A small-molecule inhibitor of MDMX activates p53 and induces apoptosis

Abstract

The p53 inactivation caused by aberrant expression of its major regulators (e.g., MDM2 and MDMX) contributes to the genesis of a large number of human cancers. Recent studies have shown that restoration of p53 activity by counteracting p53 repressors is a promising anticancer strategy. Although agents (e.g., nutlin-3a) that disrupt MDM2-p53 interaction can inhibit tumor growth, they are less effective in cancer cells that express high levels of MDMX. MDMX binds to p53 and can repress the tumor suppressor function of p53 through inhibiting its trans-activation activity and/or destabilizing the protein. Here we report the identification of a benzofuroxan derivative [7-(4-methylpiperazin-1-yl)-4-nitro-1-oxido-2,1,3-benzoxadiazol-1-ium, NSC207895] that could inhibit MDMX expression in cancer cells through a reporter-based drug screening. Treatments of MCF-7 cells with this small-molecule MDMX inhibitor activated p53, resulting in elevated expression of proapoptotic genes (e.g., PUMA, BAX, and PIG3). Importantly, this novel small-molecule p53 activator caused MCF-7 cells to undergo apoptosis and acted additively with nutlin-3a to activate p53 and decrease the viability of cancer cells. These results thus show that small molecules targeting MDMX expression would be of therapeutic benefits.

Fig. 1. Discovery of small molecules inhibitory for MDMX expression through a promoter-based drug screening

A, Schematic showing recombination between FRT fragments allowing for integration of a luciferase reporter gene driven by a MDMX promoter into a chromosomal location at Xq23 (33). B, Recombinant cells carrying the integrated MDMX promoter were plated into 96-well plates and treated with 5 µM of compounds derived from the NCI Diversity-Set chemical library for 6 h. Cells were then lysed for luciferase activity assays. The fold changes in the luciferase activity were converted into logarithm values (binary logarithm, i.e., log2) and plotted for each compound. C, Recombinant cells carrying the MDMX promoter or a CMV promoter were treated with the screening hits for 6 h and lysed for luciferase activity assays. D, HT1080 cells were treated with the indicated compounds for 24 h, and lysed for immunoblotting to measure MDMX expression levels.

Fig. 2. A Small-molecule MDMX inhibitor activates p53 leading to activation of pro-apoptotic gene expression

A, The chemical structures of XI-006 and Nutlin-3a. B, Recombinant cells carrying the MDMX promoter were treated with varying amounts of XI-006 for 6 h and lysed for luciferase activity assays. Data are depicted as average ± SD values of 3 determinations. C, MCF-7 cells were treated with XI-006 overnight and lysed for qRT-PCR assays to determine the MDMX mRNA levels. Data are depicted as average ± SD values of 3 determinations. D, MCF-7 cells were treated with DMSO (Ctrl) or XI-006 for 16 h, and lysed for immunoblotting to measure expression levels of MDMX, p53, p21 and MDM2. E, After treatment with 5 µM of XI-006 for 16 h, MCF-7 cells were incubated with 100 µg/ml of cycloheximide. Cells were lysed at the indicated time and subjected to immunoblotting assays. F, Relative p53 levels in (E) was estimated by densitometry analysis. G, MCF-7 cells were treated as in (D). Total RNA was prepared, reverse transcribed, and cDNA subjected to qRT-PCR assays for measuring levels of PUMA, BAX, PIG3, and p21 mRNA. Data are depicted as average ± SD values of 3 determinations. *, p <0.05 versus the DMSO control (Student t-test).

Fig. 3. Activation of p53 by XI-006 is a consequence of inhibition of MDMX expression

A. MCF-7 cells were treated with 0.5 µg/ml of doxorubicin (DOX) or XI-006 as indicated for 16 h and lysed to measure p53 phosphorylation levels. B, MCF-7 cells infected with Lentiviruses expressing shRNA specific to luciferase (shLuc) or MDMX (shMDMX) were treated with varying amounts of XI-006 for 24 h, and subjected to immunoblotting for p53 and MDMX expression. C, MCF-7 cells expressing shMDMX or shLuc were treated as in (B), and subjected to qRT-PCR analysis to measure the levels of the indicated mRNA. Data are depicted as the average ± SD values of 3 determinations. *, p <0.05 versus the corresponding shLuc cells (Student t-test).

Fig. 4. XI-006 induces apoptosis in MCF-7 cells

A & B, MCF-7 cells treated with 5 µM of Nutlin-3a, XI-006, or its analog compound 3268, or DMSO for 24 or 48 h were stained with PI and subjected to flow cytometry analysis. Numbers inserted in graphs in (A) indicate percentages of cells at different stages of the cell cycle. The ratios of the numbers of G1 and S phase cells are shown in (B). C, MCF-7 cells treated with XI-006 for 48 h were subjected to TUNEL staining assays. At least 300 cells were randomly chosen and the numbers of TUNEL-positive cells were counted. D, MCF-7 cells were treated with XI-006 for 2 days, and subjected to immunoblotting to detect cleaved (CL-PARP) and full-length (FL-PARP) PARP. E, MCF-7 cells were treated with 5 µM of XI-006, compound 3268, or DMSO for different days and subjected to flow cytometry to determine percentages of apoptotic cells (subG0/G1 cells). F, MCF-7 cells were treated with XI-006, compound 3268, or Nutlin-3a for 4 days. Cell viability was measured by MTT assays.

Fig. 5. XI-006 induces apoptosis through inhibition of MDMX expression and activation of p53

A, MCF-7 cells infected with Lentiviruses expressing shLuc or shMDMX were treated with XI-006 for 2 days and lysed for immunoblotting assays as in Fig 4C. B, MCF-7 cells were infected with shLuc or shRNA specific to p53 (shp53), treated with 5 µM of XI-00 of XI-006 for 2 days, and lysed for immunoblotting assays. C, Breast cancer cell lines carrying wild-type p53 were lysed and subjected to immunoblotting assays. D, Indicated cells plated in 96-well plates were treated with XI-006 for 3 days and subjected to MTT assays for measuring cell viability.

Fig. 6. XI-006 additively enhances p53 activation and anti-cancer effects of Nutlin-3a

A, The p53-responsive P2 or -nonresponsive P1 promoter of the MDM2 gene was integrated into the same chromosomal location as in Fig 1A. Recombinant cells were treated with 5 µM of Nutlin-3a with or without 2 µM of XI-006 for 6 h, and lysed for luciferase activity assays. B, MCF-7 cells were treated with 5 µM of Nutlin-3a with or without 2 µM of XI-006 for 24 h, and lysed for immunoblotting to measure MDMX, p53 and p21 levels. C, MCF-7 cells were treated with varying amounts of Nutlin-3a with or without 2 µM of XI-006 for 4 days, and subjected to MTT assays to measure cell viability. Data are depicted as average ± SD values of 3 determinations.