Evaluation of Anticancer Activities of Benzo[c]phenanthridine Alkaloid Sanguinarine in Oral Squamous Cell Carcinoma Cell Line

Materials and Methods

Materials. For these in vitro experiments, sanguinarine was purchased commercially from Sigma (St. Louis, MO, USA) and dissolved in MilliQ (Millipore, Bedford, MA, USA) at a concentration of 10 μg/ml for the stock solution and kept at −20°C prior to use.

Cell culture. SAS cells derived from a human oral squamous cell carcinoma (HSRRB, Osaka, Japan) were cultured at 37°C with 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

Proliferation assay. Cell proliferation was evaluated using the tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Dojindo, Kumamoto, Japan) assay, as described previously (19). The cells (5×10³/well) were seeded with DMEM in 96-well plates (Falcon Laboratories, McLean, VA, USA). After 24 h, the cells were treated with 0.25, 0.5, 0.75, 1.0 μM. sanguinarine and then further cultured for 3 days. Controls were medium alone or 2 mg/ml cis-diamminedichloroplatinum (CDDP; Nippon Kayaku, Tokyo, Japan) as positive control.

Anchorage-independent growth. The cells (5.0×10⁵/ml) were suspended in 0.35% agarose in DMEM containing 5% FBS. This suspension was overlaid onto a solidified layer of 0.4% agarose in a 96-well plate. Fresh DMEM was maintained on top of the plate during 5 days' incubation, at which time the cells were examined under a microscope (Eclipse TS100/TS100-F; Nicon) equipped with a digital CDD camera (DS-Fi1: Nicon) at 20× or 40× magnification. The cells were photographed and counted. Cells incubated for 5 days with or without sanguinarine (0.25-1.0 μM) or 2 μg/ml CDDP (positive control) were examined and photographed.

Migration assay. Cell migration was assayed using cell culture Boyden chambers (Cell BioLabs, San Diego, CA, USA) and vascular endothelial growth factor (VEGF) at a dose of 10 ng/ml as a chemoattractant. Following 24 hours' incubation, cells adhering to the bottom of the membrane were fixed with 70% ethanol, stained with 0.1% crystal violet and photographed. The inserts were then treated with 10% acetic acid, and absorbance was measured at 560 nm.

Adhesion assay. Assays were carried out using the CytoSelect (Cell BioLabs) 48-well cell adhesion assay extracellular matrix (ECM) array. The cells (1×10⁵) were seeded onto the wells coated with each ECM protein (Fibronectin, CollagenI, CollagenIV, Laminin, Fibrinogen) and bovine serum albumin (BSA). Cells were incubated at 37°C for 60 min in a tissue culture incubator before washing and staining. Absorbance was measured at 560 nm.

Western blot analysis. Western blot analysis was carried out according to the method described previously (8). Anti-phospho-p42/p44 mitogen-activated protein kinase (MAPK; extracellular signal-regulated kinase (ERK) 1/2) and anti-phospho-p38 were obtained from Promega (Madison, WI, USA), and anti-p42/p44 MAPK (ERK1/2), anti-phospho-c-Jun NH₂-terminal kinase and anti–c-Jun NH₂-terminal kinase were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-p38 was obtained from Calbiochem (Bad Soden, Germany). Bcl-2, Bax antibodies and peroxide-conjugated secondary antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

DNA fragmentation electrophoresis analysis. Genomic DNA was extracted from the SAS cells using a Wizard genomic DNA purification kit (Promega) according to the manufacturer's protocol. The cells were treated with sanguinarine (0.75, 1.0 μM) and CDDP (2 μg/ml) for 24 h. Approximately 50 mg of DNA was loaded into each well and 3% agarose gel electrophoresis was carried out at 100 V in a TAE buffer (Trisacetate-EDTA) for 30 min. After electrophoresis, DNA was visualized by soaking the gel in TAE buffer containing 0.1 mg/ml ethidium bromide. The gel was observed using UV light and photographed.

Terminal deoxyuridine nick-end labeling (TUNEL) assay. A TUNEL assay was carried out as described previously (20). The cells (5×10⁴) were seeded onto Lab-Tek chamber slides (Thermo Fisher Scientific, Rochester, NY, USA) and incubated for 24 h. The cells were treated with sanguinarine (0.75, 1 μM) for 24 h and then the slides were observed using phase-contrast fluorescence microscopy (ECLIPSE TS100/TS100-F; Nikon, Tokyo, Japan).

Caspase-3 and caspase-7 activities. The SAS cells were seeded in a 96-well plate at a density of 3.0×10⁴/ml. The cells were incubated for 24 h with or without various concentrations of sanguinarine. They were then lysed and the caspase-3 and caspase-7 activities in the lysates were detected by a Caspase-Glo 3/7 assay kit (Promega), according to the manufacturer's protocol. A dual luciferase system (Promega) was used for the sequential measurement of firefly and Renilla luciferase activities with beetle luciferin and coelenterazine, respectively, as the specific substrates. The quantification of both luciferase activities and the calculation of the relative ratios were carried out manually with a luminometer (GloMax; Promega).

Statistical analysis. Unless otherwise specified, all the experiments were repeated at least twice, and similar results were obtained in the repeated experiments. Statistical analysis was carried out using Student's t-test. Data are expressed as means±standard deviation.