| Background:Osteosarcoma (OS) is the most common bone tumor with a high malignant degree in osseous tissues. The occurrence of OS tends to be originated from the terminal differentiation of osteoid cells or the process of the Bone Marrow Stromal cells(BMSCs) differentiating into the osteoid cells. At the same time, the early-stage migration and local invasion ability were also developed prominently, which could cause death. Therefore, in-depth study of signaling pathways, including apoptosis, differentiation, migration and invasion process of osteosarcoma cells, and exploring the molecular targets which can effectively reverse the occurrence and development of cancer cells are crucial, meanwhile, the theory above can provide valuable theoretical and experimental evidence for osteosarcoma treatment and inhibition of tumor metastasis. Studies have shown that peroxisome proliferator-activated receptor, PPARy, plays an important role in regulating apoptosis and differentiation of a variety of tumor cells. In recent years, studies have indicated that phytoestrogens have the function of prevention and suppression of a variety of tumors, such as Breast cancer, prostate cancer, endometrial cancer, ovarian cancer, colon cancer etc. Daidzin(DAI), as a phytoestrogen ligand of PPARy agonist, is a kind of flavonoids extracted from the beans and has extensive pharmacological effects. DAI can induce tumor cell apoptosis and differentiation, and inhibit tumor migration and angiogenic gene expression in tumor, but the mechanism is not yet fully elucidated. Therefore, investigating the effect of DAI on apoptosis, differentiation and biological traits in human osteosarcoma cells and revealing its mechanism of action will provide new ideas and strategies for the clinical treatment of osteosarcoma.Objective:This study used human osteosarcoma cell line MG63as the objective to investigate the drug effect on cell proliferation and apoptosis when cells were treated with daidzin, a.k.a DAI, in different concentration. Meanwhile, the inhibition of cell’s biological characters, like migration and invasion ability, through drug induction was also discussed, and the induced-differentiation and its possible molecular mechanisms, induced by low concentration DAI, on MG63cell lines were detected at the same time. Thus, our research aimed to find more proper and low toxic solution and also provide experimental evidence for clinical treatment.Methods:1. The MG63cell proliferation was detected by MTT method within different time duration when the different concentrations of DAI treatment (OμM,1μM,10μM,20μM,40μM,80μM and160μM) were delivered.2. The AV-FITC/PI double fluorescent marker flow cytometry was used for the detection of early-stage cell apoptosis when DAI(drug concentration was OμM,20μM and80μM respectively) treated cell for24h and48h separately.3. The MG63cell apoptosis caused by DAI were investigated by Tunel method.4. The apoptosis and differentiation related gene expression were detected by real-time fluorescent quantitative PCR when cell treated with different DAI concentrations(0μM,20μM,40μM and80μM). 5. The changes of ALP expression activity of MG63cells which treated with different DAI concentration were assayed by the benzene disodium phosphate method.6. The changes of type I collagen content of MG63cells which treated with different DAI concentration were assayed by enzyme-linked immunoassay, a.k.a ELISA.7. Alizarin red staining method was used for the observation of calcification of MG63cells when DAI intervention was achieved.8. Scarification test was used for observation of MG63cell migration when DAI intervention was achieved.9. The cell invasion ability of MG63cells was detected by Transwell invasion assay when DAI intervention was delivered.10. Immunocytochemistry method was used for the detection of the cell invasion and apoptosis related gene expression when DAI intervention was occurred.Results:1) The results of MTT method showed that, the MG63cell proliferation rate could be inhibited by DAI, and with the dose concentration increased and duration prolonged, the inhibition efficiency which had a statistical significance was also increased dramatically (P<0.01).2) The results of the AV-FITC/PI double fluorescent marker flow cytometry indicated that, with the different concentrations of DAI delivery of MG63cells for24h and48h occurred, the apoptosis rate of the entire dosing group was increased (P<0.01). The apoptosis rate of the80μM dosing group was higher than the control and80μM dosing group, and the48h apoptosis rate was significant higher compared with24h when DAI concentration was80μM.3) The results of Tunel assay showed that, the apoptosis dying results of each dosing groups were positive and also higher than control group (P<0.01) when the DAI intervention of MG63was induced.4) The result of qPCR showed that, with the DAI treatment of MG63cells for48h occurred, the Runx2expression of the group with daidzin were all lower than the control group (P<0.01). However, the PPARy expression of the experiment group was all higher than the control group, meanwhile, with the concentration of the dosing group was increased, the relative expression rate of PPARy was soared (P<0.01).5) The results of ALP assay indicated that, when low concentration DAI was delivered for6d,9d,12d respectively, the activity of ALP within these three time point was increased entirely, and within the region of the drug concentration, the activity of ALP was shown a positive correlation with the DAI intervention concentration, and there was a peak value of ALP activity was observed at9d (P<0.01).6) The results of Type I Collagen assay said that, with low concentration DAI intervened MG63cells, the relative expression level of Type I Collagen was higher than the control group (P<0.01), and, when the comparison happened between each two groups at the same time point, the other groups all shown a great difference except the comparison of the20μM and40μM dosing group in Id and24d (P<0.05). Besides, the relative expression level of Type I Collagen could be increased along with the drug duration was prolonged.7) The results of alizarin red staining method showed that, the number of calcification nodules of the dosing group was significant higher that the control group after low concentration DAI, including lOμM,20μM,40μM, intervention was delivered for20d.8) The results of Scarification test told that, the migration ability of MG63cells compared with the control group was decreased after the DAI treated the cell for12h,24h and36h separately, and the migration inhibition rate became more and more significant along with the drug concentration increased continuously, in addition, the most obvious effect was observed in80μM group.9) The results of Transwell invasion assay showed that, the invasion cell number decreased dramatically compare with the control group after the DAI intervened. Moreover, given the drug group, the more drug concentration, the lesser invasion cell number it was (P<0.01).10) The results of Immunocytochemistry method indicated that, the expression level of PTEN within MG63cells was higher than the control group after the DAI intervention occurred, but, the expression level of the apoptosis inhibitory protein Survivin and the metal matrix protein MMP-9remained the opposite situation (P<0.01).Conclusions:1. The phytoestrogen DAI is able to both inhibit the proliferation of human osteosarcoma MG63cell line and induce cell apoptosis in a concentration-and time-dependent manner, and the concentration of80μM is showed a great efficiency.2. The DAI is able to activate PPARy signaling pathway, which can generate triple effects, including down-regulate apoptosis inhibitory protein Survivin, up-regulate anti-oncogene PTEN and inhibit the transcription activity of the osteogenic transcription factor Runx2, in order to both down-regulate invasion-related ECM reshape related gene protein level, a.k.a MMP-9, and suppress the invasion and migration ability of MG63cells.3. Low concentration DAI delivered on MG63cells is able to induce the ability elevation of the osteogenic specific marker ALP and the content increment of the Type I Collagen, also, the calcification deposition is observed along with such process. |
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