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冬凌草甲素诱导胃癌BGC-823细胞凋亡



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Induction of Apoptosis and G2/M Cell Cycle Arrest by Oridonin in Human Gastric Cancer BGC-823 Cells1
Han Jian, Ye Min, Qiao Xue, Wu Wanying, Qu Guiqin,

Guo Dean
The State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Xueyuan Road 38, Beijing ( 100083)
E-mail: gda@bjmu.edu.cn

Abstract
The in vitro apoptosis-induction effects of oridonin on gastric tumor cells BGC-823 and its effects on cell cycle, mitochondrial membrane potential and intracellular Ca2+ were investigated to shed light on the mode of its anticancer action. The results showed that oridonin inhibited BGC-823 cells growth with IC50 of 22.21 ?M. It induced apoptosis in a dose-dependent manner. In addition, it could decrease mitochondria membrane potential, increase intracellular Ca2+, and activate pro-caspase 3. BGC-823 cells were arrested in G2/M cell cycle phase with lower expression of cyclin A protein. The up-regulation of p53 was observed before apoptosis occurred and cell cycle arrest. It suggested that oridonin inhibits the proliferation of BGC-823 cells through G2/M cell cycle arrest and apoptosis induction, which is mediated by influx of Ca2+, up-regulation of p53, activation of caspase-3, and down-regulation of cyclin A. Keywords:Oridonin; Human Gastric Cancer; Apoptosis; Cell cycle arrest; p53; cyclin A

1. Introduction
Oridonin (Rubescensine A) is an ent-kaurane diterpenoid compound isolated from Isodon rubescens (Hemsl.) Hara, which belongs to Isodon genus of Labiatae family. It has attracted considerable attention in recent years because of its extensive activities, such as antibacterial, antiviral and anti-inflammation effects, as well as its clinic use for the treatment of human cancers. Several possible mechanisms have been proposed concerning its anticancer effects, including antiangiogenic activity [1,2], telomerase activity [3,4]. Moreover, it has been reported that oridonin inhibits growth of human melanoma A375-S2 and human cervical cancer HeLa and human hepatic carcinoma Bel-7402 cells through the induction of apoptosis [5,6,7]. However, the effects of oridonin on human gastric carcinoma cells and the involved mechanisms have not been pursued, so far. While in clinics, it exhibits specific curative effect in treatment of digestive apparatus tumor such as esophagea, gastric and liver carcinoma, as well as other type carcinoma. In the present study, we investigated the in vitro effect of oridonin on gastric cancer BGC-823 cells. Our results showed that oridonin suppressed the growth of BGC-823 cells through the induction of G2/M cell cycle arrest and apoptosis. Further investigation showed that mitochondria and the intracellular Ca2+ played important roles in oridonin-induced apoptosis of BGC-823 cells. Meanwhile the activation of caspase-3, up-regulation of p53 protein expression and down-regulation of cyclin A protein expression were also observed. The up-regulation of p53 was determined before apoptosis occurred and cell cycle arrest, suggesting that oridonin-induced apoptosis and cell cycle arrest in BGC-823 cells might be p53-dependent.

2. Experimental
Chemicals. Oridonin was a generous gift from Prof. Handong Sun, Chinese Academy of Sciences. RPMI 1640 medium was obtained from Invitrogen Corporation. MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide], RNase A, propidium iodide (PI), rhodamine 123 (rho123) and Fluo 3-AM were purchased from Sigma-Aldrich. Fluo 3-AM and
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Support by Specialized Research Fund for the Doctoral Program of Higher Education (No. 20040001136). -1-

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rho123 were prepared as stock solutions in dimethyl sulfoxide (DMSO), then diluted with phosphate-buffered saline (PBS) to the final desired concentrations. Cell cultures. BGC-823 human gastric cancer cell line was supplied by the Institute of Biochemistry and Cell Biology, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. Cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mmol.L-1 glutamine, 100 U.ml-1 penicillin and 100  g.ml-1 streptomycin, and were cultured at 37°C in a humidified atmosphere of 5% CO2. Cell proliferation study (MTT assay). BGC-823 cell suspensions (2×104 cells/ml) were seeded onto the 96-well plates (180  l/well). After 24 h of incubation at 37°C, 20  l oridonin in serial dilutions (from 0.1  M to 100  M) were added, and 0.1% DMSO was used as the control. Following 48 h of incubation with the reagent, 20  l MTT (5mg.ml-1) were added to each well. After additional 4 h of incubation, the medium was discarded and dried in the air, then 200  l of acid-isopropyl alcohol were added to dissolve the formazan crystal, and the absorbance were measured at 570 nm by a microplate reader (Model 550, Bio-Rad, Japan). The experiments were run in triplicates. Estimation of apoptosis by microscopy and flow cytometry. BGC-823 cells (5×105) were seeded onto 6-well plates with pre-placed glass slides, and treated with oridonin for 24 h. Cells were then washed twice with PBS buffer (pH=7.4), incubated with 200  l binding buffer containing 20  l Annexin V-FITC (20?g/ml) and 10 ?l PI (50?g.ml-1) for 15 min at room temperature in the dark. After washing with PBS, samples were qualitatively analyzed by confocal laser microscopy (Leica TCS SP2, Leica, Germany). Oridonin-induced apoptotic cells were quantified by Annexin V-FITC/PI double staining using a staining kit (Biosea Biotechnology CO., Beijing, China). Samples were analyzed in a FACScan flow cytometer (FACSCalibur, BD, USA) and the obtained data were processed with a Cellquest software. Cell cycle analysis. For flow cytometry analysis, BGC-823 cells (1×106) were treated with gradient concentrations of oridonin for 24 h. Cells were then collected and washed twice with PBS buffer, suspended in 70% ice-cold ethanol and kept overnight at -20°C. The next day, samples were washed with PBS and incubated with 25 ?g.ml-1 RNase A at 37°C for 30 min. Then the cells were stained with 50 ?g.ml-1 PI. Samples were analyzed on a FACScan flow cytometer using Cell ModFIT software to determine the population of cells in each cycle phase. Mitochondrial membrane potential (MMP) analysis. To examine the changes in the mitochondrial membrane potential, rho123 was used with PI [8]. Briefly, BGC-823 cells (5×105) were seeded onto 6-well plates and treated with oridonin for 24 h. After treatment, cells in 2 ml of RPMI 1640 medium were loaded with rho123 (5 ?g.ml-1) for 1 h at 37°C. Then cells were harvested and resuspended in 1 ml PBS buffer containing 5 ?g of PI. The fluorescence intensity of rho123 and PI was analyzed by flow cytometry using Cellquest software. Intracellular Ca2+ ([Ca2+]i) measurement. To estimate [Ca2+]i, Fluo 3-AM was used. BGC-823 cells (5×105) were seeded onto 6-well plates and treated with oridonin for 24 h. After treatment, cells in 2 ml of RPMI 1640 medium were incubated with Fluo 3-AM (500 nmol.L-1) for 30 min at 37°C. Then cells were harvested and resuspended in 1 ml PBS buffer. The fluorescence intensity of Fluo 3-AM was measured by flow cytometry using Cellquest software. Western blotting analysis for protein expression. At the indicated times after oridonin exposure, BGC-823 cells were lysed and protein extraction was performed. Following the determination of protein concentrations by the Bradford method [9], 50 ?g aliquots of cell protein were separated in 12% SDS-PAGE. Proteins were transferred onto a nitrocellulose membrane and blocked in
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TBS-Tween 20 buffer containing 5% non-fat dry milk. Membranes were immunoblotted with caspase-3, p53 and cyclin A (Santa Cruz, USA), respectively, and then incubated with horse-radish peroxidase conjungated secondary antibody. The bound antibody was detected by enhanced chemiluminescene (ECL) according to the manufacture’s recommendations (Santa Cruz, USA) and exposed on X-ray films.  -actin was used as an indicator for equality of lane loading.

3. Results
3.1 Inhibitory effect of oridonin on proliferation of BGC-823 cells. Oridonin
decreased cell viability in a dose-dependent manner (Fig.1). It had no inhibitory effect on BGC-823 cell proliferation below the concentration of 1 ?mol.L-1, but showed significant inhibitive effect above 10 ?mol.L-1. The IC50 value (the concentration of test sample to give 50% inhibition of the growth of cancer cells) of oridonin on BGC-823 cells was determined as 22.21±1.26 ?mol.L-1.

3.2 Assessment of oridonin-induced cell apoptosis. The change of cell morphology was
observed by Annexin V-FITC/PI double stain on confocal laser microscope (Fig.2A). Typical morphological variation of apoptotic cell was found at 60 ?mol.L-1 oridonin. These cells stain red (PI) in nuclei, which were highly condensed or fragmental, and their membranes stain green (Annexin V-FITC). The percentage of apoptotic cells was detected by Annexin V-FITC/PI double stain on FACScan (Fig.2B). Annexin V-FITC positive apoptotic fractions were detected in the first 24h after exposure to oridonin, and these fractions dramatically increased in a time-dependent manner. Annexin V-FITC and PI double-positive cells were increased at 12h after treatment, indicating that oridonin induced necrosis in addition to apoptosis in this fraction. These results showed that oridonin-induced in vitro growth inhibition of gastric cancer cells was mediated by apoptosis.

3.3 Cell cycle changes following oridonin treatment. An arrest in G2/M cell cycle phase
became evident in BGC-823 cells treated with 30 ?mol.L-1 and 40 ?mol.L-1 oridonin, when the percent of cells in G2/M phase increased to 31.80% and 36.81%, respectively, which differed from untreated controls (11.31%). In the mean time, the G1 and S phase fraction decreased (Fig.3). In contrast, with higher concentration at 60 ?mol.L-1, the G2/M fraction decreased, although it still has significant difference with control, associated with the appearance of sub-G1 peak representing apoptosis cells.

3.4 Variations of the mitochondrial membrane potential (MMP). MMP was assessed
by flow cytometry after double staining with rho123, a cationic dye, and PI. Rho123 is selectively uptaken by mitochondria, the fluorescence intensity variation of which is directly proportional to the variations of the MMP. As shown in Fig.4, BGC-823 cells had an increasing population in rho123 negative/PI negative (lower left fraction) field after treatment with oridonin for 24 h. The population in this field refers to cells that are still viable but with damaged mitochondria, indicating that oridonin induced apoptosis in BGC-823 cells through affecting the mitochondrial function. The change of the rho123 negative/PI negative fraction (LL fraction) percent is shown in Fig. 6 B in histogram.

3.5 Increase of the intracellular levels of Ca2+ ([Ca2+]i). Intracellular Ca2+ plays a key
role in apoptotic actions. Therefore, effect of oridonin on the intracellular levels of Ca2+ was examined in this study (Fig.5). With increasing concentrations of oridonin, the histogram of Fluo 3-AM fluorescence shifted to a higher intensity range, and it reached the peak value at 60 ?mol.L-1, indicating an oridonin-induced increase of [Ca2+]i. The dose-dependent effect of oridonin on Fluo
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3-AM fluorescence intensity is shown in Fig. 5B, consistent with the change trend of MMP shown in Fig. 4B.

3.6 Effects of oridonin on the expression of apoptosis- and cell cycle-related proteins. In order to clarify the molecular mechanism of oridonin-induced apoptosis effect
on BGC-823 cells, the expression of related protein caspase-3, cyclin A and p53 was investigated by western blotting analysis. The results were shown in Fig.6. Caspase-3 plays an important role in apoptosis pathway. In order to investigate the role of caspase-3 on mediating oridonin-induced apoptosis in BGC-823 human gastric cancer cells, cells were exposed to 40 ?mol.L-1 oridonin for different times or 60 ?mol.L-1 oridonin for 24h, then the cleavage and activation of caspase-3 was determined. It showed that under conditions where β-actin levels remained unchanged, levels of the 32kD pro-caspase 3 significantly decreased in time- and dose-dependent manner in treated cells as compared to control untreated cells. This demonstrated that caspase-3 is activated by cleavage in BGC-823 cells undergoing oridonin-induced apoptosis. The results of MMP detection revealed that the MMP decreased most significantly in the treatment of 60 ?mol.L-1 oridonin for 24h, which is consistent with the expression change of caspase-3. These results indicated that oridonin-induced apoptosis in BGC-823 cells might be carried out through mitochondria pathway. The progression of the cell cycle is largely controlled by cyclins, the regulatory units for CDKs. We examined the protein levels of cyclin A, one of the G2/M cell cycle-related cyclins, in BGC-823 cells by western blot analysis. The results indicated that the levels of cyclin A decreased when treated with 40 ?mol.L-1 or 60 ?mol.L-1 oridonin for 24h, a time point at which apoptotic cells appeared and G2/M phase cells increased. To further investigate molecular changes involved in oridonin-induced apoptosis and G2/M arrest in BGC-823 cells, we analyzed expression of tumor suppressor p53 protein. P53 is a cellular gatekeeper. One of its roles is to survey cellular stress and to induce apoptosis, if necessary [10]. Following the oridonin treatment, up-regulation of p53 protein was observed, and the expression of p53 increased dramatically at much earlier time points (from 3h). Increased p53 protein expression appeared before apoptosis occurred and G2/M cycle arrest, suggesting that oridonini-induced apoptosis in BGC-823 cells might be p53-dependent.

4. Discussion
From Isodon species, more than 300 ent-kaurane type diterpenoids have been isolated, and most of them exhibited a wide range of biological activities. Oridonin is an abundant constituent isolated from Isodon species and shows positive effect in cancer treatment. Some 30 years ago, it was reported that oridonin could bind to SH and NH moiety of DNA polymerase through the hydrogen bonding between the OH group at position 6 and the CO group at position 15, then inhibit the proliferation and pervasion of tumor [11]. More recently, studies have focused on its ability to induce apoptosis in a variety of human cell lines [5,6,12,13]. It was also reported that oridonin inhibited NF-κB in lymphoid malignancies cells [14] and breast cancer cells [15]. But to our knowledge, a systematic study on the effect of oridonin on gastric cancer cells has not been performed. Our studies, for the first time, demonstrated that oridonin induces apoptosis in human gastric cancer cell BGC-823. Cell cycle arrest in G2/M, the decrease of MMP and the increase of intracellular free Ca2+ took part in the regulation of apoptosis induced by oridonin in BGC-823 cells. It was also demonstrated for the first time that caspase-3, p53 and cyclin A played a crucial

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role in this progression. Our data obtained from confocal microscopy and flow cytometry showed that oridonin could induce apoptosis in BGC-823 cells in time- and dose-dependent manner. By Annexin V-FITC/PI double staining, we observed the externalization of phosphatidylserines on confocal laser microscopy and on flow cytometry. We considered that oridonin induced apoptosis in BGC-823 cells at a concentration above 30 ?mol.L-1 when treated for 24 h, while showed little effects below this concentration. The significant enzymatic feature of apoptosis is the activation of caspases, especially the caspase-3 is believed to be commonly involved in various cells at the apoptosis execution phase. Activation of caspase-3 will induce PARP cleavage, chromosomal DNA strand breakage, and finally the apoptosis occurrence. To investigate the effect of caspase-3 on oridonin-induced apoptosis, we performed the western blot analysis. The results showed that the activities of caspase-3 under the stimulation of oridonin increased in time- and dose-dependent manner. Mitochondria is considered as the apoptosis controller in cells [16]. Our results showed that oridonin could induce dose-dependent alteration of MMP in BGC-823 cells. Below the 20 ?mol.L-1 oridonin, the changes of MMP was not significant, up to 30 ?mol.L-1, it exhibited significant changes, which reflects the opening of the mitochondrial permisity transition pores (PTP), associated with apoptosis peak appearance. A concentration of 60 ?mol.L-1 led to the most remarkable changes of MMP. The loss of MMP and release of apoptogenic factors, such as cytochrome C and AIF from mitochondria into the cytosol are associated with apoptosis induced by chemotherapeutic drugs [17]. On the other side, the increased expression of Bax and the release of cytochrome C in A375-S2 [5] and NB4 cells [18] have been demonstrated by other authors. In the present study, the loss of MMP was clearly observed and the cleavage of pro-caspase 3 was also detected directly. Thus, it is reasonable to postulate that caspase-3 activation through mitochondria participates in the induction of apoptosis in oridonin-treated BGC-823 cells. Many anticancer agents arrest the cell cycle at one certain cycle phase and then induce apoptosis. Cell cycle check-points may function to ensure that cells have time for DNA repair, whereas apoptosis may function to eliminate damaged cells. Our cell cycle analysis revealed a prominent G2/M arrest of BGC-823 cells after their exposure to oridonin and this cycle arrest was associated with a down-regulation of cyclin A, a protein known to regulate cdc2 kinase activity in G2/M phase. In previous papers, cell cycle arrest in G2/M phase or G0/G1 phase by oridonin have both been reported [12,19,20]. The present results were consistent with the former ones. Interestingly, our results also showed that oridonin arrested cell cycle in G0/G1 phase in HL-60 cells (data not shown). The discrepancy in the various systems may largely be due to the difference of the apoptosis response in the different cell types to oridonin. It is well known that cells differ enormously in their apoptosis response to DNA damage, depending in part on cell type, cell cycle status and differentiation state [21]. Another conclusion about our present cell-cycle analysis is that apoptosis induced by oridonin in BGC-823 cells was cycle dependent. When the concentration of oridonin was below 30 ?mol.L-1, it did not induce apoptosis and cycle arrest, while up to 30 ?mol.L-1, it induced apoptosis and cycle arrest in G2/M phase, but at 60 ?mol.L-1, the apoptosis percent dramatically increased with the decrease cell population in G2/M. This observed decrease could be explained by an increase of apoptotic cell population. The induction of p53 levels in other types of cells exposed to oridonin has been previously reported [13, 22]. In prostate cancer cells (DU-145, LNCaP), breast cancer cells (MCF-7) and ovarian cancer cells (A2780, PTX10), oridonin induced up-regulation of p53, while in gallbladder cancer cells (GBC-SD) treated with oridonin, down-regulation of p53 was observed. The
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difference between them may be due to differences in the sensitivity and response of different cell types to oridonin. In our present studies, a dramatic increase of p53 protein levels appeared in BGC-823 cells after oridonin treatment for 3h, earlier than activation of caspase-3, apoptosis and cycle arrest in G2/M. This suggested that the apoptosis and cell cycle arrest in BGC-823 cells following oridonin exposure might be via p53-dependent pathway. Intracellular Ca2+ is commonly involved as signal transducer in chemical-induced apoptosis. In our results, oridonin induced a rapid increase in [Ca2+]i when the concentration was above 30 ?mol.L-1, associated with the apoptosis appearance and MMP decrease. A number of studies have demonstrated that increased [Ca2+]i activates various enzymes including Ca2+-dependent neutral proteases, Ca2+-dependent phosphatases (calcineurin), Ca2+-dependent trasglutamiase, Ca2+/Mg2+-dependent endonuclease (calpain), all of which are essentially involved in the induction of apoptosis [23]. Elevated [Ca2+]i induces over-expression of NF-kappa B and p53 [24], down-regulates the expression of Bcl-2 in mitochondria membrane and induce apoptosis. It could also accelerate the opening of PTP, which acts as a slow-channel of Ca2+ and releases the Ca2+ in the mitochondria, then induce apoptosis [25]. Therefore, oridonin-induced increase in [Ca2+]i may be partly responsible for the oridonin-induced apoptosis in BGC-823 cells, and this increase may originate from damaged mitochondria judged by the identical change trend between MMP and [Ca2+]i , which has not been reported in literature.

5. Conclusion
According to these results, oridonin appears to exert its anticancer properties in gastric carcinoma, and these results suggested that oridonin inhibits the proliferation of BGC-823 cells through G2/M cell cycle arrest and apoptosis induction, which is mediated by influx of Ca2+, up-regulation of p53, activation of caspase-3, and down-regulation of cyclin A, as shown in Fig.7. In conclusion, these results indicated that oridonin may be useful as one agent in the treatment of gastric carcinoma.

References
1. Meade-Tollin, L.C.; Kithsiri-Wijeratne, E.M.; Cooper, D.; Guild, M.; Jon, E.; Fritz, A.; Zhou, G.X.; Whitesell, L.; Liang, J.Y. J. Nat. Prod. 2004, 67, 2-4. 2. Sartippour, M.R.; Seeram, N.P.; Heber, D; Hardy, M. Int. J. Oncol. 2005, 26, 121-127. 3. Liu, J.J.; Wu, X.Y.; Peng, J; Pan, X.L.; Lu, H.L. Ann. Hematol. 2004, 83, 691-695. 4. Liu, J.J.; Huang, R.W.; Lin, D.J.; Wu, X.J. Neoplasma 2005, 52(3), 225-230. 5. Zhang, C.L.; Wu, L.J.; Tashiro, S.; Onodera, S.; Ikajima, T. J. Asi. Nat. Prod. Res. 2004, 6(2), 127-138. 6. Zhang, C.L.; Wu, L.J.; Tashiro, S.; Onodera, S.; Ikajima, T. Acta. Pharmacol. Sin. 2004, 25(5), 691-698. 7. Zhang, J.F.; Liu, J.J.; Lu, M.Q. Chin. Tradit. Herb. Drugs 2006, 37(10), 1517-1521. 8. Bai, J.; Rodriguez, A.M.; Melendez, J.A. J. Biol. Chem. 1999, 274, 26217-26224. 9. Bradford, M.M. Anal. Biochem. 1976, 72, 248-254. 10. Lorne, J.H.; Perwez, S.H.; Curtis, C.H. Trends Pharmacol. Sci. 2004, 25(4), 177-181. 11. Fujita, E.; Nagao, Y.; Node, M.; Kaneko, K.; Nakazawa, S.; Kuroda, H. Experientia 1976, 32(2), 203-206. 12. Ikezoe, T.; Chen, S.S.; Tong, X.J. Int. J. Oncol. 2003, 23 (4), 1187-1189. 13. Chen, S.; Gao, J.; Halicka, H.D.; Huang, X.; Traganous, F.; Darzynkiewicz, Z. Int. J. Oncol. 2005, 26(3), 579-588. 14. Ikezoe, T.; Yang, Y.; Bandobashi, K.; Saito, T.; Takemoto, S. Mol. Cancer. Ther. 2005, 4(4), 578-586. 15. Hsieh, T.C.; Wijeratne, E.K.; Liang, J.Y.; Gunatilaka, A.L.; Wu, J.M. Biochem. Biophys. Res. Commun. 2005, 337(1), 224-231. 16. Green, D.R.; Reed J.C. Science 1998, 281(5381), 1309-1312. 17. Robertson, J.D.; Orrenius S. Critical. Rev. Tox. 2000, 30, 609-627. -6-

http://www.paper.edu.cn 18. Liu, J.J.; Huang, R.W.; Lin, D.J.; Wu, X.J. Leukemia Lymphoma 2005, 46(4), 593-597. 19. Ren, K.K.; Wang, H.Z.; Xie, L.P.; Chen, D.W.; Liu, X. J. Ethnopharmacol. 2006, 103(2), 176-180. 20. Zhang, T.M.; Shou, M.G..; Wang, M.Y. Acta Pharmacol Sin, 1986, 7(5), 457-460. 21. Bratton, S.B.; Cohen, G.M. Trends Pharmacol. Sci. 2001, 22, 306-315. 22. Xue, H.W.; Pan, X.L.; Yang, S.J. J. Shandong University (Health Sciences) 2003, 43(4), 336-339. 23. Liu, J.S.; Xia, D.M.; Pan, H.Z. Signal of cell and its regulation: collection of modern biology and medicine; Joint Press of Beijing Medical University and Peking Union Medical University: Beijing, China, 1999, 334-344. 24. Dumont, A.; Hehner, S.P.; Horgmann, T.G.. Oncogene 1999, 18, 747-757. 25. Fall, C.P., Jr Bennett, J.P. Biochim. Biophy. Acta 1999, 1410, 77-84.

List of Figure:

Cell Viability(%Control)

120 100 80 60 40 20 0 1E-3 0.01 0.1 1 10 [Oridonin] (?M) 100 1000

Fig. 1. Effect of oridonin on cell viability in BGC-823 human gastric carcinoma cells: BGC-823 cells were incubated with or without each concentration of oridonin for 48h. Cell viability assay was carried out by MTT method. Results are shown as the percentage of cell viability rate compared with the control (cells were grown in medium without oridonin). Data points represent the mean values of three replications with bars indicating SEM.

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A
1 2 3

B

Fig. 2. (A) Apoptosis in BGC-823 cells using Annexin V-FITC/PI staining and evaluated by confocal laser scanning microscope. (1) Control BGC-823 cells in common transmission light; (2) Control BGC-823 cells in laser; (3) Cells treated with 60?M oridonin for 24h in laser. Apoptotic cells are pointed by arrows. Necrosis cells are pointed by n. (B) Induction of apoptosis treated by 40 ?M oridonin for the indicated times evaluated by flow cytometry. Three duplicate experiments were done with similar results.

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Fig. 3. Cell cycle analysis. Cells were treated with graded concentrations of oridonin for 24h and stained with PI, analyzed by flow cytometry. Results show a representative experiment that has been repeated three times.

Fig. 4. (A) Flow cytometry analysis of the mitochondrial membrane potential. BGC-823 cells were incubated with graded concentrations of oridonin for 24h. Results show a representative experiment that has been repeated three times. (B) The histogram indicating the percent of LL fractions which refers to cells viable but mitochondria damaged. Data represent the mean values of three replications with bars indicating SEM. *P<0.05 compared with control (without treatment by oridonin).

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Fig. 5. Effect of oridonin on Fluo 3-Am ([Ca2+]i) fluorescence of BGC-823 cells. (A) The histogram obtained from cells treated with 60?M oridonin for 24h. A shift of the Fluo 3-Am fluorescence to a higher intensity range, which corresponds to an increase of [Ca2+]i . (B) Dose-dependent effect of oridonin on mean intensity of Fluo 3-Am. Cells were treated with graded concentrations of oridonin for 24h. Data represent the mean values of three replications with bars indicating SEM. *P<0.05 compared with control (without treatment by oridonin).

A

B

Fig. 6. Effects of oridonin on expression of caspase-3, cyclin A and p53 proteins in BGC-823 cells determined by Western blot. β-actin expression was used as control for protein loading. (A) The representative blot of three independent experiments. (B) The histogram indicating the protein expression evaluated with spot densitometry analysis in Alphamager IS-2200. Protein expression in cells harvested at 0 h was taken to be 100%. Data represent the mean values of three replications with bars indicating SEM. *P<0.05 compared with control.

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oridonin

p21
dk1 1-c ocked nB l ycli esis b c h t syn

p53

mitochondrial depolarization (MMP )

G2/M arrest

cyt C/ APAF-1/caspase 9 [Ca2+]i executioner caspase 3

cyclin A
Direct modification

Apoptosis

Tentative modification

Fig. 7. Proposed mechanism of oridonin induced G2/M arrest and apoptosis in BGC-823 cells: Oridonin induced the p53 overexpression , activate caspase-3 and cyclin A down-regulation, then led to apoptosis and G2/M arrest.

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