An eIF4E-interacting peptide induces cell death in cancer cell lines

Abstract

The eukaryotic initiation factor eIF4E is essential for cap-dependent initiation of translation in eukaryotes. Abnormal regulation of eIF4E has been implicated in oncogenic transformation. We developed an eIF4E-binding peptide derived from Angel1, a partner of eIF4E that we recently identified. We show here that this peptide fused to a penetratin motif causes drastic and rapid cell death in several epithelial cancer cell lines. This necrotic cell death was characterized by a drop in ATP levels with F-actin network injury being a key step in extensive plasma membrane blebbing and membrane permeabilization. This synthetic eIF4E-binding peptide provides a candidate pharmacophore for a promising new cancer therapy strategy.

Figure 1

A1-IRS peptide inhibits in vitro translation. (a) Sequences of the eIF4E-binding motif of Angel1 (A1), the eIF4E-binding protein 4E-BP2 (BP2), the penetratin IRS domain (IRS) and the synthesized peptides (A1-IRS, A1m-IRS, A1-5 A, BP2-IRS, BP2m-IRS). The consensus eIF4E-binding motif YxxxxLϕ is indicated. (b) Capped and polyadenylated Renilla luciferase mRNA was translated in rabbit reticulocyte lysate in the presence of 50 μM of the indicated peptides. Renilla luciferase activity was then determined as described in Materials and Methods. Translation with the vehicle (DMSO) was arbitrarily set to 100%. The results presented are representative of three independent experiments performed in triplicates and expressed as mean±S.D. Versus group control (vehicle): * P<0.001

Figure 2

A1-IRS induces membrane blebbing and HeLa cell death as evaluated by plasma membrane permeability. HeLa cells were incubated with 50 μM of the indicated synthesized peptides or with the vehicle (DMSO) control. (a) HeLa cells were incubated 30 min with A1-IRS (left panel) or A1m-IRS (right panel), in presence of 5 mM Syto 13 and 12.5 μg/ml propidium iodide (PI). Images were collected on a Zeiss observer Z1 Fluorescence microscope using a × 63 objective. Scale bar=25 μm. (b) Cell viability was determined as the percentage of PI-negative and Syto 13-positive cells using the ImageJ program. At least 200 cells per well were counted. The data are given as the mean±S.D. of triplicates, and are representative of three experiments. Versus group control (vehicle): * P<0.001. (c) A field of HeLa cells were imaged under phase contrast every 10 min using a Leica SP5 microscope with a × 63 objective. The recording begins 2 min after adding A1-IRS in the cell culture medium. Scale bar=25 μm

Figure 3

A1-IRS induces cell death in various cell lines. Cell cultures were incubated with increasing concentrations of the synthetic A1-IRS peptide for 60 min and cell death was determined as described in Figure 2b. Results are expressed as the mean of three independent experiments. HEK-293, adenovirus-transformed epithelial embryonic kidney cells; MDA, mammary gland ductal carcinoma; HGT1, gastric adenocarcinoma; HeLa, cervix adenocarcinoma; Skmel, melanoma; HCT116, colorectal carcinoma; JOK-1, chronic lymphocytic leukemia

Figure 4

A1-IRS cytotoxicity requires eIF4E availability and causes necrosis via F-actin destabilization. (a) JOK-1 cells were incubated with 50 μM of the indicated peptides and analyzed as described for HeLa cells in Figure 2b. The data are given as the mean±S.D. of triplicates, and are representative of three experiments. Versus group control (vehicle): * P<0.001. (b) JOK-1 cells were incubated for 1 h with A1-IRS, A1m-IRS or the vehicle (DMSO). Protein extracts were incubated with m⁷GTP beads to purify eIF4E and its associated factors; eluted proteins were analyzed by western blot with antibodies against eIF4E and eIF4G. (c) Increasing concentrations (from 0 to 30 μM) of A1-IRS or A1m-IRS peptides were added in JOK-1 cells that were previously incubated with 200 μM BP2-IRS or BP2m-IRS for 1 h. After 10 min, cell viability was determined as described in a. Results are representative of three independent experiments. (d) JOK-1 cells were pre-treated with 0.5 ng/μl cytochalasin D for 1 h, or with jasplakinolide for 2 h. A1-IRS or A1m-IRS was then added at the indicated concentration and cell viability was determined after 10 min as described in (a). Results are representative of three independent experiments