The chemotherapeutic agent bortezomib induces the formation of stress granules

Abstract

Background: Cytoplasmic stress granules (SGs) are specialized storage sites of untranslated mRNAs whose formation occurs under different stress conditions and is often associated with cell survival. SGs-inducing stresses include radiations, hypoxia, viral infections, and chemical inhibitors of specific translation initiation factors. The FDA-approved drug bortezomib (Velcade(R)) is a peptide boronate inhibitor of the 26S proteasome that is very efficient for the treatment of myelomas and other hematological tumors. Solid tumors are largely refractory to bortezomib. In the present study, we investigated the formation of SGs following bortezomib treatment.

Results: We show that bortezomib efficiently induces the formation of SGs in cancer cells. This process involves the phosphorylation of translation initiation factor eIF2alpha by heme-regulated inhibitor kinase (HRI). Depletion of HRI prevents bortezomib-induced formation of SGs and promotes apoptosis.

Conclusions: This is the first study describing the formation of SGs by a chemotherapeutic compound. We speculate that the activation of HRI and the formation of SGs might constitute a mechanism by which cancer cells resist bortezomib-mediated apoptosis.

Figure 1

Bortezomib induces SGs formation. (A) HeLa cells were treated with 1 μM bortezomib for 3 h, fixed, permeabilized, and processed for immunofluorescence using antibodies against different SG markers. DAPI is used as a marker for nuclei. Pictures were taken using a 100× objective. (B) HeLa cells were treated with 1 μM bortezomib for the indicated times, then incubated with [³⁵S] methionine (50 μCi/ml) for another 30 min. Proteins were resolved on a SDS-polyacrylamide gel, stained with Coomassie Blue (bottom panel), and detected by autoradiography (top panel). (C) HeLa cells were treated with 1 μM bortezomib for the indicated times, and the level of phospho-eIF2α was analyzed by Western blotting using antibodies specific to the phosphorylated form (top panel). Detection of total eIF2α levels is shown in the middle panel and serves as a loading control. The activation of caspase-3 was analyzed using anti-caspase-3 antibodies (bottom panel). (D) HeLa cells were treated with 1 μM bortezomib for 10 h, fixed, permeabilized, and processed for immunofluorescence using antibodies against different SG markers. (E) The indicated histograms represent the percentage of cells harboring SGs (≥5 granules per cell) and is representative of the analysis of five different fields in three independent experiments for a total of 1000 cells. (F) Untreated HeLa cells or cells treated with bortezomib for 24 h were collected, stained with annexin V-FITC and PI, and analyzed by flow cytometry. The percentage of total dead or dying cells (indicated at the top of each panel) was defined as the sum of early (lower right box) and late (upper right box) apoptosis and is presented as the mean ± SEM from 2 independent experiments.

Figure 2

Reducing HRI levels by siRNA prevents bortezomib-induced SGs formation. (A, C) HeLa cells were transfected for 48 h with anti-HRI (HRI-1) or anti-GCN2 siRNAs (GCN2-1), or with a control siRNA. (A, B) q(RT)-PCR of HRI (A) and GCN-2 mRNAs (B). Transfected cells were collected and their mRNA content was isolated. The amount of HRI and GCN-2 mRNAs relative to GAPDH mRNA was quantified by real-time q(RT)-PCR using the ΔΔCt method. The results are presented as the mean of triplicate measurements, with error bars corresponding to the SEM. (C) Transfected cells were processed for immunofluorescence using antibodies against different SG markers, as above. (D) HeLa cells were transfected for 48 h with HRI-1, GCN2-1, or with a control siRNA, and then treated with bortezomib for 4 h. Cells were collected and protein extracts were analyzed by Western blot analysis for the amount of phospho- and total eIF2α as described in Figure 1. (E) HeLa cells were transfected for 48 h with HRI-1 siRNA or with control siRNA, and then treated with bortezomib for 3 h before a 30-min incubation with [³⁵S] methionine (50 μCi/ml). Proteins were resolved on SDS-polyacrylamide gels, stained with Coomassie Blue (bottom panel), and detected by autoradiography (top panel).

Figure 3

Reducing HRI levels by siRNA promotes bortezomib-mediated apoptosis. (A) HeLa cells were transfected for 48 h with HRI-1 or control siRNA, treated with bortezomib for 24 h, and total extracted proteins were analyzed by Western blot using anti-caspase-3 antibodies (top panel). CP: cleaved product. FMRP serves as a loading control (bottom panel). (B) Following treatment with HRI-1 or control siRNA, HeLa cells were incubated with bortezomib for 24 h, then stained with annexin V-FITC and PI, and analyzed by flow cytometry. The percentage of total dead or dying cells (indicated at the top of each panel) was defined as the sum of early (lower right box) and late (upper right box) apoptosis and is presented as the mean ± SEM from two independent experiments. (C) Following treatment with HRI-1 or control siRNA, HeLa cells were incubated with bortezomib for 24 h, trypsinized, counted, replated in the absence of drug, and incubated for 10 d. Before colony counting, cells were fixed and dried. Populations > 50 cells were counted as one surviving colony. Data were calculated as the percentage of surviving colonies relative to control (untreated) plates. The results are expressed as the mean of triplicate measurements.

Figure 4

The formation of SGs correlates with resistance to bortezomib-mediated apoptosis. HeLa, Calu-1, and Hs578T cells were treated with bortezomib for 3 h (A) or 24 h (B to D). (A) Cells were processed for immunofluorescence to detect SGs using anti-FMRP and anti-G3BP antibodies. (B) Cells were stained with annexin V-FITC and PI, and analyzed by flow cytometry. The percentage of total dead or dying cells (indicated at the top of each panel) was defined as the sum of early (lower right box) and late (upper right box) apoptosis and is presented as the means ± SEM from two independent experiments. (C) Cells were harvested and protein extracts analyzed by Western blot for the activation of caspase-3 using anti-caspase-3 antibodies. G3BP serves as a loading control. (D) Following bortezomib treatment for 24 h, cells were trypsinized, replated in the absence of drug, and incubated for 10 d. Before colony counting, cells were fixed and dried. Populations > 50 cells were counted as one surviving colony. Data were calculated as the percentage of surviving colonies relative to untreated plates. The results are presented as the mean of triplicate measurements.