Imexon induces an integrated stress response
Inhibition of protein translation initiation through phosphorylation of eIF2alpha and reduced activity of the eIF2 initiation complex is one of the hallmarks of the integrated ER-stress response [15
]. To determine if imexon treatment induces an integrated ER stress response, we first examined activation of the cis
-acting ER Stress Response Element (ERSE). Pancreatic cell lines were transfected with an ERSE dual-luciferase reportergene (24h) and incubated with the indicated concentrations of imexon for an additional 8h (). The data are expressed as a ratio of ERSE reporter luciferase to Renilla
luciferase, and standardized to untreated cells. We identified an approximate 2-fold increase in ERSE reporter activity in the three cell lines upon exposure to imexon. This reached statistical significance in the Panc-1 and BxPC3 cell lines at concentrations of 500 μM and 1000 μM imexon (). These data were validated using three well- characterized ER stress-inducing agents; thapsigargin, tunicamycin, and H2
, which each demonstrated a 3 to 5-fold increase in ERSE activity (data not shown).
Fig. 2 Imexon activates an ER stress response. (A) Panc-1, (B) BxPC3, and (C) MiaPaCa-2 were transfected with an ERSE reporter, a negative control, or a positive control for 24h, at which time the cells were washed and then exposed to drugs for an additional (more ...)
To investigate the effects of imexon on the expression of additional ER-resident stress response proteins, the three pancreatic cancer cell lines were incubated with increasing concentrations of imexon for 24h. As shown in , all 3 cell lines demonstrated increased expression of the oxidoreductase protein disulfide isomerase (PDI), but as in , no change in the expression of Ero1α. In contrast to the effects of tunicamycin, imexon did not increase levels of the molecular chaperone BiP (Grp78). This suggests that the ER stress response activated by imexon is not likely a secondary effect of misfolded protein accumulation, but rather, may be directly related to ER protein redox status.
Fig. 3 Effect of imexon on ER oxidative response proteins. Pancreatic cell lines were incubated with the indicated concentration of imexon for 24h, and cell lysates were separated by SDS-PAGE and analyzed by immunoblotting. PDI, Ero1α, BiP, and β-actin (more ...)
ER associated proteins participate in the imexon response
To identify gene products that may contribute to imexon-induced ER stress and growth inhibition, we conducted an RNA interference synthetic lethal screen in MiaPaCa-2 cells. An siRNA array was used to individually silence 21,000 genes, and cell survival was assayed by resorufin fluorescence after 48h exposure to imexon. Array plates were incubated with or without minimally toxic concentrations of imexon (72h IC10 of 90 μM). Molecular targets were identified as those resulting in at least 50% change in response to imexon, calculated as the ratio of % survival in the [siRNA + imexon] treatment population to % survival in [siRNA only] treatment. Genes whose silencing resulted in greater than 50% cell death in the [siRNA only] treatment group were eliminated from consideration. Of the remaining 18,512 genes silenced, only 20 known genes demonstrated a cell death ratio less than 50%, signifying an increased contribution to cell growth inhibition by the siRNA + imexon combination (). Three general classes of gene products emerged in this analysis: 1) the serpin family of extracellular peptidase inhibitors; 2) G-coupled receptor proteins; and 3) gene products associated with protein translation. The most significant enhancement of imexon cytotoxicity was seen with silencing of eIF2B5, a guanine nucleotide exchange factor (GEF), which is a key regulator of translation initiation.
Gene products whose silencing resulted in greater than 50% cell death in the presence of 90 μM imexon (IC10)
To validate the effects of eIF2B5 gene silencing on pancreatic cell response to imexon, we examined growth inhibition in three pancreatic cell lines by MTT assay. The MiaPaCa-2, Panc-1, and BxPC3 cells were transfected with eIF2B5 siRNA, followed by incubation with increasing concentrations of imexon for 72h. The data demonstrate that reduction of eIF2B5 expression significantly inhibits the growth of all three pancreatic cell lines, and this effect is enhanced with the addition of imexon (). We also examined the ability of MiaPaca-2 cells to form colonies after eIF2B5 silencing and subsequent exposure to imexon. As was observed with the MTT assay, there was a 70% reduction in colony formation in cells with silenced eIF2B5, and an additional 10–20% reduction in colony formation with the addition of imexon (). However, these latter results did not reach statistical significance. Western blot analysis of eiF2B5 protein expression in pancreatic cell lines following 24h transfection with si eIF2B5 is shown in . These results confirm a near total knock-down of eIF2B5 expression.
Fig. 4 Growth inhibitory effects of imexon in pancreatic cell lines with eIF2B5 silencing. Pancreatic cells were transfected with siRNA to eIF2B5 or a non-targeting sequence (si CT) followed by 72h incubation with imexon (IMX). A) Growth inhibition was analyzed (more ...)
To investigate the role of eIF2B5 in imexon-mediated protein synthesis inhibition, we next examined protein synthesis in the MiaPaCa-2 cell line with or without silencing of eIF2B5. As shown in , siRNA silencing of eIF2B5 inhibits protein synthesis by 70% (p = 0.0017). In the absence of eIF2B5 silencing, comparable inhibition of translation required exposure to 500 μM imexon for 24h. Moreover, the addition of 250 or 500 μM imexon to cells with reduced eIF2B5 expression further inhibited protein translation by only 8.5 and 5.2% respectively. This suggests that the effects of imexon on protein synthesis are mediated within the eIF2 pathway. To address this question, we examined the direct effects of imexon on the expression eIF2B5 and other proteins associated with translation initiation. MiaPaCa-2, Panc-1, and BxPC3 cells were incubated with imexon for 24h, and analyzed by Western blot. In all three cell lines, imexon did not significantly alter the levels of eIF2B5, however there was a dose-dependent increase in the phosphorylation of eIF2alpha, as well as an increase in the levels of GTP exchange protein eIF2B2 (). It is important to note that protein synthesis inhibition () was analyzed following 24h of drug exposure, which would produce minimal cell growth inhibition, whereas growth inhibitory effects () were analyzed after 72h of incubation with imexon.
Fig. 5 Fig. 5A Effects of imexon and eIF2B5 silencing on protein expression. MiaPaCa-2 cells were transfected with eIF2B5 siRNA or a non-targeting sequence (si CT) and examined for protein synthesis. Data are expressed as the percent of control, where the control (more ...)
Fig. 6 Effect of imexon on proteins associated with ER translational control. The pancreatic cell lines MiaPaCa-2, Panc-1, or BxPC3 were incubated with the indicated concentration of imexon for 24h. Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting. (more ...)
Previous studies have demonstrated inhibition of protein synthesis in cells treated with imexon, but a molecular mechanism for the inhibition was not identified [9
]. To determine if the oxidative activity of imexon is responsible for the observed inhibition of protein translation, we examined new protein synthesis under three conditions: pre-treatment with the reducing agent N-acetyl cysteine (NAC) and continuous exposure during imexon incubation period; pre-treatment with NAC and wash out prior to imexon incubation; addition of NAC 2h after imexon treatment. Imexon mediated inhibition of protein synthesis was reversed only when NAC was present during the imexon incubation period (). Furthermore, adding NAC 2h post imexon resulted in a partial reversal of imexon’s oxidative effects, demonstrating that NAC is not simply binding to imexon and preventing it’s uptake into the cell. These data were statistically significant only at the highest concentrations of imexon.
We also examined the effect of the ROS scavengers, PEG-SOD, PEG-CAT and tempol on imexon induced inhibition of protein synthesis (). Although there was a trend toward reversal of protein synthesis inhibition by PEG-CAT, especially at the 250 μM imexon dose, the difference was not statistically significant between treatment groups (p=0.26846, p=0.38651, and p=0.74059 at 100, 250 and 500 μM imexon, respectively). Thus none of these ROS scavengers, except NAC, significantly reversed protein synthesis inhibition caused by imexon.
Protein synthesis is mediated by a series of eukaryotic translation initiation factors (eIFs) that are responsive to a variety of physiological conditions and cell stresses. One of the primary regulators of protein synthesis is the interaction between the classic G-protein, eIF2α and the guanine nucleotide exchange factor (GEF) eIF2B. Phosphorylation of eIF2α in response to ER stress increases its affinity for eIF2B thereby reducing the nucleotide exchange function and resulting in decreased translation initiation [17
]. The current results show that imexon induces the phosphorylation of eIF2α and reduces protein synthesis to approximately the same extent as the silencing of the catalytic subunit of the eIF2B GEF, eIF2B5. These effects were not synergistic, suggesting a common pathway for the inhibitory activity.
The ER is the primary site of folding and post-translational modification of newly synthesized proteins. Protein folding in the ER requires the formation of disulfide bonds in a relatively oxidizing environment. This is accomplished via thiol-disulfide exchange reactions catalyzed by thiol-disulfide oxidoreductases, including protein disulfide isomerases (PDI) and the endoplasmic reticulum oxidase 1 (Ero1) family [18
]. The rate limiting step in oxidative protein folding is generally considered to be the terminal FAD-dependent electron transfer by Ero1α and regeneration of the fully oxidized PDI enzyme [19
]. In order to catalyze these oxidative reactions, the active site disulfides in these enzymes need to be reduced, highlighting the intricate balance of oxidation and reduction that must be maintained for proper protein folding. Our data demonstrate that imexon maintains Ero1α in the oxidized, and thereby, inactive state, suggesting that disruption of the redox status of the ER may be part of imexon’s mechanism of action. Since previous studies have shown that imexon readily reacts with both cysteine and GSH in vitro
], it is possible that imexon is directly binding intracellular GSH leading to a loss of reducing equivalents in the ER, and interfering with redox homeostasis.
In spite of the relatively low ratio of GSH:GSSG in the lumen of the ER (reported to range from 1:1 to 1:3), GSH has been proposed as the primary source of reducing equivalents for ER redox homeostasis [20
]. Previous work by Molteni et al. [22
] and Chakravarthi et al [23
] demonstrated the critical role of GSH as an antagonist of Ero1α. In these studies, depletion of GSH either via cell membrane permeabilization or inhibition of γ-glutamyl cysteine synthetase increased the rate of protein oxidation and lead to an accumulation of oxidized PDI and Ero1α. We cannot speculate as to whether an imexon-GSH conjugate is formed within the ER lumen, or if cytotosolic GSH depletion by imexon is sufficient to reduce the transport of GSH into the ER. Nonetheless, these data suggest that oxidative stress within the ER is induced by exposure to imexon.
The widespread introduction of RNA interference technology has provided an opportunity to systematically identify targets for drug response and resistance [24
]. Several recent studies have highlighted the use of RNA interference chemosensitization screens to identify novel targets with chemotherapeutic agents [25
]. For example, Giroux et al used a human kinome library to identify kinases that support the survival of pancreatic cell lines, and a subset that contribute to gemcitabine resistance [26
]. In a similar study, Azorsa et al used siRNA’s to identify the checkpoint kinase 1 (CHK1) as a sensitizing target in pancreatic cancer cells exposed to gemcitabine [25
]. Using a 21,000 member genome wide siRNA library, we identified molecular pathways associated with protein synthesis as potential mediators of imexon-induced growth inhibition in human pancreatic carcinoma. We initially hypothesized that this screen might highlight anti-oxidant response pathways. In contrast, the major class of gene products whose inhibition significantly enhanced the cytotoxic activity of imexon comprised genes involved in protein synthesis, and particularly eIF2B5.
Previous studies have established imexon as a pro-oxidant that can deplete intracellular thiols and induce oxidative stress [4
]. In addition, we have previously demonstrated that pancreatic cancer cell lines incubated with imexon display a marked inhibition of protein translation and degradation prior to commitment to cell death [9
]. The new findings show that the addition of NAC blocks the effects of imexon on protein synthesis, suggesting that oxidative effects of the drug may mediate the reduction in protein synthesis. The effect of imexon on eIF2α is identical to that of H2
, which typically inhibits translation by the phosphorylation of eIF2α [29
]. However, none of the enzymatic inhibitors of H2
significantly blocked imexon’s effect on protein synthesis. This indirectly suggests that the oxidative stress induced by imexon may be mediated by cellular oxidants other than H2
and superoxide, but still causing phosphorylation of eIF2α.
In summary, the current findings extend the oxidizing effects of imexon from the mitochondria to the ER compartment. The data further suggest that the alteration in ER redox status may mediate the inhibitory effects of imexon on protein synthesis. However, the degree that ER protein oxidation and reduced protein synthesis contribute to overall imexon-induced tumor cell growth inhibition is not known. Importantly, the concentrations of imexon used to inhibit protein translation in the current experiments are within the range of concentrations that can be achieved in cancer patients [30
]. For example, at the maximally tolerated phase I dose of 875 mg/m2
/day, imexon peak plasma levels of 715 μM were routinely obtained.”