A novel synthetic derivative of the natural product berbamine inhibits cell viability and induces apoptosis of human osteosarcoma cells, associated with activation of JNK/AP-1 signaling

Abstract

Osteosarcoma is the most common primary bone tumor in children and adolescents. There is a critical need to find more potent drugs for patients with metastatic or recurrent disease. Berbamine (BBM) is a natural compound derived from the Berberis amurensis plants. BBM and its derivatives have been shown to have antitumor effects in several cancers. Here, we report that a novel synthetic berbamine derivative, BBMD3, inhibits cell viability and induces apoptosis of G292, KHOS, and MG-63 human osteosarcoma cells. Induction of apoptosis in these tumor cells depends on activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP). Since pan-caspase inhibitor (Z-VAD-FMK) and caspase-9 inhibitor (Z-LEHD-FMK) could block the cleavage of PARP, the apoptosis induced by BBMD3 is through intrinsic signaling pathway. BBMD3 increased phosphorylation of c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK), resulting in increase of phosphorylated c-Jun and total c-Fos, the major components of transcriptional factor AP-1. JNK inhibitor could partially suppress antitumor effect of BBMD3 on osteosarcoma cells. BBMD3 increased the production of reactive oxygen species (ROS) and ROS scavenger, N-acetylcysteine (NAC), could block the phosphorylation of JNK and c-Jun induced by BBMD3. BBMD3 increased the expression of the pro-apototic gene Bad, associated with apoptosis induction. Finally, BBMD3 also decreased the expression of cyclin D1 and D2, the positive cell cycle regulators, which is correlated with growth inhibition in osteosarcoma cells. Collectively, these findings indicate that BBMD3 is a potentially promising drug for the treatment of human osteosarcoma.

Keywords: AP-1; JNK; apoptosis; berbamine derivative; natural product; osteosarcoma.

Figure 1. BBMD3 inhibited cell viability in KHOS, G292, and MG-63 human osteosarcoma cells. (A) Structures of berbamine (BBM) and berbamine derivative 3 (BBMD3). (B) KHOS, G292, and MG-63 cells were treated with 0, 1, 3, 5, 8, and 10 µM BBMD3 for 24 h and 48 h and viability was determined by MTS assay as described in Methods. Each experiment was performed in triplicate. Each bar graph represents the mean, and the error bars represent ± SD.

Figure 2. BBMD3 induces apoptosis of KHOS, G292 and MG-63 human osteosarcoma cells. (A) Apoptotic cells were analyzed by Annexin V-FITC and PI staining and flow cytometry. KHOS, G292, and MG-63 cells were treated with BBMD3 (0, 1, 3, 5, 10 µM) for 24 h and 48 h, respectively. Apoptotic cells represented Annexin V-FITC positive (early stage of apoptosis) or PI and Annexin V-FITC double-positive (late stage of apoptosis) cells. Each experiment was performed in triplicate or duplicate and repeated twice independently. Each bar graph represents the mean, and the error bars represent ± SD (B) Cleavages of caspase-3 and PARP were increased by 24 h BBMD3 treatment through immunoblotting analyses. (C) Caspase inhibitor, Z-VAD-FMK, blocked the effects of BBMD3 on increasing expression of cleaved caspase-3 and PARP. β-actin works as a loading control.

Figure 3. Caspase-9 inhibitor blocked the activation of caspase-3 induced by BBMD3 and effects of BBMD3 on regulatory proteins of apoptosis in osteosarcoma cells. (A) KHOS cells were first treated with 20 µM csapase-8 inhibitor (Z-IEHD-FMK) or caspase-9 inhibitor (Z-LEHD-FMK) for 30 min, and then 5 µM BBMD3 was added to cells for another 24 h. Expression of cleaved caspase-3 and PARP was analyzed by immunoblotting assays. (B) BBMD3 increased cleaved caspase-9 and in KHOS and G292 cells. (C) BBMD3 increased release of cytochrome c to cytosol in KHOS and G292 cells. Cells were treated with 5 µM BBMD3 for 24 h and cytosol was isolated. Cytochrome c was detected by immunoblotting assays. Effects of BBMD3 on anti-apoptotic proteins (D) and pro-apoptotic proteins (E) in KHOS, G292, and MG-63 cells were determined by immunoblotting assays after 24 h treatment.

Figure 4. Effects of BBMD3 on cell growth and regulatory proteins of cell cycle progression in osteosarcoma cells. (A and B) Rates of KHOS and G292 cell growth were determined by counting live cells after 24 h BBMD3 treatment with Trypan blue staining. (C) Effects of BBMD3 on cyclin D1, D2 and E, positive regulators for cell cycle, and p21Cip1 and p27Kip1, negative regulator for cell cycle in KHOS, G292, and MG-63 cells after 24 h BBMD3 treatment.

Figure 5. BBMD3 increased phosphorylation of JNK (1/2), c-Jun, and total protein of c-Fos in human osteosarcoma cells by immunoblotting assays. (A) Effects of BBMD3 on expression of total and phosphorylated JAK2 and STAT3 in KHOS, G292, and MG-63 cells after 24 h BBMD3 treatment. (B) Phosphorylated JNK (1/2), c-Jun, and total c-Fos were increased by 24 h BBMD3 treatment in KHOS, G292, and MG-63 cells. (C) KHOS and G292 cells were treated with 5 µM BBMD3 for 0, 1, 3, and 6 h. Then, expression of phosphorylated JNK (1/2) and c-Jun, and total JNK, c-Jun, and c-Fos were determined by immunoblotting analyses.

Figure 6. JNK inhibitor and ROS scavenger could block the effects of BBMD3 on human osteosarcoma cells. (A) G292 cells were first treated with 1 µM JNK inhibitor II for 30 min. Then, 2.5 and 10 µM BBMD3 were added to cells for 24 h and viability of these cells was determined. (B) Treatment of G292 cells with NAC, a ROS scavenger, blocked the increase of phosphorylated JNK (1/2), phosphorylated c-Jun and total c-Fos induced by 24 h BBMD3 treatment. (C) Effects of BBMD3 on production of ROS after 24 h BBMD3 treatment in MG-63 and G292 cells. Values of mean fluorescence intensity in ten thousand cells were presented. *P < 0.05; **P < 0.01.