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. 2017;2017:7417694.
doi: 10.1155/2017/7417694. Epub 2017 Sep 10.

NRF2 Is a Potential Modulator of Hyperresistance to Arsenic Toxicity in Stem-Like Keratinocytes

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Free PMC article

NRF2 Is a Potential Modulator of Hyperresistance to Arsenic Toxicity in Stem-Like Keratinocytes

Xiafang Wu et al. Oxid Med Cell Longev. .
Free PMC article

Abstract

Arsenic is a well-known human carcinogen. Stem cells are indicated to be involved in arsenic carcinogenesis and have a survival selection advantage during arsenic exposure with underlying mechanisms undefined. In the present study, we demonstrated that CD34high-enriched cells derived from HaCaT human keratinocytes showed stem-like phenotypes. These cells were more resistant to arsenic toxicity and had higher arsenic efflux ability than their mature compartments. The master transcription factor in antioxidant defense, nuclear factor erythroid 2-related factor 2 (NRF2) with its downstream genes, was highly expressed in CD34high-enriched cells. Stable knockdown of NRF2 abolished the hyperresistance to arsenic toxicity and holoclone-forming ability of CD34high-enriched cells. Our results suggest that skin epithelial stem/progenitor cells are more resistant to arsenic toxicity than mature cells, which is associated with the high innate expression of NRF2 in skin epithelial stem/progenitor cells.

Figures

Figure 1
Figure 1
Flow cytometric analysis of the ratio of CD34-high-expressing (CD34high) cells in HaCaT cells. (a) Representative histogram of flow cytometric analysis for CD34high cells. Left: HaCaT cells were exposed to vehicle (Veh., same volume of PBS to arsenic solution); right: HaCaT cells were exposed to 50 μM sodium arsenite for 24 h. (b) Quantification of CD34high population in HaCaT cells exposed to Veh. and arsenic. 1 × 105 cells were counted for each sample. n = 3. p < 0.05 compared with Veh.
Figure 2
Figure 2
Stem-like characteristics of CD34high-enriched cells. (a) Flow cytometric analysis of the ratio of CD34high cells. Left: isotope control of CD34 antibody; middle: the ratio of CD34high cells in the HaCaT (parent) cells; right: the ratio of CD34high cells after positive selection by CD34 magnetic beads. (b) mRNA and (c) protein levels of typical markers for undifferentiated epithelial cells or stem cells in CD34low-expressing, CD34high-enriched, and HaCaT (parent) cells. (d) Holoclone formation by CD34low-expressing, CD34high-enriched, and parent cells. Left: the number of holoclones was increased in CD34high-enriched cells compared with the CD34low-expressing or parent cells; right: representative image of holoclones (400x magnification, bar = 100 μm). n = 3. p < 0.05 compared with the CD34low-expressing cells. #p < 0.05 compared with the parent cells.
Figure 3
Figure 3
Hyperresistance of CD34high-enriched cells to acute arsenic toxicity. (a) Cell viability determined with MTS assay after 24 h exposure to sodium arsenite. n = 4. (b) Quantification and (c) representative image for Annexin V-FITC and PI double staining cells determined with flow cytometry. Apoptotic cells after 24 h exposure to sodium arsenite were counted as the cells in both Q2 and Q4. n = 3. p < 0.05 compared with CD34low-expressing compartment. #p < 0.05 compared with parent compartment.
Figure 4
Figure 4
High NRF2 activity and ROS levels in CD34high-enriched cells. (a) Representative image of Western blot for NRF2 in CD34low-expressing, CD34high-enriched, and parent cells. Cells were exposed to Veh. or 20 μM sodium arsenite for 6 h. (b) Innate mRNA levels of NRF2 downstream genes, including those encoding MRPs and antioxidant-related proteins in CD34low-expressing, CD34high-enriched, and parent cells. (c) Quantification and (d) representative histogram for intracellular ROS determined with flow cytometry. Cells were treated with Veh., 20 μM, or 40 μM sodium arsenite for 2 h. n = 3. p < 0.05 compared with CD34low-expressing compartment. #p < 0.05 compared with parent compartment.
Figure 5
Figure 5
Accumulation and efflux of arsenic in the CD34low-expressing, CD34high-enriched, and parent cells. (a) Cellular accumulation of arsenic after 24 h exposure to 10 μM of sodium arsenite. (b) 24 h cellular efflux of arsenic into the medium. n = 4. p < 0.05 compared with the CD34low-expressing cells. #p < 0.05 compared with the parent cells.
Figure 6
Figure 6
Silencing NRF2 abolished hyperresistance to acute arsenic cytotoxicity and compromised holoclone formation capacity in CD34high-enriched cells. (a) The ratio of CD34high cells in scramble and NRF2-KD HaCaT cells. n = 3. (b) mRNA levels of NRF2 determined by RT-qPCR in CD34high-enriched cells that were purified from scramble and NRF2-KD HaCaT cells. n = 3. (c) Representative protein bands of NRF2 determined by Western blot in CD34high-enriched cells that were purified from scramble and NRF2-KD HaCaT cells. Significantly reduced NRF2 protein level was observed in arsenic-treated (20 μM, 6 h) cells. (d) Cell viability of scramble and NRF2-KD CD34high-enriched cells determined with MTS assay after 24 h exposure to sodium arsenite. n = 4. (e) Holoclones formed by scramble and NRF2-KD CD34high-enriched cells. Left: representative image of holoclones (400x magnification, bar = 100 μm); right: the average number of holoclones in the 30 mm dish. n = 5. p < 0.05 compared with the scramble compartment.

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