Study The Effects Of IAA/HRP On The Cells Of MG-63

BackgroundOsteosarcoma is a common malignancy in clinical accounts for about20%of primary bone tumors. Because of the high degree of malignancy of osteosarcoma, the rapid rate of progress and the characteristics of vulnerable to relapse and metastasis, its Treatment is very poor. Although Osteosarcoma of the jaw accounted for5%of the oral and maxillofacial malignancies, with the special physiological and anatomical structure, the patient’s daily life will be difficulty after the surgical removal of the jaw. In recent years, with the development of the basic and clinical medical, especially the application of preoperative chemotherapy,5-year survival increased to60%to70%. But there are still a majority of patients prone to metastasis and recurrence. Therefore, to explore the pathogenesis of osteosarcoma, and to find effective, low toxicity of the treatment has become one of the hotspots of the studying about osteosarcoma present.Plant hormone is a class of organic substances produced by the plant’s own metabolism. It regulates the division and elongation of plant cell, the differentiation of organization and organ, the flowering and strong, the mature and aging of plant itself and the dormancy and germination.Nowadays, plant hormones have been used for the anti-tumor therapy, and has made some efficacy.Indole acetic acid (IAA) is also known as auxin.As a kind of plant hormone,it plays an important role in promoting plant growth,promoting formation of callus and inducting the rooting growth.Horseradish peroxidase(HRP) is a kind of plant peroxidase. It can be oxidized to a range of substrates, such as indole acetic acid. IAA have a higher tolerance in animals, and is less susceptible to enzymatic oxidation of the animal.So we choose HRP to oxidize IAA, and then IAA will produce toxic metabolites to induced tumor cell apoptosis. IAA and HRP will not produce any toxic effects alone.Because of of the IAA/HRP have different sensitivity in different cells, in this study, we use IAA/HRP to interfere the MG-63cells, observe the cell growth conditions, and explore the experimental and theoretical basis for using the plant hormone to treat the osteosarcoma.Aim:This study aims at the effects of IAA combined with HRP on human osteosarcoma MG-63cells through observing the inhibition of cell growth, the change of cell shape, the induction of cell apoptosis, the level of intracellular reactive oxygen,the expression of oncogene and the related gene of apoptosis.At the same time, through observating the role of antioxidant of ascorbic acid to prove the inhibition of cell proliferation on MG-63cells from the opposite side,so as to provide the experimental and theoretical basis for application at osteosarcoma treatment in clinical of IAA/HRP.Materials and methods:1. MG-63cell line was provided by Shanghai Cell Bank of Chinese Academy of Sciences.2. IAA and HRP were purchased from Sigama Company. Ascorbic acid were purchased from Sigama Company.3. The changes of number and morphology of the cells which were treated by IAA/HRP were observed under light microscope.4. The dose-and time-effect relationship of IAA/HRP role in MG-63cells was explored by CCK-8assay. Test groups:1) control group added the complete medium;2) Experimental groups added the medium which is contained HRP of1.2mg/L and 4different concentrations of IAA of25,50,100,200μmol/L. After24h,48h,72h and96h, the OD values of each group were detected. Each group was calculated cell proliferation inhibition rate. The best drug concentration and the best time of the inhibition of MG-63cells were cleared.5. With different concentrations of ascorbic acid (25,50,100,200μmol/L), to prevent this inhibitory effect of IAA/HRP. The OD values of each group were detected. The better concentration of ascorbic acid to inhibit the role of the IAA/HRP will be analyzed.6. The cells were examined by flow cytometry.The ROS content is detected by the DCFH-DA staining and the cell apoptosis is measured using Annexin V-PI kit.7. The expression of the different groups of MG-63cells PTEN protein will be detected by immunocytochemistry.8. PCR analysis is performed to find out the Caspase-3and Bcl-2mRNA expression which are responsive when MG63are treated with IAA/HRP and ascorbic acid.Results:1. Cell number and morphological changes:1) Control group:cells were fusiform and adherent well, nucleus were round, and cytoplasm were translucent;2) Experimental groups:the number of cells were fewer, suspended cells increased, cell morphology were slightly obtuse, cell nucleus become bigger, particles increased in the cytoplasm, and cytoplasm color deepened. With the increase of intervention time, the shedding of cell debris increased.2. IAA/HRP inhibit the proliferation of MG-63cells:MG-63cells proliferation was significantly increased with the increase of IAA concentration. In addition to the25μmol/L test group, other groups were significant differences (P<0.05). The longer duration of action, the more obviously did the proliferative capacity decrease. This indicates that IAA/HRP on the inhibitory effect of MG-63cells have a time-and concentration-dependent manner. The better concentration of IAA is100μmol/L, and the suitable time is48h. In addition, the application of ascorbic acid that causes cells to growth inhibition rate was significantly lower. This proves that ascorbic acid can be an effective antagonist of IAA/HRP role inhibiting the proliferation of MG-63cells.3. The content of intracellular ROS and apoptosis:After100μmol/LIAA and1.2mg/LHRP role in MG-63cells, the intracellular ROS levels were significantly increased, and the apoptosis intensified.4. Immunocytochemical analysis of PTEN protein expression:PTEN expression of the Experimental group was significantly higher than the control group. But ascorbic acid can effectively prevent this process.5. PCR detection of the expression of apoptosis-related genes:IAA/HRP can improve the content of caspase-3mRNA, and reduce the Bcl-2mRNA in MG-63cells.Conclusion:1. When MG-63cells were intervened by IAA/HRP, the cells growth will be inhibited.2. IAA/HRP can increase the intracellular ROS levels, and induce the apoptosis.3. The increasing of the tumor suppressor gene PTEN protein in MG-63cells may be one of the mechanisms of that the cells inhibited by IAA/HRP.4. The mechanism of IAA/HRP-induced MG-63cells apoptosis may be related to the expression of the pro-apoptotic genes Caspase-3and the antiapoptotic gene Bcl-2.