A549 (human lung adenocarcinoma), HEK293 (human embryonic kidney cell), HepG2 and SK-Hep1 (human hepatocarcinoma) were purchased from the American Tissue Culture Collection (Manassas, VA, USA). Cells were cultured in RPMI-1640 (A549), DMEM (HEK293), EMEM (HepG2 and SK-Hep1) (HyClone Laboratories, Logan, UT, USA) medium supplemented with 10% heat inactivated fetal bovine serum (FBS; HyClone Laboratories), 100 U/ml penicillin and 10 μg/ml streptomycin (PAA Laboratories GmbH, Pasching, Austria) in a humidified atmosphere containing 5% CO₂ at 37°C. Except the cells were used in the cell viability, A549 cells were investigated, after incubation in the FBS-free RPMI-1640 medium for 24 h, anacardic acid (AA), Calbiochem (San Diego, CA, USA) was added to the medium. Pan-caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

The exponential phase of cells were seeded into the wells of 96-well plates at an initial density of 0.5×10⁵–1×10⁵ cells in medium containing 10% FBS per well of 100 μl. Following 24 h of incubation, AA, dissolved in 100% dimethyl sulfoxide (DMSO; Sigma-Aldrich), was added to the culture medium at various concentrations. Cells were incubated for 24 h and 10 μl of EZ-Cytox Cell Viability Assay Solution (WST-1™; Daeil Lab Service, Seoul, Korea) was added and incubated for 4 h at 37°C. The intensity was measured at 450 nm using an ELISA reader (Molecular Devices, Sunnyvale, CA, USA).

A549 cell lysates were prepared in a Radioimmunoprecipitation assay buffer (RIPA, Cell Signaling Technology, Danvers, MA, USA) and proteins were visualized using enhanced chemiluminescent (ECL) detection solution (Pierce Biotechnology, Rockford, IL, USA). Antibodies include anti-cleaved caspase 3, anti-cleaved caspase 7, anti-cytochrome c, anti-Bax, anti-Bad, anti-Bak, anti-Bcl-XL, anti-cleaved PARP, anti-FoxO1, anti-FoxO3a, anti-FoxO4, anti-β-actin, secondary rabbit and mouse antibodies conjugated with HRP were purchased from Cell Signaling Technology. Anti-AIF, anti-Hsp70, anti-polyclonal Hsp27, anti-Noxa, anti-STRAP, anti-Bim and secondary antibodies conjugated with HRP rabbit goat were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

4′,6-diamidino-2-phenylindole (DAPI) staining was used for the morphological observation of apoptosome. A549 cells were seeded in plates, and the cells were treated with or without AA (3 μg/ml) for 24 h, then the cells were washed with PBS. Two-to-three milliliters of diluted DAPI was added to the cells and incubated for 15 min at 37°C. The cells were rinsed once with methanol and the result was analyzed by fluorescence microscopy using an Eclipse 50i microscope.

For observation of more detailed morphological features, transmission electron microscopy (TEM) was used. Cultured A549 cells were pre-fixed in pellets at 4°C with 2.5% glutaraldehyde in 0.1 M phosphate buffer and then post-fixed with 1% osmium tetraoxide (OsO₄) in 0.1 M phosphate buffer, pH 7.4. After fixing, cells were embedded in Epon 812 using routine procedures. Approximately 70 μm ultrasected specimens by Ultracut Reichert-jung were stained using uranyl acetate and lead citrate, and examined with Hitachi H600 TEM (Tokyo, Japan). All reagents used in the TEM experiment were purchased from Electron Microscopy Science (EMS).

A549 cells were harvested by trypsinization and fixed with ice-cold ethanol (70%) for 5 h at 4°C. Followed resuspending with PBS containing RNase A (0.2 μg/ml) and incubation for 1 h at 37°C. Cells were stained with propidium iodide (40 μg/ml) for 30 min. The distribution of sub-G1 DNA content was analyzed using the FACS Calibur apparatus (Becton-Dickinson, Mountain View, CA, USA).

For analyzing DNA fragmentation, genomic DNA was extracted using DNeasy Blood and Tissue kit purchased from Qiagen Inc. (Valencia, CA, USA) to the manufacturer’s protocol.

In order to investigate the cytotoxicity, we used a normal cells (HEK293), and lung (A549) and liver (HepG2, SK-Hep1) cancer cells were treated with AA in a dose-dependent manner. After 24 h of exposure, the results of cell viability assay showed that AA inhibits the proliferation of all cells used. Although AA inhibited cellular proliferation of HEK cells, the cytotoxicity was less than cancer cells (Fig. 2A). To study the effect of the caspase inhibitor, cells were pretreated for 2 h with the cell-permeable and irreversible pan-caspase inhibitor Z-VAD-fmk (100 μM) and then exposed to AA for 24 h in the continued presence of Z-VAD-fmk. Viability was then assessed by MTT assay using WST-1^™ and the result showed Z-VAD-fmk did not inhibit the cell viability in AA treated A549 cells (Fig. 2B). Considering our interest in lung cancer, we used A549 cells for further research and the IC₅₀ value of AA treated A549 cells was 2.75±0.25 μg/ml. Therefore, we used 3.0 μg/ml of AA for investigating the molecular mechanism of cell death in A549. Cellular morphology of AA treated A549 cells were shown under a microscope and cytotoxicity was increased dose-dependently (Fig. 2C).

In order to further elucidate the nature of AA induced cell death in A549, we investigated the nuclear morphology of AA treated cells by using 4′,6-diamidino-2-phenylindole (DAPI) staining. The results showed totally different patterns between the AA-treated and -untreated cells. AA treated cells exhibited formations of apoptosome (Fig. 3A–b), indicating that AA induces apoptosis in A549 cells. For further evidence of apoptosis, we analyzed the fragmentation of chromosomal DNA by using agarose gel electrophoresis (Fig. 3B). The fragmentation of chromosomal DNA by AA increased to the exposure time (Fig. 3B-b, c and d), while it was not observed in the untreated cells (Fig. 3B-a). In addition, to study the cell death induced by AA, we quantified the sub-G1 DNA content by fluorescence activated cell sorting (FACS) analysis. The sub-G1 genomic DNA content was gradually increased after 6, 12 and 24 h to 7.58, 14.01 and 38.79%, respectively (Fig. 3C). TEM also showed apoptotic features such as chromatin condensation and nuclear fragmentation (Fig. 3D-b). These results support the view that AA plays a major role in apoptosis induction in A549 cells.

To determine the apoptosis signaling pathway induced by AA, we analyzed the expression of several genes using western blot analysis. Cleaved caspase 3, cleaved caspase 7 and cytochrome c, known as the executioners of the intrinsic pathway, showed gradual increase in a time dependent manner on AA treated A549 cells. Caspase 9 was also analyzed (Fig. 4A). In addition, the expression of pro- and anti-apoptotic Bcl-2 family was determined. Pro-apoptotic members of the Bcl-2 family, Bim, Bad, Bak and Noxa increased, and the anti-apoptotic member Bcl-xL, decreased (Fig. 4B). The expression of Bak and Bad showed gradual increase time-dependently with AA-treatment and the expression of Bax and Noxa showed further increased level at 12 h after AA exposure. Bim has three isoforms (BimS, BimL and BimEL) with different intrinsic toxicities and promotes apoptosis (19). The cleaved form of Bim appeared at 6 h and the expression of Bcl-xl decreased gradually to 18 h after AA exposure.

Because the inhibition of the pan-caspase inhibitor, Z-VAD-fmk, failed to prevent cell death, we analyzed the possibility of a caspase-independent pathway by apoptogenic molecules, apoptosis-inducing factor (AIF). Poly (ADP-ribose) polymerase-1 (PARP-1), the mediator of AIF release, was also investigated. The expression of AIF showed gradual increase, as did the cleaved PARP-1 after 18 h (Fig. 4C).

We examined the expression of several genes that encode proteins known as molecular chaperones by assisting the correct folding of nascent and stress-accumulated misfolded proteins (20). We investigated the expression of Hsp70 and Strap, stress-responsive activator of p300 and the results showed downregulation of Hsp70 and Strap by AA (Fig. 4D).

Members of the non-phosphorylated mammalian forkhead transcription factors (FoxOs) are involved in regulating the expression of genes involved in apoptosis. FoxO1, FoxO4 and FoxO3a are known as mammalian forkhead transcription factors that trigger the up-regulation of proteins such as Bim and NOXA (21). The expression of FoxO1, FoxO4 and FoxO3a in AA treated A549 cells increased with the optimal expression time being slightly different depending on the subfamily (Fig. 4E).