The Novel Akt Inhibitor API-1 Induces c-FLIP Degradation and Synergizes with TRAIL to Augment Apoptosis Independent of Akt Inhibition

API-1 downregulates c-FLIP levels without increasing DR4 and DR5 expression. A, the given cell lines were treated with 5 μmol/L API-1 for 24 hours. B, H157 cells were treated with different concentrations of API-1 as indicated for 12 hours. C, H157 cells were treated with 5 μmol/L API-1 for the indicated times. After the aforementioned treatments, the cells were subjected to preparation of whole-cell protein lysates and subsequent Western blot analysis for the given proteins. LE, longer exposure.

API-1 enhances TRAIL-induced apoptosis as evaluated by cell survival (A), caspase activation (B), and Annexin V staining (C). A, the indicated cell lines were seeded in 96-well cell culture plates and treated the next day with the given concentrations of API-1 alone, TRAIL alone, or their respective combinations. After 24 hours, cell numbers were estimated by the SRB assay. Data are the means of 4 replicate determinations. Bars, ±SDs. B and C, the indicated cell lines were treated with 20 ng/mL TRAIL alone, 5 μmol/L API-1 alone, or their combination (A + T). After 24 hours, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis (B) or for measurement of apoptosis using Annexin V staining (C). CF, cleaved fragment. Columns, means of duplicate determinations; bars, ±SDs.

Enforced expression of ectopic c-FLIP attenuates the ability of API-1 to decrease cell survival (A) and induce apoptosis (B) including activation of caspases (C). A, the indicated cell lines were seeded in 96-well cell culture plates and treated the next day with DMSO, different concentrations of API-1 alone, 20 ng/mL TRAIL alone, or API-1 plus TRAIL. After 24 hours, cell numbers were estimated by the SRB assay. Cell survival was expressed as the percentage of control (DMSO treated) cells. Data are the means of 4 replicate determinations. Bars, ±SDs. B and C, the indicated cell lines were treated with DMSO, 5 μmol/L API-1 alone, 20 ng/mL TRAIL alone, or API-1 plus TRAIL. After 24 hours, the cells were harvested for detection of apoptosis with Annexin V staining (B) and for preparation of whole-cell protein lysates and subsequent Western blot analysis to detect the given proteins (C). Columns, means of duplicate experiments; bars, ±SDs. CF, cleaved fragment.

API-1 promotes ubiquitin/proteasome-mediated c-FLIP degradation (A and D) and decreases c-FLIP stability (B and C). A, H157 cells were pretreated with 20 μmol/L MG132 for 30 minutes and then cotreated with 5 μmol/L API-1 for another 4 hours. The cells were then harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. B and C, H157 cells were treated with DMSO or 5 μmol/L API-1 for 5 hours. The cells were then washed with PBS 3 times and refed with fresh medium containing 10 μg/mL cycloheximide (CHX). At the indicated times, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis (B). Protein levels were quantified with the NIH ImageJ software and were normalized to actin. The results were plotted as the relative c-FLIP levels compared with those at time 0 of cycloheximide treatment (C). D, H157-FLIP_L-21 cells, which stably express ectopic flag-FLIP_L, were transfected with HA-ubiquitin plasmid with FuGENE 6 transfection reagent for 24 hours. The cells were then pretreated with 20 μmol/L MG132 for 30 minutes and then cotreated with 5 μmol/L API-1 for 4 hours. Whole-cell protein lysates were then prepared for immunoprecipitation (IP) using anti-Flag antibody followed by Western blot (WB) analysis using anti-HA antibody for detection of ubiquitinated FLIP_L (Ub-FLIP_L) and anti-Flag antibody for detection of ectopic FLIP_L. LE, longer exposure.