human colorectal cancer cells. The results provide evidence of the importance of p53 and its downstream targets in aciculatin- induced cell cycle arrest and caspase-dependent apoptosis. The effects of aciculatin on MDM2 reduction and related p53 accumulation are also described. Same results were also confirmed using A549, a p53 wild-type non-small cell lung cancer cell line. Collectively, these findings suggest that aciculatin is a promising natural product for anticancer therapy. Materials and Methods Materials Aciculatin was extracted and purified from Chrysopogon aciculatus by Professor C.C. Chen, Department of Biotechnology, Hung- kuang University, Taiwan, with purity over 98%. RPMI 1640, fetal bovine serum (FBS), penicillin, streptomycin, and all other tissue culture reagents were obtained from Life Technologies (Grand Island, NY, USA). Enhanced chemiluminescence detec- tion kit was from Amersham. Trizol reagent was from Invitrogen (Carlsbad, CA, USA); random primer and M-MLV RT were purchased from Promega (Madison, WI. USA); pro-Taq was from Protech (Taipei, Taiwan). HRP polymer conjugate reagent SuperPictureTM was from ZYMED LABORATORIES INC. (San Francisco, CA, USA); Nuceolin antibody, propidium iodide (PI), 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro- mide, sulforhodamine B, and all of the other chemical reagents were obtained from Sigma Chemical (St. Louis, MO, USA). Cell culture The human colorectal cancer cell line HCT116 and human non-small cell lung cancer cell line A549 were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). p53-KO (p53 knockout) HCT116 cells were kindly provided by Dr. B. Vogelstein (Johns Hopkins). Both were cultured in RPMI 1640 with 10% heat-inactivated fetal bovine serum (v/v) and penicillin (100 units/mL)/streptomycin (100 mg/mL). Cells were maintained in a humidified incubator at 37uC in 5% CO2/95% air. MTT assay Cells were incubated with vehicle (0.1% DMSO) or compounds for 48 h. Washed once and incubated with 0.5 mg/mL 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide at 37uC for 1 h. After the incubation, cells were lysed with DMSO and the absorbance was obtained using an ELISA reader (550 nm). Results were calculated as: cell viability (%) = average O.D. of wells/average O.D. of control wells. FACScan flow cytometry After the treatment of vehicle (0.1% DMSO) or compounds for the indicated time courses, cells were harvested by trypsinization, for cell cycle analysis, cells were fixed with 70% (v/v) alcohol at 4uC overnight. After centrifugation, cells were incubated in 0.1 mol/L phosphate-citric acid buffer [0.2 mol/L NaHPO4, 0.1 mol/L citric acid (pH 7.8)] for 20 min at room temperature. Then, cells were centrifuged and resuspended with 0.5 mL PI solution containing Triton X-100 (0.1%, v/v), RNase (100 mg/ mL), and PI (80 mg/mL). DNA content was analyzed with the FACScan and CellQuest software (Becton Dickinson). For annexin V-FITC and PI double staining, the FITC Annexin V Apoptosis Detection Kit (BD Biosciences; San Jose, CA, USA) was performed. Cells were centrifuged and incubated with these reagents immediately. After incubation for 15 min, the fluores- Aciculatin Induces Apoptosis via p53 Pathway cently labeled cells were then analyzed with the FACScan and CellQuest software. TUNEL Cells seeded in 8-well slide chambers were starved overnight and treated with vehicle (0.1% DMSO) or aciculatin for 24 h. Cells and the tumor tissue slices were fixed in 4% paraformalde- hyde and washed twice with PBS. The apoptotic cells were identified in situ using terminal deoxynucleotidyl transferase to transfer biotin-deoxyuridine triphosphatase (TUNEL, Roch Di- agnostics, Mannheim, Germany) to the free 39-OH of cleaved DNA. Cleavage sites were labeled by Biotin and visualized by reaction with fluorescein conjugated avidin (avidin-fluorescein isothiocyanate). Photomicrographs were obtained by Leica TCS SP2 confocal spectral microscope. Western blot analysis Western blotting for total cell lysate and nuclear extraction were done as previously reported [15,16] with anti- p53, anti-pRb and anti-caspase-8 (BD Biosciences; San Jose, CA, USA); anti-a- tubulin, anti-actin, anti-p21, anti-p27, anti-poly(ADP-ribose) polymerase, anti-phosphorylated H2AX (Ser139), anti-MDM2, anti-PUMA, HRP-conjugated anti-mouse, and anti-rabbit IgG (Santa Cruz Biotechnology; Santa Cruz, CA, USA); anti-caspase- 3(Imgenex; San Diego, CA, USA); anti-caspase-9 and phospho- ser15-p53 (Cell Signaling Technology; Beverly, MA, USA). RNA extraction and reverse transcription-polymerase chain reaction(RT-PCR) Total RNA was extracted with Trizol reagent by the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA). Re- verse transcription was performed with 5 mg mRNA and random primer at 65uC for 5 min, then mixed with Moloney murine leukemia virus reverse transcriptase (RT) to react at 37uC for 1 h to obtain cDNA. Gene amplification was followed with RT- polymerase chain reaction (PCR). The primer sequences used for amplification were as follows: GAPDH, 59-TCCTTGGAGGC- CATGTGGGCCAT-39/59-T GATGACATCA AGA AGGT GG- TGAAG-39; p53, 59-CAGCCAAGTCTGTGACTTGCACGTA- C-39/59-CTATGTCGAAAAGTGTTTCTGTCATC-39; MD- M2, 59-GGGAGATATGTTGTGAAAGAAGC39-/59-CCCTG- CCTGATACACAGTAACTT. Standard amplification parame- ters were used with the following processes: For p53, 94uC for 3 min, 35 cycles with denaturation at 94uC for 30 s, annealing at 60uC for 45 s, and extension at 72uC for 1 min, at last, 72uC for 10 min. For GAPDH and MDM2, 95uC for 10 min; 35 cycles (MDM2: 40 cycles) with denaturation at 95uC for 15 s, annealing at 60uC for 5 s, and extension at 72uC for 12 s; followed by a final extension at 75uC for 15 s. PCR products were analyzed on 1.5% agarose gel in the presence of 1 mg/mL of ethidium bromide. Transient transfection HCT116 cells were seeded in 3.5-cm dishes overnight, and then transfected with TP53 siRNA or MDM2 plasmid using Lipofec- tamine 2000 according to the manufacturer’s protocol. TP53 siRNA and lipofectamine 2000 are from Invitrogen (Carlsbad, CA, USA) and MDM2 plasmid (No. 16233) is from Addgene (Cambridge, MA, USA). After 6 h transfection and re-serum, cells were starved and then treated with vehicle (0.1% DMSO) or aciculatin for the indicated times. Cell lysates were collected for Western blot analysis. PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e42192