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. 2022 Jan 29;23(3):1590.
doi: 10.3390/ijms23031590.

MHY2245, a Sirtuin Inhibitor, Induces Cell Cycle Arrest and Apoptosis in HCT116 Human Colorectal Cancer Cells

Yong Jung Kang  1 , Jung Yoon Jang  1 , Young Hoon Kwon  1 , Jun Ho Lee  1 , Sanggwon Lee  2 , Yujin Park  2 , Young-Suk Jung  1 , Eunok Im  1 , Hyung Ryong Moon  2 , Hae Young Chung  1 , Nam Deuk Kim  1
Affiliations

MHY2245, a Sirtuin Inhibitor, Induces Cell Cycle Arrest and Apoptosis in HCT116 Human Colorectal Cancer Cells

Yong Jung Kang et al. Int J Mol Sci. .

Abstract

Sirtuins (SIRTs), which are nicotinamide adenine dinucleotide-dependent class III histone deacetylases, regulate cell division, survival, and senescence. Although sirtinol, a synthetic SIRT inhibitor, is known to exhibit antitumor effects, its mechanism of action is not well understood. Therefore, we aimed to assess the anticancer effects and underlying mechanism of MHY2245, a derivative of sirtinol, in HCT116 human colorectal cancer cells in vitro. Treatment with MHY2245 decreased SIRT1 activity and caused DNA damage, leading to the upregulation of p53 acetylation, and increased levels of p53, phosphorylation of H2A histone family member X, ataxia telangiectasia and Rad3-related kinase, checkpoint kinase 1 (Chk1), and Chk2. The level of the breast cancer type 1 susceptibility protein was also found to decrease. MHY2245 induced G2/M phase cell cycle arrest via the downregulation of cyclin B1, cell division cycle protein 2 (Cdc2), and Cdc25c. Further, MHY2245 induced HCT116 cell death via apoptosis, which was accompanied by internucleosomal DNA fragmentation, decreased B-cell lymphoma 2 (Bcl-2) levels, increased Bcl-2-asscociated X protein levels, cleavage of poly(ADP-ribose) polymerase, and activation of caspases -3, -8, and -9. Overall, MHY2245 induces cell cycle arrest, triggers apoptosis through caspase activation, and exhibits DNA damage response-associated anticancer effects.

Keywords: DNA damage response; SIRT inhibitor; apoptosis; cell cycle arrest; colorectal cancer cells; sirtinol.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Synthetic scheme for MHY2245. Structures of sirtinol and MHY2245 [2-(naphthalen-1-yl)-2,3-dihydroquinazolin-4(1H)-one].
Figure 2
Figure 2
Effect of MHY2245 on the viability of the human colorectal cancer cell lines HCT116, HT-29, and DLD-1, and the normal human colon epithelial cell line, NCM460D. Graphs showing the viability of HCT116 (A), HT-29 (B), DLD-1 (C), and NCM460D (D) cells treated with increasing concentrations of MHY2245 for 24 h and 48 h. Results are presented as mean ± standard deviation (SD), n = 3, and are expressed as a percentage of viability in vehicle-treated control cells. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with vehicle-treated control cells.
Figure 3
Figure 3
Docking simulation of MHY2245, splitomicin, AGK2 with SIRT1/2, and pharmacophore analysis. Docking simulation results between splitomicin (A) and MHY2245 (B) toward human SIRT1. Pharmacophore results for splitomicin (C) and MHY2245 (D) obtained using LigandScout 4.1.5 showing hydrophobic (yellow) interactions. Docking simulation results between AGK2 (E) and MHY2245 (F) toward human SIRT2. Pharmacophore results for AGK2 (G) and MHY2245 (H) obtained using LigandScout 4.1.5 showing hydrophobic (yellow) interactions and aromatic interactions (violet arrow).
Figure 4
Figure 4
Effect of MHY2245 on SIRT and the expression of DNA damage response mediators and effector cascade in HCT116 cells. (A) Graph showing SIRT1 activity. Results are presented as mean ± SD, n = 3, and are expressed as a percentage of SIRT1 activity in vehicle-treated control cells. ** p < 0.01 and *** p < 0.001 compared with vehicle-treated control cells. (B) Cells were treated with the indicated concentrations of MHY2245 for 24 h and Western blot analyses were conducted to investigate the expression of SIRT1, SIRT2, Ac-p53 (Lys382), p53, γ-H2AX (Ser139), p-ATR (Ser428), p-ATM (Ser1981), p-BRCA1 (Ser1524), p-Chk1 (Ser345), and p-Chk2 (Thr68). β-actin was used as a loading control. Representative results from three independent experiments are shown.
Figure 5
Figure 5
Effect of MHY2245 on cell cycle modulation in HCT116 cells. (A) Exponentially growing cells at 60–70% confluence were treated with the indicated concentrations of MHY2245 for 24 h. Propidium iodide (PI)-stained cells were subjected to flow cytometry analysis to determine the cell distribution at each stage of the cell cycle. (B) The percentage of cells in the G0/G1, S, and G2/M phases of the cell cycle was calculated and is displayed on each bar graph. Results are presented as mean ± SD, n = 3, and are expressed as a percentage of that in vehicle-treated control cells. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with vehicle-treated control cells. (C) Effects of MHY2245 on the expression of cell cycle regulatory proteins analyzed via Western blot analysis of total lysates of cells treated with increasing concentrations of MHY2245. The membranes were probed with Cyclin B1, Cdc2, and Cdc25c. β-actin was used as a loading control. Representative results from three independent experiments are shown.
Figure 6
Figure 6
Effect of MHY2245 on the induction of apoptosis in HCT116 cells. (A) Morphological changes in MHY2245-treated cells. HCT116 cell nuclei were stained with fluorescent DNA-binding dye (Hoechst 33342). Arrows indicate apoptotic cells (×100). (B) Effect of MHY2245 on the death of cells stained with Annexin V-FITC/PI and analyzed via flow cytometry. (C) Graph showing the effect of MHY2245 on cell death. Results are presented as mean ± SD, n = 3, and are expressed as a percentage of that in vehicle-treated control cells. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with vehicle-treated control cells. (D) Representative results of DNA analyses from three independent experiments are provided. M, marker. (E) Effects of MHY2245 on the expression of Bax, Bcl-2, and PARP in HCT116 cells. Western blot analysis of total lysates of cells treated with increasing concentrations of MHY2245. β-actin was used as a loading control. Representative results from three independent experiments are shown.
Figure 7
Figure 7
Role of caspases in MHY2245-induced apoptosis. (A) The expression of pro-caspases-3, -8, and -9 in MHY2245-treated cells was detected via Western blot analysis. β-actin was used as a loading control. Results from three independent experiments are shown. (B) Cell lysates from cells treated with MHY2245 for 24 h were assayed to detect caspase-3, -8, and -9 activities using DEVD-pNA, IETD-pNA, and LEHD-pNA as respective substrates at 37 °C for 1 h. The released fluorescent products were measured. Results are expressed as mean ± SD of three independent experiments. * p < 0.05 and ** p < 0.01 compared with that in vehicle-treated control cells. (C) The presence of cells with sub-G1 DNA content following treatment with MHY2245, indicative of the onset of apoptosis, was detected via flow cytometry. ** p < 0.01 compared with that in vehicle-treated control cells. ## p < 0.01 compared with that in 1.0 μM MHY2245-treated cells. (D) Western blot analysis of PARP and pro-caspase-3 in the total lysates of cells treated with 50 μM Z-VAD-FMK (Z-VAD) and 1.0 μM MHY2245. β-actin was used as a loading control. Representative results from three independent experiments are shown.
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