Small-molecule activation of p53 blocks hypoxia-inducible factor 1alpha and vascular endothelial growth factor expression in vivo and leads to tumor cell apoptosis in normoxia and hypoxia

Abstract

The p53 tumor suppressor protein negatively regulates hypoxia-inducible factor 1alpha (HIF-1alpha). Here, we show that induction of p53 by the small-molecule RITA (reactivation of p53 and induction of tumor cell apoptosis) [2,5-bis(5-hydroxymethyl-2-thienyl) furan] (NSC-652287) inhibits HIF-1alpha and vascular endothelial growth factor expression in vivo and induces significant tumor cell apoptosis in normoxia and hypoxia in p53-positive cells. RITA has been proposed to stabilize p53 by inhibiting the p53-HDM2 interaction. However, induction of p53 alone was insufficient to block HIF-1alpha induced in hypoxia and has previously been shown to require additional stimuli, such as DNA damage. Here, we identify a new mechanism of action for RITA: RITA activates a DNA damage response, resulting in phosphorylation of p53 and gammaH2AX in vivo. Unlike other DNA damage response-inducing agents, RITA treatment of cells induced a p53-dependent increase in phosphorylation of the alpha subunit of eukaryotic initiation factor 2, requiring PKR-like endoplasmic reticulum kinase activity, and led to the subsequent downregulation of HIF-1alpha and p53 target proteins, including HDM2 and p21. Through the identification of a new mechanism of action for RITA, our study uncovers a novel link between the DNA damage response-p53 pathway and the protein translational machinery.

FIG. 1.

RITA induces p53-dependent inhibition of HIF-1α and VEGF in normoxia and hypoxia. (A to C) Western blot analysis shows HIF-1α and p53 protein levels in MCF-7 cells (A), MDA-MB-231 (B), and p53^−/− HCT116 or p53^+/+ HCT116 cells (C) treated with RITA over a concentration range for 16 h in normoxia or hypoxia (1% O₂). Actin was used as a load control. (D) The graph shows VEGF (pg/ml) secreted into cell culture medium from p53^−/− and p53^+/+ HCT116 cells treated with RITA (0 to 1 μM) for 16 h in hypoxia (1% O₂).

FIG. 2.

RITA induces p53-dependent cell death in normoxia and hypoxia. (A) Photographic images of MCF-7 and MDA-MB-231 cells treated with RITA (1 μM) for 24 h in normoxia and hypoxia (1% O₂). Cells treated with RITA have displaced into the culture medium. (B) FACS analysis of p53^−/− and p53^+/+ HCT116 cells treated with RITA (1 μM) for 24 h in normoxia or hypoxia (1% O₂). Graphs show DNA content of cells (events) measured by propidium iodide (PI) stain and FACS analysis. The percentage of sub-G₁ content (the percentage shown under the M1 bar) indicates the apoptotic population of cells. (C) Western blot analysis shows p53 protein and cleaved PARP cleavage as an indicator of apoptosis in p53^+/+ HCT116 cells transiently transfected with siRNA to p53 or a nonsilencing control (NSC) siRNA duplex. Twenty-four hours after transfection, cells were treated with RITA (+) (1 μM) for 16 h in normoxia or hypoxia (1% O₂). Actin was used as a load control. (D) Western blot analysis shows HIF-1α, cleaved PARP, and p53 proteins in p53^−/− and p53^+/+ HCT116 cells treated with RITA (+) (1 μM) for 16 h in normoxia and hypoxia (1% O₂) in the presence or absence (−) of the caspase-3 inhibitor Z-DEVD-FMK at 25 μM or 50 μM.

FIG. 3.

RITA induces phosphorylation of eIF-2α and blocks HIF-1α protein. (A) Western blot analysis shows HIF-1α and p53 protein levels in p53^+/+ HCT116 cells or p53^−/− HCT116 cells treated with the indicated concentrations of RITA for 16 h in normoxia or hypoxia (1% O₂) in the presence or absence of the proteasome inhibitor MG132 (50 μM). Actin was used as a load control. (B) Western blot analysis shows HIF-1α protein levels in RCC4 cells treated with RITA (1 μM) for 16 h. Actin was used as a load control. (C) Evaluation of HIF-1α transcripts by quantitative real-time PCR. p53^+/+ HCT116 cells were treated with the indicated concentrations of RITA for 16 h in hypoxia (1% O₂). Total RNA was prepared, and quantitative real-time PCR was performed. The graph shows the HIF-1α transcript levels relative to the level of the GAPDH control. PCR products were separated by 3% agarose gel electrophoresis and visualized by ethidium bromide. GAPDH was used as a load control. (D) Western blot analysis shows phosphorylated eIF-2α (P-eIF-2α), phosphorylated p70S6K (P-70S6K), and eIF-2α proteins in p53^−/− HCT116 and p53^+/+ HCT116 cells treated with the indicated concentrations of RITA for 16 h in normoxia or hypoxia (1% O₂). Actin was used as a load control. (E) Western blot analysis shows phosphorylated AMPK (P-AMPK) and total AMPK protein in p53^+/+ HCT116 cells treated with the indicated concentrations of RITA for 16 h in normoxia or hypoxia (1% O₂). Actin was used as a load control.

FIG. 4.

RITA induces phosphorylation of eIF-2α and downregulates p53 targets. (A and B) Western blot analysis shows HIF-1α, p53, HDM2, p21, phosphorylated eIF-2α (P-eIF-2α), total eIF-2α, cleaved PARP, and phosphorylated 4E-BP1 (P-4E-BP1) in p53^+/+ HCT116 cells treated with RITA over a concentration range (0 to 500 nM) for 16 h in normoxia (A) and hypoxia (1% O₂). (B) Actin was used as a load control. (C) The graph shows the results of densitometric analysis of the Western blot for hypoxia conditions as in panel B. HIF-1α, HDM2, and p21 protein levels are represented relative to the actin load control, and phosphorylated eIF-2α levels are represented relative to total eIF-2α protein. (D) Total RNA was prepared from p53^+/+ HCT116 cells treated with RITA and exposed to hypoxia as described above for panel A, and quantitative real-time PCR was performed. The graph shows the p21 transcript levels relative to the level of the GAPDH control and averaged for two independent experiments. Western blot analysis shows p53 and p21 proteins. Actin was used as a load control.

FIG. 5.

PERK activity is important for RITA-mediated induction of eIF-2α phosphorylation. (A) Western blot analysis shows HIF-1α, phosphorylated eIF-2α (P-eIF-2α), and total eIF-2α proteins in p53^+/+ HCT116 cells treated with RITA (1 μM) and exposed to hypoxia (1% O₂) for the times indicated (in hours). Actin was used as a load control. (B) Western blot analysis shows phosphorylated eIF-2α and total eIF-2α proteins in HCT116 cells stably expressing a pCDNA5 control plasmid (control) or a dominant-negative form of PERK (PERKΔC) under the control of the tetracycline promoter. Cells were treated with RITA (1 μM) and exposed to hypoxia (1% O₂) for 16 h. Actin was used as a load control.

FIG. 6.

RITA does not disrupt the p53-HDM2 interaction. (A) Western blot analysis of p53-HDM2 immunoprecipitated (IP) complexes. p53^+/+ HCT116 cells were treated with RITA (1 μM) or nutlin-3 (4 μM) in the presence of MG132 (10 μM) for 16 h. Cell lysates were immunoprecipitated with a monoclonal antibody to p53 (DO-1), and immunoprecipitated p53 complexes were assessed for associated HDM2 protein by Western blotting. Total immunoprecipitated p53 protein was evaluated using a polyclonal antibody. WCE, whole-cell extracts. (B) Western blot analysis shows HIF-1α and p53 proteins in TetON p53-inducible Saos-2 cells treated with doxycycline (Dox) (800 ng/ml) (+) for 24 h, then washed with PBS, and treated with RITA (1 μM) (+) for a further 16 h in normoxia or hypoxia (1% O₂). (C) Western blot analysis shows HIF-1α, HDM2, and p53 proteins in p53^+/+ HCT116 cells treated with nutlin-3 (4 μM) (+) in normoxia (N), hypoxia (H), or deferoxamine mesylate (D) for 16 h. Actin was used as a load control.

FIG. 7.

RITA induces a DNA damage response. (A) Western blot analysis shows HDM2, p53, and phosphorylated-S15-p53 (P-S15-p53) proteins in p53^+/+ HCT116 cells treated with RITA (1 μM) or nutlin-3 (4 μM) for 16 h. (B and C) Immunofluorescence analysis shows p53 (B) and phosphorylated-S139-γH2AX proteins (fluorescein isothiocyanate [FITC]) (C) in p53^+/+ HCT116 cells treated with RITA (1 μM) in normoxia or hypoxia (1% O₂) for 16 h. Nuclei (blue) were visualized with TO-PRO-3 staining. Images (×40) were captured using a confocal microscope (Leica). (D) Western blot analysis shows HIF-1α, p53, phosphorylated S15-p53, HDM2, p21, eIF-2α, phosphorylated eIF-2α, and PARP proteins in p53^−/− and p53^+/+ HCT116 cells treated with DMSO, 1 μM RITA, 1 mM hydroxyurea (HU), doxorubicin (Dox), cisplatin (Cisp), or 25 μM etoposide (Etop) for 16 h in hypoxia (1% O₂). The status of p53 is shown above the lanes: +, present; −, absent.

FIG. 8.

RITA induces p53 and γH2AX phosphorylation and blocks HIF-1α and VEGF expression in tumor xenografts. (A) Western blot analysis of p53^+/+ HCT116 tumor xenograft lysates. Mice bearing p53^+/+ HCT116 cell-derived tumor xenografts were administered with a single intraperitoneal dose of RITA at 10 mg/kg for 24 h. Tumor cell lysates from eight tumors were analyzed per group. Western blots show a representation of two independent tumors (tumor 1 [T1] and T2) from the DMSO- and RITA-treated groups, assessed for HIF-1α, HDM2, p53, phosphorylated-S15-p53 (P-S15-p53), and phosphorylated-S139-γH2AX (P-S139-H2AX) proteins. Actin was used as a load control. (B) The graph shows VEGF levels in tumor lysates. Four data points are shown for each condition. Experiments show two independent tumors (T1 and T2) in two independent experiments (diamonds and circles). Abbreviations: IP, intraperitoneally; hr, hours.