Preparation of HCT
Ethanol extract of Houttuynia cordata Thunb (yield: 6.73% of dry wt.) was obtained by 48 h incubation at room temperature. The ethanol extract was filtered through a 0.45 μm filter (Osmonics, Minnetonka, MN, USA), lyophilized and kept at 4°C. The dried extract was re-solubilized in PBS before use as previously described [32, 33].
Chemicals and reagents
RPMI-1640 cell culture medium (Gibco BRL, Life Technologies, MD, USA), DAPI (4,6-diamidino-2-phenylindole dihydrochloride), low-melting agarose, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), and DMSO (dimethyl sulfoxide) were purchased from Sigma (St. Louis, MO, USA). FBS (Fetal bovine serum), penicillin/streptomycin, PI (propidium iodide) and trypsin-EDTA were obtained from Life Technologies (Carlsbad, CA, USA). Proteinase K was purchased from Roche Diagnostics Gmbh (Mannheim, Germany). Ac-DEVE-pNA and Ac-IETD-pNA were purchased from R&D Systems Inc., (MN, USA). All other chemicals used were of analytical grade.
Human lung cancer A549 cells were obtained from the Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan), originally from the American Type Culture Collection (ATCC, USA). Cells were maintained in RPMI-1640 containing 100 mL/L FBS with 100,000 U/L penicillin and 100 mg/L streptomycin.
A549 cells were plated onto 96-well plates and incubated with HCT (0, 125, 250 and 500 μg/ml) for 24 and 48 h. MTT was added to each well then incubated for an additional 4 h at 37°C. The blue formazan product was dissolved in 100 μL of DMSO. The plates were read at O.D.570 nm using a spectrophotometric plate reader (Bio-Rad, Tokyo, Japan). The experiments were performed in triplicate (n = 3). Cell viability was calculated as O.D. of drug-treated sample/O.D. of none treated sample × 100% as previously described [32, 35].
Cell cycle transition and apoptosis determination
For cell cycle and apoptosis determination, A549 cells were plated onto 24-well plates and incubated with HCT (0, 125, 250 and 500 μg/ml) for 24 h. Cells were fixed gently in 70% ethanol at 4°C and then re-suspended in phosphate-buffered saline (PBS) containing 40 μg/ml PI, 0.1 mg/ml RNase and 0.1% Triton X-100 for 30 min at 37°C. Cell cycle transition and apoptosis were then analyzed by flow cytometry (FACS Calibur™; Becton Dickinson, NJ, USA) as previously described .
A549 cells were plated onto 24-well plates and treated with HCT (0 and 500 μg/ml) for 24 h. After HCT treatment, cells were fixed in 4% paraformaldehyde and then incubated with 1 μg/ml of DAPI staining solution in darkness. The apoptotic cells were observed by fluorescence microscopy (Zeiss, Oberköchen, Germany) as previously described [32, 35].
A549 cells were plated onto 24-well plates and incubated with HCT (0 and 500 μg/ml) for 24 h, 1 × 104 cells were mixed with 150 μL 0.75% low-melting agarose held at 37°C and layered onto a pre-treated slide with 1.5% regular agarose. After agarose were solidified on a chilled plate, the slides were transferred to the same lysis buffer, held at room temperature for 4 h and stained with propidium iodide as previously described .
A549 cells were plated onto T-75 flasks and treated with HCT (0, 125, 250 and 500 μg/ml) for 24 h. Total cell lysates were prepared as previously described. Thirty μg of total protein applied to SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (PVDF; Millipore). After blocking, the blots were incubated with the appropriate dilution of specific monoclonal antibodies for cyclin D1, cyclin A, CDK 4, CDK 2, p27, caspase-8 and caspase-3 (Santa Cruz Biotechnology, USA). Blots were washed and then incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, USA). Protein expressions were detected using enhanced chemiluminescence kits (Amersham, ECL Kits) as previously described .
Caspase-8 and caspase-3 activities assays
A549 cells were plated onto T-75 flasks and incubated with HCT (0, 125, 250 and 500 μg/ml) for 24 h. A549 cells were collected in a protein lysis buffer (50 mM Tris–HCl, 1 mM EDTA, 10 mM EGTA, 10 mM digitonin and 2 mM DTT). Cell lysates were centrifuged at 15,000 × g at 4°C and then incubated with caspase-3 and caspase-8 specific substrates (Ac-DEVE-pNA and Ac-IETD-pNA) with reaction buffer in a 96-well plate at 37°C for 1 h. Caspase activity was determined by measuring O.D. 405 nm of the released pN using a spectrophotometric plate reader (Bio-Rad, Tokyo, Japan) as previously described. The experiments were performed in triplicate .
Immunostaining assay for Fas/CD95 protein levels
Fas/CD95 cell surface antigen expression was measured by flow cytometry. A549 cells were plated onto 24 well and treated with HCT (0, 125, 250 and 500 μg/ml) for 12 h. Cells were collected and rinsed in PBS. Fas/CD95 was analyzed by direct immune-fluorescence staining. FITC-conjugated anti-Fas/CD95 and its FITC- conjugated isotype mAb (BD Biosciences Pharmingen, San Diego, CA, USA) were analyzed using a flow cytometer as previously described .
The experiments were performed in triplicate (n = 3) and all data were expressed as the mean ± standard error. Student’s t-test was used for single variable comparison. For multiple variable comparisons, data were analyzed by one-way ANOVA followed by Dunnett’s test. P < 0.05 was considered significant.