Cell Culture and Reagents
The colorectal cancer cell lines SW1116, HT29 and HCT116 from Shanghai Institutes for Biological Sciences were incubated in humidified room air containing 5% CO2 at 37°C and cultured in McCOY'S 5A medium (Sigma, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (GIBCO BRL, Grand Island,NY). Cells were routinely grown in 100 mm plastic tissue culture dishes (Nunc, Roskilde, Denmark) and harvested with a solution of trypsin-EDTA when they were in logarithmic phase of growth. Cells were maintained at these culture conditions for all experiments. Oridonin (purity > 98%) was purchased from CHENGDU MUST BIO-TECHNOLOGY CO.LTD. It was dissolved in DMSO at a stock concentration of 100 mmol/L and store at -20°C. The stock solution was further diluted with cell culture medium to yield final oridonin concentrations.
Cell Proliferation Assay
Cells were seeded into 96-well plates at 2,000 to 3,000 live cells per well and treated with Oridonin (6.25-100 μM) for 3 days. The antiproliferative effect of Oridonin was assessed using Cell Count Kit-8 (Dojindo Molecular Technologies, Inc., Gaithersburg, MD).
Cell Cycle Analysis with Flow Cytometry
Cells treated with or without Oridonin (12.5 and 25 μmol/L) were harvested for flow cytometry analysis on day 1. Cells were fixed and stained with 0.1 mg/mL propidium iodide for DNA analysis with Becton Dickinson FACScan (Franklin Lakes, NJ) as described previously .
Detection of Apoptosis
Apoptosis was evaluated with flow cytometry and on cell smears using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay (In situ Cell Death Detection kit, AP; Boehringer Mannheim GmbH, Mannheim, Germany). Samples were incubated with 50 μL of reaction mixture in a humidified chamber at 37°C for 90 minutes as described previously . The percentage of apoptotic cells was determined by counting at least 1,000 cells from 10 to 20 high-power fields (×200) under both phase-contrast and fluorescent microscopy.
Cell Senescence Assay
Senescence-associated expression of β-galactosidase activity  was done with a Senescence Detection kit (BioVision, Mountain View, CA) on fixed cells treated with or without Oridonin (12.5 and 25 μmol/L). The development of cytoplasmic blue was detected and photographed using a Nikon (Nikon Instruments, Inc., Lewisville, TX) inverted microscope equipped with a color CCD camera.
RNA Extraction and Semi-quantitative RT-PCR
Total RNA was extracted from cell cultures using TRI REAGENT (Molecular Research Center, Inc., OH) according to the manufacturer's protocol. The mRNA levels of the genes analyzed were measured by RT-PCR amplification. Sequences for mRNAs from the nucleotide data bank (National Center for Biotechnology Information) were used to design primer pairs for RT-PCR reactions (Primer Express, Applied Biosystems, CA). The following specific oligonucleotide primers were used respectively for p16 (p16-F: 5'-CAC GGC CGC GGC CCG GGG TC -3' and p16-R: 5'-GGC CCG GTG CAG CAC CAC CA -3' ), p21(p21-F: 5'-AGG CGC CAT GTC AGA ACC GGC TGG -3' and p21-R: 5'-GGA AGG TAG AGC TTG GGC AGG C-3' ), p27 (p27-F: 5'-ATG TCA AAC GTG CGA GTG TCT AAC -3' and p27-R: 5'-TTA CGT TTG ACG TCT TCT GAG GCC A-3' ), c-myc (c-myc-F: 5'-ATT CTC TGC TCT CCT CGA -3' and c-myc-R: 5'-TCT TGG CAG CAG GAT AGT -3' ) with GAPDH as internal control (GAPDH-F: 5'-TCC CAT CAC CAT CTT CCA G-3' and GAPDH-R:5'-ATG AGT CCT TCC ACG ATA CC-3';). PCR cycles were adjusted to have linear amplification for all the targets. Each RT-PCR reaction was repeated at least three times. A semiquantitative analysis of mRNA levels was carried out by the ''GEL DOC UV SYSTEM'' (Biorad Company, CA).
Attached cells were collected by scraping them off into a lysis buffer, and the detached cells in the supernatant were collected by centrifugation before resuspension in the lysis buffer. Protein concentration was determined by the bicinchoninic acid (BCA) method according to the manufacturer's (Pierce, Rockford, IL, U.S.A.) instructions after trichloroacetic acid precipitation. The protein lysates were mixed with equal volume of Laemmli buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 50 mM DTT, 10% glycerol, 0.01% bromophenol blue), boiled for 3 min at 100°C, and then resolved by SDS-PAGE on a 10 to 12% gel using a mini gel apparatus (Bio-Rad). Bromophenol Blue (0.01%) was added to the samples before an equal amount of proteins was loaded in each lane for electrophoresis and blotting. The PVDF membrane was incubated with a primary antibody against cdc2, cdc25c and cyclinB (Santa Cruz Biotechnology), p16 (PharMingen, San Diego, CA, U.S.A.), p21 (PharMingen, San Diego, CA, U.S.A.), p27 (PharMingen, San Diego, CA, U.S.A.), c-Myc (N-262; Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), Acetylated histone H3 (AcH3) and H4 (AcH4), phospho-Histone H3 (Ser10) (Upstate Biotechnology, Lake Placid, NY), histone 3, histone 4 and GAPDH (Santa Cruz Biotechnology) for 2 h. Signals were detected using a horseradish peroxidase-conjugated secondary antibody and an enhanced chemiluminescence detection kit (ECL; Amersham Biosciences, Pittsburgh, PA)  and were quantitated by an Eagle Eye II Image System with installed density-analysis software (Stratagene, La Jolla, CA, U.S.A.).
Cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature for anti-acetyl histone H4 (06-598; Upstate Biotechnology) and anti-acetyl histone H3 (06-599; Upstate Biotechnology) staining. Cells were permeabilized with 0.2% Triton X-100 (EM Science, Gibbstown, NJ) in PBS for 10 min at room temperature. FITC-labeled secondary antibody (F-0382; Sigma) were applied at the concentration of 1:500. Images were taken with Nikon E800 scope. Senescence-associated heterochromatin staining was conducted as described .
CFE in Soft Agar
Tumor cells were resuspended in DMEM with 0.3% agar and plated in 24-well plates at 2,000 per well on top of a 0.5 mL precast semisolid 1% agar underlayer following treatment with Oridonin ( 0, 6.25, 12.5, 25, 50 or 100 μmol/L) for 2 weeks as described previously . The CFE was defined as the percentage of plated cells that formed colonies relative to an untreated control.
Murine model and oridonin treatment
Five-week-old pathogen-free athymic nude mice were purchased from Experimental Animal Centre of SIBS. (Shanghai, PR China). BALB/C nude mice were bred and maintained in a specific pathogen-free environment. Mice were allowed free access to mice standard food pellets and tapwater. Twice a week cages were cleaned and water changed. Temperature was controlled at 21°C ± 2°C. The light was on a 12 hours light-12 hour dark cycle, with light on at 8 am. Xenograft model in nude mice was established by subcutaneous inoculation of 1 × 106 SW1116 cells into the right flank. The nude mice received oridonin treatment (6.25, 12.5 or 25 mg/kg per day) when tumor was measurable. Caliper measurements of the longest perpendicular tumor diameters were performed every day to estimate the tumor volume, using the following formula: 4π/3 × (width/2)2 × (length/2), representing the 3-dimensional volume of an ellipse . Animals were killed when their tumors reached 2 cm or when the mice became moribund. TUNEL assay was performed to detect in situ apoptosis on tissue section using a DeadEnd Colorimetric TUNEL System (Promega) according to the manufacturer's instructions. Senescence-associated expression of β-galactosidase activity  was detected with a Senescence Detection kit (BioVision, Mountain View, CA) in situ senescence on tissue section. Animal related experiments were performed according to the Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, revised 1996) and approved by the committee for human treatment of animals at Shanghai Jiao Tong University School of Medicine.
The effects of oridonin on cell proliferation, CFE, cell cycle arrest, apoptosis, and xenograft growth in SCID mice were analyzed with two-way ANOVA and presented as the mean ± SD.