Light Leaves Annona Diterpenoid Compounds Inhibit Hepatoma Cell Line Smmc-7721 Proliferation And Mechanisms

OBJECTIVE To study the inhibitory effects of diterpenoid compound ( drug A.drug B) isolated from Annona glabra Linn on the proliferotion of SMMC-7721 cells lines and its mechanism .METHODS SMMC-7721 cells were treated with compound drug A and drug B in various concentrations for various days,then observed and analysed by MTT assay ,Flurescent microscopy,Transmission electron microscopy (TEM), Scanning electron microscopy(SEM),Flow cytometry(FCM),Agarose gel electrophoresis and Immunohisto-chemical staining.RESULTS In vitro studies,by means of MTT assaysjt was found the more then 5umol/L drug A and the more then 10umol/L drug B could significantly inhibit the growth of SMMC-7721,the inhibitory effect of 25umol/L drug A was the most power and its 72h inhibition rate was 81.05%. After 48h of treatment with 25umol/L drug A and drug B, "Sub-G0/G1"peaks was recorded in DNA histogram of FCM . It was also showed by FCM that the cell cycle was arrested in G0/G1 phase and the apoptotic rate were43.31%and 24.95% respectively. After 72h of treatment with 25umol/L drug A under the inspection of Flurescent microscopy , SEM and TEM, most of SMMC-7721 cells displayed the typically morphological features of apoptosis, including cell shrinkage,condensation and fragmentation of nuclear chromation,etc. It was observed by Immunohistochemical staining that drug A downregulated expression of bcl-2 and upregulated expression of bax genes .CONCLUSION Diterpenoid compound (drug A,drug B) isolated from Annona glabra Linn inhibite the proliferotion of SMMC-7721 cells lines and induced SMMC-7721 cells apoptosis with downregulation of bcl-2 gene expression and upregulation of bax genes expression.