Induction of Non-apoptotic Cell Death by Odontioda Marie Noel ‘Velano’ Extracts in Human Oral Squamous Cell Carcinoma Cell Lines

Materials and Methods

Materials. The following chemicals and reagents were obtained from the indicated companies: Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA, USA); fetal bovine serum (FBS) (SAFC Biosciences, St. Louis, MO, USA); RPMI1640 mediun, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO) (Wako Pure Chemical, Osaka, Japan); melphalan (Sigma-Aldrich Inc., St. Louis, MO, USA).

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Figure 1.

The EtOAc layer of Odontioda Marie Noel ‘Velano’ extract (6.3 g) was chromatographed on a silica-gel column with different solvent ratios of CHCl₃-MeOH (100:1, 50:1, 20:1, 10:1, 5:1, 1:1) to yield five fractions.

Sequential fractionation with organic solvents. Bulbs of O. Marie Noel ‘Velano’ (Orchidaceae) were supplied from Orchid Garden Co., Ltd., Nagano Prefecture, Japan, during April of 2009. This specimen was proved and identified by Mr. H. Sumiyoshi (Orchid Garden Co., Ltd.) and also a voucher specimen (#20090617) was deposited in the Medicinal Plant Garden of Josai University.

The air-dried bulbs (533 g) of O. Marie Noel ‘Velano’ were extracted three times with MeOH under reflux for 3 hours. The MeOH extract (48.0 g) was fractionated by sequential organic solvent extraction as described previously (8). The EtOAc layer (6.3 g) was fractionated into five fractions, Fr. 1-5, by silica-gel column chromatography (Figure 1).

Cell culture. Human oral squamous cell carcinoma (OSCC) cells (HSC-2, HSC-3, HSC-4), human promyelocytic leukemia HL-60 cells, and normal human oral cells [gingival fibroblast (HGF), pulp cells (HPC) and periodontal ligament fibroblast (HPLF)] were prepared and cultured as described previously (8).

Assay for cytotoxic activity. The cells (3×10³ cells/well, 0.1 ml/well) were seeded in 96-microwell plates (Becton Dickenson, Flanklin Lakes, NJ, USA) and incubated for 48 hours to allow cell attachment. Near-confluent cells were treated for 48 hours with different concentrations (7.8-250 μg/ml) of extract fractions in fresh medium. The relative viable cell number of adherent cells (except for HL-60 cells) was then determined by the MTT method (8). The viability of the suspended cells, namely, HL-60, was determined by cell counting with a hemocytometer after staining with 0.15% trypan blue (8). The 50% cytotoxic concentration (CC₅₀) was determined from the dose–response curve. The tumor-specificity index (TS) was calculated by the following equation: TS=mean CC₅₀ (normal cells)/mean CC₅₀ (all tumor cell lines).

Assay for DNA fragmentation. The cells were lysed with 50 μl lysate buffer [50 mM Tris-HCl (pH 7.8), 10 mM EDTA, 0.5% (w/v) sodium N-lauroyl-sarcosinate]. The lysate was incubated with 0.4 mg/ml RNase A and 0.8 mg/ml proteinase K for 1 hour at 50°C, and then mixed with 50 μl NaI solution [7.6 M NaI, 20 mM EDTA-2Na, 40 mM Tris-HCl, pH 8.0], and 250 μl of ethanol. After centrifugation for 20 min at 15,000 ×g, the precipitate was washed with 1 ml of 70% ethanol. After centrifugation for 5 min at 15,000 ×g, the precipitate was dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 3-5). The samples (10-20 μl) were applied to 2% agarose gel electrophoresis in TBE buffer (89 mM Tris-HCl, 89 mM boric acid, 2 mM EDTA, pH 8.0). DNA molecular marker (Takara, Bio, Siga, Japan) and DNA from apoptotic HL-60 cells induced by UV irradiation (6 J/m²/min), followed by 3 hours incubation, were used for calibration. The DNA fragmentation pattern was examined in a photograph taken under UV illumination.

Assay for caspase activation. The cells were washed with phosphate-buffered saline without magnesium and calcium [PBS(−)] and lysed in lysis solution. After resting on ice for 10 min and centrifugation at 4000 ×g at 4°C for 3 min, the supernatant was collected. The lysate (50 μl, equivalent to 100 μg protein) was mixed with 50 μl reaction buffer (MBL) containing substrate for caspase-3 (DEVD-pNA (p-nitroanilide)). After incubation for 3 hours at 37°C, the absorbance at 405 nm of pNA produced by the cleavage of substrates was measured with microplate reader.

Assay for western blotting. HSC-2 cells were treated with 0, 25 or 50 μg/ml of Fr. 1, respectively. The cleavage of Poly ADP ribose polymerase (PARP) was measured using a Promega PARP (Asp 214) human specific antibody (Distributed by Cell Signaling Technology, Inc. Boston, MA, USA). In brief, cells were washed in ice-cold PBS, scraped, collected in lysis buffer [20 mM HEPES pH7.4, 1% Triton-X 100, 150 mM NaCl, 1.5mM MgCl₂, 12.5 mM β-glycerophosphate, 2 mM EDTA, 10 mM NaF, 2 mM dithiothreitol (DTT), 1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride (PMSF) plus 1× protease inhibitors]. The cell lysates were applied to a 8% sodium dodecyl sulfate-polyactylamide gel electrophoresis (SDS-PAGE) and the protein bands in the gels were transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% (w/v) nonfat dry milk, incubated with primary antibody [anti-cleaved PARP1 (Cell Signaling Technology), anti-β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA)], followed by incubation then with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies.

Statistical analysis. The difference between two groups was evaluated by Student's t-test.