The eIF4E/eIF4G interaction inhibitor 4EGI-1 augments TRAIL-mediated apoptosis through c-FLIP Down-regulation and DR5 induction independent of inhibition of cap-dependent protein translation

Abstract

The small molecule 4EGI-1 was identified as an inhibitor of cap-dependent translation initiation owing to its disruption of the eIF4E/eIF4G association through binding to eIF4E. 4EGI-1 exhibits growth-inhibitory and apoptosis-inducing activity in cancer cells; thus, we were interested in its therapeutic efficacy in human lung cancer cells. 4EGI-1, as a single agent, inhibited the growth and induced apoptosis of human lung cancer cells.When combined with the death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), enhanced apoptosis-induced activity was observed. As expected, 4EGI-1 inhibited eIF4E/eIF4G interaction and reduced the levels of cyclin D1 and hypoxia-inducing factor-1alpha (HIF-1alpha), both of which are regulated by a cap-dependent translation mechanism. Moreover, 4EGI-1 induced CCAAT/enhancer-binding protein homologous protein-dependent DR5 expression and ubiquitin/proteasome- mediated degradation of cellular FLICE-inhibitory protein (c-FLIP). Small interfering RNA-mediated blockade of DR5 induction or enforced expression of c-FLIP abrogated 4EGI-1's ability to enhance TRAIL-induced apoptosis, indicating that both DR5 induction and c-FLIP down-regulation contribute to enhancement of TRAIL-induced apoptosis by 4EGI-1. However, inhibition of eIF4E/eIF4G interaction by knockdown of eIF4E effectively reduced the levels of cyclin D1 and HIF-1alpha but failed to induce DR5 expression, downregulate c-FLIP levels, or augment TRAIL-induced apoptosis. These results collectively suggest that 4EGI-1 augments TRAIL-induced apoptosis through induction of DR5 and down-regulation of c-FLIP, independent of inhibition of cap-dependent protein translation.

Figure 1

4EGI-1 inhibits the growth of human lung cancer cells (A) and induces PARP cleavage (B). (A) The indicated lung cancer cell lines were seeded in 96-well cell culture plates and treated the next day with the given concentrations of 4EGI-1. After 3 days, cell numbers were estimated using the SRB assay. Cell survival was expressed as the percent of control (DMSO-treated) cells. Data are the means of four replicate determinations. Bars, ±SDs. (B) The indicated cancer cell lines were treated with the given concentrations of 4EGI-1 for 24 hours. The cells were then harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis for detecting PARP cleavage. CF indicates cleaved fragment.

Figure 2

4EGI-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 4EGI-1 alone, TRAIL alone, or their respective combinations. After 24 hours, cell numbers were estimated using the SRB assay. Data are the means of four replicate determinations. Bars, ±SDs. (B) The indicated cell lines were treated with 20 ng/ml TRAIL alone, 50 µM 4EGI-1 alone, and their combination. After 24 hours, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. CF indicates cleaved fragment. (C) A549 cells were treated with 40 ng/ml TRAIL alone, 40 µM 4EGI-1 alone, and their combination. After 24 hours, the cells were subjected to measurement of apoptosis using Annexin V staining. The percent positive cells in the upper right and lower right quadrants were added to yield the total of apoptotic cells.

Figure 3

4EGI-1 modulates the levels of DR5 and c-FLIP (A and B), which contribute to enhancement of TRAIL-induced apoptosis (C and D). (A and B) The given cell lines were treated with different concentrations of 4EGI-1 as indicated for 24 hours (A) or 50 µM 4EGI-1 for various times from 3 to 24 hours as indicated and then subjected to preparation of whole-cell protein lysates and subsequent Western blot analysis for the given proteins. (C) The H157 cell lines Lac Z-5, FLIP_L-21 and FLIP_S-1, which express different ectopic FLIPs, as detected by Western blot analysis (left panel), were treated with DMSO, 50 µM 4EGI-1 alone, 20 ng/ml TRAIL alone or 4EGI-1 plus TRAIL. After 24 hours, the cells were harvested and subjected to detection of apoptosis by Annexin V staining (right panel). Columns indicate means of duplicate experiments; bars, ±SE. (D) A549 cells were cultured in a six-well plate and, the next day, transfected with 60nMcontrol (Ctrl) or DR5 siRNA. Forty-eight hours after transfection, the cells were treated with 40 µM 4EGI-1 alone, 20 ng/ml TRAIL alone, or 4EGI-1 plus TRAIL for 24 hours and then harvested for detection of apoptosis by Annexin V staining. Columns indicate means of duplicate experiments; bars, ±SE.

Figure 4

4EGI-1 disrupts eIF4E and eIF4G interaction (A) and inhibits cyclin D1 and HIF-1α expression (B); however, eIF4E siRNA decreases cyclin D1 expression but does not induce DR5 expression, reduce c-FLIP levels (C) or enhance TRAIL-induced apoptosis (D and E). (A) H358 cells were treated with DMSO or 50 µM 4EGI-1 for 6 hours and then harvested for preparation of whole-cell protein lysates and subsequent m⁷GTP pull-down assay. (B) The given cell lines were treated with different concentrations of 4EGI-1 as indicated for 24 hours (upper panel) or 50 µM 4EGI-1 for the indicated times (lower panel) and then subjected to preparation of whole-cell protein lysates and subsequent Western blot analysis. (C) H358 cells were transfected with 20 nM control (Ctrl) or eIF4E siRNA for the indicated times and then subjected to preparation of whole-cell protein lysates and subsequent Western blot analysis. (D and E) A549 cells were transfected with 10 nM control (Ctrl) or eIF4E siRNA for 24 hours and then treated with 40 ng/ml TRAIL. After an additional 16 hours, the cells were harvested for either Western blot analysis of the given proteins (D) or Annexin V assay for detection of apoptotic cells (E). CF indicates cleaved form.

Figure 5

4EGI-1 increases DR5 expression at the transcriptional level (A-C) through a CHOP-dependent mechanism (D and E). (A) The given cell lines were treated with the indicated concentrations of 4EGI-1 for 12 hours and then subjected to preparation of total cellular RNA and subsequent RT-PCR. (B and C) The given reporter constructs were cotransfected with pCH110 plasmid into H358 cells. After 24 hours, the cells were treated with DMSO or 50 µM 4EGI-1 for 14 hours and then subjected to luciferase assay. Columns indicate means of triplicate determinations; bars, ±SDs. (D) The indicated cell lines were treated with the given concentrations of 4EGI-1 for 24 hours (upper panel) or 50 µM 4EGI-1 for the indicated times (lower panel). The cells were then harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. (E) H358 cells were transfected with 60 nM control (Ctrl) or CHOP siRNA. After 48 hours, the cells were treated with 50 µM 4EGI-1 for 14 hours and then subjected to preparation of whole-cell protein lysates and subsequent Western blot analysis. CF indicates cleaved form.

Figure 6

4EGI-1 decreases c-FLIP stability (A) and promotes ubiquitin/proteasome-mediated c-FLIP degradation (B and C). (A) H157 cells were treated with DMSO or 50 µM 4EGI-1 for 5 hours. The cells were then washed with PBS three times and refed with fresh medium containing 10 µg/ml CHX. At the indicated times, the cells were then harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. Protein levels were quantitated with NIH Image J software (Bethesda, MD) and were normalized to actin. The results were plotted as the relative c-FLIP levels compared with those at the time 0 of CHX treatment (lower panel). (B) The indicated cell lines were pretreated with 20 µM MG132 for 30 minutes and then cotreated with 50 µM 4EGI-1 for another 4 hours. The cells were then harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. NSB indicates nonspecific band. (C) H157-FLIP_L-21 cells, which stably express ectopic flag-FLIP_L, were transfected with HA-ubiquitin plasmid using FuGENE 6 transfection reagent for 24 hours. The cells were then pretreated with 20 µM MG132 for 30 minutes and then cotreated with 50 µM 4EGI-1 for 4 hours. Whole-cell protein lysates were then prepared for immunoprecipitation 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.