The effect of sulforaphane on the cell cycle, apoptosis and expression of cyclin D1 and p21 in the A549 non-small cell lung cancer cell line

Żuryń,Agnieszka; Litwiniec,Anna; Safiejko-Mroczka,Barbara; Klimaszewska-Wiśniewska,Anna; Gagat,Maciej; Krajewski,Adrian; Gackowska,Lidia; Grzanka,Dariusz

doi:10.3892/ijo.2016.3444

The effect of sulforaphane on the cell cycle, apoptosis and expression of cyclin D1 and p21 in the A549 non-small cell lung cancer cell line

Authors:
- Agnieszka Żuryń
- Anna Litwiniec
- Barbara Safiejko-Mroczka
- Anna Klimaszewska-Wiśniewska
- Maciej Gagat
- Adrian Krajewski
- Lidia Gackowska
- Dariusz Grzanka
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Affiliations: Department of Histology and Embryology, Nicolaus Copernicus University in Toruń, Collegium Medicum in Bydgoszcz, Faculty of Medicine, 85-092 Bydgoszcz, Poland, Plant Breeding and Acclimatization Institute - National Research Institute, Bydgoszcz Research Center, Department of Genetics and Breeding of Root Crops, Laboratory of Biotechnology, 85-090 Bydgoszcz, Poland, Department of Biology, The University of Oklahoma, Norman, OK 73019-4105, USA, Department of Immunology, Sexually Transmitted Diseases and Immunodermatology, Nicolaus Copernicus University in Toruń, Collegium Medicum in Bydgoszcz, Faculty of Medicine, 85-094 Bydgoszcz, Poland, Department of Dermatology, Sexually Transmitted Diseases and Immunodermatology, Nicolaus Copernicus University in Toruń, Collegium Medicum in Bydgoszcz, Faculty of Medicine, 85-094 Bydgoszcz, Poland
Published online on: March 18, 2016 https://doi.org/10.3892/ijo.2016.3444
Pages: 2521-2533

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Abstract

Sulforaphane (SFN) is present in plants belonging to Cruciferae family and was first isolated from broccoli sprouts. Chemotherapeutic and anticarcinogenic properties of sulforaphane were demonstrated, however, the underlying mechanisms are not fully understood. In this study we evaluated the expression of cyclin D1 and p21 protein in SFN-treated A549 cells and correlated these results with the extent of cell death and/or cell cycle alterations, as well as determined a potential contribution of cyclin D1 to cell death. A549 cells were treated with increasing concentrations of SFN (30, 60 and 90 µM) for 24 h. Morphological and ultrastructural changes were observed using light, transmission electron microscope and videomicroscopy. Image-based cytometry was applied to evaluate the effect of SFN on apoptosis and the cell cycle. Cyclin D1 and p21 expression was determined by flow cytometry, RT-qPCR and immunofluorescence. siRNA was used to evaluate the role of cyclin D1 in the process of suforaphane-induced cell death. We found that the percentage of cyclin D1-positive cells decreased after the treatment with SFN, but at the same time mean fluorescence intensity reflecting cyclin D1 content was increased at 30 µM SFN and decreased at 60 and 90 µM SFN. Percentage of p21-positive cells increased following the treatment, with the highest increase at 60 µM SFN, at which concentration mean fluorescence intensity of this protein was also significantly increased. The 30-µM dose of SFN induced an increased G2/M phase population along with a decreased polyploid fraction of cells, which implies a functional G2/M arrest. The major mode of cell death induced by SFN was necrosis and, to a lower degree apoptosis. Transfection with cyclin D1-siRNA resulted in significantly compromised fraction of apoptotic and necrotic cells, which suggests that cyclin D1 is an important determinant of the therapeutic efficiency of SFN in the A549 cells.

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1	Clarke JD, Dashwood RH and Ho E: Multi-targeted prevention of cancer by sulforaphane. Cancer Lett. 269:291–304. 2008. View Article : Google Scholar : PubMed/NCBI
2	Brooks JD, Paton VG and Vidanes G: Potent induction of phase 2 enzymes in human prostate cells by sulforaphane. Cancer Epidemiol Biomarkers Prev. 10:949–954. 2001.PubMed/NCBI
3	Hecht SS, Kenney PM, Wang M and Upadhyaya P: Benzyl isothiocyanate: An effective inhibitor of polycyclic aromatic hydrocarbon tumorigenesis in A/J mouse lung. Cancer Lett. 187:87–94. 2002. View Article : Google Scholar : PubMed/NCBI
4	Zhang Y, Kensler TW, Cho CG, Posner GH and Talalay P: Anticarcinogenic activities of sulforaphane and structurally related synthetic norbornyl isothiocyanates. Proc Natl Acad Sci USA. 91:3147–3150. 1994. View Article : Google Scholar : PubMed/NCBI
5	Singletary K and MacDonald C: Inhibition of benzo[a]pyrene- and 1,6-dinitropyrene-DNA adduct formation in human mammary epithelial cells bydibenzoylmethane and sulforaphane. Cancer Lett. 155:47–54. 2000. View Article : Google Scholar : PubMed/NCBI
6	Fahey JW, Haristoy X, Dolan PM, Kensler TW, Scholtus I, Stephenson KK, Talalay P and Lozniewski A: Sulforaphane inhibits extracellular, intracellular, and antibiotic-resistant strains of Helicobacter pylori and prevents benzo[a]pyrene-induced stomach tumors. Proc Natl Acad Sci USA. 99:7610–7615. 2002. View Article : Google Scholar
7	Kassie F, Uhl M, Rabot S, Grasl-Kraupp B, Verkerk R, Kundi M, Chabicovsky M, Schulte-Hermann R and Knasmüller S: Chemoprevention of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced colonic and hepatic preneoplastic lesions in the F344 rat by cruciferous vegetables administered simultaneously with the carcinogen. Carcinogenesis. 24:255–261. 2003. View Article : Google Scholar : PubMed/NCBI
8	Chung FL, Conaway CC, Rao CV and Reddy BS: Chemoprevention of colonic aberrant crypt foci in Fischer rats by sulforaphane and phenethyl isothiocyanate. Carcinogenesis. 21:2287–2291. 2000. View Article : Google Scholar
9	Verhoeven DT, Verhagen H, Goldbohm RA, van den Brandt PA and van Poppel G: A review of mechanisms underlying anti-carcinogenicity by brassica vegetables. Chem Biol Interact. 103:79–129. 1997. View Article : Google Scholar : PubMed/NCBI
10	Harper JW, Adami GR, Wei N, Keyomarsi K and Elledge SJ: The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 75:805–816. 1993. View Article : Google Scholar : PubMed/NCBI
11	Xiong Y, Hannon GJ, Zhang H, Casso D, Kobayashi R and Beach D: p21 is a universal inhibitor of cyclin kinases. Nature. 366:701–704. 1993. View Article : Google Scholar : PubMed/NCBI
12	Denicourt C and Dowdy SF: Cip/Kip proteins: More than just CDKs inhibitors. Genes Dev. 18:851–855. 2004. View Article : Google Scholar : PubMed/NCBI
13	Gartel AL, Serfas MS and Tyner AL: p21-negative regulator of the cell cycle. Proc Soc Exp Biol Med. 213:138–149. 1996. View Article : Google Scholar : PubMed/NCBI
14	Waldman T, Kinzler KW and Vogelstein B: p21 is necessary for the p53-mediated G1 arrest in human cancer cells. Cancer Res. 55:5187–5190. 1995.PubMed/NCBI
15	Wu H, Wade M, Krall L, Grisham J, Xiong Y and Van Dyke T: Targeted in vivo expression of the cyclin-dependent kinase inhibitor p21 halts hepatocyte cell-cycle progression, postnatal liver development and regeneration. Genes Dev. 10:245–260. 1996. View Article : Google Scholar : PubMed/NCBI
16	Bunz F, Dutriaux A, Lengauer C, Waldman T, Zhou S, Brown JP, Sedivy JM, Kinzler KW and Vogelstein B: Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science. 282:1497–1501. 1998. View Article : Google Scholar : PubMed/NCBI
17	Sharpless NE and DePinho RA: Telomeres, stem cells, senescence, and cancer. J Clin Invest. 113:160–168. 2004. View Article : Google Scholar : PubMed/NCBI
18	Cariou S, Donovan JC, Flanagan WM, Milic A, Bhattacharya N and Slingerland JM: Down-regulation of p21^WAF1/CIP1 or p27^Kip1 abrogates antiestrogen-mediated cell cycle arrest in human breast cancer cells. Proc Natl Acad Sci USA. 97:9042–9046. 2000. View Article : Google Scholar
19	Giannakakou P, Robey R, Fojo T and Blagosklonny MV: Low concentrations of paclitaxel induce cell type-dependent p53, p21 and G1/G2 arrest instead of mitotic arrest: Molecular determinants of paclitaxel-induced cytotoxicity. Oncogene. 20:3806–3813. 2001. View Article : Google Scholar : PubMed/NCBI
20	Baldin V, Lukas J, Marcote MJ, Pagano M and Draetta G: Cyclin D1 is a nuclear protein required for cell cycle progression in G1. Genes Dev. 7:812–821. 1993. View Article : Google Scholar : PubMed/NCBI
21	Sellers WR and Kaelin WG Jr: Role of the retinoblastoma protein in the pathogenesis of human cancer. J Clin Oncol. 15:3301–3312. 1997.PubMed/NCBI
22	Malumbres M, Sotillo R, Santamaría D, Galán J, Cerezo A, Ortega S, Dubus P and Barbacid M: Mammalian cells cycle without the D-type cyclin-dependent kinases Cdk4 and Cdk6. Cell. 118:493–504. 2004. View Article : Google Scholar : PubMed/NCBI
23	Donnellan R and Chetty R: Cyclin D1 and human neoplasia. Mol Pathol. 51:1–7. 1998. View Article : Google Scholar : PubMed/NCBI
24	Litwiniec A, Gackowska L, Helmin-Basa A, Zuryń A and Grzanka A: Low-dose etoposide-treatment induces endoreplication and cell death accompanied by cytoskeletal alterations in A549 cells: Does the response involve senescence? The possible role of vimentin. Cancer Cell Int. 13:92013. View Article : Google Scholar : PubMed/NCBI
25	Huang H, Hu YD, Li N and Zhu Y: Inhibition of tumor growth and metastasis by non-small cell lung cancer cells transfected with cyclin D1-targeted siRNA. Oligonucleotides. 19:151–162. 2009. View Article : Google Scholar : PubMed/NCBI
26	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(−Delta Delta C) method. Methods. 25:402–408. 2001. View Article : Google Scholar
27	Pledgie-Tracy A, Sobolewski MD and Davidson NE: Sulforaphane induces cell type-specific apoptosis in human breast cancer cell lines. Mol Cancer Ther. 6:1013–1021. 2007. View Article : Google Scholar : PubMed/NCBI
28	Singh AV, Xiao D, Lew KL, Dhir R and Singh SV: Sulforaphane induces caspase-mediated apoptosis in cultured PC-3 human prostate cancer cells and retards growth of PC-3 xenografts in vivo. Carcinogenesis. 25:83–90. 2004. View Article : Google Scholar
29	Herman-Antosiewicz A, Johnson DE and Singh SV: Sulforaphane causes autophagy to inhibit release of cytochrome C and apoptosis in human prostate cancer cells. Cancer Res. 66:5828–5835. 2006. View Article : Google Scholar : PubMed/NCBI
30	Mi L, Wang X, Govind S, Hood BL, Veenstra TD, Conrads TP, Saha DT, Goldman R and Chung FL: The role of protein binding in induction of apoptosis by phenethyl isothiocyanate and sulforaphane in human non-small lung cancer cells. Cancer Res. 67:6409–6416. 2007. View Article : Google Scholar : PubMed/NCBI
31	Jin CY, Moon DO, Lee JD, Heo MS, Choi YH, Lee CM, Park YM and Kim GY: Sulforaphane sensitizes tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis through downregulation of ERK and Akt in lung adenocarcinoma A549 cells. Carcinogenesis. 28:1058–1066. 2007. View Article : Google Scholar
32	Chaudhuri D, Orsulic S and Ashok BT: Antiproliferative activity of sulforaphane in Akt-overexpressing ovarian cancer cells. Mol Cancer Ther. 6:334–345. 2007. View Article : Google Scholar : PubMed/NCBI
33	Bustany S, Cahu J, Guardiola P and Sola B: Cyclin D1 sensitizes myeloma cells to endoplasmic reticulum stress-mediated apoptosis by activating the unfolded protein response pathway. BMC Cancer. 15:2622015. View Article : Google Scholar : PubMed/NCBI
34	Kuroda Y, Sakai A, Tsuyama N, Katayama Y, Munemasa S, Asaoku H, Okikawa Y, Nakaju N, Mizuno M, Ogawa K, et al: Ectopic cyclin D1 overexpression increases chemosensitivity but not cell proliferation in multiple myeloma. Int J Oncol. 33:1201–1213. 2008.PubMed/NCBI
35	Roué G, Pichereau V, Lincet H, Colomer D and Sola B: Cyclin D1 mediates resistance to apoptosis through upregulation of molecular chaperones and consequent redistribution of cell death regulators. Oncogene. 27:4909–4920. 2008. View Article : Google Scholar : PubMed/NCBI
36	Kim JE, Ryu HJ, Kim MJ and Kang TC: LIM kinase-2 induces programmed necrotic neuronal death via dysfunction of DRP1-mediated mitochondrial fission. Cell Death Differ. 21:1036–1049. 2014. View Article : Google Scholar : PubMed/NCBI
37	Zuryń A, Litwiniec A, Klimaszewska-Wiśniewska A, Nowak JM, Gackowska L, Myśliwiec BJ, Pawlik A and Grzanka A: Expression of cyclin D1 after treatment with doxorubicin in the HL-60 cell line. Cell Biol Int. 38:857–867. 2014. View Article : Google Scholar
38	Shankar S, Ganapathy S and Srivastava RK: Sulforaphane enhances the therapeutic potential of TRAIL in prostate cancer orthotopic model through regulation of apoptosis, metastasis, and angiogenesis. Clin Cancer Res. 14:6855–6866. 2008. View Article : Google Scholar : PubMed/NCBI
39	Shen G, Xu C, Chen C, Hebbar V and Kong AN: p53-independent G1 cell cycle arrest of human colon carcinoma cells HT-29 by sulforaphane is associated with induction of p21CIP1 and inhibition of expression of cyclin D1. Cancer Chemother Pharmacol. 57:317–327. 2006. View Article : Google Scholar
40	Yu J, Sun R, Zhao Z and Wang Y: Auricularia polytricha polysaccharides induce cell cycle arrest and apoptosis in human lung cancer A549 cells. Int J Biol Macromol. 68:67–71. 2014. View Article : Google Scholar : PubMed/NCBI
41	Zhang F, Zhang T, Teng ZH, Zhang R, Wang JB and Mei QB: Sensitization to gamma-irradiation-induced cell cycle arrest and apoptosis by the histone deacetylase inhibitor trichostatin A in non-small cell lung cancer (NSCLC) cells. Cancer Biol Ther. 8:823–831. 2009. View Article : Google Scholar : PubMed/NCBI
42	Lv XJ, Zhao LJ, Hao YQ, Su ZZ, Li JY, Du YW and Zhang J: Schisandrin B inhibits the proliferation of human lung adenocarcinoma A549 cells by inducing cycle arrest and apoptosis. Int J Clin Exp Med. 8:6926–6936. 2015.PubMed/NCBI
43	Yuan L, Zhang Y, Xia J, Liu B, Zhang Q, Liu J, Luo L, Peng Z, Song Z and Zhu R: Resveratrol induces cell cycle arrest via a p53-independent pathway in A549 cells. Mol Med Rep. 11:2459–2464. 2015.
44	Sikdar S, Mukherjee A and Khuda-Bukhsh AR: Anti-lung cancer potential of pure esteric-glycoside condurangogenin A against nonsmall-cell lung cancer cells in vitro via p21/p53 mediated cell cycle modulation and DNA damage-induced apoptosis. Pharmacogn Mag. 11(Suppl 1): S73–S85. 2015. View Article : Google Scholar : PubMed/NCBI
45	Singh N, Nambiar D, Kale RK and Singh RP: Usnic acid inhibits growth and induces cell cycle arrest and apoptosis in human lung carcinoma A549 cells. Nutr Cancer. 65(Suppl 1): 36–43. 2013. View Article : Google Scholar : PubMed/NCBI
46	Liu Z, Sun M, Lu K, Liu J, Zhang M, Wu W, De W, Wang Z and Wang R: The long noncoding RNA HOTAIR contributes to cisplatin resistance of human lung adenocarcinoma cells via downregualtion of p21^WAF1/CIP1 expression. PLoS One. 8:e772932013. View Article : Google Scholar