Butein was obtained from Jilin University (Changchun, China), dimethylsulfoxide (DMSO), Tris-HCl, EDTA, SDS, phenylmethylsulfonyl fluoride (PMSF), bovine serum albumin (BSA), leupeptin, Nonidet P-40, deoxycholic acid, sodium orthovanadate, aprotinin and a polyclonal antibody against β-actin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Protein assay kits were obtained from Bio-Rad Laboratories (Hercules, CA, USA). Dulbecco’s modified Eagle’s medium (DMEM) and fetal-bovine serum (FBS) were obtained from Gibco-Life Technologies (Carlsbad, CA, USA). COX-2 polyclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-mouse secondary antibodies were purchased from Qiagen (Hilden, Germany).

The A549 non-small cell lung carcinoma (NSCLC) cell line was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM supplemented with heat-inactivated FBS (10%), L-glutamine (2 mM), penicillin (100 IU/ml), and streptomycin (100 μg/ml) in a humidified incubator aerated with 5% CO₂ and 95% at 37°C. When cells reached 70–80% confluency, they were trypsinized, counted and treated with celastrol in complete cell medium. Control cells were treated with vehicle (DMSO) for the same duration.

Cells were plated in 96-well culture plates at an initial density of 1×10⁴ cells/well and allowed to adhere to the plates. The culture medium was replaced by fresh medium containing butein at concentrations ranging between 0 and 20 μmol/l and incubated for 48 h. The cell proliferation kit I (MTT) from Roche Applied Science (Mannheim, Germany) was used to measure cell viability. Briefly, 10 μl labeling solution was added to each well of the 96-well plates. After 2 h in the CO₂ incubator, 100 μl solubilization solution was added to dissolve the purple crystals, which were the products of the MTT substrates. The absorbance was measured at 570 nm by a plate reader (Perkin-Elmer, Waltham, MA, USA). Absorbance measured in the MTT assays is expressed as a percentage of the control (defined as 100%).

Cells were seeded in 25 cm² flasks and incubated overnight to allow cells to adhere to the plate. A549 cells were treated with 10 and 20 μmol/l butein for 24 h. Following treatment, control (untreated) and treated floating and adherent cells were collected by trypsinization. The cells (1×10⁶ cells/ml) were washed twice with cold phosphate-buffered saline (PBS) and fixed in 70% ethanol. Immediately prior to the analysis, the cells were washed with PBS and stained with a solution containing propidium iodide (PI; 0.2 mg/ml) for 1 h at 4°C and with RNase A (0.1 mg/ml) for 30 min at 37°C. The distribution of cells in the cell cycle was measured by flow cytometry (Becton-Dickinson, Franklin Lakes, NJ, USA). Percentages of cells in the cell cycle phases were calculated using the Cell Quest software (Becton-Dickinson).

The induction of apoptosis by butein was assayed by the Nucleosome ELISA kit (Fitzgerald Industries International, Acton, MA, USA). This kit uses a photometric enzyme immunoassay to quantitatively determine the formation of cytoplasmic histone-associated DNA fragments (mono and oligonucleosomes) following apoptotic cell death. Apoptosis was determined by ELISA and the A549 cells (1×10⁵) were treated with butein at 0, 10 and 20 μmol/l for 12, 24 and 48 h in a 96-well plate. The induction of apoptosis was evaluated by assessing the enrichment of nucleosome in the cytoplasm, and determined as described in the manufacturer’s instructions for the nucleosome ELISA kit.

Cells were seeded in 6-well plates for one day prior to treatment. The medium was removed and cells were cultured in fresh DMEM containing 20 μmol/l butein. Following 6 h treatment, total RNA was extracted from the cells using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). The concentration and purity of RNA were determined by absorbance at 260/280 nm. DNA strands were synthesized from 3 μg total RNA by oligo-dT primers and M-MLV Reverse Transcriptase (Takara Bio, Inc., Shiga, Japan). Target fragments were quantified by real-time PCR, and an ABI prism 7700 Sequence Detection system (Applied Biosystems, Foster City, CA, USA) was employed for this assay. Taqman^®/VIC^® MGB probes and primers for COX-2 [Assay ID (NM_000963.1): HS00153133_M1] and β-actin, and Real-time PCR Taqman Universal PCR Master mix were obtained from Applied Biosystems. PCR reactions were set up as described in the instructions, which was validated by the company. The signal obtained for β-actin was used as a reference housekeeping gene to normalize the quantity of total RNA amplified in each reaction. Relative gene expression data were analyzed using the 2^−ΔΔCt method.

Cells were washed once with PBS (pH 7.4) and harvested into a 1.5 ml microtube with 0.5 ml lysis buffer (PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS). The lysis buffer contained protease inhibitors (40 mg/l PMSF, 0.5 mg/l aprotinin, 0.5 mg/l leupeptin, 1.1 mmol/l EDTA and 0.7 mg/l pepstatin). The harvested cells were then lysed with a cell disruptor (Branson Ultrasonics Corp., Danbury, CT, USA) on ice for 30 sec. The protein concentration of the cell lysate was determined by a Dc protein assay (Bio-Rad, Richmond, CA, USA). Lysate protein (50 μg) was separated on 10% SDS-PAGE and transferred onto an Immobilon polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). Anti-COX-2 (Cayman Chemicals, Ann Arbor, MI, USA), and secondary anti-mouse IgG antibodies conjugated with horseradish peroxidase (HRP; Santa Cruz Biotechnology, Inc.) were used for protein detection. An enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA) provided the chemiluminescence substrate for HRP, and the targeted protein was visualized by autoradiography (Xue Wang, The First Hospital of Jilin University, Changchun, China).

Values are expressed as the mean ± standard deviation. The data were analyzed by one-way analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the antiproliferative activities of butein on A549 cells, the MTT assay was applied. As shown in Fig. 1, exposure of A549 cells to increasing concentrations of butein (0–20 μmol/l) for 72 h resulted in a dose-dependent growth inhibition. The lower butein concentrations (2.5–5 μmol/l) did not affect A549 cell viability significantly, whereas concentrations between 10 and 20 μmol/l significantly reduced cell viability. The IC₅₀ for the 48 h incubation time was ~14.18 μM. Therefore, 15–20 μmol/l doses were selected for further butein treatment studies.

The results on the effect of butein on cell cycle progression of A549 are shown in Fig. 2A. As compared with the control, 10 μmol/l butein increased the population of G1 phase from 35.3 to 42.4%. This effect was enhanced when A549 cells were treated with 20 μmol/l butein (61.5% cell population in G1 phase).

Fig. 2B shows the time course of DNA fragmentation in continuous treatment with 10 and 20 μmol/l butein. DNA fragmentation of A549 was found at 12 h and maximized at 48 h following the addition of butein. In contrast to the control, when cells were treated with butein, the number of cells undergoing apoptosis increased between ~3.3 and 8.2-fold at 10 and 20 μmol/l butein, respectively, at 48 h.

qPCR was performed to detect the mRNA expression of COX-2 in A549 cancer cells following butein treatment. As shown in Fig. 3, mRNA levels of COX-2 was significantly reduced following butein treatment as compared with the control group (P<0.05), which showed that mRNA expression of COX-2 was downregulated by butein treatment in A549 cancer cells.

A549 cells were treated with 20 μmol/l butein, and cultured for 24 h. Protein lysates were prepared for western blot analysis. As shown in Fig. 4, protein levels of COX-2 were significantly reduced in A549 cells following butein treatment compared with the control (P<0.05), which was in agreement with the result that butein treatment may downregulate COX-2 mRNA expression in A549 cancer cells.