Scutellarin Sensitizes Drug-evoked Colon Cancer Cell Apoptosis through Enhanced Caspase-6 Activation

Materials and Methods

Cell culture. Colon cancer cell line HCT116 p53^+/+ (wild-type) and p53^-/- (knockout) cells were kindly provided by Dr. B. Vogelstein (School of Medicine, the John Hopkins University, Baltimore, MD, USA). Cells homozygous for p53 (p53^+/+) were the parental cells for p53^-/-. All cells were maintained in modified McCoy's 5A medium supplemented with 10% fetal bovine serum, 1% L-glutamine and 1% penicillin-streptomycin in a humidified incubator with 5% CO₂ at 37°C. Modified McCoy's 5A, penicillin-streptomycin and L-glutamine were purchased from GIBCO (Invitrogen Singapore PTE, Singapore). Fetal bovine serum was a product of Hyclone (Logan, UT, USA). Cell cultures at approximately 70% confluence were used for all experimental treatments. For the experimental treatments involving SC, cells were pre-incubated for 30 minutes with medium containing SC (100 μM), washed once with fresh medium before being subjected to treatment with medium containing RSV (200 μM) or 5-FU (500 μM) for 10 h, 15 h or 24 h.

Chemicals and antibodies. RSV and 5-FU were purchased from Sigma (Sigma-Aldrich, Singapore). Scutellarin was purchased from the Chinese Institute of Biological Products (Beijing, China). Antibodies for β-actin, cleaved lamin-A and cleaved caspase-6 were purchased from Cell Signalling (Beverly, MA, USA). Secondary antibodies and SuperSignal kit for Western blot analysis were provided by Pierce Biotechnology (Rockford, IL, USA). Lysis buffer and fluorogenic substrate for caspase activity assay were purchased from BD Biosciences (San Jose, CA, USA).

Determination and quantification of apoptosis. Cell viability was quantified using the Cell Proliferation Kit II (XTT assay) from Roche Diagnostics (Penzberg, Germany), following the manufacturer's protocol (5). Apoptosis was evidenced and quantified using In Situ Cell Death Detection Kit (TUNEL assay) from Roche Diagnostics, followed by confocal microscopy. Briefly, HCT116 cells were grown in Labtek Chamber Slides (Rochester, NY, USA), washed once with phosphate-buffered saline (PBS) and fixed in 100% methanol at -20°C for 10 min. The fixed cells were blocked in blocking buffer (1% BSA, 0.05% Tween-20 in 1xPBS) for 1 h, followed by three washes with 1xPBS. The cells were then incubated with the TUNEL stain provided by the manufacturer, washed three times with 1xPBS before being mounted with UltraCruz Mounting Medium (Santa Cruz, CA, USA). The cells were promptly viewed under confocal microscopy for TUNEL positivity and DNA fragmentation. The cells were regularly checked to ensure that the cells remained adherent to the slides. Proof of apoptosis was also provided by flow cytometric cell cycle analyses of sub-G₁ fractions using propidium iodide (PI) staining for DNA fragmentation (5).

SDS-PAGE and Western blot analysis. Western blot analysis was performed as described elsewhere (5). Briefly, cell lysates were subjected to 15% PAGE then transferred onto PVDF membranes. After blocking 1 h with 5% dry milk in TBST solution (50 mM Tris/HCl pH 7.4, 150 mM NaCl, 0.1% Tween-20), membranes were washed three times using TBST, exposed to primary antibody overnight at 4°C with gentle shaking, washed with TBST three times, exposed to secondary antibody for 1 h, washed three times again in TBST, and eventually subjected to chemiluminescence detection using the SuperSignal Substrate Western Blotting Kit.

Determination of caspase-6 activity. Caspase-6 activity assay was performed as described previously (5). Briefly, cells were harvested and lysed with 1x cell lysis buffer (BD Biosciences Pharmingen, San Diego, CA, USA). Cell lysate was added to a 2x reaction buffer (10 mM HEPES, 2 mM EDTA, 10 mM KCl, 1.5 mM MgCl₂, 6 mM dithiothreitol). Fluorogenic caspase-6 specific substrate VEID-AFC (50 mM) was then added followed by 1 h of incubation at 37°C. The protease activity was determined by measuring the relative fluorescence intensity at 505 nm following excitation at 400 nm using a spectrofluorimeter (TECAN Spectrofluor Plus, Austria). Caspase activities were normalized with protein concentration and expressed as fold increase in activity relative to the enzymatic activity obtained from untreated control cells.

Statistical analysis. Results are presented as mean±standard deviation (SD) of data collected from three independent experiments. Statistic analysis of mean values was performed through t-tests using the statistical software package SPSS (version 14.0; Chicago, USA). A p<0.05 was considered to be significant.