1-benzyl-2-phenylbenzimidazole (BPB: Figure 1A) was synthesized at the Graduate Institute of Pharmaceutical Chemistry, China Medical University (Taichung, Taiwan), and dissolved in DMSO. Horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG, and rabbit polyclonal antibodies specific for Fas, FADD, cytochrome c, Bcl-2, Bcl-xl, Bax, Bak, Bad, Bid, caspase-8, caspase-3, caspase-9, AIF, Endo G and PARP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA).

The human chondrosarcoma cell line JJ012 was kindly provided by Dr. Sean P Scully (University of Miami School of Medicine, Miami, FL, USA) [21]. The human chondrosarcoma cell line SW1353 was obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in DMEM/α-MEM which were supplemented with 10% fetal bovine serum (FBS) and maintained at 37 °C in a humidified atmosphere of 5% CO₂.

Primary cultures of human chondrocytes were isolated from articular cartilage as previously described [22,23]. The cells were grown in plastic cell culture dishes in 95% air–5% CO₂ in DMEM supplied with 20 mM HEPES, 10% heat-inactivated FBS, 2 mM-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin.

Cell viability was determined with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After treating with BPB for 2 days, cultures were washed with PBS. Then MTT (0.5 mg/mL) were added to each well and the mixture was incubated at 37 °C for 2 h. To dissolve formazan crystals, culture medium was then replaced with an equal volume of DMSO. After the mixture was shaken at room temperature (RT) for 10 min, absorbance of each well was determined at 550 nm using a microplate reader (Bio-Tek, Winooski, VT, USA) [24].

To determine the long-term effects of BPB, cells (1000 per well) were treated with BPB at various concentrations for 3 h. After rinsing with fresh medium, cells were allowed to form colonies for 7 days before being stained with crystal violet (0.4 g/L). After washing three times with ddH₂O, acetic acid was added to a final concentration of 33% (v/v), and the absorbance was measured at 550 nm.

4′-6-diamidino-2-phenylindole (DAPI), a DNA-binding fluorescent dye, was used to determine whether the mechanism of growth inhibition after BPB treatment is through apoptosis. After treatment with BPB for 48 h, the cells were washed three times with PBS, fixed in a 3.7% formaldehyde solution for 10 min, fixed once in 1ml of methanol and then stained with DAPI for 10 min. Results were determined by visual observation of nuclear morphology through fluorescence microscopy.

Apoptosis was assessed by using Annexin V, a protein that binds to phosphatidylserine (PS) residues exposing on the cell surface of apoptotic cells, as previously described [25]. Cells were treated with vehicle or BPB for the indicated times, washed twice with PBS, and resuspended in staining buffer containing 1 μg/mL Propidium iodide (PI) and 0.025 μg/mL Annexin V-FITC. Double-labeling was performed at room temperature for 10 min in the dark, and cells were immediately analyzed by FACScan and the Cellquest program (Becton Dickinson; Lincoln Park, NJ, USA).

Quantitative assessment of apoptotic cells was also assessed by examining the cell cycle. Cells were collected by centrifugation and adjusted to 3 × 10⁶ cells/mL. Pre-chilled ethanol was added to 0.5 mL of cell suspensions and the mixture was incubated at 4 °C for 30 min. Ethanol was then removed by centrifugation, and cellular DNA was stained with 100 μg/mL PI (in PBS containing 0.1% Triton-X 100, and 1 mM EDTA) in the presence of an equal volume of DNase-free RNase (200 μg/mL). After staining, cells were analyzed immediately with a FACScan and Cellquest program. The extent of apoptosis was determined by measuring the DNA content of cells below sub G₁ peak [26].

The mitochondrial membrane potential (ΔΨ_m) was assessed using the fluorometric probe JC-1 (Calbiochem, CA, USA), a positively charged mitochondria-specific fluorophore that indicates depolarization by a fluorescence emission shift from green (525 nm) to red (610 nm) [27]. Briefly, cells were plated in 6-well culture dishes, grown to confluence, and treated with vehicle or BPB. After incubation, cells were stained with JC-1 (5 μg/mL) for 15 min at 37 °C and then analyzed by FACScan using an argon laser (488 nm). Mitochondrial depolarization, which is specifically indicated by a decrease in the red/green fluorescence intensity ratio, was analyzed by Cellquest program.

Cellular lysates were prepared as previously described [28,29]. Proteins were resolved on SDS-PAGE and transferred to Immobilon polyvinyldifluoride membranes. The blots were blocked with 4% BSA for 1 h at room temperature, and then probed with rabbit anti-human antibodies against Fas, FADD, cytochrome c, Bcl-2, Bcl-xl, Bax, Bak, Bad, Bid, caspase-8, caspase-3, caspase-9, AIF, Endo G or PARP (1:1000 dilution) for 1 h at room temperature. After washed three times, the blots were incubated with a peroxidase-conjugated donkey anti-rabbit secondary antibody (1:1000 dilution) for 1 h at room temperature. The signals were visualized by enhanced chemiluminescence with Kodak X-OMAT LS film (Eastman Kodak, Rochester, NY, USA).

The assay is based on the ability of active enzyme to cleave chromophore from enzyme substrate LEHD-pNA (for caspase-9), Ac-IETD-pNA (for caspase-8) or Ac-DEVD-pNA (for caspase-3). Cell lysates were prepared and incubated with anti-caspase-9, -8 and -3. Immunocomplexes were incubated with peptide substrate in assay buffer (100 mM NaCl, 50 mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid [HEPES], 10 mM dithiothreitol, 1 mM EDTA, 10% glycerol, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate [CHAPS], pH 7.4) for 2 h at 37 °C. The release of p-nitroaniline was monitored at 405 nm. Results are the percent change in activity compared to untreated control.

siRNA against human AIF, Endo G and control siRNA were purchased from Santa Cruz Biotechnology. Cells were transfected with siRNA (at a final concentration of 2 μg/mL) using Lipofectamine 2000 (Invitrogen Life, Carlsbad, CA, USA) according to the manufacturer’s instructions.

Male SCID mice (6 weeks old; BALB/cA-nu [nu/nu]) were purchased from the National Science Council Animal Center (Taipei, Taiwan), and maintained in pathogen-free conditions. JJ012 cells (1 × 10⁶ in 200 μL) were injected subcutaneously into the flanks of SCID mice, and tumors were allowed to develop until they reached a size of approximately 100 mm³ (~14 days). The mice were treated with vehicle or with 0.5 or 1.5 mg/kg (i.p.; total volume 200 μL) BPB every day for 21 days (10 mice/group). The volume of implanted tumors in the dorsal side of the mice was determined twice a week with a caliper and the formula V = LW²/2, where V is volume (mm³), L is largest diameter (mm), and W is smallest diameter (mm). All mice were manipulated in accordance with Animal Care and Use Guidelines of the China Medical University (Taichung, Taiwan) under a protocol approved by the Institutional Animal Care and Use Committee, and conducted in accordance with their guidelines (No.99-5-N; date: 2010/7/3).

To investigate the cell apoptotic effect of BPB in tumor tissues in vivo, paraffin-embedded tumor sections were prepared, mounted on slides, deparaffinized in xylene, rehydrated, and washed in distilled water. Protein was removed by digesting the sections with 20 μg/mL proteinase K for 15 min. After washing, labeling was performed by covering the sections with the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) reaction mixture at 37 °C for 60 min. The reaction was blocked in stop/wash buffer for 10 min. The TUNEL labeling was visualized using fluorescence microscopy. TUNEL staining was performed using the Apoptosis Detection kit (Trevigen, Gaithersburg, MD, USA).

The values reported are means ± SEM. Statistical analysis between two samples was performed using Student’s t-test. Statistical comparisons of more than two groups were performed by using one-way analysis of variance (ANOVA) with Bonferroni’s post-hoc test. In all cases, p < 0.05 was considered significant.

To investigate the potential for BPB to induce cell death in human chondrosarcoma cells, we first examined the effect of BPB on cell survival in human chondrosarcoma cells by using the MTT assay. Treatment of cells with BPB induced cell death in chondrosarcoma (JJ012 and SW1353 cells) but not primary chondrocytes (Figure 1B). The IC₅₀ values of BPB were 10.7 and 17.5 μM for JJ012 and SW1353 cells, respectively. The anti-cancer activities of BPB were further assessed with clonogenic assays, which correlated very well with previous in vivo assays of tumorigenicity in nude mice [30]. Treatment of JJ012 cells with BPB reduced colony formation dose-dependently (Figure 1C). We next investigated whether BPB induces cell death through an apoptotic mechanism by DAPI staining, PI and Annexin V/PI assay. Treatment of JJ012 cells with BPB significantly increased the condensation of chromatin by DAPI staining using immunofluorescence microscopy (Figure 1D). In addition, treating cells with BPB induced a concentration- and time- dependent increase in cell death, resulting in an increase in the percentage of cells in the sub G1 phase (Figure 2A–C). Annexin V/PI double-labeling was used to detect PS externalization, a hallmark of the early phase of apoptosis. Compared to vehicle-treated cells, a high proportion of annexin V labeling was detected in cells treated with BPB (Figure 2D,E). On the other hand, BPB also did not increase cell apoptosis in primary chondrocytes by PI and Annexin V staining (Figure 2F,G)

One of the hallmarks of the apoptotic process is the activation of cysteine proteases, which include both initiators and executors of cell death. Treatment with BPB increased expression of cleaved caspase-8 and related caspase activation (Figure 3A,C). BPB also increased the expression of cleaved caspase-8 and related activation (Figure 3A,B). Pretreatment of cells with the specific caspase-3 inhibitor (z-DEVD-FMK) or the specific caspase-9 inhibitor (z-LEHD-FMK) reduced BPB-induced cell death, as shown by PI-staining (Figure 3D). On the other hand, BPB also increased cleaved-PARP (Figure 3A). These data indicate that BPB induced cell death through an apoptosis mechanism

It is well-known that apoptosis can be activated through two main pathways: the intrinsic mitochondria-dependent pathway and the extrinsic death receptor-dependent pathway [31]. Fas, Fas-associated protein with death domain (FADD) and caspase-8 play important roles in death receptor-dependent pathway of apoptosis [32]. We examined whether BPB induced apoptosis by triggering the extrinsic apoptotic pathway. As shown in Figure 4A, BPB induced an increase in Fas and FADD protein levels. Treatment of cells with BPB also increased expression of cleaved caspase-8 and related caspase activation (Figure 4A,B). In addition, pretreatment of cells with the specific caspase-8 inhibitor (z-IED-FMK) reduced BPB-induced cell death in chondrosarcoma (Figure 4C). Therefore, extrinsic death receptor-dependent pathway is involved in BPB-induced cell apoptosis in human chondrosarcoma cells.

To explore whether BPB-induced apoptosis is mediated by mitochondrial dysfunction, we determined the mitochondrial membrane potential with the mitochondria-sensitive dye JC-1 using flow cytometry. As shown in Figure 4D, treatment of JJ012 cells with BPB for 48 h induced the loss of the mitochondrial membrane potential in a dose-dependent manner. On the other hand, treatment of JJ012 cells with BPB induced Bad, Bax and Bak protein levels (Figure 4E). In addition, BPB decreased the expression of Bid, Bcl-XL and Bcl-2, which led to an increase in the proapoptotic/antiapoptotic Bcl-2 ratio (Figure 4E). These data suggest that BPB-increased cell death is mediated by mitochondrial dysfunction in human chondrosarcoma cells.

The release of pro-apoptotic proteins (e.g., cytochrome c, AIF, and Endo G) from the mitochondrial to cytoplasm is a critical event that occurs during mitochondrial-dependent pathway [33]. BPB significantly decreased the mitochondrial cytochrome c, AIF, and Endo G as compared with the control group (Figure 5A). We next explored whether BPB increased the release of the AIF and Endo G from the mitochondria into the nuclei. Immunoblot data showed that although control cells demonstrated a lack of nuclear expression of AIF and Endo G, BPB treatment induced demonstrable translocation of AIF and Endo G to the nuclei (Figure 5A). To further investigation whether BPB induced apoptosis through AIF and Endo G, we then transfected AIF and Endo G siRNA to JJ012 cells for 24 h. Our results indicate that siRNA transfection inhibited the expression of AIF and Endo G, and blocked BPB-induced cell death (Figure 5B). These observations demonstrate that BPB induces apoptosis through AIF and Endo G translocation to the nuclei in human chondrosarcoma cells.

On the basis of the BPB-induced apoptotic effect exhibited in vitro, we decided to detect whether BPB possessed antitumor activities in vivo. Therefore, we established xenografts of JJ012 cells in SCID mice; as tumors reached 100 mm³ in size, the mice were divided into three groups and treated with either vehicle or BPB. BPB induced a dose-dependent inhibition of tumor growth (Figure 6A,B). Moreover, in these two animal models, body weights were not significantly affected by BPB (Figure 6C). On the other hand, an increase of TUNEL-positive cells was observed in tumors of the BPB-treated mice when compared with tumors taken from vehicle-treated mice (Figure 6D). Finally, ex vivo analysis of tumors excised from mice showed significantly increasing Fas, FADD, Bax, Bak, AIF and Endo G expression in the BPB-treated group compared with that in the control group, as shown by Western blot (Figure 6E). Taken together, these results suggest that BPB inhibits tumor growth by inducing JJ012 cell apoptosis in vivo.