OBJECTIVE:To prepare in-situ gelling of hydrochloride sparfloxacin eye drops and establish its quality con-trol method.METHODS:The in-situ gelling of hydrochloride sparfloxacin eye drops were prepared with boric acid as buffer to regulate the pH and osmotic pressure and with sodium polymannuronate as peptizer.The content of hydrochloride sparflo-xacin was determined by ultraviolet spectrophotometry.The stability of the sustained-release eye drops was investigated as well.RESULTS:The sustained-release eye drops were colorless or yellowish limpid liquid with its test and identification results all in conformity with the related stipulation stated in Chinese Pharmacopeia(2005 edition).There was a good linear relationship within the concentration range of 3.0～8.0 ?g?mL-1 for hydrochloride sparfloxacin,and its average recovery was 99.82%(RSD=0.236%,n=6).There was no significant change for indexes in the accelerate test and sample test,yet the temperature and lighting did have slight impact on the viscosity,osmotic pressure and pH of the solution.CONCLUSION:The preparative method is simple and feasible and the quality of in-situ gelling of hydrochloride sparfloxacin eye drops is stable and controllable.

Aim To investigate Jaridonin′s selective killing of cancer cells and explore the related molecular mechanism. Methods After treatment by Jaridonin for 24 h, the effect of Jaridonin on the cell viability was examined using MTT assay. The effect of Jaridonin on cytomorphology and mitochondrial membrane poten-tial (Δψm) was observed by a fluorescence microsco-py. The apoptosis of cell lines treated with Jaridonin, as well as the level of reactive oxygen species ( ROS ) was analyzed by flow cytometry. Expression of the pro-teins related with mitochondria apoptosis pathways was detected by Western blot. Results Jaridonin caused strong antiproliferative and apoptotic effects on MGC-803 cells, but there were not remarkable effects on GES-1 cells. Furthermore, the expression of Bax was up-regulated, and the release of cytochrome c from mi-tochondria to cytosol was also promoted in MGC-803 cells treated by Jaridonin. The cleavage of caspase-3 in MGC-803 cells was also observed. Jaridonin increased persistently intracellular levels of ROS in MGC-803 cells, whereas the level of ROS in GES-1 rose in the first stage, and then decreased, and dropped to the basic level after 6 h. More interestingly, Jaridonin-in-duced ROS accumulation and the inhibition of MGC-803 cell proliferation were almost completely attenuated in the presence of GSH. Conclusions Jaridonin se-lectively kills cancer cells and induces apoptosis in MGC-803 through ROS-mediated mitochondrial dam-age.

Objective To study the effects of Jaridonin, a novel diterpenoid from isodon rubescens, on the cell cycle of human gastric cancer cells and its molecular mechanism of action. Methods Flow cytometry was used to analyze the cell cycle distribution and expression of ataxia telangiectasia mutated kinase ( ATM) after Jaridonin treatment. Western blot was performed to detect the expression of cell cycle?related proteins. Results The results of flow cytometry showed that the percentages of MGC?803 cells in G2/M phase at 6 hours after 0, 10, 20 μmol/L Jaridonin?treatment were (10.8±2.2)%, (18.2±2.5)%, (27.3±3.2)%, respectively;those at 12 hours after Jaridonin?treatment were (12.0±1.5)%, (24.1±2.0)%and (39.7±5.2)%, respectively, indicating a G2/M phase arrest of MGC?803 cells was resulted in a time?and dose?dependent manner. The expressions of ATM, Chk1, Chk2, phosphorylated Cdc2 and CDK2 were up?regulated in the MGC?803 cells after Jaridonin treatment, while the levels of Cdc2 and CDK2 were decreased. KU?55933, an inhibitor of ATM, reversed the expression of relevant proteins and G2/M phase arrest induced by Jaridonin. Conclusions Jaridonin can significantly induce G2/M arrest in gastric cancer MGC?803 cells. Its mechanism may be related to the activation of ATM and Chk1/2, and inactivation of Cdc2 and CDK2 phosphorylation.

Objective To study the effects of Jaridonin, a novel diterpenoid from isodon rubescens, on the cell cycle of human gastric cancer cells and its molecular mechanism of action. Methods Flow cytometry was used to analyze the cell cycle distribution and expression of ataxia telangiectasia mutated kinase ( ATM) after Jaridonin treatment. Western blot was performed to detect the expression of cell cycle?related proteins. Results The results of flow cytometry showed that the percentages of MGC?803 cells in G2/M phase at 6 hours after 0, 10, 20 μmol/L Jaridonin?treatment were (10.8±2.2)%, (18.2±2.5)%, (27.3±3.2)%, respectively;those at 12 hours after Jaridonin?treatment were (12.0±1.5)%, (24.1±2.0)%and (39.7±5.2)%, respectively, indicating a G2/M phase arrest of MGC?803 cells was resulted in a time?and dose?dependent manner. The expressions of ATM, Chk1, Chk2, phosphorylated Cdc2 and CDK2 were up?regulated in the MGC?803 cells after Jaridonin treatment, while the levels of Cdc2 and CDK2 were decreased. KU?55933, an inhibitor of ATM, reversed the expression of relevant proteins and G2/M phase arrest induced by Jaridonin. Conclusions Jaridonin can significantly induce G2/M arrest in gastric cancer MGC?803 cells. Its mechanism may be related to the activation of ATM and Chk1/2, and inactivation of Cdc2 and CDK2 phosphorylation.

Objective: To investigate the effects and the underlying mechanism of DS2, a newly synthetic analog of natural ent-kaurane diterpenoid, on the proliferation and migration capabilities of human gastric cancer cells. Methods: MTT assay, colony formation assay and flow cytometry were used to measure the effects of DS2 on growth, apoptosis and cell cycle of several human gastric cancer cell lines. The function of DS2 in the migration was further detected by wound healing and transwell assays. The expression of migration related proteins were determined by western blot. Results: DS2 inhibited the growth of MGC-803, SGC-7901 and HGC-27 cells in a dose dependent manner. After treatment of DS2 at a concentration of 6.25 μmol/L for 24 h, the survival rates of MGC-803, SGC-7901 and HGC-27 cells were 53.87±3.05%, 55.91±6.97% and 32.41±2.64%, respectively. However, for the normal gastric epithelial cell GES-1, no obvious growth inhibition was observed. In addition, DS2 caused significant G₂/M arrest and induced apoptosis in MGC-803 cells. Furthermore, compared with the negative control, the colony formation, wound healing rate as well as the number of migrating cells of MGC-803 were significantly decreased in a dose dependent manner after DS2 treatment. DS2 induced the expression of E-cadherin, whereas β-catenin and N-cadherin levels were downregulated in MGC-803. Conclusion The new compound DS2 has a strong anti-cancer activity, and this study will help us to design and synthesize better diterpenoids derivatives.

OBJECTIVEThe aim of this study was to explore the molecular mechanism of apoptosis in esophageal cancer cells induced by Isodon rubescens.

METHODSThe DNA-damage effect of Jaridonin was detected by single cell gel electrophoresis (SCGE). The p53 protein was determined by Western blot. GSH assay kit was employed to determine the GSH content in human esophageal cancer EC-1 cells. Intracellular levels of hydrogen peroxide (H2O2) or superoxide (O(2).-) were determined using the redox-sensitive probes 2', 7'-dichlorodihydrofluorescein diacetate (DCF) or dihydroethidium (DHE), and the fluorescence signal was assayed by fluorescence microscopy and by flow cytometry.

RESULTSJaridonin induced DNA damage in EC-1 cells remarkably. The olive tail moments (OTM) of control and 20, 40 µmol/L Jaridonin were 3.2, 45.2 and 89.0, respectively. Compared with the control, the differences were significant (P < 0.01 for both). Jaridonin resulted in extensive p53 up-regulation in the EC-1 cells. More importantly, the p53 up-regulation occurred as early as 2 h after Jaridonin incubation, and in a time-dependent manner (P < 0.05). p53 siRNA transfection inhibited apoptosis in the EC-1 cells, and the Jaridonin-induced apoptosis rate was reduced from 38.5% to 8.8%. Intracellular level of H2O2 was increased by Jaridonin, whereas the level of O(2).- was barely changed. The GSH content in EC-1 cells was reduced from (10.3 ± 1.6) nmol/mg protein to (4.6 ± 2.1) nmol/mg protein after 20 µmol/L Jaridonin incubation for 8 h, and it was further reduced with the increase of Jaridonin concentration. Jaridonin induced DNA damage, H2O2 accumulation and apoptosis were significantly attenuated in the presence of GSH, but Jaridonin showed little effect on normal human liver L-02 cells.

CONCLUSIONSJaridonin selectively induces apoptosis in esophageal cancer EC-1 cells through H2O2-mediated DNA damage by depleting GSH.

Antineoplastic Agents ; pharmacology ; Apoptosis ; DNA Damage ; Diterpenes, Kaurane ; pharmacology ; Esophageal Neoplasms ; metabolism ; Humans ; Hydrogen Peroxide ; metabolism ; Up-Regulation