| Oridonin is a natural organic compound of the Ent-kaurene diterpenoid isolated from the Labtea plant of the genus Labiatae.As the research observed Oridonin has a unique and obvious antitumor activity and has significiant inhibitory and proliferation effects on human histiocytic lymphoma,breast cancer and gallbladder cancer cells,it has low toxicity and no obvious damage to vital organs of human body.Our previous study found that Oridonin may block human gastric cancer SGC-7901 cells in M phase.This paper is based on the results of this experiment to further verify.First of all,the effect of Oridonin on the proliferation of human gastric cancer SGC-7901 cells was investigated,followed by the mechanism of its induction of tumor cell cycle arrest,and finally the mechanism of the specific cycle arrest of the cell cycle.The main contents of this experiment are as follows:1)The effect of Oridonin on the proliferation of human gastric cancer SGC-7901 cells,including five contents,(1)The effect of Oridonin on the growth curve of human gastric cancer SGC-7901 cells was determined by MTT assay,(2)The effect of Oridonin on plate colony formation in human gastric cancer SGC-7901 cells,(3)The effect of Oridonin on the morphology of human gastric cancer SGC-7901 cells was observed with inverted microscope,(4)Effect of Oridonin on the rate of positive stained cells in human gastric cancer SGC-7901 cells by PI single staining,(5)The expression of caspase-8,caspase-9,caspase-3 and PARP in human gastric cancer SGC-7901 cells were determined by Western Blotting.2)The effect of Oridonin arrest on phase in human gastric cancer SGC-7901 cells and its mechanism of action,which contains two contents,(1)the effect of Oridonin on cell cycle arrest of human gastric cancer SGC-7901 cells by flow cytometry,(2)Western Blotting detect Oridonin on the expression of CDK4 and cyclinDl in human gastric cancer SGC-7901 cells,(3)Western Blotting detect Oridonin on the expression of cyclinA and CDK2 in human gastric cancer SGC-7901 cells;3)the effect of Oridonin arrest on M-phase of human gastric cancer SGC-7901 cells,fluorescence microscopy and Western Blotting detect effects of phosphorylated Ser10 histone H3 of Oridonin on human gastric cancer SGC-7901 cells,Western Blotting assays the effect of Oridonin on the expression of cyclinB1 and CDK1(cdc2)in human gastric cancer SGC-7901 cells.The experimental results show that 1)The effect of Oridonin on the proliferation of human gastric cancer SGC-7901 cells,(1)MTT assay detect different concentrations of Oridonin 16,32,64,128,256 ?M can inhibit the proliferation of human gastric cancer SGC-7901 cells,with the increase of drug concentration and the prolongation of action time,the inhibitory effect of SGC-7901 cells was enhanced,with time-and dose-dependent manner.(2)Plate clone experiment detects Oridonin with 2,4,8 ?M treating human gastric cancer SGC-7901 cells on 24 h,compared with the negative control group,the number of cell-forming clones significantly reduced and the formation of a single clone had a smaller diameter,(3)Inverted microscope can be seen,the negative control group cell density is big,morphology was polygonal,clear edges,culture medium almost no suspended cells.With the increase of the concentration of Oridonin,a large number of cells suspended in culture medium,cell body shrinkage,cytoplasm concentration,a small number of cells increased volume,cell rupture,cell necrosis,(4)Flow cytometry was used to detect the positive rate of PI staining of human gastric cancer SGC-7901 cells treated with different concentrations of Oridonin for 48 h.The results showed that cells trated Oridonin with the final concentrations of 30,40,50 ?M,the positive rate of PI staining of SGC-7901 cells was(7.63±4.29)%,(36.57±2.77)%and(43.20±8.27)%,respectively,compared with the negative control(4.03±2.77)%group was statistically significiant(P<0.05 or P<0.01),(5)Western blotting analysis of Oridonin on caspase-8,caspase-9,caspase-3 and PARP expression results show that compared with the control group,the levels of cleaved-caspase-8,cleaved-caspase-9,cleaved-caspase-3 and cleaved-PARP were all significiantly increased(P<0.05 or P<0.01).The above reults indicate that Oridonin can inhibit the proliferation of human gastric cancer SGC-7901 cells and induce its apoptosis;2)The effect of Oridonin on the cell cycle arrest human gastric cancer SGC-7901 cells,(1)PI single staining and flow cytometry were used to detect the periodic effects of Oridonin on human gastric cancer SGC-7901 cells.Adding to a final concentration of 8,16,32?M of Oridonin,the percentage of G2/M phase was significantly higher than the negative control(10.18±0.69)%group after 48 h,the percentage respectively:(11.64±0.33)%,(25.60±0.47)%,(37.36±1.73)%,was statistically significiant(P<0.05 or P<0.01),(2)The effect of Oridonin on the expression of CDK4 and cyclinDl in human gastric cancer cells SGC-7901 was detected by Western Blotting.With the increase of the concentration of Oridonin,the expression of CDK4 and cyclinDl could be up-regulated and cells in G0/G1 phase could be gradually transformed to S phase.(3)The protein of CDK2 and cyclinA in human gastric cancer cells SGC-7901 was detected by Western Blotting.With the increase of the concentration of Oridonin,the expression of CDK2 and cyclinA could be up-regulated and cells in S phase could be gradually transformed to G2 phase.3)The effects of Oridonin on the arrest of M phase of human gastric cancer SGC-7901 cells:(1)The effect of Oridonin on the expression of phosphorylated Ser10 histone H3 in human gastric cancer SGC-7901 cells was observed by fluorescence microscopy,after staining by fluorescence,the cells stained with PI were red,the cells stained with Alex Flour 488 were green,red cells respresented the total number of cells,green cells represented the number of M phase(p-H3 positive)cells.The results showed that Oridonin could inhibit the growth of human gastric cancer cells SGC-7901 in M phase rather than G2 phase,thus inhibiting cells proliferation.(2)The effect of Oridonin on the expression of CDK1(cdc2)and cyclinB1 in human gastric cancer cells SGC-7901 was detected by Western Blotting.The results showed that the expression of CDK1(cdc2)and cyclinB1 could be up-regulated with the increase of Oridonin concentration,suggesting that the tumor cells may be transformed from G2 phase to M phase.(3)Western Blotting detection of Oridonin on human gastric cancer cells SGC-7901 cytoplasmic phosphorylation of Ser10 histone H3 expression results show that with the increase Oridonin concentration for 48 h,the expression concention of p-H3 up-regulation,compared with negative group was statistically significiant(P<0.05 or P<0.01).The results showed that Oridonin could inhibit the proliferation of SGC-7901 cells in M phase but not in G2 phase.From the above experimental results,it was shown that Oridonin inhibits proliferation of human gastric cancer cells SGC-7901 by aresting it in M phase instead of G2 phase. |
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