Preliminary Study On The Potentiation Of 2-arsono-acetic Acid Induced Apoptosis By Zinc In SMMC-7721 Cells

Tumor's occurrence and tolerance for treatment is highly concerned with the inhibition of apoptosis (APO). As compared with the necrosis, the APO was needed less stimulation, has a good integrality of cell membrane, and the formed apoptotic body was immediately phagocytosed by the ambient phagocytes and has less affection to the organism. Therefore inducing APO has become to the new mode of treating tumor. 2-arsono-acetic acid (ASAC) is one of a serials of arsenic compounds that we synthesized. Zn2+ may be involved in regulation of cell numbers by its roles in both proliferation and death by apoptosis. In both in vitro and in vivo models, Zn2+can prevent apoptosis induced by a variety of agents and induce apoptosis. The key is the concentration of Zn2+.Objective: To screen a concentration of Zn2+ which can potentiate 2-arsono-acetic acid(ASAC) induced apoptosis on human hepatocellular carcinoma SMMC-7721 cells, and research the potential mechanism of it. Methods:SMMC-7721 cells were treated by ASAC, Zn2+ or ASAC and Zn2+ for various hours, then assays the proliferation variant by MTT and cleavage of the DNA by agarose gel electrophoresis, The changes of morphological features of cells were observed by transmission electronic microscopy and phase contrast microscopy. Flow cytometry showed that ASAC or ASAC.and Zn2+ leads to phase arrest and the mitochondrial membrane potential loss;Using SABCassays detected gene expression of Bcl-2. Results: ASAC significantly inhibited the proliferation of SMMC-7721 cells by inducing apoptosis in human hepatocellular carcinoma SMMC-7721 cells and the inhibitory effect was dose- and time-dependent. ASAC and Zn2+ caused a time-dependent inhibition of survival and growth in human hepatocellular carcinoma SMMC-7721 cells. The inhibitory effect was the best when these cells were treated with 0.1μmol/IASAC and 1μmol/1 Zn2+,Agarose gel electrophorsis showed"ladder"strand of DNA, aspecial phenomenon of cellular apoptosis.Transmission electron microscopy showed cell shrinkage, chromatin condensation, plasma membrane blebbing, and finally the breakdown of the cell into smaller units (apoptotic bodies), Flow cytometry showed that ASAC caused typical apoptotic neak and leads to G2/M arrest ,however the index value of apoptotic was maximum when these cells were treated with 0.1 μmol/lASAC and 1μmol/l Zn2+ and leads to S arrest. The gene expression of Bcl-2 were down-regulated with the extensionof times when these cells were treated, with 0.1μmol/IASAC and 1μmol/lZn2+for various times . Flow cytometry showed that the mitochondrial membrane Potential loss was time-dependent. Conclusion: First, ASAC significantly inhibited the proliferation of SMMC-7721 cells by inducing apoptosis in these cells and the inhibitory effect was dose- and time-dependent. 0.1umol/lASAC and 1μmol/1 Zn2+ significantly inhibited the proliferation of SMMC-7721 cells by inducing apoptosis in these cells and theinhibitory effect was obvious difference with 0.1μmol/IASAC inducing apoptosis; Second, Zn2+ potentiate ASAC induced apoptosis in SMMC-7721 cells and the probablely mechanism of inducing apoptosis is throug the mitochondrial membrane potential loss and the down-regulation of the gene expression of Bcl-2. Third,One of the probablely mechanisms that ASAC induced apoptosis in SMMC-7721 cells is throug the G2/M arrest.