Oridonin Induced BxPC-3Cells Into Apoptosis And Mediated Caspase Signal Pathway

Objective:To observe oridonin effect of proliferation, morphological features, cell cycles and apoptosis, and expressions of Caspase genes in BxPC-3cells, investigated relationship between oridonin-induced apoptosis and Caspase signal pathway, and found out the regulatory molecules in the signal pathways on cell proliferation or apoptosis, and explored the inhibitory mechanism of oridonin on pancreatic tumor, we compare the difference of genes expression between treatment with and without oridonin in pancreatic cancer BxPC-3cells, which provided oridonin not only possible anti-tumor accesses but also favourable clinical data.Method:1、The proliferation inhibition efforts of Oridonin were tested using (3,4,5-dimethylthliazol-2,5-dipenyltetrazolium bromid, MTT) MTT array in BxPC-3cells, we obtained data of the lowest concentration, the best concentration and timepoint for BxPC-3cells.2、BxPC-3cells were digested with trypsin, washed with cooled PBS and fixed with ethanol, then stained with PI20min without sunshine at room temperature for cells cycle using flow cytometry, after treatment with0,8and32μg/ml oridonin respectively at37℃,5%CO2,36h.3、BxPC-3cells treated with0,8and32μg/ml oridonin respectively at37℃,5%CO2,36h. morphological varieties were observed by Hoechst33258staining.4、BxPC-3cells were collected and stained with Annexin V and PI20min at room temperature away from light for cells apoptosis using flow cytometry after treatment with0,8and32μg/ml oridonin at37℃,5%CO2,36h.5、BxPC-3cells total protein were extracted using RIPA Lysis Buffer, the expressions of yH2AX, p53, CDK1(Cdc2), Caspase3,7,8,9and PARP were analyzed after treatment with0,8and32μg/ml oridonin for12h,24h and36h respectively using Western Blot. 6、Total RNA were extracted using Trizol after treated by Oug/ml and32ug/ml oridonin for36hours at37℃,5%CO2in BxPC-3cells, Agilent Quick Amp Labeling Kit was used for sample labeling. Hybridization was performed in Agilent’s SureHyb Hybridization Chambers. Slides were scanned with the Agilent DNA microarray Scanner. The differentially expressed genes were screened after data analysis.Result:1、MTT array showed that oridonin inhibited the proliferation of BxPC-3cells in a manner as dose-and time-dependent, the lowest concentration, the best concentration and timepoint were8μg/ml,32μg/ml oridonin for36hours at37℃,5%CO2in BxPC-3cells.2、Compared with control group, BxPC-3cells’cycles showed that cells of S phase treatment with8μg/ml oridonin group had no significant changes (P>0.05), but G2/M phase increased significantly (P<0.05). Meanwhile BxPC-3cells of S and G2/M phase treatment with32μg/ml oridonin increased more significantly (P<0.01).3、Hoechst33258staining showed that homogeneous and weak fluorescence in the control group cells, but concentrate and density blue fluorescence present at cell membranes and nucleuses of treatment with oridonin group cells.4、Compared with control group, BxPC-3cells after Annexin V/PI staining detected by flow cytometry showed that8μg/ml oridonin could increase the ratio of cell apoptosis (P<0.05), and32μg/ml oridonin indicated more significant cell apoptosis(P<0.01),5、Compared with control group, results showed that8μg/ml and32μg/ml oridonin could down-regulate the expressions of CDK1significantly (P<0.05) and up-regulate the expressions of yH2AX and p53significantly (P<0.05), and would have hydrolytic fragments from Caspase3,7,8,9and PARP in BxPC-3cells by Western Blot6、The result of comparing the difference of genes expression between treatment with and without oridonin in pancreatic cancer BxPC-3cells by expression microarray showed that520genes up-regulated and651genes down-regulated significantly.Conclusion:1、Oridonin could inhibit the proliferation of BxPC-3cells, down-regulated the expressions of CDK1and up-regulated the expressions of yH2AX, meanwhile arrested the cell in S and G2/M phases in BxPC-3cells.2、Oridonin could up-regulate the expressions of p53in BxPC-3cells and activate the Caspase signal pathway, it might induce BxPC-3cells into apoptosis by p53or Caspase signal pathway.3、Oridonin had a certain effect on the gene expression spectrum in BxPC-3cells, series changes of the gene expression concerning tumor and immunity may be related to the anti-tumor and immunomodulatory effect of oridonin.