The human non-small cell lung cancer (HNSCLC) A549 was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and grown in RPM-1640 medium supplemented with 10% heated-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 μg/μl streptomycin and 100 μg/μl penicillin. Human renal clear cell carcinoma Caki was 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/μl streptomycin and 100 μg/μl 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). LB42708 was purchased from TOCRIS. Anti-cIAP-1, anti-cIAP-2, anti-Bcl-2, anti-Mcl-1, anti-AIF, anti-HSC70, and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-PARP and anti-ERK antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-OxPhos Complex II subunit (QPs2) antibody was purchase from Molecular Probes (Eugene, OR, USA). Anti-cellular FLICE-like inhibitory protein (c-FLIP) antibody was purchased from Alexis (San Diego, CA, USA). Anti-XIAP and anti-cytochrome c antibodies were purchased from BD Biosciences Pharmingen (San Diego, CA, USA). Antibodies against the following proteins were purchased from the indicated suppliers: pro-caspase-3 from Santa Cruz Biotechnology. z-VAD-fmk was purchased from Biomol (Plymouth Meeting, PA, 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.

The human cDNAs encoding c-FLIP (L) and c-FLIP (s) were PCR amplified from plasmids [pCA-FLAG-hFLIP (L) and pCA-FLAG-hFLIP (s); kindly provided by Dr S.I. Park, Korea Centers for Disease Control and Prevention, Seoul, Korea] containing these sequences with the specific primers. c-FLIP (L) and c-FLIP (s) cDNA fragment were digested with KpnI and XhoI and subcloned into the pcDNA 3.1 (+) vector (Invitrogen), and the resulting constructs were confirmed by nucleotide sequencing. The human cDNA for Mcl-1 (L) was PCR amplified using the following specific primers: Mcl-1 (sense) 5′-GCGACTGGCAAAGCTTGGC CTCAA-3′ and (anti-sense) 5′-CAACTCTAGAAACTGGT TTTGGTG-3′. The human Mcl-1 (L) cDNA fragments were subcloned into the pcDNA 3.1 (+) vector (Invitrogen, Calsbad, CA, USA).

Caki cells were transfected with the following: a mammalian expression vector containing CrmA cDNA, a pMAX vector containing the human Bcl-2 gene (provided by Dr Rakesh Srivastava, NIH/NIA), a vector containing Mcl-1 (L), and a vector encoding Flag-tagged c-FLIP (L) or c-FLIP (s). Stable cell lines overexpressing CrmA, Bcl-2, Mcl-1 (L), c-FLIP (L), or c-FLIP (s) were selected with fresh medium containing 500 μg/ml G418 (Calbiochem, Madison, WI, USA) for 4 weeks. Overexpression of CrmA, Bcl-2, Mcl-1 (L), c-FLIP (L), or c-FLIP (s) was analyzed by western blotting using anti-CrmA (BD Pharmingen), anti-Bcl-2 (Santa Cruz), anti-Mcl-1 (Santa Cruz), anti-Flag (Sigma, St. Louis, MO, USA), or anti-c-FLIP (Alexis) antibody, respectively.

Approximately 0.3×10⁶ 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.

The anti-proliferative effect of the BAI or FTI on Caki 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 in the presence or absence of FTI for 24 h. During this study, JuLi Br recorded images of Caki cells at 5 minute intervals, and confluences were also measured.

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 form 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 a no template sample was used as a negative control.

The XIAP small-interfering RNA (siRNA) duplexes were obtained from Cell Signaling Technology. Control siRNA duplexes used in this study were purchased from Invitrogen and had the following sequences: green fluorescent protein (GFP), AAG ACC CGC GCC GAG GUG AAG. Cells were transfected with siRNA oligonucleotides using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s recommendations.

Rhodamine 123 (Molecular Probes) uptake by mitochondria is directly proportional to its membrane potential. Caki cells subjected to 4 and 10 h after treatment were incubated with rhodamine 123 (5 μM) for 30 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).

Caki cells (0.3×10⁶) 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, 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). The resulting cytosolic fractions were used for western blot analysis with an anti-cytochrome c antibody.

The intensities of corresponding bands were quantified using the ImageJ program (National Institutes of Health, MD, USA) according to the manufacturer’s instructions.

All data are presented as mean ± SD. Significant differences between the groups were determined using the unpaired Student’s t-test. A value of ^*P<0.005 was accepted as indication of statistical significance. All the data shown in the figures were obtained from at least two independent experiments with a similar pattern.

In order to investigate the effect of co-treatment with BAI and FTI on Caki cells, Caki cells were treated with BAI alone, LB42708 alone, or BAI plus LB42708. As shown in Fig. 1A, the combined treatment of Caki cells with BAI and LB42708 induced morphological features of apoptosis including cell shrinkage, rounding, and detachment of the cell from the plate and marked inhibited proliferation (Fig. 1A). Additionally, co-treatment with BAI and LB42708 inhibited cell viability, while BAI treatment alone or LB42708 treatment alone did not reduce cell viability (Fig. 1B). Furthermore, we analyzed the occurrence of apoptosis in Caki cells using flow cytometric analysis to detect hypodiploid cell populations. As shown in Fig. 1C, co-treatment of Caki cells with BAI and LB42708 resulted in a significantly increased accumulation of sub-G1 phase cells, whereas treatment with BAI alone or LB42708 alone rarely increased accumulation of sub-G1 phase cells. Exposure to co-treatment of BAI and LB42708 led to increased cleaved form of PARP (Fig. 1C). To generalize these phenomena, we investigated whether BAI enhances LB42708-induced apoptosis in a synergistic fashion in another human cancer cell type. The combined treatment strongly induced apoptosis in A549 cells (Fig. 1D). These results suggest that BAI can sensitize various malignant cancer cells to LB42708-induced apoptosis.

Endoplasmic reticulum (ER) stress-mediated apoptosis is a well known mechanism of cell death (20). So, to evaluate whether BAI plus LB42708-induced apoptosis is involved in ER stress, cyclosporine A (CsA) was used as a potent inhibitor of ER stress-induced apoptosis. As shown in Fig. 2A, the apoptosis induced by the combined treatment with BAI and LB42708 similarly occurred in CsA-pretreated cells. Moreover, PARP cleavage was not diminished by pretreatment of CsA (Fig. 2A). Reactive oxygen species (ROS), natural byproducts of the normal metabolism of oxygen, play an important role in apoptosis under both physiologic and pathologic conditions (21). Therefore, we examined whether ROS generation is involved in BAI plus LB42708-induced apoptosis. As shown in Fig. 2B, pretreatment with N-acetylcysteine (NAC) only slightly inhibited BAI plus LB42708-induced apoptosis and PARP cleavage. Therefore, these results clearly indicate that the combination of BAI and LB42708-induced apoptosis is not associated with ER stress and ROS generation.

To address the significance of caspase activation in the combination of BAI and LB42708- induced apoptosis, we used a general and potent inhibitor of caspases, z-VAD-fmk (benzyloxycarbonyl-Val-Ala-Aspfluoromethyl ketone). As shown in Fig. 3A, co-treatment of BAI and LB42708-induced apoptotic population was markedly inhibited by pretreatment with z-VAD-fmk. Additionally, the degradation of pro-caspase-3 and PARP was completely blocked by pretreatment of z-VAD-fmk (Fig. 3A). Not only did the BAI plus LB42708 induce increased DEVDase activity, but it also induced the degradation of pro-caspase-3 (Fig. 3B). These results suggest that BAI plus LB42708-induced apoptosis is mediated by the caspase-3-dependent pathway. To investigate which specific caspase is associated in BAI plus LB42708-induced apoptosis, CrmA overexpressing Caki cells were used. Overexpression of CrmA could not attenuate the apoptosis induced by BAI plus LB42708 (Fig. 3C). Collectively, our data suggested that BAI plus LB42708-induced apoptosis is involved in activation of caspase-3 and -7, with only slight activation of caspase-1, -4, -5, -8, -9 and -10.

The induction of cell death is generally associated with, and probably mediated, by perturbations of the mitochondrial function, a manifestation of which is the dissipation of the transmembrane potential (ΔΨ_m). Hence, we wanted to analyze ΔΨ_m during apoptosis induction in BAI plus LB42708-treated Caki cells. As shown in Fig. 4A, co-treatment of BAI and LB42708 markedly decreased transmembrane potential (ΔΨ_m). It has been reported that mitochondria play an important role in apoptosis by releasing apoptogenic effectors such as cytochrome c and apoptosis-inducing factor (AIF) (22). As shown in Fig. 4B, co-treatment of BAI and LB42708 remarkably induced time-dependent release of cytochrome c and AIF into the cytoplasm. Taken together, these results indicate that mitochondria may have an important role in BAI plus LB42708-induced apoptosis.

Next, we investigated whether co-treatment of BAI and LB42708 could modulate the expression of IAP family, Bcl-2 family, and c-FLIPs proteins. As shown in Fig. 5A, BAI plus LB42708 did not alter the expression levels of IAP family proteins such as cIAP1 and cIAP2, but XIAP. Moreover, the expression of Mcl-1 (L), Bcl-2, c-FLIP (L), and c-FLIP (s) were markedly decreased in the BAI plus LB42708-treated cells (Fig. 5A). To further examine whether the downregulation of apoptosis-related proteins by co-treatment of BAI and LB42708 is associated with activation of caspases, a pan-caspase inhibitor, z-VAD-fmk was used. As shown in Fig. 5B, pretreatment with z-VAD-fmk had no effect on reduction of those proteins by BAI plus LB42708, implying that the downregulation of those proteins induced by BAI plus LB42708 is not associated with the caspase activity. This led us to promptly identify the effect of co-treatment with BAI and LB42708 on transcriptional regulation of XIAP, Mcl-1 (L), c-FLIP (L), c-FLIP (s) and Bcl-2 in Caki cells. As shown in Fig. 5C, results of real-time PCR analysis revealed that XIAP, Mcl-1 (L), c-FLIP (L) and Bcl-2 transcripts were remarkably reduced in BAI plus LB42708-treated Caki cells. However, c-FLIP (s) mRNA expression level was not altered in BAI plus LB42708-treated Caki cells. These results suggested that co-treatment with BAI and LB42708 downregulates XIAP, Mcl-1 (L), c-FLIP (L), and Bcl-2 at their transcriptional level and c-FLIP (s) at post-transcriptional level in Caki cells.

To confirm the functional role played by downregulated apoptosis regulatory proteins in BAI plus LB42708-induced apoptosis, we employed Caki renal carcinoma cells engineered for overexpression of Mcl-1 (L), c-FLIP (L), c-FLIP (s), and Bcl-2 (Fig. 6A–D). For the control model, vector-transfected control cells (Caki/vector) were engineered (Fig. 6A–D). As shown in Fig. 6A and B, overexpression of Mcl-1 (L) and c-FLIP (s) could not attenuate the apoptosis induced by co-treatment with BAI and LB42708. Collectively, these results indicate that downregulations of Mcl-1 (L) and c-FLIP (s) is not associated with the BAI plus LB42708-induced apoptosis in Caki cells. Next, to examine the functional significance of BAI plus LB42708- induced XIAP downregulation, we employed the siRNA duplex against XIAP mRNA. Caki cells were transfected with XIAP siRNA and co-treated with or without BAI and LB42708. Immunoblot analysis demonstrated that transfection of siRNA against XIAP resulted in a suppression of XIAP expression in Caki cells as compared to cells transfected with control GFP siRNA (Fig. 6C). Under these conditions, the BAI plus LB42708-induced accumulation of the sub-G1 phase and PLC-γ1 cleavage were similar in cells transfected with XIAP siRNA as compared to control siRNA-transfected cells (Fig. 6C). Thus, these results suggest that downregulation of XIAP protein is not associated with the BAI plus LB42708- induced apoptosis in Caki cells. It has been reported that Bcl-2 is an anti-apoptotic member of the family and its aberrant expression has been linked to a variety of different cancers, and cancer cell lines (23). Moreover, c-FLIP has been found to act as a survival factor and to be overexpressed in several types of cancers (24). To investigate whether the decreased expression levels of Bcl-2 and c-FLIP (L) are important to induce apoptosis in BAI plus LB42708-co-treated Caki cells, we established Bcl-2 and c-FLIP (L) overexpressing cells. As shown in Fig. 6D, Caki/c-FLIP (L) cells treated with BAI plus LB42708 partially inhibited apoptosis in comparison with Cali/vector cells. Overexpression of Bcl-2 significantly blocked the apoptosis induced by co-treatment with BAI and LB42708. Additionally, PARP cleavage was attenuated by Bcl-2 overexpression (Fig. 6E). Therefore, these results indicate that downregulations of mainly Bcl-2 and partially c-FLIP (L) induced by BAI plus LB42708 play a critical role in the synergistic effect in growth inhibition of Caki cell line.