The Roles Of ROS And Caspases In TRAIL-induced Apoptosis And Necroptosis In Human Pancreatic Cancer Cells

Objective:Analyzing ROS and caspase family changes during apoptosis and necroptosis, we compare the fuction and relationship between apoptosis and necroptosis. The data can provide the experimental support and ideas for further research to the research of apoptosis and necroptosis and the potential mechenisim in pancreatic cancer. This provides a clinical therapeutic strategy to the pancreatic cancer.Methods:1. We analyze TRAIL-treated cell viability by using the WST-8 assay to select TRAIL sensitive cell lines. Then we examine the death receptor by FACS to prove that apoptosis is induced by TRAIL. Finally, we use FACS assess the ROS production during aopotosis in TRAIL sensitive pancreatic cancer cells.2. Using FACS analyzes TRAIL sensitive pancreatic cancer cells in condition of ROS inhibition.3. During examination of the effects of ROS inhibition on TRAIL-induced apoptosis of Mia Pa Ca-2 and Bx PC-3 cells, we found that the percentages of annexin V-/PI+ early necrotic cells were increased by TRAIL treatment after the addition of ROS inhibitors. We use FACS to determine whether the addition of necrostatin-1, an inhibitor of RIP1 and of necrosis, could decrease these early necrotic cells.4. We first examine the expression of RIP3 in two cell lines. To testify if the Annexin V-/PI+ cells mentioned above are necroptosis or not, we examined the effect of si RNA-mediated knockdown of RIP3 on necroptosis induced by coincubation with TRAIL and NAC. So that we could campare the different effects by using cell viability test WST-8, FACS.5. First we compare the caspase expression in TRAIL-treated Mia Pa Ca-2 and Bx PC-3 changes by Western Blot. Then, we examine the apoptosis and necroptosis after caspase inhibitor addition by using FACS.6. We next examined the relationship between caspase-9,-2, and ROS in TRAIL-treated pancreatic cancer cells. We first observe the caspase-2 changes in condition of caspase-9 inhibitor by Western Blot, then we observe the caspase-9 changes in condition of caspase-2 inhibitor by Western Blot. Furthermore, we examined the effects of caspase-2 or-9 inhibition on ROS production by TRAIL-treated cells using FACS.Results:1. TRAIL-sensitive pancreatic cancer cells produce ROS.2. Inhibition of ROS decreased TRAIL-induced apoptosis in Mia Pa Ca-2 cells.3. Necroptosis appears TRAIL-treated pancreatic cancer cells under ROS inhibition.4. Necroptosis in TRAIL-treated Bx PC-3 cells under ROS inhibition is RIP3-dependent.5. Caspases are involved in TRAIL-induced apoptosis and necroptosisConclusion:ROS,caspase-2,-9 are involved in apoptosis and necroptosis. As a high level ROS production cancer cell, the level of apoptosis is consistent with ROS production in Mia Pa Ca-2. Inhibition of ROS induces apopotosis and necroptosis in both cell lines, Bx PC-3 is in RIP3-dependent pathway. During the program of apoptosis and necroptosis, caspase-2 is the upstream of caspase-9. There is no crosstalk between caspase-2,-9 and ROS.