Tolerance Mechanism Of Tumor Cells And Normal Cells To TRAIL Induced Apoptosis

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines. TRAIL induces apoptotic cell death in a variety of tumor cells, while sparing most normal cells and tissues that are of great concerns in the current cancer therapy. However, the majority of tumor cells are resistance to TRAIL-induced apoptosis. Studies have shown that the mechanisms of TRAIL resistance are not the same in different tumor cells. While TRAIL has no toxic apoptotic effects on normal cells, there are no reports on the mechanism of its lack of apoptotic effects in normal cells.TRAIL binds to death receptors and thus recruits FADD and procaspase-8/-10 to promote the formation of DISC. On the DISC, procaspase-8/-10 dimerizes and initializes self-enzymatic cutting to release active caspases and subsequent caspase cascade of apoptosis in cancer cells. TRAIL-induced apoptosis is independent on the status of the p53. Unlike the mechanisms of radiotherapy and chemotherapy-induced apoptosis that are mainly related to involvement of the Bcl-2 family in the mitochondrial pathway of apoptosis. Therefore, the adjustment of the level of functional DISC that can activate the initiator caspases and executor caspases may be the key to the resistance of cancer cells to TRAIL-induced apoptosis. c-FLIP which contains two DED domains similar to procaspase-8/-10, can inhibit procaspase-8/10 activation by competitively binding to DED domain of FADD. High levels of inhibitor of apoptosis protein (IAPs) directly bind to executive caspase-3/-7/-9 to inhibit their actvities and the apoptosis in TRAIL resistant cancer cells.This study was first to investigate TRAIL-induced apoptosis in several tumor cells and normal cells for the understanding of tolerance mechanisms. Different tumor cells and normal cells were treated with c-FLIP translational inhibitor Rocaglamide, the IAPs pan-antagonist AT 406 and the rhTRAIL protein alone and/or in combination. The survival rates of cells were determined with the MTT assay to understand tolerance mechanisms of each cell type.Our results show that c-FLIP and IAPs are important causes in tumor cells to TRAIL resistance. In the presence of two these proteins inhibitor or antagonist, a relatively sensitive cell HCT 116 and two relative tolerant cells U2-OS and SK-OV-3 were shown to be far more sensitive to TRAIL, while primary cultures of normal cells did not show significant effects. These results exclude the possible causes of c-FLIP and IAPs on the lack of TRAIL-induced apoptosis in normal cells. However, Rocaglamide the inhibitor of c-FLIP induced 50% mortality and potntiated the apoptotic effect of TRAIL in the immortalized normal cell line HEK 293, suggesting that the primary normal cells and the immortalized normal cells have different tolerance mechanisms.The second part of the study, we would like to establish a fluorescence labeled cell model with c-FLIPs as a target for the high-throughput screening of specific anti-cancer drugs. It is based on the protein-protein interactions between c-FLIPs and FADD to achieve drug discovery. Specifically, after double digesting the full sequence of the c-FLIPs DNA obtained from PCR program, we inserted c-FLIPs DNA into the eukaryotic expression vector pEGFP-N2, and then transfected the vector by liposome transfection method to stably express c-FLIPs fused with fluorescently labeled GFP in U2-OS cell. By collecting cell fluorescence imaging, to analyze the distribution of fluorescence change before and after the administration of TRAIL, we can be able to screen the death receptor agonists, the modifier or antagonists and c-FLIP antagonists. The experimental results showed that the fluorescence distributed throughout the cell after transfecting the control empty vector into U2-OS, and cells divided and growed normally. While the fluorescence aggregated without death signaling stimulation after transfecting c-FLIPs-pEGFP-N2 into U2-OS. A few days later these cells could not survive, and the fluorescence decreased with increasing incubation.Since there were reports that high level of expression of c-FLIPs lead to necrosis and cell death, different concentrations of inhibitor of necrosis necrostatin-1 were added to the cultures. The cultured cells were not survived either after a number of days of culture, and fluorescence gradulally reduced with the culture time increased until all gone. Cells transfected with control empty vector could grow normally, suggesting vector and transfection reagent are non-toxic to cells. c-FLIPs in itself may be involved in the negative signal U2-OS cells leading to cell death, the specific cause of the mechanism needs to be further investigated.Our studies have revealed different tolerance mechanisms of tumor cells and normal cells to TRAIL induced apoptosis. Inhibitory proteins to TRAIL induced apoptosis in tumor cells, such as c-FLIP or IAPs, are potential targets for the development of specific anti-cancer agents. The combination therapy of rhTRAIL or agonistic humanized DR monoclonal antibodies or small molecule DR agonists, a c-Flip antagonist and an IAP anatagonist could be the ideal strategy for specific treatment of cancer. Since there is no specific c-FLIP antagonist thus far having been discovered, we have focused on the development of a specific fluorescence labeled cell model for screening c-FLIP antagonists. Further works are required to refine this c-FLIPs-GFP model.