Tumor necrosis factor associated apoptosis inducing ligand is a person in the tumor necrosis factor family and is being tested in phase I oncology trials because of its unique capability to trigger apoptosis in a variety of types of cancer cells with restricted toxicity toward normal cells. After washing 3 times with water and once with PBS, cover slips were mounted on cover slides with VECTASHIELD? mounting medium containing diamino 2 phenylinodole. Fluorescent images were acquired with a Zeiss Axiovert 200M florescence microscope equipped with an apotome using AxioVision Rel. Pictures shown in figure were taken using a Zeiss Plan Apochromat ubiquitin conjugation 1. 4 Oil Dic goal. API 1 is a novel small molecule inhibitor of Akt, which acts by binding to Akt and preventing its membrane translocation, and has encouraging preclinical antitumor activity. In this study, we reveal a novel purpose of API 1 in regulation of c FLIP levels and tumor necrosis factor related apoptosis inducing ligand induced apoptosis, independent of Akt inhibition. API 1 effectively induced apoptosis in tested cancer cell RNAP lines including activation of caspase 8 and caspase 9. It paid off the levels of c FLIP without increasing the term of DR4 or DR5. Appropriately, it synergized with TRAIL to induce apoptosis. Enforced expression of ectopic c FLIP did not attenuate API 1 induced apoptosis, but inhibited its ability to enhance TRAILinduced apoptosis. These data indicate that down-regulation of c FLIP mediates enhancement of TRAIL induced apoptosis by API 1, but is not adequate for API 1 induced apoptosis. API 1 induced reduction of d FLIP might be blocked by the proteasome inhibitor MG132. More over, API 1 improved c FLIP ubiquitination and decreased c FLIP security. These information together suggest that API 1 downregulates c FLIP by facilitating its proteasome mediated degradation and ubiquitination. Since other Akt inhibitors including pifithrin a API 2 and MK2206 had minimal effects on reducing c FLIP and enhancement of TRAIL induced apoptosis, it’s likely that API 1 decreases c FLIP and increases TRAIL induced apoptosis independent of its Akt inhibitory activity. API 1 is really a recently discovered small molecule inhibitor of Akt, which functions through binding to Akt and blocking its membrane translocation. A previous study shows that API 1 possesses promising anticancer action, evidenced by its power to suppress cell growth, induce apoptosis and hinder the growth of cancer xenografts, particularly individuals with activated Akt, in nude mice. Nevertheless, several primary tumors are inherently resistant to TRAIL mediated apoptosis and need additional sensitization. PATH triggers apoptosis by binding to cell surface death receptor four or five, this induces oligomerization of the death receptors and development of the death inducing signaling complex, involving recruitment of the adaptor molecule FADD and subsequent caspase 8.