Sirtinol, a class III HDAC inhibitor, induces apoptotic and autophagic cell death in MCF-7 human breast cancer cells

Wang,Jing; Kim,Tae Hyung; Ahn,Mee  Young; Lee,Jaewon; Jung,Jee  H.; Choi,Wahn  Soo; Lee,Byung  Mu; Yoon,Kyuing  Sil; Yoon,Sungpil; Kim,Hyung  Sik

doi:10.3892/ijo.2012.1534

Sirtinol, a class III HDAC inhibitor, induces apoptotic and autophagic cell death in MCF-7 human breast cancer cells

Authors:
- Jing Wang
- Tae Hyung Kim
- Mee Young Ahn
- Jaewon Lee
- Jee H. Jung
- Wahn Soo Choi
- Byung Mu Lee
- Kyuing Sil Yoon
- Sungpil Yoon
- Hyung Sik Kim
View Affiliations

Affiliations: College of Pharmacy, Pusan National University, Busan 609-735, Republic of Korea, Department of Immunology, College of Medicine, Konkuk University, Chungju-Si 380-701, Republic of Korea, Division of Toxicology, School of Pharmacy, Sungkyunkwan University, Suwon, Gyeonggi-do, Republic of Korea, Research Institute, National Cancer Center, Goyang-si, Gyeonggi-do, Republic of Korea
Published online on: June 26, 2012 https://doi.org/10.3892/ijo.2012.1534
Pages: 1101-1109

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Sirtuins (SIRTs), NAD+-dependent class III histone deacetylases (HDACs), play an important role in the regulation of cell division, survival and senescence. Although a number of effective SIRT inhibitors have been developed, little is known about the specific mechanisms of their anticancer activity. In this study, we investigated the anticancer effects of sirtinol, a SIRT inhibitor, on MCF-7 human breast cancer cells. Apoptotic and autophagic cell death were measured. Sirtinol significantly inhibited the proliferation of MCF-7 cells in a concentration-dependent manner. The IC50 values of sirtinol were 48.6 µM (24 h) and 43.5 µM (48 h) in MCF-7 cells. As expected, sirtinol significantly increased the acetylation of p53, which has been reported to be a target of SIRT1/2. Flow cytometry analysis revealed that sirtinol significantly increased the G1 phase of the cell cycle. The upregulation of Bax, downregulation of Bcl-2 and cytochrome c release into the cytoplasm, which are considered as mechanisms of apoptotic cell death, were observed in the MCF-7 cells treated with sirtinol. The annexin V-FITC assay was used to confirm sirtinol-induced apoptotic cell death. Furthermore, the expression of LC3-II, an autophagy-related molecule, was significantly increased in MCF-7 cells after sirtinol treatment. Autophagic cell death was confirmed by acridine orange and monodansylcadaverine (MDC) staining. Of note, pre-treatment with 3-methyladenine (3-MA) increased the sirtinol-induced MCF-7 cell cytotoxicity, which is associated with blocking autophagic cell death and increasing apoptotic cell death. Based on our results, the downregulation of SIRT1/2 expression may play an important role in the regulation of breast cancer cell death; thus, SIRT1/2 may be a novel molecular target for cancer therapy and these findings may provide a molecular basis for targeting SIRT1/2 in future cancer therapy.

View Figures

View References

1.	International Agency for Research on Cancer (IARC): World Cancer Report. http://globocan.iarc.fr/factsheets/populations/factsheet.asp?uno=900, 2008.
2.	Yoo KY, Kang D, Park SK, et al: Epidemiology of breast cancer in Korea: occurrence, high-risk groups, and prevention. J Korean Med Sci. 17:1–6. 2002. View Article : Google Scholar : PubMed/NCBI
3.	Esteve JM and Knecht E: Mechanisms of autophagy and apoptosis: Recent developments in breast cancer cells. World J Biol Chem. 26:232–238. 2011. View Article : Google Scholar
4.	Cook KL, Shajahan AN and Clarke R: Autophagy and endocrine resistance in breast cancer. Expert Rev Anticancer Ther. 11:1283–1294. 2011. View Article : Google Scholar : PubMed/NCBI
5.	Aguilar H, Solé X, Bonifaci N, et al: Biological reprogramming in acquired resistance to endocrine therapy of breast cancer. Oncogene. 29:6071–6083. 2010. View Article : Google Scholar : PubMed/NCBI
6.	Yue W, Fan P, Wang J, Li Y and Santen RJ: Mechanisms of acquired resistance to endocrine therapy in hormone-dependent breast cancer cells. J Steroid Biochem Mol Biol. 106:102–110. 2007. View Article : Google Scholar : PubMed/NCBI
7.	Notte A, Leclere L and Michiels C: Autophagy as a mediator of chemotherapy-induced cell death in cancer. Biochem Pharmacol. 82:427–434. 2011. View Article : Google Scholar : PubMed/NCBI
8.	Glick D, Barth S and Macleod KF: Autophagy: cellular and molecular mechanisms. J Pathol. 221:3–12. 2010. View Article : Google Scholar : PubMed/NCBI
9.	Minucci S and Pelicci PG: Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat Rev Cancer. 6:38–51. 2006. View Article : Google Scholar : PubMed/NCBI
10.	Xu WS, Parmigiani RB and Marks PA: Histone deacetylase inhibitors: molecular mechanisms of action. Oncogene. 26:5541–5552. 2007. View Article : Google Scholar : PubMed/NCBI
11.	Blander G and Guarente L: The Sir2 family of protein deacetylases. Annu Rev Biochem. 73:417–435. 2004. View Article : Google Scholar : PubMed/NCBI
12.	Frye RA: Characterization of five human cDNAs with homology to the yeast SIR2 gene: Sir2-like proteins (sirtuins) metabolize NAD and may have protein ADP-ribosyltransferase activity. Biochem Biophys Res Commun. 260:273–279. 1999. View Article : Google Scholar : PubMed/NCBI
13.	Liszt G, Ford E, Kurtev M and Guarente L: Mouse Sir2 homolog SIRT6 is a nuclear ADP-ribosyltransferase. J Biol Chem. 280:21313–21320. 2005. View Article : Google Scholar : PubMed/NCBI
14.	Liu T, Liu PY and Marshall GM: The critical role of the class III histone deacetylase SIRT1 in cancer. Cancer Res. 69:1702–1705. 2009. View Article : Google Scholar : PubMed/NCBI
15.	Fraga MF and Esteller M: Epigenetics and aging: the targets and the marks. Trends Gene. 23:413–418. 2007. View Article : Google Scholar : PubMed/NCBI
16.	Lim CS: Human SIRT1: a protein biomarker for tumorigenesis? Cell Bio Int. 31:636–637. 2007. View Article : Google Scholar
17.	Vaquero A, Scher MB, Lee DH, et al: SirT2 is a histone deacetylase with preference for histone H4 Lys 16 during mitosis. Genes Dev. 20:1256–1261. 2006. View Article : Google Scholar : PubMed/NCBI
18.	Jeong J, Juhn K, Lee H, et al: SIRT1 promotes DNA repair activity and deacetylation of Ku70. Exp Mol Med. 39:8–13. 2007. View Article : Google Scholar : PubMed/NCBI
19.	Yeung F, Hoberg JE, Ramsey CS, et al: Modulation of NF-κB-dependent transcription and cell survival by the SIRT1 deacetylase. EMBO J. 23:2369–2380. 2004.
20.	Brunet A, Sweeney LB, Sturgill JF, et al: Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase. Science. 303:2011–2015. 2004. View Article : Google Scholar : PubMed/NCBI
21.	Motta MC, Divecha N, Lemieux M, et al: Mammalian SIRT represses forkhead transcription factors. Cell. 116:551–563. 2004. View Article : Google Scholar : PubMed/NCBI
22.	Bouras T, Fu M, Sauve AA, et al: SIRT1 deacetylation and repression of p300 involves lysine residues 1020/1024 within the cell cycle regulatory domain 1. J Bio Chem. 280:10264–10276. 2005. View Article : Google Scholar : PubMed/NCBI
23.	Vaziri H, Dessain SK, Ng Eaton E, et al: Hsir2(SIRT1) functions as an NAD-dependent p53 deacetylase. Cell. 107:149–159. 2001. View Article : Google Scholar : PubMed/NCBI
24.	Bedalov A, Gatbonton T, Irvine WP, Gottschling DE and Simon JA: Identification of a small molecule inhibitor of Sir2p. Proc Natl Acad Sci USA. 98:15113–15118. 2001. View Article : Google Scholar : PubMed/NCBI
25.	Grozinger CM, Chao ED, Blackwell HE, Moazed D and Schreiber SL: Identification of a class of small molecule inhibitors of the sirtuin family of NAD-dependent deacetylases by phenotypic screening. J Biol Chem. 276:38837–38843. 2001. View Article : Google Scholar : PubMed/NCBI
26.	Avalos JL, Bever KM and Wolberger C: Mechanism of sirtuin inhibition by nicotinamide: altering the NAD(+) cosubstrate specificity of a Sir2 enzyme. Mol Cell. 17:855–868. 2005.
27.	Bitterman KJ, Anderson RM, Cohen HY, Latorre-Esteves M and Sinclair DA: Inhibition of silencing and accelerated aging by nicotinamide, a putative negative regulator of yeast sir2 and human SIRT1. J Biol Chem. 277:45099–45107. 2002. View Article : Google Scholar : PubMed/NCBI
28.	Ota H, Akishita M, Eto M, Iijima K, Kaneki M and Ouchi Y: Sirt1 modulates premature senescence-like phenotype in human endothelial cells. Mol Cell Cardiol. 43:571–579. 2007. View Article : Google Scholar : PubMed/NCBI
29.	Heltweg B, Gatbonton T, Schuler AD, et al: Antitumor activity of a small-molecule inhibitors of human silent information regulator 2 enzymes. Cancer Res. 66:4368–4377. 2006. View Article : Google Scholar : PubMed/NCBI
30.	Olaharski AJ, Rine J, Marshall BL, et al: The flavoring agent dihydrocoumarin reverses epigenetic silencing and inhibits sirtuin deacetylases. PLoS Genet. 1:e772005. View Article : Google Scholar : PubMed/NCBI
31.	Napper AD, Hixon J, McDonagh T, et al: Discovery of indoles as potent and selective inhibitors of the deacetylase SIRT1. J Med Chem. 48:8045–8054. 2005. View Article : Google Scholar : PubMed/NCBI
32.	Saunders LR and Verdin E: Sirtuins: critical regulators at the crossroads between cancer and aging. Oncogene. 26:5489–5504. 2007. View Article : Google Scholar : PubMed/NCBI
33.	Millot C, Millot JM, Morjani H, Desplaces A and Manfait M: Characterization of acidic vesicles in multidrug-resistant and sensitive cancer cells by acridine orange staining and confocal micro-spectrofluorometry. J Histochem Cytochem. 45:1255–1264. 1997. View Article : Google Scholar
34.	De Duve C, de Barsy T, Poole B, Trouet A, Tulkens P and van Hoof F: Commentary. Lysosomotropic agents. Biochem Pharmacol. 23:2495–2531. 1974.
35.	Paglin S, Hollister T, Delohery T, et al: A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles. Cancer Res. 61:439–444. 2001.PubMed/NCBI
36.	Petiot A, Ogier-Denis E, Blommaart EF, Meijer AJ and Codogno P: Distinct classes of phosphatidylinositol 3-kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. J Biol Chem. 275:992–998. 2000. View Article : Google Scholar : PubMed/NCBI
37.	Fraga MF, Agrelo R and Esteller M: Cross-talk between aging and cancer: the epigenetic language. Ann NY Acad Sci. 1100:60–74. 2007. View Article : Google Scholar : PubMed/NCBI
38.	Mahlknecht U and Hoelzer D: Histone acetylation modifiers in the pathogenesis of malignant disease. Mol Med. 6:623–644. 2000.PubMed/NCBI
39.	Michishita E, Park JY, Burneskis JM, Barrett JC and Horikawa I: Evolutionarily conserved and nonconserved cellular localizations and functions of human SIRT proteins. Mol Biol Cell. 16:4623–4635. 2005. View Article : Google Scholar : PubMed/NCBI
40.	Arico S, Petiot A, Bauvy C, et al: The tumor suppressor PTEN positively regulates macroautophagy by inhibiting the phosphatidylinositol 3-kinase/protein kinase B pathway. J Biol Chem. 276:35243–35246. 2001. View Article : Google Scholar : PubMed/NCBI