A549 human non-small cell lung cancer cells and HCT116 human colorectal carcinoma cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and grown in RPMI-1640 medium (WelGENE Inc., Daegu, Korea) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 μg/ml streptomycin and 100 μg/ml penicillin. Caki human renal clear cell carcinoma cells were obtained from the ATCC and grown in Dulbecco’s modified Eagle’s medium (DMEM), containing 10% heat-inactivated FBS, 20 mM HEPES buffer and 100 μg/ml streptomycin and 100 μg/ml penicillin.

2-[1,1′-biphenyl]-4-yl-N-[5-(1,1-dioxo-1λ⁶-isothiazolidin-2-yl)-1H-indazol-3-yl]acetamide (BAI) was kindly supplied by Dr J.H. Lee (Keimyung University, Daegu, Korea). Anti-Bcl-xL (sc-634, 1:700), anti-AIF (sc-5586, 1:700), anti-p53 (sc-126, 1:1,000), anti-PUMA (sc-19187, 1:700), anti-cytochrome c oxidase subunit II (sc-23983, 1:700), and anti-Bcl-2 (sc-783, 1:700) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-β-actin (A5441, 1:2,000) antibody was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Anti-poly(ADP-ribose) polymerase (PARP) (#9542, 1:1,000) antibody was purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-caspase-3 (610322, 1:1,000), anticytochrome c (556433, 1:700), and anti-Bax (554104, 1:700) antibodies were purchased from BD Biosciences (Bedford, MA, USA). Benzyloxy carbony-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk) was purchased from R&D Systems (Minneapolis, MN, USA). PD-98059 (MEK inhibitor, PD), SP600125 (JNK inhibitor, SP), and SB-203580 (p38 MAP kinase inhibitor, SB) were purchased from Enzo Life Sciences (Farmingdale, NY, USA).

Cellular lysates were prepared by suspending 0.3×10⁶ cells in 80 μl of lysis buffer (137 mM NaCl, 15 mM EGTA, 0.1 mM sodium orthovanadate, 15 mM MgCl₂, 0.1% Triton X-100, 25 mM MOPS, 100 μM phenylmethylsulfonyl fluoride and 20 μM leupeptin, adjusted to pH 7.2). The cells were disrupted by vortexing and extracted at 4°C for 30 min. The proteins were electrotransferred to Immobilon-P membranes (Millipore Corp., Bedford, MA, USA). Detection of specific proteins was carried out with an ECL Western blotting kit according to the manufacturer’s instructions (Millipore Corp.).

The anti-proliferative effect of the BAI on cancer cells was investigated using a live cell movie analyzer, JuLI™ Br (NanoEnTek Inc., Seoul, Korea). Briefly, the cells were plated in 6-well culture plates at a density of 0.3×10⁶ cells/well in medium and allowed to attach for 10 h. The cells treated with BAI for 24 h. During this study, JuLi Br recorded images of the cells at 5 min intervals, and confluences were also measured.

Approximately 0.5×10⁶ cells were suspended in 100 μl PBS, and 200 μl of 95% ethanol was added while vortexing. The cells were incubated at 4°C for 1 h, washed with PBS, and resuspended in 250 μl of 1.12% sodium citrate buffer (pH 8.4) together with 12.5 μg RNase. Incubation was continued at 37°C for 30 min. The cellular DNA was then stained by applying 250 μl propidium iodide (50 μg/ml) for 30 min at room temperature. The stained cells were analyzed by a FACScan flow cytometer for relative DNA content based on red fluorescence.

To evaluate caspase-3 activity, cell lysates were prepared after their respective treatment with various drugs. Assays were performed in 96-well microtiter plates by incubating 20 μg cell lysates in 100 μl reaction buffer [1% NP-40, 20 mM Tris-HCl (pH 7.5), 137 mM NaCl, and 10% glycerol)] containing the caspase 3 substrate (DEVD-pNA) at 5 μM. Lysates were incubated at 37°C for 2 h. Thereafter, the absorbance at 405 nm was measured with a spectrophotometer.

Total cellular RNA was extracted from tissues using the TRIzol reagent (Molecular Research Center, Inc., Cincinnati, OH, USA). RNA was quantified using Nanodrop 1000 (Thermo Scientific, Wilmington, DE, USA). Each cDNA was synthesized from 2 μg of total RNA using M-MLV reverse transcriptase (Promega, Madison, WI, USA) according to the manufacturer’s protocol. By using the specific primer pairs described in Table I and SYBR Green Premix (Toyobo, Japan). Quantitative real-time PCR (qPCR) was performed on the LightCycler^® 480 real-time PCR system (Roche Diagnostics, Mannheim, Germany). β-actin was used as a housekeeping gene for normalization, and no-template sample was used as a negative control. Then, the qPCR data were analyzed by the 2^−ΔΔct method (11).

Rhodamine 123 (Invitrogen, Molecular Probes, Inc., Eugene, OR, USA) uptake by mitochondria is directly proportional to its membrane potential. Caki cells subjected to 2 h after treatment were incubated with rhodamine 123 (20 μM) for 10 min in the dark at 37°C. The cells were harvested and suspended in PBS. The mitochondrial membrane potential was subsequently analyzed using a flow cytometer (BD Bioscience).

Approximately 0.3×10⁶ Caki cells were harvested, washed once with ice-cold PBS and gently lysed for 2 min in 80 μl ice-cold lysis buffer (250 mM sucrose, 1 mM EDTA, 20 mM Tris-HCl pH 7.2, 1 mM DTT, 10 mM KCl, 1.5 mM MgCl₂, 5 μg/ml pepstatin A, 10 μg/ml leupeptin, 2 μg/ml aprotinin). Lysates were centrifuged at 12,000 g at 4°C for 10 min to obtain the supernatants (cytosolic extracts free of mitochondria) and the pellets (fraction that contains mitochondria). Cytosolic protein (30 μg) was resolved on 12% SDS-PAGE and then transferred to nitrocellulose, and probed with specific anti-cytochrome c antibody.

The cells were suspended by conjugation buffer (PBS with 10 mM EDTA). The cell lysates were incubated with 0.2 mM bismaleimide (Thermo Scientific, Hudson, NH, USA) at room temperature for 1 h and then extracted by lysis buffer for western blot analysis.

Caki cells were exposed to 60 nM BAI for the indicated time periods and cell lysates were prepared in 1× RIPA buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1% NP-40, 1% deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 mg/ml leupeptin, #9806, Cell Signaling Technology]. The cells were disrupted by sonication and centrifuged (13,000 rpm) at 4°C for 15 min. Cell lysates were then subjected to immunoprecipitation with an anti-Bcl-xL antibody. Protein G PLUS-agarose were added and then the cell lysates were rotated at 4°C for 2 h. The cell lysates were centrifuged (13,000 rpm) at 4°C for 10 min. The presence of p53 and PUMA in the anti-Bcl-xL immunoprecipitate (IPs) and lysates was then evaluated by immunoblot analysis using the specific antibodies.

The data were analyzed using a one-way ANOVA followed by post-hoc comparisons (Student-Newman-Keuls) using the Statistical Package for Social Sciences version 22.0 (SPSS Inc., Chicago, IL, USA).

Previous reports demonstrated that BAI induces apoptosis of various human cancer cell lines. To investigate the anticancer effects of BAI in detail, we first analyzed the growth inhibitory effect of BAI in the same human cancer cell lines using an automated cell counter. As shown in Fig. 1A, BAI markedly inhibited proliferation of various human cancer cell lines dose- and time-dependently. To examine the apoptotic effects of BAI, the cells were next treated with various concentrations of BAI for the indicated times and then apoptosis was assessed using flow cytometry to detect hypodiploid cell populations. Treatment of the cells with BAI resulted in a remarkably increased accumulation of cells in the sub-G1 population and an increase in PARP cleavage in a dose-dependent manner (Fig. 1B and C) and a time-dependent manner (Fig. 1B and D). Furthermore, BAI induced caspase-dependent apoptosis in various cancer cell lines, including A549, HCT116, and Caki, in a dose- and time-dependent manner (Fig. 2). Taken together, these data demonstrate that BAI induces caspase-3-dependent apoptosis.

Mitogen-activated protein kinases (MAPKs) are key participants in cell proliferation, survival, and differentiation (12,13). To explore the signaling events regulated during BAI-induced apoptosis, we used specific inhibitors. Our results showed that specific MAPK inhibitors (PD, MEK inhibitor; SP, JNK inhibitor; SB, p38 MAPK inhibitor) did not affect BAI-induced apoptosis in Caki and A549 cells (Fig. 3A). Reactive oxygen species (ROS), natural byproducts of the normal metabolism of oxygen, play a crucial role in apoptosis under both physiologic and pathologic processes (14). Therefore, we investigated whether ROS generation is involved in BAI-induced apoptosis in Caki cells. As shown in Fig. 3B, BAI-induced apoptosis was not attenuated by pretreatment with N-acetylcysteine (NAC) or glutathione (GEE). These data indicate that BAI-induced apoptosis is not associated with MAPK pathways or ROS generation.

In general, apoptosis induction is correlated with, and probably mediated by, perturbations of mitochondrial function, a manifestation of which is the dissipation of the transmembrane potential (ΔΨ_m). Therefore, we evaluated ΔΨ_m during apoptosis induction in BAI-treated human cancer cells. As shown in Fig. 4A, treatment with BAI markedly decreased ΔΨ_m in Caki cells. Mitochondria mediates apoptosis by releasing apoptogenic effectors such as cytochrome c and apoptosis-inducing factor (AIF) (15,16). As shown in Fig. 4B, BAI remarkably induced time-dependent release of cytochrome c and AIF into the cytoplasm in Caki cells. Several lines of evidence strongly support the notion that activation of the pro-apoptotic Bcl-2 protein, Bax, plays a critical role in apoptosis by changes of MMP levels and release of cytochrome c (17). Therefore, we next evaluated the effect of BAI on Bax activation. As shown in Fig. 4C, BAI markedly promoted Bax oligomerization. Taken together, these results suggest that BAI induces loss of MMP levels and release of cytochrome c through activation of Bax.

We next determined the effect of BAI on Bcl-2 regulation in Caki cells. As shown in Fig. 5A, data from kinetic analysis showed that treatments with BAI for various time-points (6–24 h) led to a marked downregulation of Bcl-2. To identify the Bcl-2 regulating mechanisms by BAI, we treated Caki cells with or without BAI in the presence or absence of z-VAD-fmk, a pan-caspase inhibitor, for 24 h, and then measured sub-G1 populations and the cellular levels of PARP, Bcl-2, and β-actin by FACS and western blot analysis, respectively. BAI induced cleavage of PARP and increased the population of Caki cells in the sub-G1 phase, which were largely suppressed by pre-treatment with z-VAD-fmk (Fig. 5B). However, BAI-induced downregulation of Bcl-2 was not blocked by pre-treatment with z-VAD-fmk, suggesting that the downregulation of Bcl-2 protein is not involved in caspase activity (Fig. 5B). Therefore, we next investigated the effect of BAI on the transcriptional regulation of Bcl-2 by RT-qPCR analysis. As shown in Fig. 5C, BAI reduced levels of Bcl-2 transcripts in a time-dependent manner. To further investigate the role of Bcl-2 in BAI-induced apoptosis, we used Caki renal carcinoma cells engineered for overexpression of Bcl-2. As shown in Fig. 5D, overexpression of Bcl-2 could not attenuate the apoptosis induced by BAI. Collectively, these results indicate that downregulation of Bcl-2 is not associated with BAI-induced apoptosis in Caki cells.

Bcl-xL is a widely studied factor of resistance to cytotoxic anticancer agents. We first examined whether Bcl-xL is associated with BAI-induced apoptosis, cancer cells were treated with BAI at different times. As shown in Fig. 6A, BAI treatment of cancer cells for various time-points resulted in markedly decreased expression levels of Bcl-xL in A549 and Caki cells. We explored the possible link between loss of Bcl-xL protein and activation of caspases in BAI-treated A549 cells. As shown in Fig. 6B, pretreatment with z-VAD-fmk had no effect on the reduction of Bcl-xL protein by BAI, implying that the BAI-induced downregulation of Bcl-xL protein is not associated with caspase activity. This led us to investigate the effect of BAI on transcriptional regulation of Bcl-xL. Notably, results of RT-qPCR analysis, as shown in Fig. 6C, demonstrated a marked reduction of Bcl-xL transcripts in BAI-treated cells, suggesting that BAI downregulates Bcl-xL at the transcriptional levels. To evaluate the functional significance of BAI-induced Bcl-xL downregulation, we transfected A549 cells with siRNA targeting Bcl-xL mRNA and treated cells with or without BAI for 24 h. The concentrations of BAI were sub-cytotoxic in comparison with the results of previous experiments. Immunoblot analysis confirmed that transfection with Bcl-xL siRNA resulted in suppression of Bcl-xL expression in A549 cells compared with cells transfected with control GFP siRNA (Fig. 6D). Notably, the BAI-induced accumulation of sub-G1 phase was markedly increased in cells transfected with Bcl-xL siRNA as compared with control siRNA-transfected cells (Fig. 6D). In addition, the expression of cleaved PARP was induced only in cells transfected with Bcl-xL siRNA (Fig. 6D).

Bcl-2 family members regulate survival/death decisions through a network of interactions among the pro-survival member Bcl-xL, the pro-apoptotic member PUMA, and p53 (9,10,18). We next investigated whether BAI affects the expression levels of p53 and PUMA proteins in cancer cells. As shown in Fig. 6E, Caki cells treated with BAI showed upregulation of p53 and PUMA in a time-dependent manner. We then determined whether BAI modulates the interactions between specific Bcl-2 families in Caki cells using co-immunoprecipitation assays. As shown in Fig. 6F, BAI not only efficiently disrupted the Bcl-xL/p53 interaction but also induced the binding between PUMA and Bcl-xL in Caki cells in a time-dependent manner. Additionally, A549 and Caki cells treated with BAI showed induction of Bax cleavage in a time dependent manner (Fig. 6G). Taken together, these results suggest that downregulation of Bcl-xL protein is importantly associated with the BAI-induced apoptosis and that BAI modulates interactions among p53 and Bcl-2 family proteins in human cancer cells.