Oridonin induces apoptosis and senescence in colorectal cancer cells by increasing histone hyperacetylation and regulation of p16, p21, p27 and c-myc

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.

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).

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 [14].

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 [15]. 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.

Senescence-associated expression of β-galactosidase activity [16] 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.

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) [17] 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 [18].

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 [15]. The CFE was defined as the percentage of plated cells that formed colonies relative to an untreated control.

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 [19]. 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 [20] 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.

To investigate the possible effect of oridonin on the proliferation of colorectal cancer cells, three colorectal cancer cell lines HCT116, HT29, SW1116 were used. As shown in Figure 1, oridonin could inhibit proliferation of the three colorectal cancer cells in a time- and dose-dependent manner. These tumor cells showed different sensitivity to the oridonin treatment. It seemed that HT29 cells are more sensitive to oridonin treatment than HCT116 and SW1116 cells. The growth of HT29 cells was greatly inhibited by oridonin at 6.25 μM for 24 hours, which become more obvious on day 3. However, 12.5 μM oridonin was needed to obtain 50% inhibition in HCT1116 and SW1116 cells on day 3. At 25 μM, growth of all three colorectal cancer cell lines was completely inhibited.

We next examined the effects of oridonin on cell cycle distribution and apoptosis. HCT116 and SW1116 cells were treated with oridonin at low dose (12.5 μM for HCT116 or 25 μM for SW1116) or at higher dose (25 μM for HCT116 or 50 μM for SW1116) for 24 hours. Low dose oridonin causes obvious G2/M arrest in HCT116 and SW1116 cells. As shown in Figure 2A and 2B, the population of G2/M cells increased from 23.5% and 18.2% to 44.1% and 39.6%, respectively in HCT116 and SW1116 cells. Further, the G2/M related protein cyclin B, cdc2, cdc25c and phosphorylated histone H3 were detected in HCT116 and SW1116 cells (Figure 2C and 2E). Oridonin treatment for 12, 24 h causes down-regulation of all these proteins, indicating that oridonin induces G2 arrest in these cells.

While at higher concentration (25 μM for HCT116 or 50 μM for SW1116), a marked increase of subdiploid peak was observed, indicating the increase of apoptotic cells. This apoptosis inducing effect of oridonin was further confirmed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay (Figure 2D and 2F). These results suggested cell cycle arrest and apoptosis induction involved in oridonin induced cell proliferation inhibition.

Cellular senescence has been identified as one of the mechanisms mediating the anticancer effects of chemotherapies [16]. One of the morphologic changes that were frequently observed in the oridonin-treated cells is the flattening of the adherent cells with increased granularity, which is a typical morphologic change associated with cellular senescence (Figure 3A and 3C). By examining senescence-associated expression of β-galactosidase activity, we confirmed that cellular senescence was indeed induced in those flattened HCT116 and SW1116 cells treated by oridonin (12.5 or 25 μM) for three days (Figure 3B and 3D), indicating that senescence inducing also contribute to the proliferation inhibition effect of oridonin.

To perform detailed temporal analysis of gene expression alteration, SW1116 cells were treated with 25 μM oridonin for 0, 24, 48 and 72 hours. Then, the mRNA and protein levels of p16, p21, p27 were examined. As shown in Figure 4A and 4C, after treated oridonin for 24 hours, the mRNA and protein level of p21 but not p16 and p27 were significantly increased. Treatment with oridonin for 48 and 72 hours resulted in increase of the p16, p21 and p27. A series of studies have documented that c-myc regulates a wide range of genes involved in cell proliferation, differentiation, and apoptosis [21]. We also detected the expression of c-myc in oridonin treated SW1116 cells. Treatment with oridonin (25 μM) dramatically reduced c-myc mRNA transcription and c-myc protein levels as early as 24 hours. These data suggested that suppression of c-myc and up-regulation of p16, p21 and p27 correlated with oridonin responsiveness of colorectal cancer cells.

Cellular senescence is known to be associated with changes in chromatin structure [18, 22]. Therefore, we examined global chromatin modifications associated with cellular senescence in SW1116 cell lines treated with oridonin (25 μM) for 3 days in vitro. As shown in Figure 5A, after 72 h of oridonin treatment, heterochromatin formation were observed with DAPI staining, and a marked global increase of total acetylation of histone H3 and histone H4 were detected by immunofluorescence. Time course analysis showed that protein levels of acetylation of histone H3 and histone H4 gradually increased, especially for histone H4 (Figure 5B and 5C). Hence, oridonin treatment resulted in global changes of histone modifications that have been associated with cellular senescence.

To examine the suppressive effects of oridonin on CFE, SW1116 cells were treated with 0, 6.25, 12.5, 25, 50, 100 μM oridonin for 14 days. Our results showed that oridonin exerted dose-dependant suppression of CFE in SW1116 cells (P < 0.01; Figure 6A). Treatment with 6.25 μM oridonin for 2 week causes over 50% inhibition of CFE. With the increase in drug concentration, more significant suppressive effects were observed. At higher concentration (50 and 100 μM), the formation of colony was completely inhibited (Figure 6A).

We next further assessed the in vivo anti-colorectal cancer effects of oridonin. BALB/C nude mice xenografts were treated with oridonin by daily i.p injection at 6.25 or 12.5 or 25 mg/kg for up to 28 days. Such treatment resulted in significant suppression of xenograft growth , compared with the PBS or DMSO (1% in PBS) treated group (P < 0.05; Figure 6B and 6C). In the low-dose (6.25 mg/kg) group, significant tumor growth inhibition effect was observed until 21 days, while in the middle dose (12.5 mg/kg) group, significant tumor growth inhibition effect was observed on day 14. More interesting, the high dose (25 mg/kg) administration almost completely inhibited tumor growth at the onset phase. It is worth noting that no obvious changes in body weight were observed in the oridonin treated groups, compared with the PBS or DMSO (1% in PBS) treated groups (Figure 6D). In H & E stained tumor section, sparse tumor cells and areas of necrosis foci can be seen in oridonin treated mice, while massive tumor cells were observed in PBS or DMSO (1% in PBS) treated mice (Figure 7A). TUNEL assay (Figure 7B) and Senescence-associated-β-galactosidase (SA-β-Gal) staining (Figure 7C) were also performed to detect the degree of apoptosis and senescence. The results showed that oridonin induces apoptosis and senescence of colorectal cells in vivo in a dose dependent manner.