1-benzyl-2-phenylbenzimidazole (BPB), a benzimidazole derivative, induces cell apoptosis in human chondrosarcoma through intrinsic and extrinsic pathways

Abstract

In this study, we investigated the anticancer effects of a new benzimidazole derivative, 1-benzyl-2-phenyl -benzimidazole (BPB), in human chondrosarcoma cells. BPB-mediated apoptosis was assessed by the MTT assay and flow cytometry analysis. The in vivo efficacy was examined in a JJ012 xenograft model. Here we found that BPB induced apoptosis in human chondrosarcoma cell lines (JJ012 and SW1353) but not in primary chondrocytes. BPB induced upregulation of Bax, Bad and Bak, downregulation of Bcl-2, Bid and Bcl-XL and dysfunction of mitochondria in chondrosarcoma. In addition, BPB also promoted cytosolic releases AIF and Endo G. Furthermore, it triggered extrinsic death receptor-dependent pathway, which was characterized by activating Fas, FADD and caspase-8. Most importantly, animal studies revealed a dramatic 40% reduction in tumor volume after 21 days of treatment. Thus, BPB may be a novel anticancer agent for the treatment of chondrosarcoma.

Figure 1

The effect of 1-benzyl-2-phenylbenzimidazole (BPB) on cell viability and colony formation in human chondrosarcoma cells (A) Chemical structure of BPB; (B) JJ012, SW1353 and chondrocyte cells were incubated with various concentrations of BPB for 48 h, and the cell viability was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay; (C) For the colony-forming assay, the clonogenic assay was performed as described in Materials and Methods. The quantitative data are shown; (D) JJ012 cells were treated with BPB for 48 h, apoptotic cells were determined by 4′-6-diamidino-2-phenylindole (DAPI) staining and fluorescence microscopy. Results are expressed as the means ± SEM of four independent experiments. ^*p < 0.05 as compared with control group.

Figure 2

BPB-induced apoptosis of human chondrosarcoma cells. (A,B,F) JJ012 cells or primary chondrocytes were treated with vehicle or BPB for 48 h, and the percentage of apoptotic cells was analyzed by flow cytometry of Propidium iodide (PI)-stained cells. (C) JJ012 cells were treated with vehicle or BPB (10 μM) for 48 h, and the percentage of apoptotic cells was analyzed by flow cytometry of PI-stained cells (D,E,G) JJ012 cells or primary chondrocytes were treated with vehicle or BPB for 48 h, and the percentage of apoptotic cells was also analyzed by flow cytometric analysis of Annexin V/PI double staining. Results are expressed as the means ± S.E.M. ^*p < 0.05 as compared with control group.

Figure 2

BPB-induced apoptosis of human chondrosarcoma cells. (A,B,F) JJ012 cells or primary chondrocytes were treated with vehicle or BPB for 48 h, and the percentage of apoptotic cells was analyzed by flow cytometry of Propidium iodide (PI)-stained cells. (C) JJ012 cells were treated with vehicle or BPB (10 μM) for 48 h, and the percentage of apoptotic cells was analyzed by flow cytometry of PI-stained cells (D,E,G) JJ012 cells or primary chondrocytes were treated with vehicle or BPB for 48 h, and the percentage of apoptotic cells was also analyzed by flow cytometric analysis of Annexin V/PI double staining. Results are expressed as the means ± S.E.M. ^*p < 0.05 as compared with control group.

Figure 3

BPB induced the activation of caspases in human chondrosarcoma cells. (A) JJ012 cells were incubated with BPB (10 μM) for different time intervals, and the PARP, caspase-3 and caspase-9 expression were examined by Western blot analysis; (B,C) JJ012 cells were incubated with BPB for 24 h, and then caspase-3 and caspase-9 activities were examined by caspase ELISA kit; (D) Cells were pre-treated for 30 min with z-DEVD-FMK (caspase 3 inhibitor) or z-LEHD-FMK (caspase 9 inhibitor), and then followed by stimulation with BPB for 48 h, and the percentage of apoptotic cells was analyzed by flow cytometry of PI-stained cells. Results are expressed as the means ± SEM. ^*p < 0.05 as compared with control group. # p < 0.05 compared with BPB-treated group.

Figure 4

Intrinsic and extrinsic pathways are involved in BPB-induced cell apoptosis in human chondrosarcoma cells. (A) JJ012 cells were incubated with BPB (10 μM) for different time intervals, and Fas, Fas-associated protein with death domain (FADD) and caspase-8 expression were examined by Western blotting; (B) JJ012 cells were incubated with BPB for 24 h, and then caspase-8 activation was examined by caspase-8 ELISA kit; (C) Cells were pretreated for 30 min with z-IED-FMK (caspase-8 inhibitor) followed by stimulation with BPB (10 μM) for 48 h, and the percentage of apoptotic cells was determined by flow cytometric analysis of cell cycle; (D) JJ012 cells were incubated with various concentration of BPB for 48 h, and the mitochondrial membrane potential was examined by flow cytometry; (E) JJ012 cells were incubated with BPB (10 μM) for different time intervals, and Bax, Bak, Bad, Bcl-2 and Bcl-xl expression were examined by Western blotting. Results are expressed as the means ± SEM. ^*p < 0.05 compared with control group. # p < 0.05 compared with BPB-treated group.

Figure 4

Intrinsic and extrinsic pathways are involved in BPB-induced cell apoptosis in human chondrosarcoma cells. (A) JJ012 cells were incubated with BPB (10 μM) for different time intervals, and Fas, Fas-associated protein with death domain (FADD) and caspase-8 expression were examined by Western blotting; (B) JJ012 cells were incubated with BPB for 24 h, and then caspase-8 activation was examined by caspase-8 ELISA kit; (C) Cells were pretreated for 30 min with z-IED-FMK (caspase-8 inhibitor) followed by stimulation with BPB (10 μM) for 48 h, and the percentage of apoptotic cells was determined by flow cytometric analysis of cell cycle; (D) JJ012 cells were incubated with various concentration of BPB for 48 h, and the mitochondrial membrane potential was examined by flow cytometry; (E) JJ012 cells were incubated with BPB (10 μM) for different time intervals, and Bax, Bak, Bad, Bcl-2 and Bcl-xl expression were examined by Western blotting. Results are expressed as the means ± SEM. ^*p < 0.05 compared with control group. # p < 0.05 compared with BPB-treated group.

Figure 5

Apoptosis-inducing factor (AIF) and endonuclease G (Endo G) are involved in BPB-induced cell death. (A) JJ012 cells were incubated with BPB (10 μM) for different time intervals, and the levels of cytochrome c, AIF and Endo G in mitochondria and nuclei were examined by Western blot analysis. (B) Cells were transfected with AIF, Endo G or control siRNA for 24 h, and the AIF and Endo G expression were examined by Western blot analysis (upper panel). Cells were transfected with AIF, Endo G or control siRNA for 24 h, and then followed by stimulation with BPB (10 μM) for 48 h, and the percentage of apoptotic cells was analyzed by flow cytometry of PI-stained cells (lower panel). Results are expressed as the means ± SEM. ^*p < 0.05 compared with control; # p < 0.05 compared with BPB-treated group.

Figure 6

Effects of BPB on tumorigenicity and in vivo growth of xenografts in SCID mice. (A,B) The chondrosacoma cells (1 × 10⁶) were injected subcutaneously into the 5-week-old SCID mice. After the tumors reached 100 mm³ in size, the animals were treated with an intraperitoneal injection of BPB (1 or 2 mg/kg) or vehicle daily for 3 weeks. The mean tumor volume was measured at the indicated number of days after implantation (n = 8–10). (C) Mean body weight was measured at the indicated number of days after implantation. (D) Transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assay in tissues from chondrosarcoma cells xenografts. BPB-treated tumors show marked green staining of fragmented nuclei, indicative of apoptosis. (E) The expression of Fas, FADD, Bax, Bak, AIF and Endo G were evaluated by Western blot analysis in tumor with and without treatment. (F) The proposed signaling pathways of BPB-induced cell apoptosis in human chondrosarcoma cells.