Ca9-22, a human gingival squamous carcinoma cell line, was maintained in RPMI 1640 (GIBCO BRL, Grand Island, NY, USA) with 10% heat-inactivated fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 mg/ml) at 37˚C in 5% CO₂ and air.

Cyclic hydroxamic acid-containing peptide (CHAP)15, renamed Ky-2, cyclo (Asu(NHOH)-Aib-Phe-D-Pro), and suberoylanilide hydroxamic acid (SAHA; Cayman Chemical, Ann Arbor, MI, USA) (Figure 1(a)) [8] were prepared as stock solutions at concentrations of 80 and 75 mM, respectively. The caspase-9 inhibitor benzyloxycarbonyl-Leu-Glu(OMe)-His-Asp(OMe)-CH2F (ZLEHD-fmk), was purchased from Calbiochem Co. (San Diego, CA, USA).

Cell proliferation was evaluated using a colorimetric WST-1 assay. Ca9-22 cells were seeded in flat-bottomed 96-well plates at a concentration of 1 × 10⁴ cells/ml. After the cells were treated with Ky-2 for 44 hours, 10 ml of WST-1 reagent (Dojindo, Kumamoto, Japan) was added to each well followed by incubation for 4 hours. Absorbance at 450 nm was measured using a Multiskan JX microplate reader (Thermo Fisher Scientific, Rockford, IL, USA)

After treatment with Ky-2 or SAHA for 24 hours, Ca9-22 cells were examined under a light microscope (IX71; OLYMPUS, Tokyo, Japan) for changes in cell morphology and photographs were obtained using a DP71 (OLYMPUS, Tokyo, Japan).

For immunofluorescence analysis, cells were cultured in 8-well Lab-Tek^TMÒ chamber slides (Nagle Nunc International, Rochester, NY, USA) at a density of 1 × 10⁴ cells/well. After Ky-2 (100 nM) treatment for 3 hours, cells were fixed with 4% formaldehyde, then permeabilized with 0.2% Triton X-100 in phosphate buffered saline (PBS, pH 7.2) for 15 minutes at room temperature. After blocking with 1% bovine serum albumin (BSA) in PBS, the slides were treated overnight at 4˚C with rabbit polyclonal antibodies against acetyl-histone H3 (Lys14) (1:1000; Cell Signaling Technology, Beverly, MA, USA), then washed and treated with goat anti-rabbit IgG antibody Alexa Fluor^® 488 (1:200; Molecular Probe, Invitrogen, Carlsbad, CA, USA) for 2 hours at room temperature, followed by the addition of an actin staining reagent, Alexa Fluor^® 568 phalloidin (1:40; Molecular Probe, Invitrogen, Carlsbad, CA, USA). Cells were visualized with a Fluorescence Microscope BZ-9000 (KEYENCE CORP., Osaka, Japan). Images were captured digitally in real time and processed using BZ-II imaging software (KEYENCE CORP., Osaka, Japan).

The samples were lysed in SDS lysis buffer (50 mM Tris-HCl, 2% SDS; pH 6.8). The protein contents were determined using a protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). Each protein sample (30 mg) was subjected to electrophoresis on a 7.5% or 15% SDS-polyacrylamide gel, and electro-blotted onto a PVDF membrane. After incubation with 5% non-fat skim milk in PBS containing 0.1% Tween-20 for 1 hour, the membranes were treated with the primary antibody, as described below. Polyclonal antibodies against acetyl-histone H3 (Lys14), lamin A/C, poly (ADP-ribose) polymerase (PARP), caspase-3, caspase-6, and caspase-7 were purchased from Cell Signaling Technology (Beverly, MA, USA), while a monoclonal antibody against caspase-9 was purchased from Medical & Biological Laboratories (Woburn, MA, USA) and a polyclonal antibody against Actin (C-2) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Immunodetection was performed using an ECL-plus Western blotting detection system (Amersham Bioscience, Little Chalfont, Buckinghamshire, UK) according to the manufacturer’s instructions. The band densities were measured with a Molecular Imager® ChemiDocTM XRS Plus system (Bio-Rad Laboratories, Hercules, CA, USA). Blots were stained with Coomassie Brilliant Blue and each lane was confirmed to contain a similar amount of protein extract.

Ca9-22 cells were suspended in a hypotonic solution (0.1% Triton X-100, 1 mM Tris-HCl; pH 8.0), 3.4 mM sodium citrate, 0.1 mM ethylenediaminetetraacetic acid) and stained with 5 mg/ml of propidium iodide (PI), then analyzed with a FACScalibur flow cytometer (EPICS XL; Beckman Coulter, Fullerton, CA, USA). For Annexin V and PI staining, Ca9-22 cells were washed with PBS and resuspended in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2; pH 7.4). Fluoresceinconjugated Annexin V and PI solutions were added, and mixed gently according to the manufacturer’s instructions (Molecular Probe, Invitrogen, Carlsbad, CA, USA). Cells were incubated at room temperature and then analyzed with an EPICS XL. To detect apoptotic nuclei, Ca9-22 cells were fixed with 1% glutaraldehyde for 1 hour, washed with PBS, and stained with 1 mM of Hoechst dye 33342 (Ana Spec, Fremont, CA, USA). Nuclei were visualized by fluorescence microscopy (BX51; OLYMPUS, Tokyo, Japan), with an excitation wavelength of 355 nm and emission wavelength of 465 nm.

All experiments were performed 3 times and the results are presented as the mean ± standard deviation (S.D) of each.

We investigated the effects of Ky-2 on the viability of Ca9-22 cells using a WST-1 assay. As shown in Figure 1(b), Ky-2 dose-dependently inhibited cell proliferation with estimated half maximal inhibitory concentrations (IC₅₀) ranging from 12.5 to 25.0 nM. In contrast, SAHA had a lower effect on the growth of Ca9-22 cells. Both Ky-2 and SAHA induced a significant decrease in the number of viable cells as compared with the control, and also caused dramatic changes to the cells to develop an elongated shape with filamentous protrusions (Figure 1(b)).

To clarify whether Ky-2 is an effective inhibitor of HDAC enzymes in Ca9-22 cells, we examined the acetylation of histone H3 by immunoblot analysis. Histone H3 acetylation was expressed in Ca9-22 cells treated with Ky-2, as shown by immunoblot analysis (Figure 2(a)), while immunofluorescence analysis showed that Ky-2 treatment enhanced that acetylation (Figure 2(b)).

When Ca9-22 cells were treated with Ky-2 (100 nM) for 24 hours, the percentage of cells in the sub-G1 phase was 32.9% (Figure 3(a)). To investigate the nature of Ca9-22 cell death following Ky-2 treatment, we analyzed the cells after exposing them to a combination of

Annexin V and PI. Flow cytometric analysis revealed that Ky-2 treatment enhanced the surface expression of phosphatidylserine after being cultured for 24 and 36 hours, as shown by an increase of Annexin V binding (Figure 3(b)). We also examined apoptotic nuclei in Ky-2-treated Ca9-22 cells using Hoechst’s staining. As shown in Figure 3(c), apoptotic cells were indentified according to their characteristic cell morphology, such as condensation and degradation of the nuclei, after Ky-2 treatment. Taken together, these results indicate that Ky-2 treatment induces apoptosis in Ca9-22 cells.

To clarify the mechanism of the cytotoxic effects of Ky-2 (100 nM) on Ca9-22 cells, we investigated the involvement of caspases in apoptotic cell death. Ca9-22 cells were treated with Ky-2, and the expression of procaspase-9, -3, -6, and -7 were examined using immunoblot analysis. Procaspase-9, -3, -6, and -7 were detected in the cells as protein bands with a molecular mass of 45, 35, 35, and 35 kDa, respectively. Cleaved forms of caspase-9 (35 kDa), -3 (17 kDa), -6 (15 kDa) and -7 (20 kDa) were detected at 18 hours after Ky-2 treatment (Figure 4). Furthermore, immunoblot analysis revealed that treatment with Ky-2 caused cleavage of PARP, an enzyme involved in DNA repair, and a substrate for caspase-3 and -7, in a time-dependent manner as an 89-kDa fragment. Also, PARP-cleaved products appeared at 18 hours and their levels were increased

after 24 hours (Figure 4). Proteolytic lamin A/C, a major structural protein of the nuclear envelope and substrate for caspase-6, was cleaved as a small (28 kDa) fragment at 18 hours after Ky-2 treatment.

Next, we examined the effect of a caspase-9 inhibitor (Z-LEHD-fmk; 100 nM) on Ky-2-induced apoptosis in Ca9-22 cells. When Ca9-22 cells were treated with Ky-2 (100 nM) for 24 hours, the percentage of cells in the sub-G1 phase was 32.9%. On the other hand, when those cells were cultured with caspase-9 inhibitor the percentage was 20.4%, as shown by flow cytometric analysis (Figure 5(a)). At 18 hours after beginning Ky-2 treatment, the caspase-9 inhibitor blocked the expressions of cleaved caspase-9, -3, -6, and -7 (Figure 5(b)). In addition, cleavage of lamin A/C and PARP were not detected in Ky-2 treated-cells incubated with caspase-9 inhibitor (Figure 5(b)).