The Effects Of Berbamine Derivative BBD24 On The Growth In Vitro And In Vivo Of Human Osteosarcoma And Corresponding Molecular Mechanisms

Osteosarcoma is one of the most common types of primary bone tumors for children and adolescents with the incidence of 4-5*10-6 and no difference among various ethnic groups. Current common chemotherapeutic agents for osteosarcoma are adoxorubicin (ADM), cisplatin (DDP), methotrexate (MTX) and the recently-introduced ifosfamide (IFO). Chemotherapy improves outcome of patients with osteosarcoma and to some extent regimens of osteosarcoma have been progressing recently. However, the 5-year survival rate is about 60% during last 2 decades. An endless stream of new drugs emerged in the past 20 years. Patients with lung cancer, for example,have been greatly relieved by new agents. However, few novel effective candidates for osteosarcoma can used for osteosarcoma treatment, except that the combination of Gemcitabine and Yew terpene displays anti-tumor activity for patients with metastasis of bone and soft tissue sarcomas with good tolerance. So the major concern in osteosarcoma treatment is to develop novel chemotherapy agents with more potent anti-tumor activity and low toxicity.Recent studies showed that berbamine, a natural product extracted from Berber is plants, inhibited the cell growth of a variety of human tumors such as SMMC7721 (hepatoma), HeLa (cervical cancer) and K562 (chronic myeloid leukemia, CML) and HL-60(acute myeloid leukemia, AML), but little is known about the effects of berbamine and derivatives on osteosarcoma. In this study, we investigated the effect and mechanism of BBD24, a novel berbamine derivative, on osteosarcoma in vivo and in vitro. MTT assay showed that BBD24 remarkably inhibited the growth of cell lines MNNG/HOS and MG63 with time-and dose-dependent manners. Cell cycle analysis showed that treatment of tumor cells with BBD24 resulted in cell arrest at G2/M phase. Interestingly, BBD24 could trigger a variety of cell death pathways including apoptosis, necrosis and autophagy. Subcellular distribution investigation using immunofluorescence assay (IFA) with biotinylated berbamine revealed that berbamine was mainly distributed in cytoplasm at 24 hours, but berbamine was predominantly localizated in nucleus at 48 hours. Studies of tumor-related signaling pathways indicated that berbamine derivative BBD24 activated caspase-dependent pathway, and down-regulated NF-kB, AKT, and ERK pathways. Combined treatments of BBD24 with cisplatin (DDP), a commomly-used chemotherapeutic agent for osteosarcoma, on cell line MNNG/HOS confirmed their synergistic effect.Primary osteosarcoma cells were collected from eight untreated patients and MTT assay showed BBD24 effectively killed these primary cells in a obvious dose-dependent manner with IC50 1.0-2.0μg/ml, meanwhile, PBMCs were also collected from 4 health adults, and MTT assay showed that the IC50 was up to 8.0-10.0μg/ml in general, suggesting that BBD24 selectively inhibit the growth of osteosarcoma cells.In this study, ostersarcoma xenograft model of nude mice (BALB/c-nu/nu) was used to evaluate the anti-tumor activity of BBD24 in vivo.3×106/0.2ml MNNG/HOS cells were injected subcutaneously near the scapular of mice, and osteosarcoma appeared in all experimental animals at 5 days after injection. BBD24 was orally administered 24 hours after being implanted with MNNG/HOS at 10mg/kg bid for 20 days. Experimental results showed that xenografts of MNNG/HOS were significantly inhibited and no obviously toxic or side effects were observed during experiments.Taken together, our study results demonstrate berbamine derivative BBD24 posses a potent antitumor activity against osteosarcoma in vivo and in vitro, suggesting that it might be a potential agent for osteosarcoma, and deserving further investigation.