HAC-Y6 was synthesized in the laboratory of the Graduate Institute of Pharmaceutical Chemistry, China Medical University (Taichung, Taiwan, R.O.C.). MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), Hoechst 33258, propidium iodide (PI), Tris-HC1, Triton X-100, and RNase A were purchased from Sigma Chemical Co. (St. Louis, MO, USA). RPMI-1640 medium, L-glutamine, fetal bovine serum, penicillin-streptomycin, and trypsin-EDTA were obtained from Invitrogen Corp (Carlsbad, CA, USA). Antibodies for AIF, Apaf-1, aurora A, aurora B, phospho-aurora A, phospho-aurora B, Bcl-xL, BubR1, caspase-9, caspase-3, Endo G, phospho-H3, α-tubulin, β-tubulin, and poly(ADP-ribose) polymerase (PARP) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies for β-actin, Bax, Bcl-2, cyclin B1, CDK1, cytochrome c, horseradish peroxidase (HRP)-linked goat anti-mouse IgG, and goat anti-rabbit IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The Annexin V-FITC Apoptosis Detection kit was obtained from Strong Biotech Corporation (Taipei, Taiwan, R.O.C.). The Flow Cytometry Mitochondrial Membrane Potential Detection kit was obtained from BD Biosciences (Los Angeles, CA, USA).

The human colon cancer cell line COLO 205 was obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan, R.O.C.). Cells were seeded into plates or dishes in RPMI-1640 medium supplemented with 10% fetal bovine serum, L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin in a humid atmosphere of 5% CO₂ and 95% air at 37°C. Cells were plated at a density of 2.5×10⁴ cells per well in 96-well plates for the cell viability assay, 1×10⁵ cells per well in 24-well plates with cover slips for Hoechst staining, 2×10⁵ cells per well in 12-well plates for cell cycle assay, 1×10⁶ cells per well in 6-well plates for mitochondrial membrane potential assay, and 1×10⁷ cells per plate in 10 cm dishes for western blot analysis. Cells were allowed to adhere for 24 h before use.

COLO 205 cells were plated at a density of 2.5×10⁵ cells per well in 12-well plates and then incubated with 0.5 μM of HAC-Y6 for 24 to 72 h. Cells were directly examined and photographed under a phase contrast microscope.

COLO 205 cells were plated at a density of 2.5×10⁴ cells per well in 96-well plates, and cell survival was evaluated using MTT reduction assays. The reduction status of the cells was measured by a colorimetric assay, indicating cell survival (28). MTT was dissolved in phosphate-buffered saline (PBS, 500 ml contains 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.47 mM KH₂PO₄, pH 7.4) at a concentration of 1 mg/ml and filtered. After 48 h exposure to HAC-Y6, 50 μl MTT was added to each well and incubated for 2 h at 37°C in the dark. When absorbed by living cells, MTT is converted to a water-insoluble blue product (formazan). The formazan product was dissolved by adding 150 μl dimethylsulf-oxide (DMSO) to each well. The absorption value at 570 nm was determined using an ELISA plate reader. Data are presented as the percentage of survival relative to vehicle-treated control culture. All measurements were performed in triplicate and each experiment was repeated at least three times.

Nuclei were stained with Hoechst 33258 (bis-benzimide, Sigma) to detect chromatin condensation or nuclear fragmentation, morphological characteristics of apoptosis. HAC-Y6 treatment cells were stained with 5 μg/ml Hoechst 33258 for 10 min. After washing twice with PBS, cells were fixed with 4% paraformaldehyde (PFA) in PBS for 10 min at 25°C. Fluorescence of the soluble DNA (apoptotic) fragments was measured in a Varian Fluorometer at an excitation wavelength of 365 nm and emission wavelength of 460 nm.

Cells were fixed in 70% ethanol overnight and re-suspended in PBS containing 20 μg/ml PI, 0.2 mg/ml RNase A, and 0.1% Triton X-100 in a dark room. After 30 min incubation at 37°C, cell cycle distribution was analyzed using ModFit LT Software (Verity Software House, Topsham, ME, USA) in a BD FACSCanto flow cytometer (Becton-Dickinson, San Jose, CA, USA).

COLO 205 cells (2×10⁵ cells/well) were fluorescently labeled for detection of apoptotic and necrotic cells by adding 100 μl of binding buffer, 2 μl of Annexin V-FITC, and 2 μl of PI to each sample. Samples were mixed gently and incubated at room temperature in the dark for 15 min. Binding buffer (300 μl) was added to each sample immediately before flow cytometric analysis. A minimum of 10,000 cells within the gated region were analyzed.

Cells were plated on 6-well at 1.0×10⁶ cells/well and treated with 1 μM HAC-Y6 for 6–24 h. Mitochondrial membranes were stained with 0.5 ml JC-1 working solution (BD MitoScreen Kit, Becton-Dickinson) to each sample. Samples were incubated for 10–15 min at 37°C in the dark. Mitochondrial membrane potential was measured using the BD FACSCanto flow cytometer (Becton-Dickinson).

After treatment, cells were fixed with 4% PFA, blocked with 2% bovine serum albumin, stained with anti-tubulin monoclonal antibody, and then with FITC conjugated anti-mouse IgG antibody. PI was used to stain the nuclei. Cells were visualized using a Leica TCS SP2 Spectral Confocal System.

Tubulin assembly was measured through turbidimetry, as described previously (29). Assay mixtures containing 1.0 mg/ml (10 μM) tubulin and varying drug concentrations were preincubated for 15 min at 30°C in the absence of GTP. The samples were then placed on ice, and 0.4 mM GTP was added. Reaction mixtures were transferred to cuvettes held at 0°C, and turbidity development was monitored for 20 min at 30°C after a rapid temperature increase. Drug concentrations that inhibited the increase in turbidity by 50% relative to a control sample were determined. Inhibition of the binding of [³H]colchicine to tubulin was measured as described previously (29). A total of 1.0 μM tubulin was incubated with 5.0 μM [³H]colchicine and 5.0 μM inhibitor for 10 min at 37°C. At this time, approximately 40 to 60% of maximum colchicine binding occurred.

The treated cells were collected and washed with PBS. After centrifugation, cells were lysed in a lysis buffer. The lysates were incubated on ice for 30 min and centrifuged at 12,000 x g for 20 min. Supernatants were collected, and protein concentrations were then determined using the Bradford assay. After adding a 5X sample loading buffer containing 625 mM Tris-HCl, pH 6.8, 500 mM dithiothreitol, 10% SDS, 0.06% bromophenol blue, and 50% glycerol, protein samples were electrophoresed on 10% SDS-polyacrylamide gels and transferred to a nitrocellulose membrane. Immunoreactivity was detected using the western blot chemiluminescence reagent system (PerkinElmer, Boston, MA, USA). β-actin was used as a loading control.

Statistical analysis was performed with an analysis of variance (ANOVA) followed by the Tukey’s test. All data were expressed as mean ± SD from at least three independent experiments. ^*P<0.001 was indicative of a significant difference.

Evaluation of the HAC-Y6 inhibitory activity against the NCI panel of 60 human cancer cell lines provided the results shown in Fig. 2. The mean graph midpoint (MID) values for the log GI₅₀, log TGI, and log LC₅₀ values were −7.04, −5.26 and −4.17, respectively. For total growth inhibition (TGI), HAC-Y6 also demonstrated significant inhibitory activity (log TGI <−6.5) against 12 tumor cell lines: HL-60, RPMI-8228, NCI-H522, COLO 205, HCC-2998, HCT-116, HT29, MDA-MB-435, OVCAR-3, NCI/ADR-RES, PC-3 and DU145.

Exposure of COLO 205 cells to HAC-Y6 for 48 h and performance of MTT metabolism assays confirmed the effects of HAC-Y6 on cell viability. The IC₅₀ value was 0.52±0.035 μM with HAC-Y6 decreasing COLO 205 cell viability in a dose-dependent manner. Exposure of COLO 205 cells to 0.1, 0.25, 0.5, 0.75 and 1 μM HAC-Y6 reduced the survival to 90.8±1.9, 69.2±0.9, 49.4±1.7, 32.3±3.4 and 14.1±1.1% of the control (0.1% DMSO), respectively (Fig. 3A). HAC-Y6 inhibited COLO 205 cell growth dose- and time-dependently (Fig. 3B).

Morphological analysis confirmed the HAC-Y6 cytotoxic effects. As shown in Fig. 4, apoptotic morphological changes included cell rounding and shrinkage after 24 h incubation with 0.5 μM of HAC-Y6 (the white arrowhead indicates an apoptotic nucleus). Fig. 4D and F show COLO 205 cells with more than one nucleus per cell (the black arrowheads indicate multinucleate cells).

Staining COLO 205 cells with Hoechst 33258 confirmed apoptosis as the cause of reduced cell viability. As shown in Fig. 5, control cells without HAC-Y6 treatment exhibited uniformly dispersed chromatin, normal organelles, and intact cell membranes. Cells treated with 0.5 μM of HAC-Y6 for 24, 36, 48, 60 and 72 h demonstrated typical characteristics of apoptosis, including the condensation of chromatin, the shrinkage of nuclei and the appearance of apoptotic bodies (the white arrowhead indicates an apoptotic nucleus).

Annexin V-FITC/PI double-labeling detected phosphatidylserine externalization, a hallmark of the early phase of apoptosis (Fig. 6). Cells incubated in the absence of HAC-Y6 for 12, 24, 36 and 48 h were undamaged and were negative for both Annexin V-FITC and PI staining (Q3). After incubation with 1 μM HA-Y6 for 24 to 48 h, the numbers of advanced apoptotic cells stained by positive Annexin V-FITC and negative PI (Q4) were significantly increased as the incubation time grew longer. The numbers of advanced apoptotic cells stained by positive Annexin V-FITC and PI (Q2) also increased significantly with incubation time.

During apoptosis, the mitochondrial membrane potential (Δψm) decreases. Cells treated with 1 μM of HAC-Y6 for 6, 12 and 24 h, stained cells with JC-1 confirmed apoptosis as the cause of decreased Δψm. As shown in Fig. 7, JC-1 fluorescence is seen in both the FL2 and FL1 channel (P2) in the control cell population (0 h). There is a significant increase in the number of cells with reduced red fluorescence (P3), indicative of a change in the Δψm, in the population induced to undergo apoptosis (6–24 h). These data demonstrate that HAC-Y6 induced cell apoptosis in COLO 205 cells.

Treatment of COLO 205 cells with 1 μM of HAC-Y6 for 0, 12, 24, 36, 48 and 60 h, followed by flow cytometric analysis to determine cell cycle distribution of the treated cells, was used to investigate the effects of HAC-Y6 on disruption of the cell cycle and provide further insights into the apoptotic effects of the compound. As shown in Fig. 8, HAC-Y6 induced a time-dependent accumulation of G₂/M (4N) cells, as well as causing formation of octoploid cell (8N) population and apoptotic (sub-G₁) cells. These flow cytometric findings with HAC-Y6-treated COLO 205 cells are in accord with the multinucleated cells presented above (Figs. 4D, F and 5; the black arrowheads indicate multinucleate cells).

COLO 205 cells were treated with 1 μM of HAC-Y6 for 24 h and then stained appropriately and examined in a confocal microscope to determine the effects of the compound on the cellular microtubule cytoskeleton. As shown in Fig. 9A, treatment with HAC-Y6 resulted in microtubule changes similar to those induced by colchicine. Both compounds caused cellular microtubule depolymerization with short microtubule fragments scattered throughout the cytoplasm. Taxol, in contrast, induced significantly increased tubulin polymerization. As shown in Fig. 9B, after cells were treated with HAC-Y6 for 6–48 h, HAC-Y6 caused inhibition of microtubule (α- and β-tubulin) assembly. Therefore, our data demonstrated that HAC-Y6 induced microtubule depolymerization.

Further examination of HAC-Y6 in tubulin assays and comparison with combretastatin A-4 (CA-4) provided the results presented in Table I. HAC-Y6 potently inhibited tubulin assembly, with an IC₅₀ value of 0.81±0.03 μM. Although HAC-Y6 inhibited tubulin assembly more potently than CA-4 (IC₅₀ 1.3±0.1 μM), it was slightly less effective than CA-4 in inhibiting colchicine binding to tubulin.

Cyclin B1 and CDK1 are markers for induction of mitotic arrest. Treatment of COLO 205 cells with 1 μM of HAC-Y6 increased cyclin B1 protein levels (Fig. 10A). BubR1, an essential component of the mitotic check-point, localizes in kinetochores (8). Treatment of COLO 205 cells with 1 μM HAC-Y6 increased the levels of BubR1 (Fig. 10A). This result suggests that BubR1 contributed to activation of the mitotic checkpoint induced by HAC-Y6. The compound directly causes microtubule disassembly, and the cell cycle protein changes, including increased BubR1 expression, are secondary to the failure to form a spindle, which results from disruption of tubulin assembly.

Treatment of COLO 205 cells with 1 μM HAC-Y6 was used to investigate the effects of HAC-Y6 on aurora kinase function. As shown in Fig. 10B, HAC-Y6 decreased aurora A, phospho-aurora A, aurora B and phospho-aurora B expression. Histone H3 is one of the substrates of aurora B kinase. During mitosis, aurora B is required for phosphorylation of histone H3 on serine 10, and this might be important for chromosome condensation (15). We therefore examined whether HAC-Y6 inhibited phosphorylation of histone H3 in COLO 205 cells by western blot analysis. As shown in Fig. 10B, HAC-Y6 decreased phospho-H3 expression after a 6 h treatment. This finding suggests that inactivation of aurora kinases A and B is involved in HAC-Y6-induced G₂/M arrest and multinucleation.

Following our observations that HAC-Y6 caused apoptosis in COLO 205 cells, we next determined levels of selected proteins associated with apoptosis. The activation of caspases is a hallmark of apoptosis. Activated caspases cleave a variety of target proteins including poly(ADP-ribose) polymerase, DNA-dependent protein kinase, and sterol regulatory-dependent binding protein, and thereby disable cellular processes and break down the cellular structure (23). To investigate whether HAC-Y6-induced apoptosis was involved in the activation of caspase cascades, COLO 205 cells were exposed to 1 μM of HAC-Y6 for 6, 12, 24, 36 and 48 h. The activities of caspase-3 and caspase-9 were then determined using Western blot analysis, which revealed activation of caspase-3 and caspase-9 within 12 h of HAC-Y6 treatment. Cleavage of PARP, a substrate for caspase-3, also occurred (Fig. 11A). HAC-Y6 also increased levels of Endo G, AIF, Apaf-1 and cytochrome c (Fig. 11B). These results suggest that the mitochondrial signaling pathways of COLO 205 cells mediate HAC-Y6-induced apoptosis.

The Bcl-2 family proteins are key regulators of mitochondrial-related apoptotic pathways (30). Some of these proteins (such as Bcl-2 and Bcl-xL) are anti-apoptotic, whereas others (such as Bad, Bax and Bid) are pro-apoptotic. The balance of pro- and anti-apoptotic bcl-2 proteins influences the sensitivity of cells to apoptotic stimuli (31). Exposure of COLO 205 cells to 1 μM of HAC-Y6 for 6, 12, 24, 36 and 48 h verified the involvement of Bcl-2 protein activity in HAC-Y6-induced apoptosis. As shown in Fig. 12, results indicated that HAC-Y6 reduced anti-apoptotic Bcl-2 and Bcl-xL levels, as well as increased pro-apoptotic Bax levels and the release of Endo G, AIF, Apaf-1, cytochrome c and procaspase-9 from the mitochondria to the cytosol. Release of Apaf-1, and cytochrome c leads to the activation of caspase-9. Activated caspase-9, in turn, cleaves and activates caspase-3.