The Inhibitory Effect And Mechanism Of Melittin On Human Osteosarcoma Cell Line MG63

Background and objectiveOsteosarcoma is one of the most common malignant bone tumor in clinic. The incidence of osteosarcoma ranks first in the primary malignant bone tumor in our country. Chemotherapy plays a very crucial role in the treatment of osteosarcoma. Modern treatment of osteosarcoma mainly adopts the combined therapy based on operation, including operation, chemotherapy before operation and postoperative regular chemotherapy. Effect of chemotherapy in the treatment process should not be replaced, but the side effects of chemotherapy has become a puzzling problem in recent years.This reality constantly drives people to explore new chemotherapeutic agents for the treatment of osteosarcoma. Melittin (MEL, melittin) is the main active component in bee venom and has high biological activity. In recent years, the antitumor effect of melittin has been paid more and more attention. Research shows that, melittin has broad antitumor activity, its antitumor mechanisms include direct killing, immune regulation, cell apoptosis, inhibition of metastasis and angiogenesis. Although many experiments had demonstrated that melittin can induce apoptosis of tumor cells, but the mechanism of its effect on osteosarcoma cells is not clear. This experiment selects the human osteosarcoma cell line MG63as the research object, to observe the effects of melittin on MG63cell and take a further research on its possible mechanism, and provide theoretical and experimental basis for its application in clinic.Experimental materials1.Cells and melittin Human osteosarcoma cell line MG63, human fetal osteoblast cell line Hfobl.19provided by cell bank Chinese Academy of sciences. Melittin provided by the laboratory of traditional Chinese medicine, Changhai Hospital of Second Military Medical University.2.The main reagents and instruments MTT-Huamei biotech company, RTPCR Kit-USA Invitrogen company, Annexin V-FITC/PI apoptosis Kit-KGI Nanjing company, Western blot experimental device.-GE America, Flow cytometry-America BD companyExperimental method1.Cell culture:human osteosarcoma cells MG63and human fetal osteoblasts Hfobl.19grow in DMEM containing10%heat inactivated bovine serum, penicillin(100U/ml), streptomycin(100μg/ml) in the culture bottles in37℃,5%CO2saturated humidity incubator, logarithmic growth phase cells for experiments2.Constructs containing melittin gene Plasmid and transfects experiment cells: Extracted total RNA from melittin peptide, reverse transcription reaction synthesis of cDNA, the primers were designed according to genebank, MEL gene cloning. Connect the MEL gene and the vector. Construct the eukaryotic expression vector, transfect the experimental cell.3.The cells were divided into three groups. Control group:no intervention. Melittin transfection group:to construct the plasmid containing melittin gene which were transfected into human osteosarcoma cell line MG63and the human fetal osteoblastic Hfob1.19cells. Melittin incubation group;use the final concentration (32μg/ml) of melittin to incubate human osteosarcoma MG63cells and human fetal osteoblastic Hfobl.19cells.4.The four methyl thiazolyl tetrazolium (MTT) assay detects the inhibitory effect of melittin on human osteosarcoma MG63cells and human fetal osteoblastic Hfobl.19cells.The logarithmic growth phase cells were made into single cell suspension, adding MTT and DMSO, use the optical density determination of full automatic enzyme immunoassay instrument to calculate the living cell rate. The experiment was repeated3times5.The cell apoptosis was detected by flow cytometry (Annexin V-FITC/PI notation).6.Using Western-blot method to detect the intracellular IRE-a, p-perk, XBP1, EIF2-a, AFT-6, Caspase-12and CHOP protein expression.7.Using RT-PCR to detect XBP1, EIF2-a, TRAF2mRNA level to explore the possible molecular mechanism.ResultsThe human osteosarcoma cell line MG63and human fetal osteoblastic Hfob1.19, after melittin transfection or incubations,a high level of melittin was in cells.MTT showed that human fetal osteoblastic Hfobl.19cells transfected by melittin or incubation, The cell survival rate compared with the control group had no significant difference. While the MG63human osteosarcoma cells transfected or incubated by melittin, the cell activity was significantly lower than that of the control group, the difference was statistically significant. Flow cytometry results showed that human fetal osteoblastic Hfob1.19cells transfected or incubated by melittin, the proportion of apoptotic cells compared with the control group had no significant difference, but MG63human osteosarcoma cells treated by melittin, the proportion of apoptotic cells was significantly higher than that in the control group, the difference was statistically significant. Western-blot display, IRE-a, CHOP proteins in human osteosarcoma MG63cells were significantly increased, compared with the control group, the difference was significant. RT-PCR detect the endoplasmic reticulum stress unfolded protein response downstream proteins XBP1, EIF2-a, TRAF2mRNA,results showed, MG63human osteosarcoma cells treated by melittin,the expression of XBP1mRNA was significantly higher than the control group, the difference was statistically significant. Conclusion1.Melttin can specifically inhibit the activity of MG63cells and induce apoptosis.2.This process may be realized through activating IRE-a signal transduction protein in the endoplasmic reticulum stress reaction.3.CHOP protein is involved, and play an important roleSignificance1. Confirmed that melittin can specifically induce human osteosarcoma MG63cells apoptosis2. Investigated the possible mechanism of apoptosis of human osteosarcoma cell line MG63induced by melittin and provide a possible research direction for the further elucidation of the molecular mechanism.