NSC146109 is small molecule strongly activates p53 while selectively inhibiting growth of transformed cells without inducing genotoxicity, indicating its potential as a drug lead for p53-targeted therapy. However, the mechanism(s) by which NSC146109 activates p53 and the effects of NSC146109 on growth of breast cancer cells are currently unknown. Here, we report that NSC146109 promoted breast cancer cells to undergo apoptosis through activating p53 and inducing expression of proapoptotic genes. Importantly, we found that activation of p53 by this small molecule was achieved through a novel mechanism, that is, inhibition of MDMX expression. NSC146109 repressed the MDMX promoter, resulting in down-regulation of MDMX messenger RNA level in MCF-7 cells. In line with these results, NSC146109 decreased the viability of breast cancer cells expressing low levels of MDMX in a less-efficient manner. Interestingly, NSC146109 acted additively with the MDM2 antagonist Nutlin-3a to inhibit growth of breast cancer cells. We conclude that NSC146109 belongs to a novel class of small-molecule p53 activators that target MDMX and could be of value in treating breast cancer.

NSC146109 Activates p53 in Breast Cancer Cells
The pseudourea derivative NSC146109 was identified as one of the most potent active hits in a reporter-based screening for small-molecule p53 activators . We therefore sought to determine whether this small molecule could activate p53 in p53 wild-type MCF-7 breast cancer cells. Toward this end, we treated MCF-7 cells with NSC146109 and measured expression levels of p53 and its downstream target gene p21 using immunoblot assays. As controls, we also treated cells with Nutlin-3a and RITA—two well-characterized small-molecule p53 activators. Interestingly, NSC146109 increased the cellular p53 protein level as efficient as the two known p53 activators. Moreover, NSC146109 treatments resulted in a dose-dependent increase in the p21 protein level, suggesting that p53 was indeed activated by the small molecule . To corroborate these results, we carried out quantitative reverse transcription-PCR (qRT-PCR) assays and found that NSC146109 dramatically increase messenger RNA (mRNA) levels of p21 as well as p53-targeted proapoptotic genes including PUMA, BAX, and PIG3. These results indicated that NSC146109 could strongly activate p53 in breast cancer cells. Moreover, NSC146109 significantly extended the half-life of p53 , consistent with a notion that this small-molecule p53 activator could increase the stability of p53 in breast cancer cells.[1]

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NSC146109 Induces Apoptosis in Breast Cancer Cells
p53 activation can lead to cell cycle arrest or apoptotic cell death. To determine the consequence of p53 activation induced by NSC146109, we treated MCF-7 cells with 0.5 µM of NSC146109 and carried out flow cytometry analysis to examine cell cycle progression. It has been shown that activation of p53 by the MDM2 inhibitor Nutlin-3a results in only cell cycle arrest but not apoptosis in cancer cells overexpressing MDMX (e.g., MCF-7) . Indeed, we found that Nutlin-3a induced a significant decrease in the number of S-phase cells. In contrast, NSC146109 did not appear to diminish the S-phase subpopulation . However, NSC146109 treatments resulted in apparent accumulation of cells in sub-G0/G1 phase , suggesting that this small molecule could rather induce MCF-7 cells to undergo apoptosis. To confirm this important finding, we used TUNEL staining to specifically label apoptotic cells. In line with the notion that NSC146109 could induce apoptosis, numbers of TUNEL-positive cells were largely increased after NSC146109 treatments. This finding was further confirmed by the observations that cleavage of PARP—a biochemical marker for apoptosis—was induced by NSC146109 in a dose-dependent manner. Indeed, more than 40% of MCF-7 cells were apoptotic after treatments with 0.5 µMNSC146109 for 5 days . These results thus indicate that one of the major effects of NSC146109 on breast cancer cells was to induce apoptosis. This effect was in sharp contrast to the effect conferred by Nutlin-3a, suggesting that NSC146109 might activate p53 through a mechanism distinct from the MDM2 inhibitor.[1]

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NSC146109 Induces Apoptosis through Activating p53
To demonstrate that the apoptosis-inducing activity of NSC146109 was a consequence of p53 activation, we knocked down p53 expression in MCF-7 cells using a p53-specific shRNA and determined PARP cleavage induced by NSC146109. Indeed, NSC146109-induced PARP cleavage was largely impaired in p53-downregulated cells . Moreover, the NSC146109-induced expression of proapoptotic genes (i.e., PUMA, BAX, and PIG3) was significantly diminished by knockdown of p53 expression.[1]

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NSC146109 Activates p53 and Induces Apoptosis through Inhibiting MDMX Expression
It has been suggested that the failure in induction of apoptosis by Nutlin-3a in MCF-7 cells was likely due to MDMX overexpression. Because we have shown that NSC146109 was distinct from Nutlin-3a and could induce MCF-7 cells to undergo apoptosis, we tested a hypothesis that NSC146109 activates p53 through targeting MDMX. Interestingly, accompanied by p53 activation, the MDMX expression level was dramatically decreased after MCF-7 cells were treated with NSC146109 . Inhibition of MDMX expression by NSC146109 occurred 4 to 8 hours after treatment when p53 activation started to become notable. These results suggest a strong possibility that p53 activation by NSC146109 could be a consequence of MDMX inhibition. To explore this possibility, we knocked down MDMX expression in MCF-7 cells with an MDMX-specific shRNA and determined p53 activation by immunoblot analysis and qRT-PCR assays. Indeed, activation of p53 and induction of expression of p53 target genes (i.e., PUMA, BAX, and PIG3) were largely impaired in cells expressing a low level of MDMX. Moreover, the cleavage of PARP induced by NSC146109 was also impaired by knockdown of MDMX expression . Furthermore, a small interfering RNA (siRNA) specific to MDMX but targeting a sequence different from the shRNA used above also impaired p53 activation induced by NSC146109 . These results thus indicate that NSC146109 activated p53 and induced apoptosis through inhibiting MDMX expression. Of note, knockdown of MDMX expression by shRNA alone seemed insufficient to activate p53 and induce apoptosis —an observation in agreement with several but in contrast to other reports . The reason for this apparent discrepancy might be related to the fact that the p53 activity is regulated by a complicated network and thus effects of MDMX shRNA might be cell context-dependent.[1]

[1]. A small-molecule p53 activator induces apoptosis through inhibiting MDMX expression in breast cancer cells. Neoplasia. 2011 Jul;13(7):611-9.

See also: (10-Methyl-9-anthryl)methyl imidothiocarbamate (annotation moved to).

C17H16N2S

280.38734

316.08

C, 64.44; H, 5.41; Cl, 11.19; N, 8.84; S, 10.12

59474-01-0

740031-90-7;59474-01-0 (HCl);

16759161

White to yellow solid powder

1.23g/cm3

493.6ºC at 760 mmHg

252.3ºC

1.665

5.985

3

2

3

21

333

0

N=C(N)SCC1=C2C=CC=CC2=C(C)C3=CC=CC=C13.[H]Cl

VIBMUYOXJUCEMA-UHFFFAOYSA-N

InChI=1S/C17H16N2S.ClH/c1-11-12-6-2-4-8-14(12)16(10-20-17(18)19)15-9-5-3-7-13(11)15;/h2-9H,10H2,1H3,(H3,18,19);1H

(10-methylanthracen-9-yl)methyl carbamimidothioate;hydrochloride

NSC-146109; NSC 146109; NSC146109; NSC 146109 HCl

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~83.3 mg/mL (~263.00 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.89 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (6.56 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (6.56 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)