Butein (3,4,2′,4′-tetrahydroxychalcone) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). MG132 and cycloheximide were purchased from Calbiochem (La Jolla, CA, USA). The caspase inhibitor, Z-VAD-FMK, was purchased from Promega (Madison, WI, USA). The KBM5 and K562 cell lines were kindly given by Dr Bharat B Aggarwal (University of Texas M.D. Anderson Cancer Center, Houston, TX, USA) and from Dr Dong-Hoon Jin (Asan Medical Center, Seoul, Korea), respectively. The cells were cultured in Iscove's modified Dulbecco's medium, supplemented with 10% fetal bovine serum and 1% antibiotics (Welgene, Inc., Daegu, Korea).

A total of 2×10⁴ cells (for either the KBM5 or the K562 cell line) were seeded into each well of 96-well plates and were subsequently treated with butein at different concentrations for 24 h. The cell viability was measured using an EZ-Cytox Enhanced Cell Viability assay kit (DoGen, Seoul, Korea), according to the manufacturer's instructions. Trypan blue assays were performed to measure cell growth. The cells were treated with various concentrations of butein for 72 h and the viable cell numbers were quantified daily.

Whole cell extracts were lysed in cell lysis buffer (Biosesang, Inc., Seongnam, Korea). Equal quantities of protein (30 µg) were separated on 8–12% SDS-PAGE gels and were subsequently transferred onto a polyvinylidene difluoride membrane (GE Healthcare Life Sciences, Freiburg im Breisgau, Germany). After blocking the membranes with 1% bovine serum albumin and 2% skimmed milk for 1 h, the membranes were incubated at 4°C overnight with the appropriate primary antibody, and were washed three times in phosphate buffered saline with 0.01% Tween-20. The membranes were incubated at room temperature for 1 h with horseradish peroxidase-conjugated secondary antibodies. In order to visualize the protein bands, the membranes were treated with enhanced chemiluminescence kit solution (DoGen) and exposed to X-ray film (AGFA Healthcare, Mortsel, Belgium). Anti-PARP, caspase-3, caspase-9, cyclin-dependent kinase (CDK)4, phosphorylated (p-)p53 and p-murine double minute 2 (MDM2) antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-CDK1, CDK2, cyclin E, cyclin A, cyclin B, p21, p53 and Bcl-2 antibodies were purchased from Santa Cruz Biotechnology, Inc. Anti-cyclin D and Bcl-xL antibodies were obtained from BD Biosciences (San Jose, CA, USA). The anti-tubulin antibody was obtained from Sigma-Aldrich, Inc. (St. Louis, MO, USA).

The total RNA was extracted using a total RNA Extraction kit (Intron Biotechnology, Inc., Seongnam, Korea). The cDNA was synthesized using a cDNA synthesis kit (Takara Bio, Inc., Shiga, Japan), according to manufacturer's instructions. MDM2 mRNA amplification was then performed with cDNA (l µg/µl) and the following primers: forward, 5′-CTGGGGAGTCTTGAGGGACC-3′ and reverse, 5′-CA GGTTGTCTAAATTCCTAG-3′ (21). The cycling conditions used were as follows: denaturation at 94°C for 30 sec, annealing at 53°C for 1 min, and elongation at 68°C for 2 min. After 35 cycles, the MDM2 mRNA band was visualized using a Davinch-Chemi™ Chemiluminescence Imaging system (Davinch-K Co., Ltd., Seoul, Korea).

To assess the cell cycle profile, the cells were treated with butein and were subsequently fixed in 95% ethanol with 0.5 % Tween-20 at −20°C overnight. The fixed cells were stained with 50 µg/ml propidium iodide (Santa Cruz Biotechnology, Inc.). For apoptotic cell death, the cells were treated with butein and subsequently stained with annexin V and 7-aminoactinomycin D (7-AAD; Santa Cruz Biotechnology, Inc.). The cells were analyzed using a FACSCalibur (BD Biosciences, San Jose, CA, USA) with CellQuest Pro, version 5.2 software.

Student's t-test (Microsoft Excel version, 2007) was used to determine statistically significant differences between the cell lines. P<0.05 was considered to indicate a statistically significant difference.

Butein is a polyphenolic compound obtained from plants (Fig. 1A). To determine whether the cytotoxic effect of butein on CML cells was p53-dependent, CML cells expressing p53 (KBM5) or not (K562) were treated with butein at different concentrations and were subjected to viability assays. The expression patterns of p53 were confirmed in the KBM5 and K562 cells, to ensure that the p53 status was as described (Fig. 1B). Treatment with 5 µg/ml butein for 24 h reduced the viability of the KBM5 cells by ~48.1%, whereas it caused no effect on the viability of the K562 cells (Fig. 1C). Cell growth was subsequently measured for 72 h treatment. In accordance with the data from the cell viability assays, butein reduced KBM5 cell numbers, however, not K562 cell numbers. Additionally, the treatment prevented cell growth, even at 0.5 µg/ml, while butein at 5 µg/ml marginally reduced K562 cell growth (Fig. 1D). Therefore, these data indicated that the expression of p53 in CML cells was crucial for butein sensitivity.

Next, whether butein affected the cell cycle of CML cells was assessed, since an abnormal cell cycle is tightly associated with cell death. Butein arrested the KBM5 cells in S-phase of the cell cycle (Fig. 2A). Furthermore, butein reduced the expression levels of CDK1, CDK4, cyclin A, cyclin B and cyclin E, and caused no affect on the expression levels of CDK2 and cyclin D (Fig. 2B). Therefore, butein-mediated changes in the expression levels of CDK/cyclin appeared to cause S-phase arrest. Furthermore, butein increased the phosphorylation of p53 and in turn, increased the expression of p21, while reducing phosphorylated and total expression levels of MDM2 in the KBM5 cells (Fig. 2C). In the K562 cells, butein marginally reduced the expression levels of p21 and MDM2 (Fig. 2C). Therefore, these data suggested that butein may impair the CML cell cycle by targeting p53 and its downstream effectors.

Butein reduced the expression of MDM2 independent of the p53 status (Fig. 2B). Therefore, the present study further examined the effect of butein on MDM2. When the KBM5 and K563 cells were treated with butein, the mRNA expression of MDM2 was increased (Fig. 3A). Since butein reduced the protein expression of MDM2, whether butein affected MDM2 protein stability was assessed. When the KBM5 cells were pretreated with MG132 and subsequently treated with butein, the protein expression of MDM2 was reduced (Fig. 3B), as earlier. Additionally, butein reduced the protein expression of MDM2 even in KBM5 cells treated with cycloheximide (Fig. 3C). Therefore, the MDM2 protein may be degraded in a proteasome-independent manner. Notably, treatment with MG132 rescued the butein-mediated MDM2 reduction in K562 cells (Fig. 3B). In addition, the protein expression of MDM2 was reduced even in the K562 cells treated with butein and cycloheximide (Fig. 3C). Therefore, these data suggested that butein-mediated MDM2 degradation may differ between the KBM5 and K562 cells.

To confirm that butein induced apoptotic cell death, the cells were stained with annexin V and 7-AAD. Treatment with butein for 24 h induced the apoptosis of KBM5 cells. This apoptotic effect was not as severe in the K562 cells (Fig. 4A). These data are consistent with the cell growth and viability assays, and support that p53 sensitizes butein-mediated apoptotic cell death.

Next, the effect of butein on the expression levels of proteins involved in apoptosis was assessed. Assessing Bcl-2 reduction, cleavage of PARP and the levels of caspase-3, it was demonstrated that KBM5 cells were more sensitive to butein compared with the K562 cells (Fig. 4B). Next, the effect of butein on apoptotic cell death at different treatment durations was determined. Butein caused apoptotic responses in KBM5 cells 6 h after treatment, as indicated by reduced levels of Bcl-2 and Bcl-xL, and increased cleaved forms of caspase-9, caspase-3 and PARP (Fig. 4C). To determine whether butein-mediated apoptosis involved the activation of caspases, Z-VAD-FMK, a caspase inhibitor was used. This inhibitor failed to reduce PARP cleavage, while it inhibited the cleavage of caspase-9 and caspase-3 (Fig. 4D). Therefore, these data suggested that butein may cause CML apoptosis via a p53-dependent and caspase-independent signaling pathway.