Zhang Ting, Yao Guangze, Chen Huiting, Yang Qianlei, An Yan
School of Public Health, Medical College of Soochow University, Suzhou 215123, China.
The First Clinical School of Medicine, Soochow University, Suzhou 215123, China.
Wei Sheng Yan Jiu. 2024 Sep;53(5):763-789. doi: 10.19813/j.cnki.weishengyanjiu.2024.05.012.
To explore the role of nuclear transcription factor E2-related factor 2(NRF2)-mediated reductive stress in arsenite induced malignant transformation in human keratinocytes.
HaCaT cells and fluorescent labeled mitochondrial glutathione HaCaT cells(Mito-Grx1-roGFP2 HaCaT) were cultured to 35 passages in medium containing 0.0 and 1.0 μmol/L NaAsO_2 to establish a model of malignant transformation of cells. Cellular and mitochondrial reduced glutathione/oxidized glutathione(GSH/GSSG) and reduced coenzyme II/oxidized coenzyme II(NADPH/NADP~+) ratios were measured in HaCaT cells. Cell doubling time, cell migration ability, soft agar clone formation ability and GSH/GSSG at different times in the 0 passage, the early stage(1st, 7th and 14th passages) and later stage(21st, 28th and 35th passages) were measured in Mito-Grx1-roGFP2 HaCaT cells. NaAsO_2 induced malignant transformation cells were transfected with NRF2 siRNA, and detected the expression level of NRF2 and the redox-related indexes and malignant transformation indexes.
Compared with the control group, the GSH/GSSG ratio in 1.0 μmol/L NaAsO_2 treated HaCaT cells significantly decreased in the 1st and 7th generations, but significantly increased after the 21st generation, and the NADPH/NADP+ ratio significantly increased in the 1st, 14th, 21st, 28th and 35th generations; The levels of GSH/GSSG in mitochondria significantly increased from 1st to 35th generation, and the levels of NADPH/NADP+ in mitochondria significantly increased at 1st, 7th, 21st, 28th and 35th generation. After continuous treatment of Mito-Grx1-roGFP2 HaCaT cells with 0.0 or 1.0 μmol/L NaAsO_2 to 35 passages, the doubling time of cells treated with 1.0 μmol/L NaAsO_2 was significantly shortened, the cell migration rate was increased greatly, and more clones with larger volumes than the control cells formed. The GSH/GSSG ratio in mitochondria of Mito-Grx1-roGFP2 HaCaT cells showed a significant decrease in the 1st generation and increased from the 7th generation onwards(all P<0.05). After transfection of NaAsO_2 treated cells with NRF2 siRNA, the levels of hydrogen peroxide and superoxide increased compared with the siRNA controls. The levels of cell and mitochondrial NADPH/NADP~+ and GSH/GSSG decreased and the level of mitochondrial GSH/GSSG in Mito-Grx1-roGFP2 HaCaT cells decreased. Cell doubling time increased, cell migration rate and soft agar clone formation ability decreased(all P<0.05). The malignant phenotype was reversed.
In the early stage(1st, 7th and 14th passages) of NaAsO_2 treated HaCaT cells, oxidative stress occurred with continuous high NRF2 expression. Later(21st, 28th and 35th passages), NRF2 induced reductive stress, leading to malignant transformation.
探讨核转录因子E2相关因子2（NRF2）介导的还原应激在亚砷酸盐诱导人角质形成细胞恶性转化中的作用。
将HaCaT细胞和荧光标记的线粒体谷胱甘肽HaCaT细胞（Mito-Grx1-roGFP2 HaCaT）在含有0.0和1.0 μmol/L NaAsO₂的培养基中培养至35代，建立细胞恶性转化模型。检测HaCaT细胞中的细胞内及线粒体还原型谷胱甘肽/氧化型谷胱甘肽（GSH/GSSG）和还原型辅酶II/氧化型辅酶II（NADPH/NADP⁺）比值。检测Mito-Grx1-roGFP2 HaCaT细胞在0代、早期（第1、7和14代）及后期（第21、28和35代）不同时间点的细胞倍增时间、细胞迁移能力、软琼脂克隆形成能力及GSH/GSSG。用NRF2 siRNA转染NaAsO₂诱导的恶性转化细胞，检测NRF2表达水平、氧化还原相关指标及恶性转化指标。
与对照组相比，1.0 μmol/L NaAsO₂处理的HaCaT细胞在第1和7代时GSH/GSSG比值显著降低，但在第21代后显著升高，且NADPH/NADP⁺比值在第1、14、21、28和35代时显著升高；线粒体中GSH/GSSG水平从第1代到第35代显著升高，线粒体中NADPH/NADP⁺水平在第1、7、21、28和35代时显著升高。用0.0或1.0 μmol/L NaAsO₂连续处理Mito-Grx1-roGFP² HaCaT细胞至35代后，1.0 μmol/L NaAsO₂处理的细胞倍增时间显著缩短，细胞迁移率大幅增加，形成的克隆比对照细胞更多且体积更大。Mito-Grx1-roGFP² HaCaT细胞线粒体中的GSH/GSSG比值在第1代时显著降低，从第7代起升高（均P<0.05）。用NRF2 siRNA转染NaAsO₂处理的细胞后，与siRNA对照组相比，过氧化氢和超氧阴离子水平升高。细胞及线粒体中NADPH/NADP⁺和GSH/GSSG水平降低，Mito-Grx1-roGFP² HaCaT细胞线粒体中GSH/GSSG水平降低。细胞倍增时间增加，细胞迁移率及软琼脂克隆形成能力降低（均P<0.05）。恶性表型得到逆转。
在NaAsO₂处理的HaCaT细胞早期（第1、7和14代），随着NRF2持续高表达出现氧化应激。后期（第21、28和35代），NRF2诱导还原应激，导致恶性转化。
