Arsenic-induced Toxicity In vitro and In vivo : 세포 및 생체에서의 비소 독성

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수의과대학 수의학과
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서울대학교 대학원
ArsenicToxicityHepG2 cellsMonkey liverATOChemotherapy
학위논문 (박사)-- 서울대학교 대학원 : 수의학과(수의병인생물학 및 예방수의학(환경위생학)전공), 2015. 8. 류덕영.
Arsenic is an environmental pollutant, and its toxicity has long been recognized. Arsenic has been associated with cancers of the skin, bladder, lung, kidney, and liver as well as with noncancerous conditions, diabetes and hepatopathy. Arsenic acts on cells through a variety of mechanisms, influencing numerous signal transduction pathways. It induces a variety of cellular effects such as apoptosis, growth inhibition, promotion or inhibition of differentiation and angiogenesis. Arsenic-induced responses vary depending on cell type, dose and its chemical form.
In the present study, the effect of arsenic on liver protein expression was analyzed by a proteomic approach in monkeys. Monkeys were orally administered sodium arsenite (SA) for 28 days. The 2D-PAGE in combination with MS showed that the expression levels of 16 proteins were quantitatively changed in SA treated monkey livers compared to those of control-treated monkey. Specifically, the levels of two proteins, mortalin and tubulin beta chain, were significantly increased, and decreased were 14 proteins including plastin-3, cystathionine-beta-synthase, selenium-binding protein 1, annexin A6, alpha-enolase, phosphoenolpyruvate carboxykinase-M, erlin-2, and arginase-1. With regards to their functional roles, differential expression of these proteins may contribute to arsenic-induced liver toxicity, including cell death and carcinogenesis. Among the 16 identified proteins, four were selected for validation by Western blot and immunohistochemistry and many changes in the abundance of the toxicity-related proteins were also demonstrated in SA-treated human hepatoma HepG2 cells. In addition, to examine the involvement of c-Met and PI3K pathways in the SA-induced down-regulation of catalase, expression levels of catalase mRNA and protein were analyzed in HepG2 cells treated with SA and either an inhibitor of c-Met (PHA665752 (PHA)) or of PI3K (LY294002 (LY)). SA treatment markedly activated Akt and decreased the expression levels of both catalase mRNA and protein. Both PHA and LY attenuated SA-induced activation of Akt. PHA and LY treatment also prevented the inhibitory effect of SA on catalase protein expression but did not affect the level of catalase mRNA. These findings suggest that SA-induced inhibition of catalase expression is regulated at the mRNA and post-transcriptional levels in HepG2 cells, and that the post-transcriptional regulation is mediated via c-Met- and PI3K-dependent mechanisms.
Arsenic is also known as an anticancer agent. Arsenic trioxide (ATO) was reported to induce remission in patients with acute promyelocytic leukemia (APL). Arsenic trioxide (ATO) has been used clinically to treat acute promyelocytic leukemia, but is less effective in solid tumors because the doses required to exert anti-cancer effects are extremely high. It is important to identify the mechanism associated with anti-cancer effects of ATO to reduce the side effects caused by high dose. This study was performed to elucidate the role of down-regulated Akt in the cell death induced by high dose ATO treatment. High-dose ATO caused a marked suppression of Akt expression in human cancer cell lines, including HepG2, HCT116, HeLa, and PC3 cells. In HepG2 cells, ATO induced apoptosis, which was prevented by pre-treatment with antioxidants, N-acetylcysteine (NAC) and ascorbic acid. Antioxidants attenuated the inhibitory effects of ATO on Akt expression at both the mRNA and protein levels. Down-regulation of Akt expression by ATO extended the suppression of Akt phosphorylation, and then activates GSK3β function by decreasing its phosphorylation. GSK3β silencing using GSK3β-specific siRNA effectively prevented ATO-induced apoptosis, suggesting that activation of GSK3β via the suppression of Akt phosphorylation was critical in the ATO-induced apoptosis. The results indicate that GSK3β may be expected to become a new approach to use ATO for the treatment of solid tumors, such as hepatocellular carcinoma.
The present study about the arsenic-induced regulation of proteins with their critical roles may provide the specific mechanisms underlying SA-induced toxicity. Moreover, comprehensive study on the biology of ATO could help in developing ATO-based therapeutic interventions against solid tumors including hepatocellular carcinoma.
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College of Veterinary Medicine (수의과대학)Dept. of Veterinary Medicine (수의학과)Theses (Ph.D. / Sc.D._수의학과)
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