The prohibitin-binding compound fluorizoline affects multiple components of the translational machinery and inhibits protein synthesis

Abstract

Fluorizoline (FLZ) binds to prohibitin-1 and -2 (PHB1/2), which are pleiotropic scaffold proteins known to affect signaling pathways involved in several intracellular processes. However, it is not yet clear how FLZ exerts its effect. Here, we show that exposure of three different human cancer cell lines to FLZ increases the phosphorylation of key translation factors, particularly of initiation factor 2 (eIF2) and elongation factor 2 (eEF2), modifications that inhibit their activities. FLZ also impaired signaling through mTOR complex 1, which also regulates the translational machinery, e.g. through the eIF4E-binding protein 4E-BP1. In line with these findings, FLZ potently inhibited protein synthesis. We noted that the first phase of this inhibition involves very rapid eEF2 phosphorylation, which is catalyzed by a dedicated Ca²⁺-dependent protein kinase, eEF2 kinase (eEF2K). We also demonstrate that FLZ induces a swift and marked rise in intracellular Ca²⁺ levels, likely explaining the effects on eEF2. Disruption of normal Ca²⁺ homeostasis can also induce endoplasmic reticulum stress, and our results suggest that induction of this stress response contributes to the increased phosphorylation of eIF2, likely because of activation of the eIF2-modifying kinase PKR-like endoplasmic reticulum kinase (PERK). We show that FLZ induces cancer cell death and that this effect involves contributions from the phosphorylation of both eEF2 and eIF2. Our findings provide important new insights into the biological effects of FLZ and thus the roles of PHBs, specifically in regulating Ca²⁺ levels, cellular protein synthesis, and cell survival.

Keywords: calcium; cancer; cell death; eEF2; eIF2; elongation; endoplasmic reticulum stress (ER stress); eukaryotic initiation factor 2 (eIF2); eukaryotic translation initiation; initiation; prohibitin; protein synthesis; translation elongation factor.

Figure 1.

FLZ evokes eEF2 and eIF2α phosphorylation. A, A549 cells were treated with indicated concentrations of different PHB ligands for 15 or 60 min. Lysate proteins were analyzed by SDS-PAGE/Western blotting using indicated antibodies. B–D, A549 (B), MDA-MB-231 (C), and HeLa (D) cells were treated with 20 μm FLZ for the indicated periods of times. Protein lysates were subjected to SDS-PAGE and Western blot analysis using the indicated antibodies. * = eEF2 band.

Figure 2.

FLZ inhibits protein synthesis in cancer cell lines. A–C, A549 (A), MDA-MB-231 (B), or HeLa cells (C) were treated with vehicle (DMSO) control (C), 20 μm FLZ, or 10 μm CHX for 15 min or 45 min. Cells were labeled with puromycin (SunSET assay) to assess the rate of protein synthesis (analyzed by immunoblotting). Puromycin was then added for a further 15 min. The time windows indicated the periods during which puromycin was present, i.e. 15–30 min and 45–60 min. D, eEF2K^+/+ and eEF2K^−/− MDA-MB-231 cells were treated with 20 μm FLZ; puromycin was present for the indicated time windows, after which cells were lysed and samples subjected to immunoblot analysis. E, eEF2K^−/− MDA-MB-231 cells were treated with 20 μm FLZ for the indicated periods of time before being subjected to SunSET assays. F, quantification of E. For A–C and F, results are shown as mean ± S.D., n = 3. *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01; ***, P < 0.001.

Figure 3.

FLZ increased the affinity of 4E-BP1 to m⁷GTP. A, A549 cells were treated with vehicle (DMSO) or 20 μm FLZ for 15 or 60 min. 4E-BP1 and eIF4E were then isolated by affinity chromatography on immobilized m⁷GTP, followed by analysis using SDS-PAGE/Western blot analysis. C = control. B, 4E-BP1/eIF4E ratio in m⁷GTP beads eluate. C, P-4E-BP1/actin ratio (input). D, 4E-BP1/actin ratio (input). E, P-eEF2/actin ratio (input) from A are quantified. Results are shown as mean ± S.D., n = 3. *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01; ***, P < 0.001.

Figure 4.

FLZ evokes eEF2K activation and induces ISR. A, A549 cells were cultured in the absence or presence of 20 μm FLZ for the indicated periods of time. Cells were then lysed and proteins were subjected to SDS-PAGE and Western blot analysis. B and C, data were quantified in B (P-eEF2:eEF2 ratio) and C (P-eIF2α:eIF2α ratio). D, splicing of XBP1 mRNA upon FLZ (20 μm) and Tg (1 μm) treatment in A549 and MDA-MB-231 cells was assessed by RT-PCR. E, MDA-MB-231 cells were cultured in the absence or presence of 20 μm FLZ for the indicated periods of time. Cells were then lysed and proteins were subjected to SDS-PAGE and Western blot analysis. F, quantification of P-eIF2α:eIF2α ratio from E. B, C, and F, results are given as mean ± S.D.; n = 3. *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01; ***, P < 0.001.

Figure 5.

FLZ increases intracellular calcium levels. Intracellular calcium changes were assessed in A549 cells. Fluorescence from the Fluo-4 calcium sensor was measured with time-lapse image sequences (1 image every 2 min) under 40× magnification while alternating perfusates with or without 20 μm FLZ. Time-lapse image sequences were recorded across 528 cells from 12 FOV in three independent experiments. ROI were drawn over the cell bodies and then averaged to determine changes in fluorescence intensity over time in different perfusates. A, the mean intracellular calcium sensor fluorescence (n = 168 cells from four FOV) in basal medium containing 2 mm CaCl₂ was stable over time in the absence of FLZ. B, the mean intracellular calcium sensor fluorescence (n = 186 cells from four FOVs) in basal medium containing 2 mm CaCl₂ was monitored after applying 20 μm FLZ. FLZ was washed out after 30 min. C, As in (B), but using medium lacking CaCl₂. A–C, data shown as mean ± S.E. Dotted lines represent the time at which the perfusate reached the bath (left) and the time taken to fill the bath entirely (right). Baseline Fluo-4 fluorescence measurements shown between −10 (before addition of FLZ) and 0 min. D, representative images of Fluo-4 fluorescence at 40× magnification in basal medium with and without 2 mm CaCl₂, before and after application of 20 μm FLZ. E, A549 cells were cultured in KRB buffer in the absence or presence of CaCl₂ for 30 min, before incubated with vehicle (DMSO) or 20 μm FLZ. After 15 min of FLZ treatment, cells were lysed and proteins were subjected to SDS-PAGE and Western blot analysis. F, quantified results are given as mean ± S.D.; n = 3. ***, P < 0.001.

Figure 6.

FLZ evokes eEF2K activation and induces ISR to promote cell death. A, A549 cells were pretreated with IPTG (to induce shRNA expression to knock down eEF2K) for 5 days before experiment. Cells were then cultured in the absence or presence of 1 μm ISRIB or/and 20 μm FLZ. 48 h later cells were dispersed, stained with Annexin V (FITC-A) and PI (PI-A), and then analyzed by flow cytometry. B, A549 cells were treated as described for A, and then subjected to cytotoxicity assay using CellTox Green kit. C, eEF2K^+/+ and eEF2K^−/− MDA-MB-231 cells were cultured in the absence or presence of 1 μm ISRIB or/and 20 μm FLZ. 48 h later cells were dispersed, stained with Annexin V (FITC-A) and PI (PI-A), and then analyzed by flow cytometry. D, A549 cells were treated with vehicle (DMSO, control), 20 μm FLZ, or/and 10 μm CHX for the indicated periods of time before subjected to cytotoxicity assay using CellTox Green kit. Results are given as mean ± S.D.; n = 3. *, 0.01 ≤ P < 0.05; **, 0.001 ≤ P < 0.01; ***, P < 0.001.

Figure 7.

Schematic presentation of intracellular signaling pathways evoked by FLZ to promote cell death. The figure recognizes the possibility that the effects of FLZ observed might reflect the interaction of FLZ with targets other than PHBs, although the available data indicate PHBs are the only binding partners for FLZ (27).