As shown in Physique 2D, simvastatin at various concentrations (5C20 M) induced the activation of caspase 3 and PARP, suggesting that simvastatin might cause not only cell proliferation inhibition but also apoptosis in ATC cells. and CCND1 proteins. Adding GGpp abolished the simvastatin-increased levels of p27kip protein, and the GGpp-caused effect was abolished by Skp2 inhibition. Introduction of Cyr61 siRNA into ATC cells prevented the epidermal growth factor (EGF)-enhanced cell migration. The EGF-induced increases of Cyr61 protein expression and cell migration were prevented by simvastatin. Taken together, these results suggest that simvastatin induced ATC proliferation inhibition through the deactivation of RhoA/Rac1 protein and overexpression of p21cip and p27kip, and migration inhibition through the abrogation of Cyr61 protein expression. = 3). * < 0.05, different from corresponding control. 2.2. Effects of MEV and Its Metabolites around the Simvastatin-Induced ATC Cell Proliferation Inhibition To study the involvement of the MEV pathway Nikethamide in simvastatin-induced cell proliferation inhibition, SW1736 and 8305C cells were co-incubated with simvastatin (10 M) and MEV (50 M) or MEV-derived metabolites. As illustrated in Physique 2A, the add-in squalene (SQ; 10 M), lanosterol (LS; 10 M) or cholesterol (Chol; 10 M) had no significant effect on the simvastatin-inhibited cell proliferation, suggesting that this anti-proliferative effects of simvastatin might be due to the depletion of cholesterol and its intermediates. Both MEV and GGpp (20 M), but not FPP (20 M), can significantly rescue the simvastatin-induced anti-proliferation effect (Physique 2B). We further verified the effect of MEV and isoprenoids depletion on cell cycle progression. As exhibited in Physique 2C, simvastatin treatment led to G1 arrest, and this effect was abrogated by co-treatment with MEV or GGpp, but not Fpp. These results suggest Rabbit Polyclonal to CADM2 that the depletion of MEV or GGpp might contribute to the anti-proliferative activity of simvastatin, and implied that this geranylgeranylation pathway Nikethamide might play a role in the simvastatin-induced anti-proliferation in SW1736 and 8305C cells. Since simvastatin might increase the apoptotic cell populations at the sub-G1 phase (Physique 2C), we examined the protein expression levels of the apoptotic markers caspase 3 and poly (ADP-ribose) polymerase (PARP). As shown in Physique 2D, simvastatin at various concentrations (5C20 M) induced the activation of caspase 3 and PARP, suggesting that simvastatin might cause not only cell proliferation inhibition but also apoptosis in ATC cells. Open in a separate window Physique 2 Effects of MEV-derived metabolites around the simvastatin-induced ATC cell proliferation inhibition. (A) Simvastatin Nikethamide (10 M)-inhibited proliferation of SW1736 and 8305C cells was not affected by the add-in SQ (10 M) or LS (10 M) or Chol (10 M). Cells were pre-incubated with SQ, LS or Chol for 30 min followed by simvastatin for additional 48 h. The simvastatin (10 M) induced cell proliferation inhibition (B) and accumulation of cells at the G1-phase (C) of SW1736 and 8305C cells were abolished by MEV (50 M) and GGpp (20 M), but not Fpp (20 M). Cells were pre-incubated Nikethamide with MEV, GGpp or Fpp for 30 min followed by simvastatin for additional 48 h. The relative cell number was estimated using MTT assays. Values represent the means SEM (= 3). The DNA content was measured by PI staining. Arrows indicated sub-G1 cell population. (D) Simvastatin brought on apoptosis in SW1736 and 8305C cells. Cells were incubated with 0C20 M simvastatin for 48 h, Nikethamide and then the protein expression levels of full-length and active form of caspase 3 and PARP were examined by immunoblotting analysis. * 0.05, different from corresponding control. # 0.05, different from simvastatin-incubated group. C, control; Veh: simvastatin-incubated group. 2.3. Simvastatin Reduces the Acitivity of Rho GTPases The involvement of geranylgeranylation pathway was examined in an anchorage-dependent and anchorage-independent cell growth of SW1736 cells. As presented in Physique 3A, GGTI-298, a specific geranylgeranyl transferase inhibitor, significantly and concentration-dependently.