Thus, targeting AKT with CTD may partially induce apoptosis via DNA damage, in contrast to previous reports stating that AKT fails to cause DNA damage . phosphorylated AKT (ser473), total AKT, phosphorylated MDM2 (Ser166), phosphorylated p53 (ser315), and total p53 were purchased from Cell Signaling Technology (Beverly, MA, USA total MDM2, phosphorylated CDC25C, total CDC25C, cyclin B1, Bcl-2, and -actin from Santa Cruz Biotechnology (Santa, Cruz, CA, USA). Open in a separate windows Fig. 1 CTD inhibits the proliferation of the CRC cells. a The structure of the costunolide. b MTT assay was conducted to measure the effect of CTD with numerous concentrations around the proliferation of the CRC cells at 24, 48, 72, and 96?h, respectively. c IC50 was analyzed via the cell viability assay. d The anchorage-independent colony formation assay was performed to detect the proliferative ability of the CRC cells treated with different concentrations of CTD Mevastatin for 2?weeks. e The cell cycle of the CRC cells treated with different concentrations of CTD for 24?h was detected using circulation cytometry. f The effects of CTD around the expression of the biomarkers with the cell cycle. g The cell apoptosis of the treated with numerous CTD concentrations for 24?h was detected using circulation cytometry. h The effects of CTD around the expression of biomarkers of apoptosis. The data are shown as mean??SD of values from triplicate samples. (*p?0.05, **p?0.01, ***p?0.01) indicate a significant difference compared to the control Cell lines and culture Human colorectal malignancy cell lines (HCT-15, HCT-116, and DLD1) were purchased from your Korea Cell Lines Lender (KCLB). Professor Mee-Hyun Lee (Dongshin University or college, KOREA) kindly provided us with the HCT-116 (p53+/+ and p53?/?) cell lines. The human immortalized healthy colon cell CCD-18Co (cultivate in MEM) from KCLB. Cells were cultivated in the following medium (obtained from Gibco by Life Technologies): MEM (CCD-18Co), RPMI 1640 (HCT-15 and DLD1), and McCoys 5A (HCT-116 and HCT-116 p53+/+ or p53?/?) supplemented with penicillinCstreptomycin (1%) (Solarbio Technology Co., Ltd. Mevastatin Korea) and 10% fetal bovine serum (Biological Industries) in a humidified atmosphere at 37?C with 5% CO2. All the cells were cytogenetically tested and authenticated before being frozen. Each vial of frozen cells was thawed and managed in culture for a maximum of 8 passages. Cell viability assay CRC cells (2??104 cells) and healthy colon cells (5??104 cells) were seeded into 96-well plates and treated them with different doses of CTD or dimethyl sulfoxide (DMSO, 99.7%, Sigma-Aldrich Co. LLC). Cell proliferation was decided using the MTT reagent every 24?h for 4 consecutive days. Absorbance was measured at 570/690?nm. For anchorage-independent cell growth assessment, the cells (8??103 cells/well) suspended in total medium were added to Mevastatin 0.3% agar with CTD (0, 2.5, 5, and 10?M) for anchorage-independent cell growth assessment GPR44 in the top layer, over a base layer of 0.6% agar with CTD. The cultures were managed at 37?C in a 5% CO2 incubator for 2?weeks, and the colonies were then visualized under a microscope and counted using the Image-Pro Plus software (v.6.1) (Media Cybernetics). Circulation cytometry analyses Cells (2??105) were treated with CTD or DMSO in 60-mm dishes and incubated for 24?h before analysis. The cells were trypsinized, washed with PBS, and fixed in 70% ethanol at ??20?C overnight, followed by incubation with RNase (1?mg/mL) and staining with propidium iodide (PI) (50?g/mL). DNA content was decided via circulation cytometry using a BD FACS Calibur instrument. Cell death was quantified using the AnnexinV Alexa Fluor 488 & Propidium Iodide (PI) Dead Cell Apoptosis kit from Invitrogen. In brief, cells were harvested and analyzed with a BD FACS Calibur instrument according to the manufacturers instructions. The cells that were positive for Annexin V alone and for Annexin V and PI were counted. Transwell migration, invasion assay, and wound-healing assay Transwell chambers (8-m pore size, COSTAR) were used to conduct the migration and invasion assays as per the manufacturers procedure and published methods . Mevastatin For the cell transmembrane migration assay, all processes were performed similarly to those in the invasion assay except Mevastatin for matrigel (1:5) covering. In brief, cells (1??105) were treated with CTD or DMSO in chambers for 24?h. Cells were fixed in 4% formaldehyde, permeabilized with 100% methanol, and stained with 0.5% crystal violet. The cells were photographed using a Leica SP2 confocal microscope (Leica Microsystems, Exton,.