Study Of Effects Of 4-p-amino-benzonic Acid-4'-demethyle Pipod-phyllotoxin Ester (PDE) On K562 Cells Apoptosis And Mechanisms Of PDE Inducing Tumor Cells Apoptosis

ObjectiveTo observe the apoptosis of K562 cell line induced by 4-p-amino-benzonic acid-4'-demethylepipodophyllotoxin ester (PDE), proving its antitumor effects.To assay ions concentration, reactive oxygen species and mitochondrial membrane potential changes and Bcl-2/Bax expression, thus discussing mechanisms of PDE inducing tumor cells apoptosis. MethodsThe cells growth and proliferation were measured by MTT assay method. Flow cytometry(FCM) measured the percentage of apoptosis. Laser scanning confocal microscopes(LSCM) assayed K562 cells within [Ca²⁺],[Mg²⁺],[H⁺],reactive oxygen species and mitochondrial membrane potential changes.Bcl-2/Bax expression of apoptotic associated genes were examined by inmunocytochemistry. Results1. PDE of different concentration can inhibited the growth of K562 cells, and this role had time and dose dependent. There were significantly different in OD value between over 0.39 /*g/ml of the concentration of PDE and control group (p<0.05). And with the extension of the time, cellular growth inhibitory rate increased.2. After different concentrations of PDE dealed with K562 cells 24 hours, FGM measured the percentage of apoptosis that increased with the concentration increase.We can observe apoptosis peak in the experiment groups,too.3. Comparing with control group, apoptotic intracellular [Ca2+],[H+],[Mg2+] fluorescent intensity assayed by LSCM increased with increased concentration of PDE, so did reactive oxygen species.But mitochondrial membrane potential fluorescent intensity declined with test concentration increase.4. Immunocytochemistry of methods to measure cells Bcl-2/Bax Group ratio changes, the result showed that the expression of Bcl-2 was depressed in apoptotic cells .On the contrary, the expression of Bax was intensive in apoptotic cells.Conclusions1. Derivative of podophyllotoxin—4-p-amino-benzonic acid-4'-demet-hylepipodophyllotoxin ester(PDE) can significantly inhibit the growthof K562 cells and induce apoptosis. With an increase in the concentration of PDE and the extension of the time, cellular growth inhibitory rate increased.2. In the role of PDE, the concentration of intracellular Ca ion ,H ion and Mg ion increased. Reactive oxygen species increased ,too. Mitochondrial membrane potential disappeared. Which educed that one of mechanisms of PDE inducing tumor cells apoptosis maybe be by increasing the number of intracellular Ca ion , Mg ion and reactive oxygen species, damaging mitochondrial membrane potential stability and eliciting apoptotic genes release.3. The imbalance of the expression of Bcl-2 gene families maybe play a certain role in PDE inducing tumor cells apoptosis .