3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), bafilomycin A1, 4,6-diamidino-2-phenylindole (DAPI), monodansylcadaverine (MDC) and doxorubicin were purchased from the Sigma-Aldrich Chemical Co. (St. Louis, MO). Antibodies specific for actin, Bad, Bax, Bcl-2, Bcl-xL, Bid, caspase-3, caspase-8, caspase-9, cIAP-1, cIAP-2, death receptor (DR) 4, DR5, Fas, Fas legend (FasL), Fas-associated death domain (FADD), cellular FLICE-like inhibitory protein (c-FLIP), nuclear factor erythroid 2-related factor 2 (Nrf2), poly(ADP-ribose) polymerase (PARP), survivin, XIAP, heme oxygenase 1 (HO-1) and goat anti-rabbit IgG-FITC were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies specific for Atg3, Atg5, Atg7, Atg12, Beclin-1 and microtubule-associated protein 1 light chain 3 (LC3) were purchased from Cell Signaling (Beverly, MA). The antibody specific for phospho (p)-Nrf2 was purchased from Epitomics (Burlingame, CA). Peroxidase-labeled donkey anti-rabbit and sheep anti-mouse immunoglobulins and an enhanced chemiluminescence (ECL) kit were purchased from Amersham (Arlington Heights, IL). Caspase activity assay kits were purchased from R&D Systems (Minneapolis, MN). z-VAD-fmk was purchased from Calbiochem (San Diego, CA). OB was kindly provided by Professor Hyung-In Moon of Dong-A University (Busan, Republic of Korea) (18) and dissolved in dimethyl sulfoxide (DMSO) as a stock solution at 30 mM concentration. Dilutions were made in Dulbecco’s modified Eagle’s medium (DMEM, Gibco-BRL, Gaithersburg, MD).

The A549 cell line was obtained from the American Type Culture Collection (Rockville, MD) and cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco-BRL). Cell viability was measured based on formation of blue formazan metabolized from colorless MTT by mitochondrial dehydrogenases, which are active only in live cells.

To analyze the percentage of apoptotic cells, cells were collected, washed with cold phosphate-buffered saline (PBS), and fixed in 75% ethanol at 4°C for 30 min. The DNA content of the cells was measured using a DNA staining kit (CycleTEST PLUS Kit, Becton-Dickinson, San Jose, CA). Propidium iodide (PI)-stained nuclear fractions were obtained by following the kit protocol. The cells were then filtered through 35-mm mesh, and DNA content fluorescence was determined using a FACSCalibur flow cytometer within 1 h. The cellular DNA content was analyzed with CellQuest software (Becton-Dickinson).

After OB treatment, the cells were lysed in a buffer containing 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA and 0.5% Triton X-100 for 1 h at room temperature. The lysates were vortexed and cleared by centrifugation at 15,000 rpm for 10 min at 4°C. The DNA in the supernatant was extracted using a 25:24:1 (v/v/v) equal volume of neutral phenol:chloroform:isoamyl alcohol. To assay the DNA fragmentation pattern, samples were loaded onto 1.0% agarose gel containing 0.1 μg/ml ethidium bromide (EtBr) and electrophoresis was carried out.

The enzymatic activity of the caspases was assayed using a colorimetric assay kit according to the manufacturer’s protocol. The cells were incubated in the absence and presence of OB for the indicated times. The cells were harvested and lysed in a lysis buffer for 30 min on an ice bath. Lysed cells were centrifuged at 14,000 rpm for 20 min, and equal amounts of protein (100 μg per 50 μl) were incubated with 50 μl reaction buffer and 5 μl colorimetric tetrapeptides, Asp-Glu-Val-Asp (DEVD)-p-nitroaniline (pNA) for caspase-3, Ile-Glu-Thr-Asp (IETD)-pNA for caspase-8, and Leu-Glu-His-Asp (LEHD)-pNA for caspase-9, at 37°C for 2 h in the dark. Caspase activity was determined by measuring the changes in absorbance at 405 nm using an ELISA reader.

To observe autophagy formation, A549 cells were grown on glass coverslips for 24 h. The cells were incubated in the absence and presence of OB for the indicated times, and then the cells were treated with 0.05 mM MDC at 37°C in 5% CO₂ for 1 h. The cells were then fixed with 4% paraformaldehyde in PBS for 10 min. The cellular changes were analyzed with a fluorescence microscope.

Whole-cell protein extracts from A549 cells were prepared with cell lysis buffer (20 mM sucrose, 1 mM EDTA, 20 μM Tris-HCl, pH 7.2, 1 mM DTT, 10 mM KCl, 1.5 mM MgCl₂ and 5 μg/ml aprotinin) for 30 min. Cells were disrupted by sonication and extracted at 4°C for 30 min. The protein extracts were quantified using the Bio-Rad kit (Pierce Biotechnology, Rockford, IL). For western blot analysis, lysate proteins (30–50 μg) were resolved over sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred onto nitrocellulose transfer membranes (Schleicher & Schuell, Keene, NH). Specific proteins were detected with an ECL Western blotting kit according to the recommended procedure. In a parallel experiment, cells were washed with ice-cold PBS and collected. Then cytoplasmic and nuclear proteins were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology).

Cells were grown on coverslips and treated as indicated. Cells were washed twice in PBS, fixed with 4% paraformaldehyde in PBS at room temperature for 30 min, and then permabilized with 0.25% Triton X-100 solution for 10 min. The cells were subsequently incubated in a blocking solution of 1% bovine serum albumin (BSA) and incubated with primary antibodies at room temperature for 1 h. After the incubation period, the samples were rinsed four times with PBS and then incubated with the secondary anti-rabbit-FITC diluted 1:200 in buffer for 1 h at room temperature. Nuclei were stained with 1 μg/ml DAPI, and then captured using a Zeiss LSM 510 laser scanning confocal device (Carl Zeiss).

All data are expressed as mean ± SD. The significant differences between the groups were determined using an unpaired Student’s t-test. A value of p<0.05 was considered significant. All figures shown represent results from at least two independent experiments with a similar pattern.

To investigate the effects of OB on cell viability in A549 cells, cells were treated with OB and subjected to an MTT assay. As shown in Fig. 1A and B, treatment with OB significantly reduced cell viability in a concentration- and time-dependent manner. Subsequently, to examine whether OB inhibits the proliferation of A549 cells by inducing apoptosis, genomic DNA was extracted from cells and agarose gel electrophoresis were assessed. As indicated Fig. 1C, treatment with OB time-dependently induced DNA fragmentation, a hallmark of apoptosis, in a time-dependent manner. In addition, flow cytometric analysis also revealed that treatment with OB increased the accumulation of cells at the apoptotic sub-G1 phase in a time-dependent manner (Fig. 1D). Taken together, these results indicate that the cytotoxic effects observed in response to OB are associated with the induction of apoptotic cell death in A549 cells.

To determine which pathway was involved in the apoptosis induction of OB-treated A549 cells, the expression levels of death receptor-related proteins and the Bcl-2 and IAP family of proteins were determined with western blot analysis to measure the expression of the proteins. As shown in Fig. 2, exposure to OB led to a significant reduction in the anti-apoptotic protein cIAP-2, survivin and c-FLIP in a time-dependent fashion. However, OB treatment resulted in a time-dependent increase in the level of the pro-apoptotic FasL proteins. Under these conditions, although we did not detect the truncated form of the pro-apoptotic protein Bid, a BH3-only pro-apoptotic member of the Bcl-2 family, our results indicate that OB treatment caused time-dependent downregulation of the whole form of the Bid proteins, which reflects Bid cleavage and activation.

To investigate whether OB-induced apoptosis in A549 cells involves the caspase cascade pathway, the caspase expression levels and activity were determined. As shown in Fig. 3A, western blot analyses indicated that the active forms of caspase-3 and -8 increased and the expression of pro-caspase-9 decreased in a time-dependent manner following OB treatment. For further quantification of the proteolytic activation of caspases, protein in the lysates of cells treated with OB was normalized and then assayed for in vitro activities using fluorogenic substrates. As indicated in Fig. 3B, treatment with OB resulted in a time-dependent increase in caspase activity (−3, −8 and −9) compared with the control cells, which was associated with the progressive proteolytic cleavage products of PARP, an activated caspase-3 substrate protein. To further investigate the significance of caspase activation in OB-induced apoptosis, A549 cells were pretreated with z-VAD-fmk, a broad-spectrum caspase inhibitor, for 1 h, followed by treatment with 30 μM OB for 24 h. Interestingly, pretreatment with z-VAD-fmk did not restore cell viability compared with control (Fig. 4A), and failed to suppress the OB-induced apoptosis (Fig. 4B). However, under the same condition, z-VAD-fmk significantly suppressed doxorubicin-induced growth inhibition and apoptosis (Fig. 4). Taken together, these results suggest that OB-induced apoptosis was independent of caspase activation in A549 cells.

As shown in Fig. 5A, based on our finding that extensive cytoplasm vacuolization compared with control cells appeared in cytosol after OB treatment, we hypothesized that the vacuoles might be associated with autophagy. For this study, we used MDC, a commonly used autophagic dye, as a more specific marker for autophagy. The results indicated that untreated control cells presented diffused staining, but OB treatment clearly resulted in an extensive punctuate MDC staining pattern (Fig. 5B). However, LC3, another typical marker of autophagy, exists in the cytosol and is called LC3-I, after its C-terminal region, which is cleaved through post-translational modification. When autophagosomes are formed, LC3-I changes to LC3-II through conjugation with phosphatidylethanolamine and is recruited into the autophagosome membrane. That is why LC3-II accumulation is a critical marker of autophagy (19). As shown in Fig. 6C, OB treatment induced an increase in LC3-II expression. In addition, treatment with OB strongly increased the essential autophagosome-regulatory gene expression of Atg3, and slightly increased the gene expression of Atg5, but not Atg7 and Atg12. We also found an accumulation of LC3 puncta following OB treatment through fluorescence microscopic observation (Fig. 7A). Taken together, these results indicate that OB-induced cytoplasmic vacuoles are related to autophagy induction.

Because many recent studies have reported that Nrf2 is involved in cancer cell survival and in the acquisition of drug resistance against anticancer therapies, we investigated whether the Nrf2 signaling pathway is involved in OB-caused cell death. As shown in Fig. 6, although exposure of cells to OB led to dephosphorylation of Nrf2 proteins without altering their total levels, Nrf2 and p-Nrf2, the active form of Nrf2, were translocated to the nucleus in response to OB treatment, which was associated with decreased expression of HO-1 whose transcription is regulated by Nrf2. Therefore, OB-induced cell death appeared to be responsible for nuclear translocation of p-Nrf2.

To elucidate the molecular mechanism controlling the relationship between OB-induced apoptosis and autophagy, we next investigated whether OB-induced autophagy functioned as a survival mechanism or a death mechanism. As shown in Fig. 7A, pretreatment with bafilomycin A1, a well-known inhibitor of autophagosomal lysosome degradation, blocked the formation of OB-induced LC3 puncta, representing recruited LC3-II in the cytosol. Western blot analysis also showed that PARP cleavage, caspase-3 activation, and p-Nrf2 translocation were restored to levels seen in the control cells in response to bafilomycin A1 pretreatment, which was connected with significant blockage of OB-induced apoptosis and growth inhibition (Fig. 7C and D). Taken together, these results clearly show that OB treatment induces autophagy as a cell death mechanism in A549 cells.