Construction Of Expression Plasmids Of ASPP Family And Its Role In ROS-induced Apoptosis In HepG2

Apoptosis, also called programmed cell death, includes exogenous and endogenous pathways. Accurate apoptosis model is very essential for the homeostasis of cell numbers, development of organs, and differentiation of body system. Reactive oxygen species (ROS), as a product of metabolism, are constantly generated and eliminated in the biological system, and play important roles in apoptotic pathway. ASPP family can induce or impede apoptosis by specifically activating or inhibiting p53, and is closely related to tumorigenesis. In addition, overexpression of the ASPP family can affect the tolerance of tumor cells to anticancer drugs, and many anticancer drugs are shown to induce production of intracellular ROS. This implies that close relationship may exist between the ASPP family and ROS.The study attempted to explore the signal transduction pathway of ROS-induced apoptosis, and the role of the ASPP family in ROS-induced apoptosis in tumor cells. It aims to further improve the signal transduction pathway of intracellular ROS, and better understand the mechanisms of ASPP family affecting cells, which can provide new ideas for anti-tumor therapy.Firstly we constructed two expression plasmids:pEGFP-N1-ASPP2and pEGFP-N1-iASPP, and then used H2O2to construct the high oxidative stress environment in HepG2, and overexpressed the ASPP family proteins by cell transfection, to study the mechanism of H2O2-reduced cell activity, and influence of overexpression of ASPP family proteins on H2O2-reduced cell activity. The results are as follows:(1) H2O2could effectively increase the intracellular oxidative stress, induce apoptosis and reduce the cell activity. It also indicated that H2O2up-regulated the expression of p53, Bax, Bak, cytochrome c, down-regulated the expression of Bcl-2, Bcl-xL, and increased activity of Caspase-3and Caspase-9;(2) Overexpression of the ASPP family proteins had no influence on intracellular redox state. Overexpression of ASPP2promoted the H2O2-induced apoptosis. It also enhanced upregulation of Bax, Bak, downregulation of Bcl-2, and increase of Caspase activity induced by H2O2. While the overexpression of iASPP showed the opposite results.The results showed that:(1) H2O2could increase intracellular ROS, and induce apoptosis in HepG2cells by mitochondria-mediated pathway, meanwhile affecting the expression of apoptotic proteins, leading to cytochrome c releasing from mitochondria and activating activity of Caspase family;(2) Respectively, ASPP2acted as a facilitator and iASPP acted as a inhibitor in ROS-induced apoptosis in tumor cells expressing wild-type p53. ROS is closely related to ASPP family in apoptotic pathway. The balance between ASPP2and iASPP is very important to cells.