NCTD of analytical grade purity was from the Ju-nan Pharmaceutical Works (Junan, China) and dissolved in RPMI-1640 (HyClone, U.S.A.). Caspase-8 inhibitor (z-IETD-fmk) was from Enzyme Systems (CA, U.S.A.). Caspase-3 inhibitor (z-DEVD-fmk) and pan-caspase inhibitor (z-VAD-fmk) were from Calbiochem (CA, U.S.A.). Caspase-9 inhibitor (Ac-LEHD-CHO), rabbit polyclonal antibodies against ICAD, cytochrome c, Bax and Bcl-x_L, mouse polyclonal antibodies against Bcl-2, horseradish peroxidase-conjugated secondary antibody (goat-anti-rabbit or goat-anti-mouse) were from Santa Cruz Biotechnology (Santa Cruz, CA). Caspase-3, -8 and -9 Apoptosis Detection Kits were from Santa Cruz Biotechnology (Santa Cruz, CA).

A375-S2, melanoma cells, were obtained from American Type Culture Collection (ATCC, #CRL, 1872, MD, U.S.A.) and were cultured in RPMI-1640 medium (HyClone, U.S.A.) supplemented with 10% heat inactivated (56℃, 30 min) fetal calf serum (Beijing Yuanheng Shengma Research Institution of Biotechnology, Beijing, China), 2 M L-glutamin (GIBCO, U.S.A.), 100 kU/L penicillin and 100 g/L streptomycin (GIBCO, U.S.A.) at 37℃ in 5% CO₂.

A375-S2 cells (1.0×10⁸ cells/L) seeded in 96-well plate (NUNK^TM, Roskilde, Denmark) were cultured for 24 hr, then various concentrations of NCTD (60-480 µM) were added and cultured for 12, 24, 36, 48 hr further. MTT (thiazolyl blue, Sigma, MO, U.S.A.) test were carried out to detect cell growth using an enzyme-linked immunosorbent assay plate reader (TECAN, Austria) (18). After preincubation with given concentrations of pan-caspase inhibitor (z-VAD-fmk), caspase-8 inhibitor (z-IETD-fmk), caspase-9 inhibitor (Ac-LEHD-CHO), caspase-3 inhibitor (z-DEVD-fmk) for 2 hr, 60 µM NCTD were added and cultured for further 24 hr. Growth inhibition was evaluated by MTT method. The percentage of cell growth inhibition was calculated as follows:

Relative viability (%)={1-[A₄₉₂ (control)-A₄₉₂ (NCTD)]}/A₄₉₂ (control)×100

Apoptotic nuclear morphology was assessed using Hoechst 33258 (Sigma, U.S.A.) as previously described (19). A375-S2 cells, containing adherent and floating, were collected by centrifugation at 1,000 g for 5 min, washed two times with PBS. The cells were fixed with 3.7% paraformaldelyde at room temperature for 2 hr, then washed and stained with Hoechst 33258 167 µM at 37℃ for 30 min. At the end of incubation, the cells were washed and resuspended in PBS for observation of nuclear morphology using fluorescence microscope (Nikon, Osaka, Japan).

The cells were cultured with NCTD for 12, 24 or 36 hr. Floating dead cells were collected from culture medium by centrifugation (240 g for 10 min at 4℃), and the lactate dehydrogenase (LDH) content from the pellets lysed in 1% NP-40 for 15 min was used as an index of apoptotic cell death (LDHp). The released LDH in the culture medium (extracellular LDH or LDHe) was used as an index of necrotic cell death. The adherent and viable cells were lysed in 1% NP40 for 15 min to release LDH (intracellular LDH or LDHi). Then the substrate reaction buffer of LDH (L (+)-lactic acid 0.5 mM, indonitrotetrazolium 0.66 mM, phenazine methosulfate 0.28 mM, β-nicotinamide adenine dinucleotide 1.3 mM in pH 8.2 Tris-HCl) was added. The OD value at 492 nm of reaction for 1 and 5 min were assayed and LDH activities were determined by the average difference between 1 min and 5 min. The percentage of apoptotic and necrotic cell death was calculated as follows:

% apoptosis=LDHp/(LDHp+LDHe+LDHi)×100

% necrosis=LDHe/(LDHp+LDHe+LDHi)×100

DNA extraction and electrophoresis were performed as described previously (22). In brief, A375-S2 cells, containing adherent and floating, were collected by centrifugation at 1,000 g for 5 min. The cell pellet was suspended in cell lysis buffer [Tris-HCl 10 mM (pH 7.4), EDTA 10 mM (pH 8.0), Triton-100 0.5%) and kept at 4℃ for 10 min. The lysate was centrifuged at 25,000 g for 20 min. The supernatant was incubated with RNase A 40 µg/L (Sigma) at 37℃ for 1 hr, then incubated with proteinase K 40 µg/L (Merck) at 37℃ for 1 hr. The supernatant was mixed with NaCl 0.5 M and 50% 2-propanol at -20℃ overnight, then centrifuged at 25,000 g for 15 min. After drying, DNA was dissolved in TE buffer [Tris-HCl 10 mM (pH 7.4), EDTA 1 mM (pH 8.0)] and separated by 2% agarose gel electrophoresis at 100 V for 1 hr.

A375-S2 cells were treated with or without NCTD. Analysis of caspase-3, caspase-8 and caspase-9 activities was performed using Caspase Apoptosis Detection Kit (Santa Cruz, CA, U.S.A.) according to the manufacturer's instruction. In brief, harvested cells at various time points were washed with PBS two times and centrifuged at 150 g for 5 min. The supernatant was aspirated off and 100 µL cell lysis buffer (provided) was added to an Eppendorf centrifuge at 500 µL per 1×10⁶ cells. Cells in the lysis buffer were incubated on ice for 10 min. Reaction buffer containing 10 µL DTT, 10 µL of DEVD-AFC, IEVD-AFC or LEHD-AFC substrates and 380 µL H₂O was added to each aliquot of cell lysate. The reaction mixtures were incubated at 37℃ for 1 hr. The fluorescence of the cleaved substrates was determined with a spectrofluorometer set at 400 nm excitation wavelength and at 505 nm emission wavelength. The unit of enzyme activity corresponds to the activity that cleaves the respective substrate in 1 min/mg protein at 37℃.

A375-S2 cells were treated with 60 µM NCTD for 0, 12, 24, 36 hr. Both adherent and floating cells were collected and frozen at -80℃. Western blot analysis was performed as previously described (23) with some modification. Briefly, A375-S2 cells were lysed for 1 hr on ice in lysis buffer [50 mM HEPES (pH 7.4), 1% Triton X-100, 2 mM sodium orthovanadate, 100 mM sodium fluoride, 1 mM EDTA, 1 mM phenylmethanesulfonyl fluoride (PMSF)], supplemented with proteinase inhibitors: 100 µg/mL aprotinin, 10 µg/mL leupeptin, and 100 µg/mL pepstatin. Protein concentration was determined by the Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, CA). The lysate was centrifuged at 16,000 g at 4℃ for 10 min. Equivalent amounts of protein lysates were mixted in 2×loading buffer [50 mM Tris-HCl (pH 6.8), 2% SDS, 10% 2-mercaptoethanol, 10% glycerol, and 0.002% bromphenol blue], heated at 100℃ for 5 min, and then analyzed by electrophoresis in 12% SDS polyacrylamide gel and blotted onto nitrocellulose membrane (Amersham Biosciences, U.K.). After blocked with Tween 20-Tris-buffer saline [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.02% Tween 20] containing 5% nonfat milk at room temperature with the primary antibodies at 1:500 dilution in blotting buffer. After washed 3 times for 10 min each in Tris-buffered saline, the membrane was incubated with a diluted horseradish peroxidase-labeled secondary antibody (1:500) in blotting buffer at room temperature for 1 hr. After 3 more washes, proteins were detected by chemiluminescence, according to the manufacturer's instructions (Bio-Rad Laboratories, Hercules, CA).

All results were confirmed in at least three separate experiments. Data are expressed as mean±SD. Data of the representative were analyzed for statistical significance by Student's t test. p-value of less than 0.05 was considered statistically significant.

NCTD 15 to 240 µM exerted potent inhibitory effect on A375-S2 cell growth. By 24 hr after NCTD 60 µM treatment, cell death rate reached to almost 50% (Fig. 2).

In control group, A375-S2 cells were round in shape and stained homogeneously. After 24 hr treatment with NCTD, blebbing nuclei and granular apoptotic bodies appeared (Fig. 3, arrows).

DNA fragmentation as a hallmark of apoptosis was observed in NCTD-treated A375-S2 cells (Fig. 4).

The ratio of apoptotic A375-S2 cells increased from 4.6% at 0 µM NCTD to 38.5% at 240 µM NCTD, however, that of necrotic cells were still negligible (Fig. 5).

A375-S2 cells were treated with 60 µM NCTD for 24 hr in the absence or presence of various caspase inhibitors: pancaspase inhibitor (z-VAD-fmk, 40 µM), caspase-3 inhibitor (z-DEVD-fmk, 20 µM), caspase-8 inhibitor (z-IETD-fmk, 20 µM), caspase-9 inhibitor (Ac-LEHD-CHO, 20 µM). Z-VAD-fmk, z-DEVD-fmk and Ac-LEHD-CHO partially blocked NCTD-induced A375-S2 cell apoptosis. Inhibitory ratio was 30.98%, 38.96%, and 51.50%, respectively. However, caspase-8 inhibitor did not affect the death ratio (Fig. 6). Caspase-3 activity was significantly enhanced after the cells were treated with NCTD 60 µM at different time points, however, caspase-9 activity increased moderately and increase of caspase-8 activity was almost negligible (Fig. 7).

The ICAD protein degradation was significant after 24 hr incubation with NCTD (Fig. 8), and this change was blocked by caspase-3 inhibitor.

After incubation with NCTD, protein expression ratio of Bcl-x_L/Bax and Bcl-2/Bax was down-regulated, and this change was blocked by caspase-3 inhibitor. At the same time, the protein of cytchrome c was increased in cytosol and caspase-3 inhibitor had no influence on the increase (Fig. 9).