Phenethyl isothiocyanate (PEITC), a natural product, exists in biological activities, including anticancer activity in many human cancer cells. No information shows that PEITC affects DNA damage in human retinoblastoma (RB) cells in vitro. In this study, the aim of experiments was to determine whether PEITC decreased total viable cell number or not by inducing protein expressions involved in DNA damage and repair in Y79 RB cells in vitro. Total cell viability was measured by PI exclusion assay, and PEITC reduced the total Y79 viable cell numbers in a dose‐dependent manner. DNA condensation and DNA impairment were conducted by DAPI staining and comet assays, respectively, in Y79 cells. The findings show that PEITC induced DNA condensation dose‐dependently based on the brighter fluorescence of cell nuclei stained by DAPI staining. PEITC‐induced DNA damage showed a more extended DNA migration smears than that of the control, which was performed by a comet assay. Western blotting was performed to measure the protein expressions involved in DNA damage and repair, which showed that PEITC at 2.5–10 μM increased NRF2, HO‐1, SOD (Mn), and catalase; however, it decreased SOD (Cu/Zn) except 10 μM PEITC treatment, and decreased glutathione, which were associated with oxidative stress. Furthermore, PEITC increased DNA‐PK, MDC1, H2A.XpSer139, ATMpSer1981, p53, p53pSer15, PARP, HSP70, and HSP90, but decreased TOPIIα, TOPIIβ, and MDM2pSer166 that were associated with DNA damage and repair mechanism in Y79 cells. The examination from confocal laser microscopy shows that PEITC increased H2A.XpSer139 and p53pSer15, and decreased glutathione and TOPIIα in Y79 cells. In conclusion, the cytotoxic effects of PEITC on reducing the number of viable cells may be due to the induction of DNA damage and the alteration of DNA repair proteins in Y79 cells in vitro.

中文翻译：

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PEITC 在体外诱导人视网膜母细胞瘤细胞中的 DNA 损伤并抑制 DNA 修复相关蛋白


异硫氰酸苯乙酯 （PEITC） 是一种天然产物，存在于生物活动中，包括许多人类癌细胞中的抗癌活性。没有信息表明 PEITC 在体外会影响人视网膜母细胞瘤 （RB） 细胞中的 DNA 损伤。在本研究中，实验的目的是确定 PEITC 是否通过诱导体外 Y79 RB 细胞中参与 DNA 损伤和修复的蛋白质表达来降低总活细胞数。通过 PI 排斥试验测量总细胞活力，PEITC 以剂量依赖性方式减少 Y79 活细胞总数。分别通过 DAPI 染色和 Comet 测定在 Y79 细胞中进行 DNA 浓缩和 DNA 损伤。研究结果表明，PEITC 诱导 DNA 浓缩呈剂量依赖性，基于 DAPI 染色染色的细胞核的更亮荧光。PEITC 诱导的 DNA 损伤显示比对照的 DNA 迁移弥散条带更广泛，对照组通过彗星测定进行。进行蛋白质印迹以测量参与 DNA 损伤和修复的蛋白质表达，结果显示 2.5-10 μM 的 PEITC 增加 NRF2、HO-1、SOD （Mn） 和过氧化氢酶;然而，除 10 μM PEITC 处理外，它降低了 SOD （Cu/Zn），并减少了与氧化应激相关的谷胱甘肽。此外，PEITC 增加了 DNA-PK、MDC1、H2A。XpSer139 、 ATMpSer1981 、 p53 、 p53pSer15 、 PARP 、 HSP70 和 HSP90 ，但降低与 Y79 细胞中 DNA 损伤和修复机制相关的 TOPIIα 、 TOPIIβ 和 MDM2pSer166。共聚焦激光显微镜检查显示 PEITC 增加了 H2A。Y79 细胞中的 XpSer139 和 p53pSer15 以及谷胱甘肽和 TOPIIα 降低。 总之，PEITC 对减少活细胞数量的细胞毒作用可能是由于体外 Y79 细胞中 DNA 损伤的诱导和 DNA 修复蛋白的改变。