Aciculatin Induces Apoptosis via p53 Pathway a FACScan flow cytometric assay. The curve chart shows that aciculatin increased the G1 population of cells in a time-dependent manner. Mean 6 SE values from 3 independent experiments. *P,0.05 and ***P,0.001, compared with non-treated cells. D, HCT116 cells were treated with vehicle (0.1% DMSO) or aciculatin (7.5 mM) and then double stained with TUNEL and DAPI. Increased green fluorescence indicated that the cells underwent apoptosis after aciculatin treatment (TUNEL, right panel). The nuclei were stained with DAPI (left panel). The bar chart shows the proportions of TUNEL positive cells in each treatment normalized to DAPI. ***P,0.001. doi:10.1371/journal.pone.0042192.g001 Immunohistochemistry analysis The formalin-fixed paraffin-embedded (FFPE) tumor tissue slices were deparaffinized in xylene and rehydrated in a graded series of ethanol. Submerge tissue slices with 3% hydrogen peroxide to quench endogenous peroxidase activity. To unmask antigen, tissue slices were heated in 95uC Tris-EDTA Buffer (10 mM Tris Base, 1 mM EDTA Solution, 0.05% Tween 20, pH 9.0) for 30 min. Slices were blocked with 4% nonfat milk for 40 min and incubated with indicated antibody for 1 h at room temperature. HRP polymer conjugate reagent A (SuperPic- tureTM) were applied to the slices and reagent B for subsequently peroxidase catalyzation which visualizes the location of the antigen. Mayer’s Hematoxylin solution was for counterstaining. The results were captured by Zeiss Axioskop-2 microscope. Tumor xenograft models Male severe combined immunodeficient (SCID) mice were implanted s.c. with HCT116 tumor cells. When the tumors reached the average volume of 90 mm3, the mice were divided into two groups (n = 5) and the agent treatment was initiated. Vehicle (Cremophor EL/ethanol, 1:1; 0.2 mL/mouse) or acicu- latin (30 mg/kg) were administered intraperitoneally (i.p.) for five times each week. The length (L) and width (W) of the tumor were measured every 3 to 4 days, and the tumor volume was calculated as LW2/2. The protocols of the in vivo study were approved by the Animal Care and Use Committee at National Taiwan University. Statistical analysis Data were expressed as mean 6 SEM of the indicated number for separate experiments. Statistical analysis of data was performed with one-way ANOVA followed by the t test, and p,0.05 were considered significant. Results Aciculatin induces G0/G1 arrest and subsequent apoptotic cell death in human cancer cells Aciculatin is a novel C-glycoside flavonoid derived from C. aciculatus extract (Figure 1A).The tight carbon-carbon bond makes a C-glycoside more stable than an O-glycoside in the acidic environment or the presence of glycosidases in vitro and in vivo. We used a sulforhodamine B assay (SRB) to demonstrate that aciculatin caused growth inhibition, not only in the HCT116 human colorectal cancer cell line (GI50 = 2.81 mM) but also in other cancer cell lines (Table S1). The cytotoxicity of aciculatin in HCT116 cells was determined by mitochondrial 3-(4,5-di- methylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) bromide re- duction assay with an IC50 of 5.88 mM (Figure 1B). To determine the cell cycle distribution, synchronized cells were treated with aciculatin and analyzed with FACScan flow cytometry and propidium iodide (PI) staining. The results showed that aciculatin can induce G0/G1 arrest and subsequent accumulation of sub-G1 phase cells in a time-dependent manner. The increased G1 proportions after treatment at the indicated time points are shown in the curve chart (Figure 1C). We next investigated the induction of apoptosis by aciculatin using TUNEL assay. Figure 1D shows that DNA fragmentation increased after 24 h of 7.5 mM aciculatin treatment. The percentages of TUNEL positive cells in this assay were calculated and quantification data was shown. These data indicate that aciculatin can significantly induce apoptosis in HCT116 cells. Figure 2. Effect of aciculatin on G0/G1-related proteins and apoptotic factors in HCT116 cells. A, HCT116 cells were treated with aciculatin (10 mM) for the indicated periods and then harvested for detection of G0/G1 arrest-related proteins (p21, pRb, and p27) by immunoblotting. B, HCT116 cells were treated with aciculatin (10 mM) for the indicated periods and then harvested for detection of caspase activation and PARP cleavage. C, HCT116 cells were treated with various concentrations of aciculatin (5, 7.5 and 10 mM) for 24 h. After aciculatin treatment, apoptotic proteins were detected for each treatment concentrations. The star marks a non-specific band. doi:10.1371/journal.pone.0042192.g002 PLOS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e42192