Inhibition of protein synthesis by imexon reduces HIF-1alpha expression in normoxic and hypoxic pancreatic cancer cells

Abstract

Hypoxia-inducing factor-1 alpha (HIF-1alpha), is a major survival factor for tumor cells growing in a low oxygen environment. The anti-cancer agent imexon binds thiols and causes accumulation of reactive oxygen species (ROS) in pancreatic cancer cells. Unlike many cytotoxic agents, imexon is equi-cytotoxic in human MiaPaCa-2 and Panc-1 cells grown in normoxic (21% O(2)) and hypoxic (1% O(2)) conditions. Western blot analyses of imexon-treated cells demonstrated that imexon reduces HIF-1alpha protein levels in both normoxic and hypoxic conditions in a time- and concentration-dependant fashion. Gemcitabine did not similarly affect HIF-1alpha levels. Imexon did not reduce transcription of new HIF-1alpha mRNA, but did reduce the synthesis of new proteins, including HIF-1alpha, measured by (35)S methionine/cysteine (Met/Cys) incorporation. Concurrently, the half-life of existing HIF-1alpha protein was increased by imexon, in association with a marked inhibition of chymotryptic activity in the 20S proteasome. The inhibition of HIF-1alpha translation was not specific, rather it was part of a general decrease in protein translation caused by imexon. This inhibitory effect on translation did not involve phosphorylation of eukaryotic initiation factor-2alpha (eIF-2alpha) and was not closely correlated to cell growth inhibition by imexon, suggesting that mechanisms other than protein synthesis inhibition contribute to the drug's cytotoxic effects. In summary, imexon blocks the translation of new proteins, including HIF-1alpha, and this effect overcomes an increase in the stability of preformed HIF-1alpha due to proteasome inhibition by imexon. Because net HIF-1alpha levels are reduced by imexon, combination studies with other drugs affected by HIF-1alpha survival signaling are warranted.

Fig. 1

Concentration dependent decrease in active HIF-1α protein after 24 h imexon under normoxic (a and b) and hypoxic (c and d) conditions. Cells were cultured under normoxic or hypoxic conditions (1% O₂) for 16 h prior to exposing to imexon or gemcitabine for 24 h, harvested and nuclear lysates prepared. Nuclear lysates were separated by SDS-PAGE and immunoblotted for HIF-1α protein. Lamin A/C serves as a loading control. a Panc-1, normoxia; b MiaPaCa-2, normoxia; c Panc-1, hypoxia; d MiaPaCa-2, hypoxia

Fig. 2

Proteasome inhibitor MG132 prevents imexon induced decrease in HIF-1α protein in MiaPaCa-2 cells. Cells were pre-treated with 50–500 nM of MG132 for 15 min prior to incubation with imexon for 24 h. Nuclear lysates were separated by SDS-PAGE and immunoblotted for HIF-1α protein. Lamin A/C serves as a loading control

Fig. 3

a Direct inhibition of 20S proteasome activity by imexon in a cell-free system. Purified human erythrocyte 20S proteasome enzyme (0.2 μg) was incubated with increasing concentrations of imexon or epoxomicin, and 75 μM Suc-LLVY-AMC substrate. Fluorescence readings were taken at 1 min intervals and the specific activity (SA) was calculated as follows: SA (pmol/min)=slope (AFU/min)×conversion factor (μM/AFU)×assay volume (μl), where the conversion factor=1/slope of the standard curve, which was prepared using 7-amino-4-methylcoumarin (AMC). b Inhibition of 20S proteasome activity in imexon treated MiaPaCa-2 cells. Cells were treated with increasing concentrations of imexon or MG132 for 24 h, lysed, and equivalent amounts of total cellular protein assayed for 20S proteasome activity. Cells cultured under normoxic conditions (left) are compared to cells cultured under hypoxic conditions (right)

Fig. 4

Imexon inhibits the synthesis and delays the degradation of HIF-1α protein. Cells were pre-treated with 1,000 μM of imexon for 2 h prior to depletion in methionine- and cysteine-free media (“starve”) for 1 h. 100 μCi/ml of ³⁵S-Met/Cys protein labeling mix was added (“pulse”) for 1 h, after which time the “pulse” media was removed, cells washed, and replaced by “chase” media containing 10% FBS/RPMI for up to 3 h. Exposure to imexon was maintained throughout the experiment. Cells were collected at 0, 1, 2, and 3 h, and lysates immunoprecipitated for HIF-1α protein. Immunoprecipitated proteins were separated by SDS-PAGE, gels dried, and band density quantitated using ImageQuant. Data shown is the densitometric quantitation of HIF-1α protein normalized to Lamin A/C

Fig. 5

Imexon increases HIF-1α protein half-life. MiaPaCa-2 cells were treated under normoxic or hypoxic conditions, with or without imexon (0–24 h) concurrently with cycloheximide (20 μg/ml), a standard inhibitor of protein synthesis. After 30, 60, 90 or 120 min of cycloheximide exposure, cells were harvested and nuclear lysates prepared, proteins separated using SDS-PAGE, and immunoblotted for HIF-1α protein. Data shown is the densitometric quantitation of HIF-1α protein normalized to Lamin A/C, and presented as percent control (0 h). Filled squares normoxia control (0 μM imexon), t_1/2=20 min; filled circles hypoxia control (0 μM imexon), t_1/2=60 min; filled triangles hypoxia (500 μM imexon×4 h), t_1/2>120 min; filled diamonds hypoxia (500 μM imexon×24 h), t_1/2>120 min

Fig. 6

Imexon blocks cellular protein synthesis. Cells were seeded in duplicate 96 well plates and exposed to imexon for 24 h, followed by the addition of 0.5 μCi/ml of ¹⁴C valine (to measure protein synthesis) or MTT dye (to measure cytotoxicity) in tandem. Data are expressed as mean±SEM. a MiaPaCa-2, b Panc-1. Open bars represent ¹⁴C valine incorporation, closed bars represent cytotoxicity by MTT