Novel Inhibitor of Geranylgeranyltransferase-I Figure 4. P61-E7 exhibits improved potency to inhibit cell cycle progression and cell proliferation. Panc-1 (A) and MiaPaCa-2 (B) cells were treated with DMSO, or the indicated concentrations of P61-E7 or P61-A6 for 48 h. Cell cycle profiles were monitored by flow cytometry. The percentages of cells in each phase of the cell cycle are indicated by different shades. Data shown are averages 6 S.D. of three independent experiments. (C) Panc-1 cells were treated with DMSO, or various concentrations of P61-E7 or P61-A6, and whole cell lysates were collected and resolved on SDS-PAGE for immunoblotting using antibodies against p21CIP1/WAF1. Results shown are representative of three independent experiments. The p21CIP1/WAF1 and RhoGDI (loading control) bands were quantified using ImageJ. Intensities of p21CIP1/WAF1 bands were normalized to their respective loading control, and the results are given above the images as fold change compared to the DMSO control. (D) Panc-1 cells were treated with DMSO, P61-A6, or P61-E7 under low-serum conditions (0.5% FBS) for 72 hours. Cell Proliferation relative to the DMSO control (100%) is plotted. Data shown are averages of two independent experiments, each in quadruplet. doi:10.1371/journal.pone.0026135.g004 Besides inducing p21CIP1/WAF1 expression, P61-E7 increased p27Kip1 protein level in Panc-1 cells (Figure S2A). Phosphorylation of p27Kip1 on Thr187 by cyclin E/CDK2 results in the association of p27Kip1 with SCF ubiquitin ligase, targeting it for degradation. It has been shown that Cdk2 phosphorylates p27Kip1 on Thr187 and promotes its nuclear degradation, and that the inhibition of the phosphorylation results in nuclear accumulation of p27Kip1 [36]. Therefore, we investigated whether P61-E7 inhibits phos- phorylation of p27Kip1 on Thr187 in Panc-1 cells. P61-E7 increased p27Kip1 protein level in Panc-1 cells (Figure S2B and S2C), while inhibiting the phosphorylation of p27Kip1 on Thr187 (Figure S2B). The abilities of P61-A6 and P61-E7 to inhibit proliferation of Panc-1 cells under low-serum conditions (0.5%) were examined and compared. Consistent with findings discussed above, P61-E7 is more effective at inhibiting cell proliferation (Figure 4D). For example, at 1.25 mM, P61-A6 treatment inhibited proliferation by 9.72%611.5% in comparison to DMSO treatment, while treatment with 1.25 mM of P61-E7 inhibited proliferation by 48.6%64.49%. The improved potency of P61-E7 to inhibit cell proliferation when compared to P61-A6 reflects its increased ability to inhibit protein geranylgeranylation inside the cells (Figure 2). P61-E7 Causes Actin Cytoskeleton Disorganization, Abolishes Formation of Focal Adhesions, and Inhibits Anchorage-Independent Growth of Panc-1 Cells To further investigate cellular effects of P61-E7, we carried out experiments using Panc-1 cells. First, we examined the effects of P61-E7 on the cell morphology of Panc-1 cell. Previous studies have shown that GGTase-I deficiency results in disrupted actin cytoskeleton in fibroblasts [7], and that GGTI treatment causes disorganization of actin cytoskeleton and inhibits formation of vinculin-containing focal adhesions [37]. Panc-1 cells were treated with DMSO or P61-E7 and the PLoS ONE | www.plosone.org 6 October 2011 | Volume 6 | Issue 10 | e26135