Studies On Suppression Of Growth, Induction Of Apoptosis And Inhibition Of Metastasis Of MG-63 Cells By Oridonin

Background:Osteosarcoma is the most common high-grade primary bone tumor and usually occurs in adolescents and children.The treatment of choice for osteosarcoma is neoadjuvant chemotherapy. However,despite substantial improvements in patient survival,no effective therapy is available for metastatic osteosarcoma.The frequent acquisition of drug-resistant phenotypes and the occurrence of second malignancies often associated with chemotherapy remain serious problems.New therapeutic strategies must be evaluated to improve survival.Oridonin is a naturally active substance isolated from Rabdosia rubescens.The medicinal value of oridium has been well recognized due to its anti-inflammation and anti-DNA-damaging effects.In recent years, many studies have shown that oridonin possesses anti-proliferative and apoptotic activities against various cancer cells with little toxicity.In our study,we observe whether Oridonin suppresses proliferation,induces apoptosis and inhibits metastasis of MG-63 cells and to explore its possible mechanisms. PartⅠProliferation-inhibiting effect of Oridonin on MG-63 cellsObjective:To determine proliferation-inhibiting capability of Oridonin on MG-63 cells.Methods:MG-63 cells were treated with different concentrations of Oridonin,then cell viability was analyzed with trypan blue exclusion test,MTT assay and drug concentration-time survival curve test;Cell cycle was tested by flow cytometry.Results:(1)Oridonin effectively inhibited the proliferation of MG-63 cells in a concentration-time dependent way,and the IC50 of MG-63 cells was 13μmol/L.(2)Oridonin increased the percentage of G0/G1 phase and decreased S phase of MG-63 cells.Conclusion:Oridonin can inhibit proliferation of MG-63 cells effectively with a concentration-time dependent manner. PartⅡApoptosis-inducing effect of Oridonin on MG-63 cellsObjective:To ensure whether Oridonin can induce the apoptosis of MG-63 cells.Methods:After MG-63 cells were incubated with different concentrations of Oridonin,(1)morphologic character of apoptosis was evaluated by Hochest33258 staining;(2)the percentage of earlier apoptosis cell was analyzed by flow cytometry;(3)the molecular character of apoptosis was analyzed by Western blot detecting Caspase-8 expression.Results:(1)Hochest33258 staining and flow cytometry detection powerfully testify Oridonin can induce the apoptosis of MG-63 cells in a concentration-dependent way;(2)The apoptosis of MG-63 cells,which was induced by Oridonin,kept company with up-regulation and cleavage of Caspase-8.Conclusion:Oridonin can induce the apoptosis of MG-63 cells with a concentration-dependent manner. PartⅢMolecular mechanisms of the proliferation-inhibiting and apoptosis-inducing effects of Oridonin on MG-63 cellsObjective:(1)To reveal the relation between the proliferationinhibiting effect of Oridonin and the change of MG-63 cells cycle;(2)To partly explore the mechanism of apoptosis-inducing effects of Oridonin on MG-63 cells;(3)To determine the important role Caspase-8 plays in the apoptosis of MG-63 cells induced by Oridonin.Methods:(1)The change of cell cycle was analyzed by flow cytometry;(2)The protein expression of CyclinD1,P21,Survivin,Bcl-2 and Bax in MG-63 cells were detected by Western blot;(3)The telomerase activities were detected with TRAP-PCR-ELISA;(4)After the activation of Caspase-8 was inhibited,the changes of proliferation-inhibiting and apoptosis-inducing effects of Oridonin were detected by Z-IETD-fmk blocking tests.Results:(1)Oridonin increased the percentage of G0/G1 phase and decreased S phase of MG-63 cells;(2)Oridonin down-regulated CyclinD1,Survivin and Bcl-2,and up-regulated P21 and Bax protein in a concentration-dependent way in MG-63 cells;(3)Oridonin downregulated telomerase activities in a concentration-dependent way in MG-63 cells;(4)The apoptosis -inducing effects of Oridonin were partly suppressed after Z-IETD-fmk blocking the Caspase-8 activation of MG-63 cells. Conclusion:Oridonin inhibits proliferation and induces apoptosis of MG-63 cells through regulating cell cycle protein,blocking "checkpoint" of G1/S phase,down-regulating CyclinD1,Survivin and Bcl-2, up-regulated P21 and Bax and activating Caspase-8. PartⅣMetastasis-inhibiting effect of Oridonin on MG-63 cells and its mechanismObjective:To determine metastasis-inhibiting capability of Oridonin on MG-63 cells and to explore its mechanisms.Methods:After MG-63 cells were incubated with different concentrations of Oridonin,(1)the metastasis ability was analyzed with experiment of chemotaxic migration;(2)the mRNA expressions of VEGF and MMP-9 were analyzed by RT-PCR;(3)the expressions of VEGF and MMP-9 were analyzed by ELISA.Results:(1)Oridonin effectively inhibited the metastasis of MG-63 cells in a concentration-time dependent way.(2)Oridonin effectively inhibited the expressions of VEGF and MMP-9 in a concentration-time dependent way.Conclusion:Oridonin can inhibit metastasis of MG-63 cells effectively with a concentration dependent manner and the molecular mechanism may relate to Oridonin down-regulating VEGF and MMP-9 expression.