Żuryń Agnieszka, Litwiniec Anna, Safiejko-Mroczka Barbara, Klimaszewska-Wiśniewska Anna, Gagat Maciej, Krajewski Adrian, Gackowska Lidia, Grzanka Dariusz
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.
Int J Oncol. 2016 Jun;48(6):2521-33. doi: 10.3892/ijo.2016.3444. Epub 2016 Mar 18.
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.
萝卜硫素（SFN）存在于十字花科植物中，最初是从西兰花芽苗菜中分离出来的。萝卜硫素的化疗和抗癌特性已得到证实，但其潜在机制尚未完全明确。在本研究中，我们评估了经萝卜硫素处理的A549细胞中细胞周期蛋白D1和p21蛋白的表达，并将这些结果与细胞死亡程度和/或细胞周期改变相关联，同时确定了细胞周期蛋白D1对细胞死亡的潜在作用。用浓度递增的萝卜硫素（30、60和90 μM）处理A549细胞24小时。使用光学显微镜、透射电子显微镜和视频显微镜观察形态学和超微结构变化。应用基于图像的细胞术评估萝卜硫素对细胞凋亡和细胞周期的影响。通过流式细胞术、RT-qPCR和免疫荧光法测定细胞周期蛋白D1和p21的表达。使用小干扰RNA（siRNA）评估细胞周期蛋白D1在萝卜硫素诱导的细胞死亡过程中的作用。我们发现，用萝卜硫素处理后，细胞周期蛋白D1阳性细胞的百分比降低，但同时反映细胞周期蛋白D1含量的平均荧光强度在30 μM萝卜硫素处理时增加，在60和90 μM萝卜硫素处理时降低。处理后p21阳性细胞的百分比增加，在60 μM萝卜硫素处理时增加最多，在此浓度下该蛋白的平均荧光强度也显著增加。30 μM剂量的萝卜硫素诱导G2/M期细胞群体增加，同时细胞多倍体比例降低，这意味着功能性G2/M期阻滞。萝卜硫素诱导的细胞死亡主要模式是坏死，凋亡程度较低。用细胞周期蛋白D1-siRNA转染导致凋亡和坏死细胞比例显著降低，这表明细胞周期蛋白D1是萝卜硫素对A549细胞治疗效果的重要决定因素。
