trans-3,4,5'-Trihydroxystilbene (resveratrol) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). 4-(6-Hydroxy-2-naphthyl)-1,3-benzendiol (HS-1793) was supplied by Professor Hongsuk Suh (Pusan National University, Busan, Korea), and dissolved at a concentration of 100 mM in ethanol as a stock solution, and stored −20°C. The stock solution was diluted with cell culture medium to the desired concentration prior to use. The maximal concentration of ethanol did not exceed 0.1% (v/v) in the treatment range, where there was no influence on the cell growth.

MCF-7 (wild-type p53) and MDA-MB-231 (mutant type p53) cells were obtained from American Type Culture Collection (Manassas, VA, USA). MCF-7 and MDA-MB-231 cells were maintained in DMEM medium (HyClone, Logan, UT, USA) in humidified atmosphere of 37°C with 5% CO₂. DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS, HyClone), 2 mM glutamine (Sigma-Aldrich), 100 U/ml penicillin (HyClone) and 100 μg/ml streptomycin (HyClone).

Cell survival was determined by colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay which measures mitochondrial activity in viable cells. Cells (1.5×10⁵) were plated in each well of a 6-well plate, allowed to adhere overnight and then the culture medium was replaced with fresh media. Cells were treated with resveratrol or HS-1793 at concentrations of 12.5, 25 and 50 μM for 24 h. Control groups were treated with ethanol equal to the highest percentage of (<0.1%) solvent used in experimental conditions for MTT assay. After 24 h the medium was replaced with fresh medium. MTT was freshly prepared at 5 mg/ml in PBS and passed through a filter (pore size, 0.2 μm). An aliquot of 2 ml of MTT stock solution was added to each well, and the plate was incubated at 37°C for 4 h in humidified 5% CO₂ incubator. After 4 h, media were removed and formazan crystals were dissolved in 2 ml of DMSO for 10 min with gentle agitation. The optical density of each well was measured with a spectrophotometer equipped with a 540-nm filter.

Cells were harvested and solubilized in lysis buffer (40 mM Tris, pH 8.0, 120 mM NaCl, 0.5% NP-40, 0.1 mM sodium orthovanadate, 2 μg/ml aprotinin, 2 μg/ml leupeptin and 100 μg/ml phenylmethylsulfonyl fluoride), and the supernatant was collected and protein concentrations were then measured with a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as the standard. Equal amount of protein extracts were denatured by boiling at 100°C for 5 min in sample buffer (0.5 M Tris-Cl, pH 6.8, 4% SDS, 20% glycerol, 0.1% bromophenol blue, 10% β-mercaptoethanol) in ratio of 1:1. Equal amount of the total proteins were subjected to 6–15% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 5% non-fat dry milk in TBS-T buffer (20 mM Tris, 100 mM NaCl, pH 7.5 and 0.1% Tween-20) for 1 h at room temperature. The membrane was incubated with diluted primary antibody in TBS-T buffer at 4°C overnight. The membranes were washed once for 10 min 3 times with TBS-T buffer and incubated for 1 h with secondary antibody in TBS-T buffer at room temperature.

MCF-7 and MDA-MB-231 cells were incubated with 12.5, 25 and 50 μM resveratrol or HS-1793 for 24 h and then cells were washed twice with PBS containing 1% bovine serum albumin (PBS-B) and fixed with 70% ethanol containing 0.5% Tween-20 at 4°C for 30 min. Fixed cells were washed with PBS-B, and stained with 4 μg/ml Hoechst 33342 for 30 min at room temperature. The stained cells were washed twice with PBS-B and observed under Zeiss Axiophot microscope (Göttingen, Germany) at ×400 magnification.

Cells were trypsinized, washed with PBS, and fixed in 95% ethanol with Tween-20 at 4°C overnight. Prior to analyses, cells were again washed with PBS, suspended in cold propidium iodide (PI, Sigma-Aldrich) solution, and incubated at room temperature for 30 min in the dark. Before analysis cell suspensions were filtered with 40 μM pore nylon mesh to remove debris. Flow cytometry analysis was performed on a FACScan (Becton Dickinson, San Jose, CA, USA).

Total cell lysates were lysed in lysis buffer. The supernatant was collected and protein concentration determined with Bio-Rad protein assay kit (Bio-Rad). For immunoprecipitation, cell extracts were incubated with immunoprecipitating antibody in lysis buffer at 4°C for 1 h. The immuno-complexes were precipitated with protein A-sepharose beads (Sigma-Aldrich) for 1 h, and washed five times with extraction buffer prior to boiling in SDS sample buffer. Immunoprecipitated proteins or aliquots containing 40 μg protein were separated on SDS-PAGE and transferred to PVDF membranes. Western blot analysis was performed. Primary antibodies to p53, MDM2, p21^WAF1/CIP1, cyclin D1, CDK4, CDK6, cyclin B1, Cdc2, Cdc25C, Fas, Fas-L, PARP, Bax, Bcl-2, ERK1/2, pERK, JNK and pJNK were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Primary antibody to β-actin was purchased from Sigma-Aldrich. The proteins were visualized with enhanced chemiluminescence (ECL) detection system (GE Healthcare Biosciences, Pittsburgh, PA, USA).

Cells were harvested and washed twice in PBS at 4°C. Total cells were lysed with the lysis buffer at 4°C for 30 min with vortexing. Cell lysate (200 μg) was mixed with assay buffer in a final volume of 100 μl, followed by addition of 10 μl of 2 mM of p-nitroaniline (pNA)-conjugated caspase-8 (Z-IETD-pNA), caspase-9 (Ac-LEHD-pNA), or caspase-3 (Z-DEVD-pNA) substrates, respective, for the caspase assay. The reaction mixture was incubated at 37°C for 30 min and the liberated pNA was measured at 405 nm.

Results are expressed as the mean ± SD of three separate experiments and analyzed by Student's t-test. Means were considered significantly different at p<0.05 or p<0.01.

To investigate the effects of resveratrol and HS-1793 on the viability of MCF-7 and MDA-MB-231 cells, the MTT assay was performed. Resveratrol did not show any prominent effects, with IC₅₀ values not being measurable at the concentration of 12.5, 25 and 50 μM. However, the IC₅₀ values of HS-1793 in MCF-7 and MDA-MB-231 cells were 25 μM and 50 μM, respectively (Fig. 2A and B). Therefore, HS-1793 seems to induce more efficient inhibition of cell viability than resveratrol. Cell proliferation was also evaluated by counting dead and live cell numbers by the trypan blue exclusion method. Results indicated that resveratrol and HS-1793 exerted time- and concentration-dependent inhibition of cell proliferation in both MCF-7 and MDA-MB-231 cells (Fig. 2C and D). Although both resveratrol and HS-1793 exhibited anti-proliferative effect on both breast cancer cells, HS-1793 was more potent than resveratrol.

We next investigated whether resveratrol and HS-1793 affect cell cycle progression. Cells were treated with either resveratrol (12.5, 25 and 50 μM) or HS-1793 (3, 6.25 and 12.5 μM) for 24 h and then the cell cycle was analyzed by flow cytometric analysis. As shown in Table I, HS-1793 inhibited the cell growth through G2/M phase arrest, while resveratrol led to G1 phase arrest in MCF-7 (16) and MDA-MB-231 (17) cells as already published.

Generally, p53 is known as a tumor suppressor gene and controls the expression of p21^WAF1/CIP1, a potent cyclin-dependent kinase (CDK) inhibitors in G1 and G2/M phases. MDM2, an oncogene, negatively regulates p53 through inhibiting the transactivation activity of p53 by binding to its transactivation domain. MDM2 has also a ubiquitin ligase activity that leads to the degradation of p53 (18,19). In MCF-7 cells, p53 and p21^WAF1/CIP1 were increased while MDM2 was decreased by both resveratrol and HS-1793. In contrast, in MDA-MB-231 cells, p21^WAF1/CIP1 was increased without a change in the level of MDM2. Given that MDA-MB-231 cells are p53 mutant, there might be a p53-independent stimulus inducing the increase in p21^WAF1/CIP1 (Fig. 3A and B). Western blot analyses were conducted to further characterize the molecular mechanisms by which resveratrol or HS-1793 inhibits cell growth. The levels of intracellular proteins of G1 phase, such as cyclin D1, CDK4 and CDK6, were not significantly changed (Fig. 4A). Immunoprecipitation was conducted to investigate binding activity of the cyclin D1-CDK4 complex which showed a decrease of the complex in both cell types (Fig. 4B). Cyclin B1, Cdc2 and Cdc25C, which are proteins of G2 phase, were decreased in both cell lines by HS-1793 treatment in a concentration-dependent manner (Fig. 4C). Cdc2 and cyclin B1 interaction was also inhibited by HS-1793 treatment (Fig. 4D).

To determine whether the growth inhibitory effects of resveratrol and HS-1793 could be attributed to apoptotic cell death in MCF-7 and MDA-MB-231 cells, the morphological changes were assessed with Hoechst 33342 staining (Fig. 5A and B). The control cells displayed typical normal nuclear structure in a concentration-dependent manner, while cells treated with resveratrol and HS-1793 exhibited chromosomal condensation and formation of apoptotic bodies, indicating apoptotic cell death. At 12.5 μM, HS-1793 was effective in inducing chromosomal condensation in both cell types, whereas resveratrol did not (Fig. 5A and B). Therefore, these results also showed that HS-1793 exhibited more efficient induction of apoptosis than resveratrol in both cell lines.

The degradation of polypeptides, such as poly(ADP-ribose) polymerase (PARP), was examined to further study the possible involvement of apoptosis-associated caspases in the induction of apoptotic cell death (Fig. 6). Treatment with resveratrol and HS-1793 caused increase in cleavage of PARP in both cell types (Fig. 6A and B). To determine whether the expression levels of death receptors and death receptor-mediated apoptotic proteins were changed by resveratrol and HS-1793, western blot analysis was performed and expression levels of Fas, Fas-ligand (Fas-L), Bcl-2 and Bax were measured. The expression of Fas and Fas-L was increased in a concentration-dependent manner in both cell lines. In addition, in both HS-1793 treated cell lines, the expression level of Bcl-2 protein was markedly downregulated, while Bax was upregulated in a concentration-dependent manner. These data suggest that resveratrol and HS-1793 induce apoptosis through the alteration in expression levels of death receptor proteins as well as altered the expression ratio of Bax/Bcl-2 protein (Fig. 6A and B).

In an attempt to further characterize the molecular mechanisms of apoptosis induced by resveratrol or HS-1793, the activity of caspases (-3, -8, and -9) was determined by colorimetric assay. In case of MCF-7 cells (caspase-3 null type), the activity of caspase-8 and -9 was increased with the treatment of resveratrol or HS-1793 (Fig. 7A and B). In MDA-MB-231 cells, however, caspase-3, -8, and -9 were all activated with the treatment of both compounds (Fig. 7C and D). Overall, these results implicate that both HS-1793 and resveratrol induce caspase-dependent apoptotic cell death in MCF-7 and MDA-MB-231 cells.

The mitogen-activated protein kinase (MAPK) signaling pathway has been shown to play important roles in cell cycle and apoptosis (20,21). Thus, to investigate whether the MAPK pathway is involved in HS-1793 and resveratrol-induced apoptosis, MCF-7 and MDA-MB-231 cells were treated with resveratrol or HS-1793 at 12.5, 25 and 50 μM for 24 h and then the expression levels of MAPKs [i.e., extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK)] were compared by western blot analysis. As shown in Fig. 8, both resveratrol and HS-1793 induced phosphorylation of ERK and JNK in MCF-7 cells (Fig. 8A). In contrast to this, only ERK phosphorylation was increased in MDA-MB-231 cells (Fig. 8B). These results suggest that apoptosis induced by resveratrol or HS-1793 may be mediated by different pathways in p53 wild and mutant type cell lines.