Evaluation Of Anticancer Activity Of Nitidine Chloride In Vitro And Investigation Of Its Molecular Mechanisms

Objective:1.To screen out the compounds with superior antitumor activity in vitro from sixteen candidates;2.To evaluate anticancer activity of nitidine chloride in vitro and explore its molecular mechanisms.Methods:1.Screening of compounds with superior antitumor activity:By combining MTT assay and observation under microscope,compounds displaying marked inhibition of proliferation of tumor cell were selected;2. Evaluation of the effect of nitidine chloride on the tumor cell cycle distribution: The cell cycle were measured by using flow cytometry;3.Detection of genes expression in G₂/M phase and their coding protein:The expressions of CyclinB1 and cdc2 in treated and untreated cells with nitidine chloride were detected by employing semi-quantitative polymerase chain reaction;the expressions of p21 and p27 in treated and untreated cells with nitidine chloride were detected by using real time fluorescence quantitative polymerase chain reaction;the expressions of protein CyclinB1,cdc2,p21 and p27 were detected by applying immunocytochemical technique;4.Evaluation of the effect of nitidine chloride on the apoptosis of tumor cell:Cell apoptotic morphology change was detected by observation under electron microscope and using Hoechst 33258 technique;5.Evaluation of the effect of nitidine chloride on the reactive oxygen species in tumor cell:The change of reactive oxygen species in tumor cell was analyzed by applying flow cytometry and fluorescent microscope.Results:1.Screening of compounds with superior antitumor activity: According to our activity evaluation results,nitidine chloride,liriodendrin coordination compound with Au,Ag,Pt,plumbagin and its coordination compound with Cu,Co were qualified as the positive compounds.All their IC₅₀ values were less than 10μg/ml and nitidine chloride showed stronger antitumor activity than other selected compounds;the IC₅₀values for MDA-MB-231 cell, for KB cell and for its multidrug resistant KBv200 cell was(2.85±0.45)μg/ml, (2.36±0.22)μg/ml and(2.43±0.19)μg/ml,respectively.2.Evaluation of the effects of nitidine chloride on the tumor cell cycle distribution:After treated with nitidine chloride of 3μg/ml for 12 hours,the proportion of cells in G_0/G₁ phase and S phase was significantly less than that in untreated group(all P＜0.01),the number of cells in G₂/M phase dramatically increased(all P＜0.01).Moreover,with the extension of treatment of nitidine chloride,the tendency became more apparent.Although the proportion of cells in G₂/M phase was more than 70%at 48 h,there was no statistical difference (P＞0.05)in this value between at 24h and at 48h.In summary,nitidine chloride showed significant inhibiting effect on tumor cells G₂/M phase and this effect reached the maximum after 24h.3.Detection of genes expression in G₂/M phase and their coding protein: After treated with nitidine chloride of 3μg/ml for 12 hours,a degradation of the relative expression amount of CyclinB1 mRNA was detected(P＜0.05);for cdc2 mRNA and p27 mRNA,the expression remained at the same level(P＞0.05); whereas the mRNA of p21 immediately displayed a dramatically strong expression(P＜0.01).After 24 h,CyclinB1 mRNA showed further depression (P＜0.01);no change of expressions were observed for cdc2 mRNA and p27 mRNA(P＞0.05)and p21 mRNA still showed strong expression(P＜0.01).After 48 h,the expression of CyclinB1 mRNA reached the lowest level(P＜0.01);no difference of the expressions for cdc2 mRNA and p27 mRNA from beginning were observed(P＞0.05);however,the expression of p21 mRNA nearly restored to the level of 0 h group(P＞0.05).The similar observations were obtained for the corresponding proteins in the cell cycle.Protein CyclinB1 showed weak expression(P＜0.01),protein p21 was reinforced(P＜0.01),but the expression of protein cdc2 and p27 showed no significant change.4.Evaluation of the effect of nitidine chloride on the apoptosis of tumor cell:The result obtained from Hoechst 33258 test indicated that the apoptotic ratio increased with enhancing dosage of nitidine chloride;compared with 0μg/ml group,a statistical difference was obtained in 6μg/ml group and 9μg/ml group(P＜0.01);interestingly,the apoptotic ratio in 9μg/ml group was lower than that in 6μg/ml group.These facts were also confirmed by the observation for all groups under electron microscope.Many necrotic cells with few apoptotic cells were observed in 9μg/ml group.Therefore,in order to induce tumor cell apoptosis,nitidine chloride should be controlled in a certain dosage range,but necrosing tumor cell in high dose.5.Evaluation of the effect of nitidine chloride on the reactive oxygen species in tumor cell:It is generally agreed that the level of reactive oxygen species in tumor cell is higher than normal cell.The content of reactive oxygen species was clearly attenuated with increasing dosage of nitidine chloride, which was reflected by the left-shift of the corresponding peak in flow cytometry.Namely,the proportion of positive cells decreased.Compared with 0μg/ml group,all the treated groups showed statistical difference(P＜0.01).The results were also confirmed by a reduction of the fluorescence intensity under fluorescence microscope.Conclusion:1.Nitidine chloride is capable of degrading the level of reactive oxygen species in tumor cell,and markedly inhibits the proliferation of tumor cell.The effectiveness of nitidine chloride reasonably depends on its dosage: arresting G₂/M phase at low concentration;apoptosising tumor cells at medium concentration;necrosing tumor cells at high concentration.2.Its mechanism of G₂/M phase arrest could be explained by the down-regulation of transcription and expression of CyclinB1 and the up-regulation of transcription and expression of p21,thereby,decreased the formation of maturation promoting factor.