Studies On Inhibiting Proliferation And Inducing Apoptosis Effects On A549 Human Lung Carcinoma Cells By Rhizoma Polygonum Cuspidatum Extract

Objective: To investigate the effect of the rhizoma polygonum cuspidatum extracton proliferation and apoptosis on A549 human lung cancer cell line in vitro andexplore its probable molecular mechanisms.Methods: A549 cells were treated with different does and time. The growthinhibition rates and cytotoxic effect of A549 cells were measured with MTT assay.Morphological changes of the apoptosis of cancer cells were observed by HE stainingthrough invert microscope, light microscope, AO/EB double fluorescent stainingunder fluorescent microscope. The TdT-mediated dUTP nick end labeling(TUNEL)method was used to detect the apoptosis-induced by Rhizoma polygonum cuspidatumextract. The distribution of cells cycle and the rate apoptosis were observed by flowcytometry. The bcl-2,ki67,p21ras,Caspase-3,Caspase-8 and Caspase-9 proteinswere determined by immunohistochemical S-P technique.Results: (1)MTT assay showed that the effect of rhizoma polygonum cuspidatumextract was in a does-dependent and time-dependent manner(p＜0.01);(2)Underinverted microscope the A549 cells showed the classical apoptosis changes as the cell membrane blurred and apoptic bodies appeared by treated with 40μg/ml of the rhizompolygonum cuspidatum extract before 48 hours; after 48 hours cell show necrosis.(3)HE stain showed that the observation was the same with in invert microscope, theapoptotic bodies were stained by the classical navy blue and pompadour color, thechanges could be observed under the optical microscope;(4)Apoptosis in theexperimental group was observed by AO/EB double fluorescent staining underfluorescent microscope.(5)TUNEL showed that the apoptotic cells increased withtime raised; the apoptosis rate of experiment groups had different significantlycompared with the control groups(p＜0.01);the apoptosis index was in adoes-dependent and time-dependent manner;(6)Flow cytometry showed the rhizomapolygonum cuspidatum extract could induce the lung cells apoptosis; The percentageof cells in G0/G1,phase increased and the percentage of calls in S and G2/M phasedecreased after the cells treated by the rhizoma polygonum cuspidatum extract; Therhizoma polygonum cuspidatum extract could arrest A549 cells in G0/G1 phase.(7)Immunocytochemistry showed that the expression of Caspase-3,Caspase-8 andCaspase-9 proteins increased, the expression of bcl-2,Ki-67 and p21ras proteinsdecreased after the A549 cells were treated by the rhizoma polygonum cuspidatumextract, there were different significantly compared with control.Conclusions: 1.The proliferation ofA549 human lung cancer cell line was inhibitedsignificantly and was induced apoptosis by the rhizoma polygonum cuspidatumextract in vitro; 2.The mechanism of inhabiting proliferation of A549 cells by therhizoma polygonum cuspidatum extract might be related with down-regulationexpression of Ki-67,p21ras and might be related with arresting cell cycle in G₀/G₁phase; 3.Apoptosis of A549 cell line induced by the rhizome polygonum cuspidatumextract may be related with down-regulation expression of bcl-2 and up-regulationexpression of Caspase-3,8,9;It might be induced apoptosis in A549 cell line by theFas-dependent pathway and mitochondria pathway; 4.The rhizoma polygonumcuspidatum extract had a significant anti-tumor effect, it will be exploded a new kindof anti-tumor medicine;...