Induction of Non-apoptotic Cell Death in Human Oral Squamous Cell Carcinoma Cell Lines by Rhinacanthus nasutus Extract

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; RPMI-1640 medium, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO), Wako Pure Chemical, Osaka, Japan; melphalan, Sigma-Aldrich Inc., St. Louis, MO, USA.

Extracts and sequential fractionation with organic solvents. The root of R. nasutus was supplied from the Chiayi Grass-Produce Cooperation Farm (Chiayi County, Taiwan, ROC) during the autumn of 2009. This specimen was identified by one of the authors (Professor Y. Shirataki) and also a voucher specimen (#201006060) is deposited in the Medicinal Plant Garden of Josai University.

The root (1.2 kg) of R. nasutus was extracted three times with MeOH under reflux for 3 hours each. The MeOH extract (83 g) was fractionated by sequential organic solvent extraction as described previously (10). The EtOAc layer (16.0 g) was chromatographed on a silica-gel column with different solvent ratios of CHCl₃-MeOH (50:1, 20:1, 10:1, MeOH) to yield five fractions: Fr. 1 (eluted with CHCl₃-MeOH=50:1, 9.1 g), Fr. 2 (eluted with CHCl₃-MeOH=50:1, 2.4 g), Fr. 3 (eluted with CHCl₃-MeOH=20:1, 1.3 g), Fr. 4 (eluted with CHCl₃-MeOH=10:1, 1.5 g) and Fr. 5 (eluted with MeOH, 1.5 g) (Figure 2).

Cell culture. Human oral squamous cell carcinoma (OSCC) cell lines (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 (10). HL-60 cells were cultured at 37°C in RPMI-1640 supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin G and 100 μg/ml streptomycin sulfate under a humidified 5% CO₂ atmosphere. Other cells were cultured in DMEM supplemented with 10% FBS and antibiotics.

Assay for cytotoxic activity. The cells (3 × 10³ cells/well, 0.1 ml/well) were seeded in 96-microwell plates (Becton Dickenson, Franklin Lakes, NJ, USA) and incubated for 48 hours to allow cell attachment. Near-confluent cells were treated for 48 hours with different concentrations of extracts in fresh medium. The relative viable cell number of adherent cells (except for HL-60 cells) was then determined by the MTT method (10). The viability of the suspended cells, i.e. HL-60, was determined by cell counting with a hemocytometer after staining with 0.15% trypan blue (10). 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. 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 1-2 hours 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 dried for 10 min, and then dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 3-5). Fr. 1 (0-60 μg/ml) was applied to a 2% agarose gel electrophoresis in TBE buffer (89 mM Tris-HCl, 89mM boric acid, 2 mM EDTA, pH 8.0). The DNA molecular marker (Takara, Bio, Otsu, Shiga, Japan) and DNA from apoptotic HL-60 cells induced by ultraviolet (UV) irradiation (6 J/m²/min), followed by 3 hours incubation, were used for calibration. The DNA fragmentation pattern was examined in photographs taken under UV illumination.

Assay for caspase activation. Cells were washed with phosphate-buffered saline without magnesium and calcium [PBS(–)] and lysed in lysis solution. After resting for 10 min on ice centrifugation for 5 min at 10,000 ×g, the supernatant was collected. The lysate (50 μl, equivalent to 100 μg protein) was mixed with 50 μl reaction buffer (MBL) containing substrates for caspase-3 [DEVD-pNA (p-nitroanilide)]. After incubation for 3 hours at 37°C, the absorbance at 405 nm of the liberated chromophore pNA was measured with microplate reader.

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Figure 1.

The shrub Rhinacanthus nasutus (L.) Kurz (Acanthaceae).

Assay for western blotting. HSC-2 cells were treated with 15, 30 and 60 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, US). 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, 1.5 mM MgCl₂, 12.5 mM β-glycerophosphate, 2 mM EGTA, 10 mM NaF, 2 mM dithiothreitol (DTT), 1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride (PMSF) plus 1× protease inhibitor]. The cell lysates were applied to 8% Poly-Acrylamide 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, USA)], and 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.