| Obsjective:Oridonin is a kind of ent-kaurane diterpenoids isolated from the aerial parts of Rabdosia rubeseens,and it has much more potent cytotoxic effects on many kinds of tumor cells. Oridonin are the main source of plants , such as Rabdosia amethystoides, Rabdosia nervosa, Rabdosia jabdsia, Rabdosia downsis and Isodon japonicus. Its molecular formula is C20H28O6 and the relative molecular mass is 364.4. Research has proved that oridonin has the biological effect. It can both limit proliferation of tumor cells and induce the apoptosis of tumor cells.So it has a reputation of "the second Taxol". In the experimental study on the treatment of various types of cancers, discovered that it has some biological effect ,especially to liver cancer, leukemia, lymphoma, and so on . There are not the reports about apoptosis of breast cancer MDA-MB-231 cells induced by the oridonin .In the research,we study the inhibitory effect and molecular mechanism of MDA-MB -231 induced by oridonin. We think it could provide experimental basis for clinical treatment of breast cancer .Methods:1. Cultured human breast cancer MDA-MB-231 cells were randomly divided into the control group (the negative control group), oridonin-treated groups (5, 10,20,40μmol/L) .2. The inhibitory rate and IC50 values were evaluated by MTT assay after treatment with oridonin for 24h, 48h and 72h.3. After being treated with oridonin for 48h, morphological changes of apoptosis were observed with fluorescance microscope.4. After being treated with oridonin for 48h DNA ladder was examined by DNA agarose gel electrophoresis in oridonin -treated groups。5. After being treated with oridonin for 48h, the expression of bcl-xl mRNA ,bid mRNA ,caspase-3 mRNA and heparanase mRNA were detected by semi-quantitive RT-PCR in 5,10, 20 and 40μmol/L oridonin -treated groups.Results:1. oridonin displayed strong proliferation inhibitory effect in a dose-and-time- dependent manner against MDA-MB-231 cells. The inhibitory rates which were treated with 5, 10, 20 and 40μmol/L oridonin for 24h were (10.223±3.176)%,(12.799±2.335)%,(44.924±5.58) and (56.654±2.315)% , respectively. After treatment for 48h was (11.662±4.253)%,(16.367±1.032)%,(52.762±3.390)% and (60.908±3.211)%, respectively.After treatment for 72h were (22.092±5.960)%,(77.992±1.524)%,(91.716±0.458)% and (95.176±0.780)%,respectively. The inhibitory rate was much higher in oridonin groups than that of control group (P<0.001). Besides, after treatment with oridonin for 24, 48 and 72 h, the IC50 value were 30.62 , 25.45 and 7.210μmol/L, respectively.2. There were obvious changes of morphology, biochemistry. Exposure of MDA-MB-231 cells to oridonin induced nuclear shrinkage, chromatin condensation and margination, nuclear fragmentation, apoptotic bodies and DNA ladder on agarose gel electrophoresis, as seen in typical apoptotic cells. No changes were observed in control group.3. Bcl-xl mRNA was down-regulated, While bid mRNA and caspase-3 mRNA was up-regulated. After being treated with 5, 10, 20 and 40μmol/L oridonin for 48h ,the ratios of RT-PCR products of bcl-xl to GAPDH were 0.347±0.016, 0.267±0.019, 0.137±0.045 and 0.073±0.020, respectively. There was significant difference in oridonin groups compared with control group (0.370±0.026). The ratios of RT-PCR products of bid to GAPDH were 0.237±0.044, 0.397±0.026, 0.650±0.050 and 0.870±0.030, respectively. There was significant difference in oridonin groups compared with control group (0.183±0.021). The ratios of RT-PCR products of caspase-3 to GAPDH waere 0.123±0.059, 0.223±0.015, 0.283±0.021and 0.357±0.025,respectively. There was significant difference in oridonin groups compared with control group (0.073±0.015).4.After treated by oridonin for 48 h, the apoptosis ratio induced by oridonin is 75%.5. Heparanase mRNA was down-regulated. After being treated with 5, 10, 20 and 40μmol/L oridonin for 48h ,the ratios of RT-PCR products of heparanase to GAPDH were 0.616±0.032, 0.565±0.019, 0.434±0.021and 0.348±0.028, respectively. There was significant difference in oridonin groups compared with control group (0.654±0.033).6. Oridonin can reduce the action of MMP-2 and MMP-9. After being treated with 5, 10, 20 and 40μmol/L oridonin for 48h ,the product ratios of MMP-2 to control group were 0.920±0.020, 0.790±0.026, 0.610±0.035 and 0.277±0.025,. The product ratios of MMP-9 to control group were 0.917±0.065, 0.797±0.015, 0.483±0.032 and 0.210±0.026, respectively.Conclusion:1. Oridonin has strong proliferation inhibitory effect aginst human breast cancer MDA-MB-231 cells and it can induce the apoptosis of MDA-MB-231 cells .2. Oridonin could down-regulate the expression of gene bcl-xl, and up-regulate the expression of gene bid and caspase-3. It indicated that the effect of oridonin on inducing-apoptosis of MDA-MB-231 cells may be related to the passageway of chondrosome, that bid and bcl-xl may be the regulating-target for oridonin .3. Oridonin could down-regulate the expression of gene heparanase and reduce the action of MMP-2 and MMP-9. |
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