| Background:Osteosarcoma is a kind of highly malignant tumor. Based on its biological features of different osteosarcoma cell types, the origin of osteosarcoma is assumed to come from the processes of bone marrow stromal stem cell differentiation into osteoblast or that of osteoblast terminal differentiation. Terminal differentiation of osteoblast is a precisely coordinated process which requires the involvement of osteoblast specific transcription factor Runx2(core binding factor a, cbfa) and Rb protein. Hypophosphorylated Rb prevents cell entering S phase by inhibiting synthesis of S phase associated proteins while Runx2promotes osteoblast differentiation by binding to osteoblast specific cis acting elements which increases transcription of osteoblast differentiation associated genes. The main behavior of malignant tumors is its deregulated cell cycle presented by limitless proliferation and this process is mediated by CDK (cyclin-dependent kinase) and Cyclin. In osteosarcoma, CDK-Cyclin complex are either over-expressed or derailed of its normal function and CDKs such as CDK4can mediate the ubiquitin dependent degradation of Runx2and Rb protein phosphorylation simultaneously. So inhibition of CDK activity and restoration of cell differentiation key factor such as Runx2may orient it to its terminal differentiation and reverse the malignant phenotype of osteoblast tumor cells. Tumor apoptosis might be achieved in this way and this might be beneficial for osteoblast tumor treatment.Purpose:In this project, osteosarcoma cell line MG63was chosen and treated with varied concentrations of flavopiridol. The effects of flavopiridol on MG63cell’s proliferation and apoptosis were studied and the mechanism of its ability in induction of MG63cell differentiation was explored.Methods:1. MTT assay was used to study flavopiridol’s effect on MG63cell proliferation.2. Cell cycle changes were recorded by FCM with PI staining after FP treatment.3. Annexin V/PI double staining was used to detect early apoptotic activity.4. RT-PCR was used to detect the changes of cell apoptosis related genes expression after FP intervention.5. Alkaline phosphatase (ALP) activity was determined after FP treatment.6. Type I collagen expression after FP intervention was assayed with commercial ELISAkit.7. Protein amount changes of CDK2and Runx2after FP intervention were determined by Western blottingResults:1. MTT assay showed that FP exhibited apparent proliferation inhibition effect in the range of200-2000nM and a linearity of strengthened inhibition was observed from200nM to400nM. Inhibition was also observed with a upward trend following prolonged time interval and the corresponding IC50s for each time interval(24h,48h,72h) were580nM,390nM,260nM respectively.2. PI staining results showed that FP at200nM and400nM arrested cells at G2phase after24hour’s treatment as exhibited by increased G2phase cells and reduced S phase cells compared with control group.(P<0.01).3. Apoptosis rate at24hours after FP treatment for control group,200nM and400nM were:0.96±0.08%,17.29±1.15%,24.39±2.74%respectively. One-way ANOVA analysis showed significance among the three groups and multiple comparison indicates concentration dependent apoptosis effect as400nM induced increased apoptosis activity. Apoptosis rate at48hours after FP treatment for control group,200nM and400nM were0.55±0.10%,24.91±5.09%and20.21±1.96%respectively. One-way ANOVA analysis showed significance between control and treated groups but not the200nM and400nM groups.4. ALP activity test shows the ALP activity of all experimental group including control group was rising gently during1day to12days and the peak value appeared at12d then the activity start to fall.The statitics analysis shows the ALP activity of all the flavopiridol groups including100nM,200nM and300nM group was higher that control group (P<0.05),300nM group was higher than100nM and200nM group (P<0.05).5. Type I collagen expression was increased following FP treatment at24days and300nM FP treated group showed highest expression level(P<0.05)..6. Gray scan was performed to the images of RT-PCR assay.The result shows the concentration of2000nM flavopiridol downregulated the mRNA expression of CDK2(P<0.01), P27KIP1(P<0.01), P21WAF(P<0.02), Survivin(P<0.05) and Rb (P<0.05) obviously in MG63cells at96h, meanwhile downregulated the CDK4mRNA expression gently(P<0.05).The concentration of300nM flavopiridol upregulated the mRNA expression of Runx2(P<0.01) and OCN(P<0.05).7. Western blotting results shows100nM,200nM and300nM flavopiridol downregulated the CDK2protein level at10days (P<0.02),and upregulated the level of Runx2protein(P<0.01).Conclusions:1. Flavopiridol inhibit MG63cells proliferation significantly with concentration and time depend effect.Flavopiridol induce MG63cells G2phase arrest, and400nM flavopiridol was most significant and induce MG63cells apoptosis.2. Flavopiridol downregulated the expression level of cell cycle related gene like CDK2, CDK4, P27KIP1, P21WAF, Survivin and Rb in mRNA level as well as the protein expression level of CDK2.3. FP at low concentrations (100nM～300nM) induced increased activity of ALP and increased protein expression of type I collagen, OCN and Runx2as well as increased Runx2expression in mRNA level. |
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