Comparison On Toxicological Effects Of Bombys Batryticatus Ethanol Extract Against Insect Cell HzAM1 And Human Tumor Cell SMMC-7721

This paper was undertaken to discuss the differences on toxicological effects of Bombyx batryticatus ethanol extract(BBE)against insect cell HzAM1?human tumor cell SMMC-7721 on a celluar level,and deeply discuss the mechanism of inducing apoptosis of tumor cells.MTT assay indicated that BBE had unconspicuous effect on cell viability of HzAM1 and no significant toxicity on HzAM1.However,the inhibition of cell viability by BBE on SMMC-7721 cells was correlated with treatment time and concentration.When the concentration of BBE ranged in 0.1mg/mL-1.0 mg/mL,the inhibition rate was increasing with the concentration of BBE increasing.The IC50 value of BBE were 0.8886 mg/mL,0.5998 mg/mL,0.3988 mg/mL,0.1812 mg/mL respectively when SMMC-7721 cells were treated for 12 h,18 h,24 h and 30 h.When BBE was 1.0 mg/mL,the inhibition rate reached to more than ninety percent.Cell morphology observed that cell morphology of HzAM1 kept normal when HzAM1 cells were treated with BBE.However,when SMMC-7721 cells were treated by 0.6 mg/mL BBE for 24 h,inverted microscopy demonstrated the majority of SMMC-7721 cells were severely destroyed,grew slowly,got off from the wall of bottle,overflowed content,turned round and became shrinkage.In order to further clarify the specific induction of apoptosis on tumor cells by BBE,this paper was undertaken to discuss the specific pathways of BBE-induced apoptosis on SMMC-7721.The following were the main results:Laser confocal fluorescence microscopy analysis demonstrated that only cytomembrane was stained bright green fluorescence and cells appeared early apoptosis when cells were treated with 0.6 mg/mL BBE for 3 h.When treatment time lasted to 6 h and 12 h,owing to PI coming into the cell nucleus,not only the cytomembrane was stained bright green fluorescence but also the cell nucleus was stained red green fluorescence and cells appeared late apoptosis.It was stated that BBE induced cell apoptosis via destroying cell membrane firstly and then destroying cell nucleus.Flow cytometry detecting apoptosis rate indicated that treatment of SMMC-7721 cells with 0.1 mg/mL-0.6 mg/mL BBE for a period of 18 h significantly increased the percentages of apoptotic cells from 17.2%,30.3%,35.1%and 38.6%respectively.DNA fragmentation assay indicated that BBE could induce chromosomal DNA into small fragments of DNA breaks.When the concentration of BBE ranged in 0.2 mg/mL-0.5 mg/mL,the extent of DNA fragmentation revealed a progressive increase with the concentration of BBE increasing.The proapoptotic activity of BBE influencing Bax,Bcl-2 and P21 gene expression analysis showed that the expression profiles of Bax and P21 at protein and mRNA level are dramatically increased whereas the synthesis of Bcl-2 is decreased when cells are treated with 0.1 mg/mL-0.6 mg/mL BBE for 18 h.These results suggested that BBE-induced apoptosis on SMMC-7721 cell was achieved by its ability to modulate the high expression of Bax/Bcl-2 and P21 at protein and mRNA level.