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. 2013 Jul;4(7):984-94.
doi: 10.18632/oncotarget.1070.

Targeting mutant p53 by a SIRT1 activator YK-3-237 inhibits the proliferation of triple-negative breast cancer cells

Yong Weon Yi  1 , Hyo Jin Kang, Hee Jeong Kim, Yali Kong, Milton L Brown, Insoo Bae
Affiliations

Targeting mutant p53 by a SIRT1 activator YK-3-237 inhibits the proliferation of triple-negative breast cancer cells

Yong Weon Yi et al. Oncotarget. 2013 Jul.

Abstract

Many types of mutations in tumor suppressor p53 are oncogenic through gain-of-function. Therefore, targeting mutant p53 (mtp53) is a promising therapeutic approach to fight against many types of cancers. We report here a small molecule compound YK-3-237 that reduces acetylation of mtp53 and exhibits anti-proliferative effects toward triple-negative breast cancer (TNBC) cells carrying mtp53. YK-3-237 activates SIRT1 enzyme activities in vitro and deacetylation of both mtp53 in a SIRT1-dependent manner. Deacetylation of mtp53 resulted in depletion of mtp53 protein level and up-regulated the expression of WTp53-target genes, PUMA and NOXA. YK-3-237 also induces PARP-dependent apoptotic cell death and arrests the cell cycle at G2/M phase of mtp53 TNBC cells. Taken together, our data suggest that targeting acetylation of mtp53 is a potential target to treat human cancers.

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Conflict of interest statement

Patent applications have been filed by Georgetown University on the behalf of the inventors that are listed as authors in this work.

Figures

Figure 1
Figure 1. YK-3-237 reduces the proliferation and acetylation of mtp53 in breast cancer cell lines
A, structure of YK-3-237. B, relative sensitivities of breast cancer cell lines to YK-3-237. Breast cancer cells were treated with increasing amount of YK-3-237 for ~72 hr and the viable cells were measured by MTT assay. Relative sensitivities were calculated as described in Materials and Methods. The EC50 value of each cell line was plotted in Sup Figure 1. Data are presented as mean ± SEM from two independent experiments performed in triplicate. C, western blot analysis of breast cancer cell lines. Cell lysates from each cell line were analysed by indicated antibodies. D, effect of YK-3-237 on the level of mtp53. Cells were treated with 1 µM of YK-3-237 for 24 hr and western blot analysis was performed with indicated antibodies. E, YK-3-237 reduces acetyl-mtp53. Cells were treated with increasing concentrations of YK-3-237 for 24 hr and cell lysates were subjected to western blot analysis with indicated antibodies. C~E, β-actin was used as a loading control.
Figure 2
Figure 2. The effect of YK-3-237 on the activity of SIRT1
A, the effect of YK-3-237 on the enzyme activity of purified human SIRT1 enzyme in vitro. SIRT1 enzyme assay was performed as described in the Materials and Methods. Data are presented as mean ± SD. *** P < 0.001. B, the effect of YK-3-237 on the SIRT1-mediated repression of WTp53 transcriptional activity. MCF7 cells were transfected with indicated plasmid DNAs and further treated with YK-3-237 for 24 hr. Luciferase reporter activity was measured as described in the Materials and Methods. Data are presented as mean ± SD. **P < 0.01 and ***P < 0.001. C, SIRT1-KD reverses the YK-3-237-mediated repression of WTp53 transcriptional activity. MCF7 cells were transfected with siRNA and p53-Luc and further treated with YK-3-237 for 24 hr. Relative luciferase activity was determined as in B. Data are presented as mean ± SD.*P < 0.05, **P < 0.01, and *** P < 0.001. D, suramin reduces the YK-3-237-mediated deacetylation of mtp53. HS578T cells, pretreated with suramin for 1 hr, were further treated with YK-3-237 for 23 hr. Western blot analysis was performed as indicated. β-actin was used as a loading control. E, the effect of YK-3-237 on the SIRT1-mediated deacetylation of mtp53. SUM149PT cells were transfected with either control (C)- or SIRT1 (S1)-siRNA for 3 days, re-seeded and further treated with YK-3-237 for 4 hr. Western blot analysis was conducted with indicated antibodies. β-actin was used as a loading control.
Figure 3
Figure 3. The effect of mtp53 depletion and YK-3-237 on the expression of WTp53-target genes in TNBC cell lines
A, the effect of mtp53 knockdown on the expression of WTp53-target genes. Cells were transfected with either control (C)- or p53-siRNA and subjected to quantitative real time-PCR (qRT-PCR) as described in Materials and Methods. Data are shown as mean ±SD. Knockdown of mtp53 was assessed by western blot analysis.**P < 0.01 and ***P < 0.001. B, qRT-PCR analysis of WTp53-target genes in cells treated with YK-3-237. Cells were treated with YK-3-237 for 24 hr and qRT-PCR was performed as in A. Data are presented as mean ±SD. *P < 0.05 and ***P < 0.001.
Figure 4
Figure 4. Induction of apoptotic cell death and G2/M arrest by YK-3-237 in TNBC cell lines
Cells were treated with YK-3-237 for 24 hr and both floating and attached cells were harvested for western blot analysis (A) and cell cycle analysis (B). In A, β-actin was used as a loading control.
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