通过诱导孤儿核受体 NR4A2（Nurr1），用 1,1-双（3'-吲哚基）-1-（芳基）甲烷类似物预防皮肤癌。
Chemoprevention of skin cancer with 1,1-Bis (3'-indolyl)-1-(aromatic) methane analog through induction of the orphan nuclear receptor, NR4A2 (Nurr1).
机构信息
College of Pharmacy and Pharmaceutical Sciences, Florida A&M University, Tallahassee, Florida, United States of America.
出版信息
PLoS One. 2013 Aug 7;8(8):e69519. doi: 10.1371/journal.pone.0069519. eCollection 2013.
BACKGROUND
The objective of this study was to demonstrate the anti-skin cancer and chemopreventive potential of 1,1-bis(3'-indolyl)-1-(p-chlorophenyl methane) (DIM-D) using an in vitro model.
METHODS
In vitro cell cytotoxicity and viability assays were carried out in A431 human epidermoid carcinoma cell line and normal human epidermal keratinocytes (NHEK) respectively by crystal violet staining. Apoptosis induction in A431 cells (DIM-D treated) and NHEK cells pretreated with DIM-D (2 hr) prior to UVB irradiation, were assessed. The accumulation of reactive oxygen species (ROS) in DIM-D pretreated NHEK cells (2 hr) prior to UVB exposure was also determined. Immunocytochemistry and western blot analysis was performed to determine cleaved caspase 3 and DNA damage markers in DIM-D treated A431 cells and in DIM-D pretreated NHEK cells prior to UVB irradiation.
RESULTS
The IC50 values of DIM-D were 68.7 ± 7.3, 48.3 ± 10.1 and 11.5 ± 3.1 μM whilst for Epigallocatechin gallate (EGCG) were 419.1 ± 8.3, 186.1 ± 5.2 and 56.7 ± 3.1 μM for 24, 48 and 72 hr treatments respectively. DIM-D exhibited a significantly (p<0.05) greater induction of DNA fragmentation in A431 cells compared to EGCG with percent cell death of 38.9. In addition, DIM-D induced higher expression in A431 cells compared to EGCG of cleaved caspase 3 (3.0-fold vs. 2.4-fold changes), Nurr1 (2.7-fold vs. 1.7-fold changes) and NFκB (1.3-fold vs. 1.1-fold changes). DIM-D also exhibited chemopreventive activity in UVB-irradiated NHEK cells by significantly (p<0.05) reducing UVB-induced ROS formation and apoptosis compared to EGCG. Additionally, DIM-D induced expression of Nurr1 but reduced expression of 8-OHdG significantly in UVB-irradiated NHEK cells compared to EGCG and UV only.
CONCLUSION
Our results suggest that DIM-D exhibits Nurr1-dependent transactivation in the induction of apoptosis in A431 cells and it protects NHEK cells against UVB-induced ROS formation and DNA damage.
背景
本研究旨在利用体外模型证明 1,1-双(3'-吲哚基)-1-(对氯苯基)甲烷(DIM-D)的抗皮肤癌和化学预防潜力。
方法
通过结晶紫染色，分别在 A431 人表皮癌细胞系和正常人表皮角质形成细胞（NHEK）中进行体外细胞细胞毒性和活力测定。评估 DIM-D 处理的 A431 细胞和 DIM-D 预处理（2 小时）后再用 UVB 照射的 NHEK 细胞中的细胞凋亡诱导。还测定了 DIM-D 预处理的 NHEK 细胞（暴露于 UVB 前 2 小时）中活性氧（ROS）的积累。对 DIM-D 处理的 A431 细胞和 DIM-D 预处理的 NHEK 细胞（照射 UVB 之前）中 cleaved caspase 3 和 DNA 损伤标志物进行免疫细胞化学和 Western blot 分析。
结果
DIM-D 的 IC50 值分别为 68.7 ± 7.3、48.3 ± 10.1 和 11.5 ± 3.1 μM，而表没食子儿茶素没食子酸酯（EGCG）的 IC50 值分别为 419.1 ± 8.3、186.1 ± 5.2 和 56.7 ± 3.1 μM，分别为 24、48 和 72 小时处理。与 EGCG 相比，DIM-D 显著（p<0.05）诱导 A431 细胞中的 DNA 片段化增加，细胞死亡率为 38.9%。此外，与 EGCG 相比，DIM-D 诱导 A431 细胞中 cleaved caspase 3（3.0 倍与 2.4 倍变化）、Nurr1（2.7 倍与 1.7 倍变化）和 NFκB（1.3 倍与 1.1 倍变化）的表达更高。与 EGCG 相比，DIM-D 还在 UVB 照射的 NHEK 细胞中表现出化学预防活性，通过显著（p<0.05）减少 UVB 诱导的 ROS 形成和细胞凋亡。此外，与 EGCG 和仅 UV 相比，DIM-D 诱导 NHEK 细胞中 Nurr1 的表达，但降低 8-OHdG 的表达。
结论
我们的结果表明，DIM-D 在诱导 A431 细胞凋亡中表现出 Nurr1 依赖性反式激活，并且它可保护 NHEK 细胞免受 UVB 诱导的 ROS 形成和 DNA 损伤。
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