title: 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.
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.
authors.affiliation: Department of Histology and Embryology, Nicolaus Copernicus University in Toruń, Collegium Medicum in Bydgoszcz, Faculty of Medicine, 85-092 Bydgoszcz, Poland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Żuryń
authors.affiliation: 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.
authors.forename: Anna
authors.initials: A
authors.lastname: Litwiniec
authors.affiliation: Department of Biology, The University of Oklahoma, Norman, OK 73019-4105, USA.
authors.forename: Barbara
authors.initials: B
authors.lastname: Safiejko-Mroczka
authors.affiliation: Department of Histology and Embryology, Nicolaus Copernicus University in Toruń, Collegium Medicum in Bydgoszcz, Faculty of Medicine, 85-092 Bydgoszcz, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Klimaszewska-Wiśniewska
authors.affiliation: Department of Histology and Embryology, Nicolaus Copernicus University in Toruń, Collegium Medicum in Bydgoszcz, Faculty of Medicine, 85-092 Bydgoszcz, Poland.
authors.forename: Maciej
authors.initials: M
authors.lastname: Gagat
authors.affiliation: Department of Histology and Embryology, Nicolaus Copernicus University in Toruń, Collegium Medicum in Bydgoszcz, Faculty of Medicine, 85-092 Bydgoszcz, Poland.
authors.forename: Adrian
authors.initials: A
authors.lastname: Krajewski
authors.affiliation: Department of Immunology, Sexually Transmitted Diseases and Immunodermatology, Nicolaus Copernicus University in Toruń, Collegium Medicum in Bydgoszcz, Faculty of Medicine, 85-094 Bydgoszcz, Poland.
authors.forename: Lidia
authors.initials: L
authors.lastname: Gackowska
authors.affiliation: Department of Dermatology, Sexually Transmitted Diseases and Immunodermatology, Nicolaus Copernicus University in Toruń, Collegium Medicum in Bydgoszcz, Faculty of Medicine, 85-094 Bydgoszcz, Poland.
authors.forename: Dariusz
authors.initials: D
authors.lastname: Grzanka
mesh_terms: D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology*; D017209:Apoptosis; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D019938:Cyclin D1 / Q000235:genetics* / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics* / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D017879:Isothiocyanates / Q000494:pharmacology*; D008175:Lung Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism; D013454:Sulfoxides
chemical_list: D000972:Antineoplastic Agents, Phytogenic; C530104:CCND1 protein, human; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D017879:Isothiocyanates; D013454:Sulfoxides; D019938:Cyclin D1; C016766:sulforaphane