Mutant p53 targeting by the low molecular weight compound STIMA-1

Abstract

Reactivation of mutant p53 in human tumor cells should induce cell death by apoptosis and thus eliminate the tumor. Several small molecules that reactivate mutant p53 have been identified. Here we show that STIMA-1, a low molecular weight compound with some structural similarities to the previously identified molecule CP-31398, can stimulate mutant p53 DNA binding in vitro and induce expression of p53 target proteins and trigger apoptosis in mutant p53-expressing human tumor cells. Human diploid fibroblasts are significantly more resistant to STIMA-1 than mutant or wild type p53-carrying tumor cells. STIMA-1 may provide new insights into possible mechanisms of mutant p53 reactivation and thus facilitate the development of novel anticancer drugs that target mutant p53-carrying tumors.

Figure 1

(A) Chemical structure of STIMA‐1 and CP‐31398. (B) STIMA‐1‐induces mutant p53‐dependent growth suppression in Saos‐2‐His273 and H1299‐His175 cells. Cell growth was assessed using the WST‐1 proliferation agent. Data represent mean±standard error (n=3).

Figure 2

Chemical and biological reactivity of STIMA‐1 and CP‐31398. (A) STIMA‐1 (upper panel) and CP‐31398 (lower panel) form adducts with N‐acetylcysteine (NAC) as shown by HPLC analysis. (B) STIMA‐1‐induced growth suppression in H1299‐His175 cells is completely blocked by NAC according to the WST‐1 assay. The effect of CP‐31398 was only partially blocked by NAC. (C) STIMA‐1, MIRA‐1 and CP‐31398 block thiol groups in recombinant GST‐His175 mutant p53.

Figure 3

Competition for thiol groups in H1299‐His175 cells. Treatment with 10 and 20μM STIMA‐1 caused reduced staining of the FITC‐maleimide conjugate in the cells as compared to control treatment. Pretreatment with NAC inhibited this competition and did not lead to a reduction of the staining after STIMA‐1 treatment.

Figure 4

STIMA‐1 induces cell death by apoptosis. (A) FACS analysis of Saos‐2‐His273 cells before (upper panel) and after (lower panel) treatment with 15μM STIMA‐1. Doxycycline treatment shuts off expression of mutant p53 in Saos‐2‐His273 cells. (B) Quantification of the sub‐G1 cell fraction after STIMA‐1 treatment. (C) Quantification of caspase activation assays in H1299‐His175 and H1299 cells (left panel), in Saos‐2‐His273 and Saos‐2 cells (middle panel) and in Saos‐2‐His273 and Saos‐2‐His273 (+dox) cells (right panel). Data represent mean±standard error (n=2 or 3).

Figure 5

The effect of STIMA‐1 and cisplatin in cells with different p53 status. (A) Diagram showing the ratio between the IC50 values for the mutant p53‐expressing cells/wild type p53 expressing cells and the IC50 values for corresponding p53 null cells. While the Saos‐2‐His273 cells and H1299‐His175 cells are more sensitive to STIMA‐1 treatment, indicating growth suppression in a mutant p53 dependent manner, the wild type p53‐carrying HCT116 cells are more sensitive to cisplatin treatment. (B) IC50 values as determined by the WST‐1 proliferation assay. The mutant p53‐expressing H1299‐His175 and Saos‐2‐His273 cells are more sensitive to STIMA‐1 treatment than their p53 null counterparts and HCT116 p53+/+ and HCT116 p53−/− cells. Human diploid fibroblasts showed the lowest sensitivity to STIMA‐1 treatment. Data represent mean±standard error (n=2).

Figure 6

Expression of p53 and p53 target genes. (A) Western blot analysis of p53 in doxycycline‐treated H1299‐His175 and Saos‐2‐His273 cells. (B–D) Western blot analysis of the expression of p21 (B) and PUMA (C), and analysis of Bax expression (D) by immunofluorescence staining in STIMA‐1‐treated H1299 and H1299‐His175 cells. (C) PUMA protein expression is also induced by treatment with CP‐31398.

Figure 7

Effect of STIMA‐1 on p53 DNA binding as assessed using the TransAM assay. (A) The diagram shows p53 DNA binding activity in MCF7 cells carrying wild type p53 (positive control) and STIMA‐1‐treated H1299‐His175 and untreated parental p53 null H1299 cells. Treatment with 8μM STIMA‐1 enhances p53 DNA binding in H1299‐His175 cells to levels comparable to those in MCF7 cells.