Kimura Masayuki, Fujii Yuta, Yamamoto Ryuichi, Yafune Atsunori, Hayashi Shim-mo, Suzuki Kazuhiko, Shibutani Makoto
Laboratory of Veterinary Pathology, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan.
Exp Toxicol Pathol. 2013 Nov;65(7-8):979-88. doi: 10.1016/j.etp.2013.01.012. Epub 2013 Mar 7.
Thioacetamide (TAA) induces oxidative stress and hepatocarcinogenicity in rats. We previously reported that TAA promotion caused various disruptions in cell cycle protein expression in rats, including downregulation of p16(Ink4a), which is associated with intraexonic hypermethylation in hepatocellular proliferative lesions. This study further investigated the contribution of cell cycle aberrations associated with early hepatocarcinogenic processes induced by TAA using antioxidants, enzymatically modified isoquercitrin (EMIQ) and α-lipoic acid (ALA), in a two-stage rat hepatocarcinogenesis model. TAA-promotion after initiation with N-diethylnitrosamine increased the number and area of hepatocellular foci immunoreactive for glutathione S-transferase placental form (GST-P) and the numbers of proliferating and apoptotic cells. Co-treatment with EMIQ and ALA suppressed these increases. TAA-induced formation of p16(Ink4a-) foci in concordance with GST-P(+) foci was not suppressed by co-treatment with EMIQ or ALA. TAA-promotion increased cellular distributions of cell proliferation marker Ki-67, G2/M and spindle checkpoint proteins (phosphorylated checkpoint kinase 1 and Mad2), the DNA damage-related protein phosphorylated histone H2AX, and G2-M phase-related proteins (topoisomerase IIα, phosphorylated histone H3 and Cdc2) within GST-P(+) foci, and co-treatment with EMIQ or ALA suppressed these increases. These results suggest that downregulation of p16(Ink4a) may allow selective proliferation of preneoplastic cells by TAA promotion. However, antioxidants did not counteract this gene control. Moreover, effective suppression of TAA-induced cellular population changes within preneoplastic lesions by antioxidants may reflect facilitation of cell cycling and accumulation of DNA damage causing the activation of cell cycle checkpoints, leading to G2 and M phase arrest at the early stages of hepatocarcinogenesis promoted by TAA.
硫代乙酰胺（TAA）可诱导大鼠产生氧化应激和肝癌致癌性。我们之前报道过，TAA促进作用会导致大鼠细胞周期蛋白表达出现各种紊乱，包括p16（Ink4a）的下调，这与肝细胞增殖性病变中的外显子内高甲基化有关。本研究使用抗氧化剂、酶促修饰异槲皮苷（EMIQ）和α -硫辛酸（ALA），在两阶段大鼠肝癌发生模型中进一步研究了与TAA诱导的早期肝癌致癌过程相关的细胞周期异常的作用。用N -二乙基亚硝胺启动后进行TAA促进，增加了对谷胱甘肽S -转移酶胎盘型（GST - P）免疫反应阳性的肝细胞灶的数量和面积，以及增殖细胞和凋亡细胞的数量。EMIQ和ALA联合处理可抑制这些增加。与EMIQ或ALA联合处理并未抑制TAA诱导的与GST - P（+）灶一致的p16（Ink4a -）灶的形成。TAA促进作用增加了细胞增殖标志物Ki - 67、G2/M和纺锤体检查点蛋白（磷酸化检查点激酶1和Mad2）、DNA损伤相关蛋白磷酸化组蛋白H2AX以及G2 - M期相关蛋白（拓扑异构酶IIα、磷酸化组蛋白H3和Cdc2）在GST - P（+）灶内的细胞分布，而EMIQ或ALA联合处理可抑制这些增加。这些结果表明，p16（Ink4a）的下调可能使TAA促进作用导致的癌前细胞选择性增殖。然而，抗氧化剂并未抵消这种基因调控。此外，抗氧化剂有效抑制TAA诱导的癌前病变内细胞群体变化可能反映了细胞周期的促进以及DNA损伤的积累，从而导致细胞周期检查点的激活，导致在TAA促进的肝癌发生早期出现G2和M期阻滞。
