Background Sinularin isolated from the cultured soft coral has been reported to exert potent cytotoxic effects against particular types of cancer. p-cdc2 (Tyr(15)), and p53 coupled with increased expression of downstream proteins p21 buy Phenylephrine hydrochloride and down-regulation of p-cdc25 (Ser(216)). Moreover, the results of the apoptosis assay and western blot analysis indicated that the cytotoxic activity could be related to mitochondrial apoptosis, characterized by decrease of Bcl-2 expression, disruption of mitochondrial membrane potential, and sequential activation of caspases and Poly (ADP-ribose) polymerase (PARP). Conclusions This study reveals for the first time the anti-HCC activities of sinularin, the active compound isolated from the cultured soft coral as described [5] and kindly provided by Dr. Jui-Hsin Su (National Museum of Marine Biology & Aquarium, Pingtung, Taiwan); sorafenib was purchased from Cayman Chemical (Ann Arbor, MI USA). Sinularin and sorafenib were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO USA) at Speer4a 100?mM as stock solution for in vitro assays. Henceforth, DMSO and sorafenib were used as vehicle control and positive control, respectively. Cell lines and culture The human hepatocellular carcinoma cell lines, HepG2 and Hep3B, were purchased from American Type Culture Collection (ATCC?) (Manassas, VA USA). The growth medium is DMEM with 10% (v/v) fetal bovine serum (FBS), streptomycin (100?g/mL) and penicillin (100 U/ml) (Gibco/BRL, Gran Island, NY USA). Cells were cultured in incubators with 5% CO2 at 37?C. MTT assay Cell viability was monitored by the MTT colorimetric assay (BioVision, Milpitas, CA USA). Briefly, 200?L of MTT (0.5?mg/mL) was added to each well and incubated for 4?h at 37?C after removing the culture supernatants. MTT crystals were dissolved by DMSO and the absorbance was measured at 540?nm wavelength with an ELISA microplate reader (Tecan Sunrise, San Jose, CA USA). All MTT assays were performed in 96-well plates and repeated at least three times. Cell cycle assay Sinularin- or sorafenib- treated cells were harvested, washed buy Phenylephrine hydrochloride with PBS, and fixed with 70% ethanol at ?20?C overnight. The fixed cells were stained by propidium iodide (PI; Sigma-Aldrich, St. Louis, MO buy Phenylephrine hydrochloride USA) containing RNase A (Sigma-Aldrich, St. Louis, MO USA) for 30?min in the dark at room temperature. The cells were then analyzed by an AccuriTM C5 cytometer (BD Biosciences, San Jose, CA USA). Data were further analyzed by C6 Accuri system software (BD Biosciences, San Jose, CA USA). Annexin V-FITC/PI staining assay The Annexin V-FITC/PI staining assay was performed using the FITC Annexin V Apoptosis Detection Kit (BioVision, Milpitas, CA USA) in accordance with manufacturers instructions. In brief, sinularin-treated cells were trypsinized and gently washed with cold PBS twice followed by resuspension in binding buffer. Equal volumes of Annexin V-FITC and propidium iodide were added to stain cells for 10?min at room temperature in the dark prior to determining the percentage of apoptotic cells with an AccuriTM C5 cytometer. Measurement of mitochondrial membrane potential (MMP) HepG2 cells treated with sinularin for 24?h were trypsinized, washed with PBS, and resuspended with culture medium followed by staining with JC-1 solution (10?ng/L) (Life Invitrogen, Carlsbad, CA USA) for 10?min at 37?C. Changes in mitochondrial membrane potential were then examined by an AccuriTM C5 cytometer. Western blotting Cells were gathered with lysis buffer after treatments at the indicated time points. BCA Protein Assay Reagent (Thermo Fisher Scientific, Waltham, MA USA) was utilized to measure the protein concentrations before carrying out SDS-polyacrylamide skin gels electrophoresis. Proteins were then transferred to PVDF membranes for subsequent antibody hybridization. The membranes were clogged with 5% non-fat milk at space temp for 1?h and blotted with main antibody at 4?C for over night incubation, followed by secondary antibodies conjugated with horseradish peroxidase (HRP) (Jackson Laboratory, Pub Harbor, ME USA) at buy Phenylephrine hydrochloride 4?C overnight. The immunoactive groups were recognized with an enhanced chemiluminescence (ECL) system and developed using the LAS3000 system (Fujifilm, Valhalla, NY USA). In this study, the main antibodies acquired from Cell Signaling Technology Inc. (Beverly, MA USA) were anti-phospho-cdc25-ser-216, anti-cdc25, anti-cyclin M1, anti-p53, anti-p21, anti-cleaved-caspase-3, anti-BCL-2, anti-cleaved-caspase-8, anti-cleaved-caspase-9, anti-cleaved-PARP, anti-Bax, anti-phospho-ATM-ser-1981, anti-ATM, anti-CHK-2, anti-ATR-ser-428, anti-ATR anti-phospho-CHK-2-Thr-68, anti-phospho-CHK-1-ser-345, anti-CHK-1 antibodies, from Epitomics (Burlingame, CA USA) were anti-cdc2-Tyr-15 and anti-cdc2 antibodies, from GeneTex (Sann Antonio, TX USA) were anti-cyclin M1 and anti-GAPDH antibodies, and from Millpore (Billerica, MA USA) were anti-p-H2A.Times and anti-GAPDH antibodies. Statistical analysis The results were indicated as the mean??SD. All statistical analyses were performed with the GraphPad Prism software bundle version 5.0. Experimental data arranged by one variable were.