人類角質細胞中活性氧化物參與在三氧化二砷透過 c-Src 轉錄活化 EGFR 所調控 p21WAF1/CIP1 基因表現

Abstract

Although arsenic is a well-known carcinogen, it has been used in the treatment of acute promyelocytic leukemia (APL) as it induces cell apoptosis in many cancer cell lines. Arsenic induced cell carcinogenesis or apoptosis is dependent on the stimulation dose. Previously, arsenic was shown to induce apoptosis via upregulation of p21WAF1/CIP1 (p21) expression through the c-Src/EGFR/ERK pathway and reactive oxygen species (ROS) generation. However, in arsenic trioxide (ATO, 20 M) stimulation, the N-terminal domain of c-Jun phosphorylation by JNK recruits TG-interacting factor (TGIF)/histone deacetylases 1 (HDAC1) to the Sp1 sites and then represses p21 expression, which could antagonize ATO-induced cellular apoptosis in human keratinocytes. Firstly, we aimed to study the relationships among ROS generation, c-Src/EGFR/ERK signaling and cell apoptosis/arrest induced by ATO (20 M). Our data revealed that EGFR-Y845 and EGFR-Y1173 could be phosphorylated by c-Src in response to ATO. Pretreatment with apocynin, DPI, and tiron could remove ATO-induced ROS production. Furthermore, ATO-induced cytosolic p67phox expression increase and p67phox translocation to the membrane resulted in increased NADPH oxidase activity. Knockdown of p67phox could abolish ATO-induced ROS production. Therefore, we suggest that NADPH oxidase-produced superoxide is 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. Overexpression of c-Src, as well as treatment with ATO, could stimulate EGFR-Y845/ERK phosphorylation, p21 expression, and growth arrest, which could be attenuated by pretreatment with apocynin or the knockdown of p67phox. We suggest that NADPH oxidase is involved in the ATO-induced c-Src/EGFR/ERK pathway. Secondly, based on the ATO-induced antagonistic signaling pathway, we aimed to study the roles of TGIF in ATO stimulation. We found that low-dose ATO (0.2 M) induced cell proliferation and TGIF expression in various cancer cells. Low-dose ATO stimulation induced anchorage-independent growth, cell invadopodia formation, migration, invasion abilities, induced n-cadherin (CDH2), matrix metalloproteinase 2 (MMP2) and decreased e-cadherin (CDH1) protein expression. In addition, TGIF promoter activity which was induced by low-dose ATO stimulation could be reduced by PP1 pretreatment or knockdown p67phox expression. We suggest that, taken together, the Src/NADPH oxidase/EGFR signaling pathway is involved in ATO-induced cell apoptosis and malignant transformation. Finally, we found that overexpression of TGIF also induced cell invadopodia formation, migration and invasion. Collectively, we suggest that NADPH oxidase is involved in the ATO (20 M)-induced arrest/apoptosis and is regulated by c-Src activation. Moreover, blockage of TGIF expression may enhance the ATO therapy effect.