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. 2016 Dec 6;12(12):1555-1567.
doi: 10.7150/ijbs.13833. eCollection 2016.

Anticancer Effects of a New SIRT Inhibitor, MHY2256, against Human Breast Cancer MCF-7 Cells via Regulation of MDM2-p53 Binding

Eun Young Park  1 , Youngwoo Woo  1 , Seong Jin Kim  1 , Do Hyun Kim  1 , Eui Kyung Lee  1 , Umasankar De  2 , Kyeong Seok Kim  2 , Jaewon Lee  1 , Jee H Jung  1 , Ki-Tae Ha  3 , Wahn Soo Choi  4 , In Su Kim  2 , Byung Mu Lee  2 , Sungpil Yoon  2 , Hyung Ryong Moon  1 , Hyung Sik Kim  2
Affiliations

Anticancer Effects of a New SIRT Inhibitor, MHY2256, against Human Breast Cancer MCF-7 Cells via Regulation of MDM2-p53 Binding

Eun Young Park et al. Int J Biol Sci. .

Abstract

The sirtuins (SIRTs), a family of NAD+-dependent class III histone deacetylase, are involved in various biological processes including cell survival, division, senescence, and metabolism via activation of the stress-response pathway. Recently, inhibition of SIRTs has been considered a promising anticancer strategy, but their precise mechanisms of action are not well understood. In particular, the relevance of p53 to SIRT-induced effects has not been fully elucidated. We investigated the anticancer effects of a novel SIRT inhibitor, MHY2256, and its efficacy was compared to that of salermide in MCF-7 (wild-type p53) and SKOV-3 (null-type p53) cells. Cell viability, SIRT1 enzyme activity, cell cycle regulation, apoptosis, and autophagic cell death were measured. We compared sensitivity to cytotoxicity in MCF-7 and SKOV-3 cells. MHY2256 significantly decreased the viability of MCF-7 (IC50, 4.8 μM) and SKOV-3 (IC50, 5.6 μM) cells after a 48 h treatment period. MHY2256 showed potent inhibition (IC50, 0.27 mM) against SIRT1 enzyme activity compared with nicotinamide (IC50, >1 mM). Moreover, expression of SIRT (1, 2, or 3) protein levels was significantly reduced by MHY2256 treatment in both MCF-7 and SKOV-3 cells. Flow cytometry analysis revealed that MHY2256 significantly induced cell cycle arrest in the G1 phase, leading to an effective increase in apoptotic cell death in MCF-7 and SKOV-3 cells. A significant increase in acetylated p53, a target protein of SIRT, was observed in MCF-7 cells after MHY2256 treatment. MHY2256 up-regulated LC3-II and induced autophagic cell death in MCF-7 cells. Furthermore, MHY2256 markedly inhibited tumor growth in a tumor xenograft model of MCF-7 cells. These results suggest that a new SIRT inhibitor, MHY2256, has anticancer activity through p53 acetylation in MCF-7 human breast cancer cells.

Keywords: MDM2; MHY2256; SIRT inhibitor; apoptosis; autophagy.; p53.

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Conflict of interest statement

The authors have no competing interests to declare.

Figures

Figure 1
Figure 1
Basal expression levels of p53 and MDM2 proteins in human cancer cell lines. (A) The cells were cultured in the absence of any drug treatment and were analyzed using Western blot analysis with antibodies to p53, MDM2, and β-actin (as a loading control). (B) Immunofluorescence for p53 proteins levels and fluorescence detection of p53 using rhodamine red-tagged secondary antibody was observed using confocal microscopy (Magnification x400).
Figure 2
Figure 2
Effect of MHY2256 on sirtuin (SIRT) and p53 expression in MCF-7 and SKOV-3 cells. (A) Chemical structure of MHY2256 used in the present study. (B) Effects of MHY2256 and nicotinamide on SIRT1 activity. SIRT1 enzyme activity was measured using the SensoLyte® 520 FRET SIRT1 assay kit. The results are presented as the percentage of activity relative to the control in each group. Statistical analysis was performed using one-way analysis of the variance (ANOVA) followed by Bonferroni's multiple comparison tests. **p <0.01 indicates significant differences between control and treatment groups. (C) Expression levels of SIRT1-3 in MHY2256- and salermide-treated cells were detected using Western blot analysis. (D) Effects of MHY2256 on p53, acetylated p53 (Ac-p53), and MDM2 expression. MCF-7 and SKOV-3 cells were treated with MHY2256 and salermide for 48 h, and then Western blot analysis was performed.
Figure 3
Figure 3
Effects of MHY2256 and salermide on cell viability. (A) The cells were treated with MHY2256 and salermide at various concentrations (0.1-50 μM) for 48 h. Viable cells were detected using MTT assay, and viability was determined as the ratio between treated cells and untreated controls. Data are presented as the mean ± SEM of three independent experiments. Statistical analysis was performed using one-way analysis of the variance (ANOVA) followed by Bonferroni's multiple comparison test. *p<0.05, **p <0.01 indicate significant differences between control and treatment groups. (B) Morphological changes in human cancer cells after MHY2256 and salermide treatment. MCF-7 and SKOV-3 cells were incubated for 48 h with the indicated drug concentrations and the cell morphology was examined microscopically (Magnification x100).
Figure 4
Figure 4
Effects of MHY2256 or salermide on cell cycle regulation. MCF-7 (A) and SKOV-3 (B) cells were treated with the indicated concentrations for 48 h. Cells stained with propidium iodide (PI) were subjected to flow cytometric analysis to determine the cell distributions in each phase of the cell cycle. Statistical analysis was performed using one-way analysis of the variance (ANOVA) followed by Bonferroni's multiple comparison tests. *p<0.05 indicate significant differences between control and treatment groups.
Figure 5
Figure 5
Effects of MHY2256 on expression levels of cell cycle regulatory proteins. MCF-7 and SKOV-3 cells were treated with MHY2256 (0, 0.2, 1, and 5 μM) or salermide (50 μM) for 48 h, and then protein levels were detected using Western blot analysis. The cells were homogenized, and the proteins were isolated. Aliquots of proteins were immunoblotted with specific primary antibodies against p21, cyclin D1, cyclin E, cyclin A, CDK4, CDK6 and CDK2. Equal loading and transfer were verified by re-probing the membranes with β-actin antibody.
Figure 6
Figure 6
Effect of MHY2256 on apoptosis. (A) Assay for apoptosis of MHY2256 and salermide treatment in MCF-7 (a) and SKOV-3 (b) cells. Cells were treated with MHY2256 or salermide for 48 h at the indicated concentrations. MHY2256-induced apoptosis was examined using annexin V/7-AAD double staining. Representative flow cytometry scatter plots indicate the percentage of cells in the early and late phases of apoptosis after drug treatment. (B) Effect of MHY2256 on expression of proteins related to apoptotic cell death. Cells were treated with MHY2256 or salermide for 48 h at the indicated concentrations, and Western blot analysis was performed to determine apoptosis-related protein levels. The protein levels were normalized by comparison to β-actin levels.
Figure 7
Figure 7
Effects of MHY2256 on the expression of autophagy-related proteins in MCF-7 and SKOV-3 cells. (A) Cells were treated with MHY2256 and salermide for 48 h at the indicated concentrations, and Western blot analysis was performed with LC3-I/II, Beclin-1, Atg5, and Atg7. Protein levels were normalized by comparison to β-actin levels. (B) Immunofluorescence microscopy of acridine orange-stained MCF-7 and SKOV-3 cells treated for 48 h at the indicated drug concentration (magnification, x200). (C) Histogram profiles of control and drug-treated MCF-7 (a) and SKOV-3 (b) cells analyzed with flow cytometry.
Figure 8
Figure 8
Effects of MHY2256, salermide, and doxorubicin on the growth of MCF-7 tumors in nude mice. Mice with established tumors were randomized into four groups. Vehicle control, doxorubicin (4 mg/kg/week, i.p.), salermide (30 mg/kg/week, i.p.) or MHY2256 (5 mg/kg, twice/week, i.p.) were administered to the tumor-bearing mice. (A) The mean tumor volumes for each treatment group are indicated. (B) Each bar represents the inhibition rate (% of control) of mean tumor weight. The results are presented as the mean ± SEM per group. Statistical analysis was performed using one-way analysis of the variance (ANOVA) followed by Bonferroni's multiple comparison test. *p<0.05, **p <0.01 indicate significant differences between control and treatment groups. (C) The tumors were fixed in 10% formalin and embedded in paraffin. Immunohistochemical staining for Ki-67 were measured in tumors. Magnification x200. Scale bar = 50 μm. The representative images were recorded under a 40x objective lens. (D) Expression levels of p53, acetylated p53, MDM2, Bax, and Bcl-2 were measured in tumor tissues using Western blot analysis. Western blots are representative of three independent experiments.
Figure 9
Figure 9
A scheme depicting the proposed role of MHY2256 as a p53 activator via reduction of MDM2 expression and SIRT inhibitors. MHY2256, a new SIRT inhibitor, has bifunctional effects on SIRT and MDM2. MHY2256 acetylates p53, and acetylated p53 allows for the disruption of MDM2-p53 interaction and promotes the activation of cell cycle-related genes and proapoptotic genes. Furthermore, MHY2256 increased p53 by reducing MDM2 expression levels.
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