Abstract
Arsenic is a well-known poison and carcinogen in humans. However, it also has been used to effectively treat some human cancers and non-carcinogenic ailments. Previously, we demonstrated in keratinocytes that arsenic trioxide (ATO)-induced p21WAF1/CIP1 (p21) expression leading to cellular cytotoxicity through the c-Src/EGFR/ERK pathway and generation of reactive oxygen species (ROS). In this study, we found that EGFR-Y845 and EGFR-Y1173 could be phosphorylated by ATO. Using confocal microscopy and flow cytometry, we found that pretreatment with apocynin, DPI, and tiron could remove ATO-induced ROS production. Furthermore, to increase NADPH oxidase activity, ATO could induce cytosolic p67phox expression and translocation to membrane. In addition, knockdown of p67phox could abolish ATO-induced ROS production. Therefore, we suggest that NADPH oxidase-produced superoxide was a major source of ATO-induced ROS production. Conversely, ATO-induced NADPH oxidase activation and superoxide generation could be inhibited by the c-Src inhibitor PP1, but not by the EGFR inhibitor PD153035. In addition, overexpression of c-Src as well as treatment with ATO could stimulate EGFR-Y845/ERK phosphorylation, p21 expression, and cellular arrest/apoptosis, which could be attenuated by pretreatment with apocynin or knockdown of p67phox. Collectively, we suggest that NADPH oxidase was involved in the ATO-induced arrest/apoptosis of keratinocytes, which was regulated by c-Src activation.
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This work has been granted by the National Science Council of Taiwan (grant numbers: NSC95-2320-B-006-055-MY3 and NSC98-2320-B-006-008-MY3).
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204_2012_856_MOESM1_ESM.pdf
Requirement of c-Src activation in ATO-induced EGFR/ERK phosphorylation and p21 expression. a HaCaT cells were treated with 20 μM ATO for the indicated time periods. Total cell lysates were analyzed by Western blot with specific antibodies as indicated. (PDF 59 kb)
204_2012_856_MOESM2_ESM.pdf
Biosynthesis of p67phox in response to ATO. a Cells were treated with 20 μM ATO for various time periods as indicated. Total RNA was extracted and p67Phox mRNA expression was examined by real-time PCR. b Cell lysates were also collected to detect p67phox and β-actin protein expression by Western blot.(PDF 26 kb)
204_2012_856_MOESM3_ESM.pdf
Overexpression of wtSrc can induce EGFR-Y845 phosphorylation and p21 expression. a A431 cells or (b) HaCaT cells were transfected with wtSrc for 24 h, then the total cell lysates were collected and analyzed by Western blot with specific antibodies as indicated. c A431 cells were transfected with wtSrc for 24 h, followed by treatment with various doses of apocynin for 1 h. Total cell lysates were collected and analyzed by Western blot with specific antibodies as indicated. (PDF 201 kb)
204_2012_856_MOESM4_ESM.pdf
Involvement of c-Src/NADPH oxidase signaling in ATO-induced cellular arrest/apoptosis of HaCaT. Cells were transfected with various doses of wtSrc for different time periods. a Cell numbers were counted after staining with trypan blue in HaCaT cells. Cells were transiently transfected with empty vectors or 2 μg of shRNA of p67Phox for 24 h, followed by treatment with or without 20 μM ATO for 24 h as indicated. Then, the cell death assay was performed as described previously. (PDF 132 kb)
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Tseng, HY., Liu, ZM. & Huang, HS. NADPH oxidase-produced superoxide mediates EGFR transactivation by c-Src in arsenic trioxide-stimulated human keratinocytes. Arch Toxicol 86, 935–945 (2012). https://doi.org/10.1007/s00204-012-0856-9
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DOI: https://doi.org/10.1007/s00204-012-0856-9