| Background:Osteosarcoma is a common primary malignant bone tumor that occurs mainly in children and adolescents. Distal metastasis is of frequent occurrence and it has a poor prognosis. Nowadays, surgery combined with chemotherapy is the main treatment for osteosarcoma. But it is a pity that surgery can not prevent metastases. Therefore, chemotherapy plays a very important role in the treatment of osteosarcoma. From the late of 1970s, neoadjuvant chemotherapy has been put forward and widely used in clinic. Because of this scheme, the prognosis of non-matastatic osteosarcoma has made great progress, which the 5-year survival rate is now about 70%. But recent 30 years, the main chemotherapy drugs for osteosarcoma are still cisplatin, doxorubicin and high dose of methotrexate. Though these agents have good efficiency, they often have extensive side effects, such as ototoxicity, nephrotoxicity and myelosuppression. In addition, multidrug resistant cases are common, which is the main cause of treatment failure of chemotherapy. Thus, there is an urgent need for some new and effective chemotherapy agents that may improve the clinical outcome of osteosarcoma patients. Nitidine chloride (NC) is a kind of benzophenanthrene alkaloids isolated from the dryer root of Zanthoxylum nitidum(Roxb.)DC. Nitidine as a traditional Chinese medicinal plant, is widely distributed in southwestern part of China. The extract of nitidine is frequently used as anti-inflammatory, easing pain and releasing from hyperkinesias, etc. Nitidine chloride is the main active component of nitidine. Recent years, it has been reported that nitidine chloride also has some antitumor activity. In vitro, nitidine chloride could evidently inhibit the growth of human lung cancer cells, human tongue cancer cells and human oral cancer cells, etc. But for now, little is known about the effect of nitidine chloride on human osteosarcoma cells. In this work, we used human osteosarcoma cell line MG-63 as the research object, to investigate the effect of nitidine chloride on proliferative inhibition of MG-63 cells and explore the related molecular mechanisms.Objectives:To investigate the anti-proliferative activity and apoptosis-inducing effect of nitidine chloride on human osteosarcoma cell line MG-63, and further explore the related molecular mechanisms.Methods:MG-63 cells were treated with indicated nitidine chloride (0,0.25,0.5,1,2,4 and 8μmol/L) for 24,48 and 72 h, and the cell viability rate was assessed using the MTT assay. In addition, the half-maximal inhibitory concentration (IC50) of nitidine chloride was calmulated, respectively. The morphological changes of cells treated with nitidine chloride were observed using ordinary optical microscope, fluorescence and electron microscope. Flow cytometry was performed not only to analyze the apoptotic rate of the cells, but also to detect the cell cycle distribution. The protein expression levels of caspase-3, caspase-9, Bax, Bcl-2, cdc2, cdc25c, cyclin B1 and survivin in MG-63 cells were detected by Western blotting.Statistical analysis was done using SPSS software (version 16.0 for windows). The data was expressed as mean±standard deviation. The statistical significance between the groups was evaluated by Student's t test. Values of P<0.05 were considered to be statistically significant. Values of P＜0.01 was considered as dramatically statistic significant.Results:From the data of MTT assay, nitidine chloride could remarkably inhibit the proliferation of MG-63 cells in a dose-and time-dependent manner. The differences between nitidine chloride treated groups (beyond 0.5μmol/L for 48 and 72 h) and control group (0μmol/L) had dramatically statistic significance (P＜0.01). The IC50 values of nitidine chloride on MG-63 cells at 24,48 and 72 h were 8.29,2.80 and 1.34μmol/L, respectively.Based on the findings of the light microscopy, the density of MG-63 cells treated with nitidine chloride decreased in a dose dependent manner and plenty of cells were detached from the plate. Distinct apoptotic changes of the cells after nitidine chloride exposure were revealed through fluorescence and electron microscopy. For example, stained with Hoechst 33258, cells in control group showed normal and healthy shape, while cells in treated group displayed condensed and fragmented nuclei.In ultrastructure, the microvilli on the cells treated with nitidine chloride were no longer detectable and the cytoplasmic vacuoles were appear.The data of flow cytometry indicated that nitidine chloride induced the apoptosis of MG-63 cells in a dose dependent manner. The early apoptotic rates of MG-63 cells treated with 0,0.5,1 and 2μmol/L nitidine chloride for 48 h were 2.82%,8.37%, 23.75% and 49.96%, respectively. Nitidine chloride also influenced the cell cycle distribution of MG-63 cells. It could increase the proportion of MG-63 cells in G2/M phase, while decreased the proportion of cells in S phase. Exposed to high concentrations of nitidine chloride, most of MG-63 cells were arrested at G2/M phase.After incubation with indicated nitidine chloride for 48 h, as shown by Western blotting, the expression levels of Bax in MG-63 cells were up-regulated, while the expression levels of Bcl-2, cdc2, cdc25c, cyclin B1 and survivin were down-regulated. In addition, nitidine chloride also made caspase-9 and caspase-3 activated in a dose-dependent manner.Conclusions:Nitidine chloride could remarkly inhibit the proliferation of human osteosarcoma cell line MG-63. The anti-osteosarcoma effect might be related with induction apoptisis of MG-63 cells and G2/M cell cycle arrest. As a new antitumor agent, nitidine chloride may have a good potential for the treatment of osteaosarcoma. Further research work should be needed to investigate the activity of nitidine chloride on osteosarcoma.
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