessed every 24 h until day 10. ♦: control; ▪: 1 μM; ▴: 5 μM; ×: 10 μM; *: 20 μM.

FIG. 3.

Cell cycle analysis was performed by flow cytometry. CCI-779 induced G₁/S cell cycle arrest in GFb cells. Ctr: control (48 h without CCI-779); 1: treatment with 11 μM of CCI-779 for 48 h.

FIG. 4.

Activities of mTOR and its downstream targets were inhibited by CCI-779 in GFb cells. CCI-779 inhibited the activity of the mTOR and S6. Expression levels of the mTOR, p70S6K, and S6 were slightly suppressed in GFb cells at 48 h after treatment. CCI-779 induced a dose-dependent decrease in the expression and activities of mTOR and S6. mTOR, mammalian target of rapamycin.