University of Louisville School of Medicine, Kentucky 40202, USA.
Mol Carcinog. 2010 Jan;49(1):44-53. doi: 10.1002/mc.20575.
The loss of manganese superoxide dismutase function has been associated with increased incidence of Barrett's esophagus and esophageal adenocarcinoma. In previous studies, we have demonstrated that loss of MnSOD resulted in severe esophageal damage by both endogenous and exogenous bile. However, the alterative manner of MnSOD in esophageal epithelium is largely unknown. In this study, we investigated the expression and localization of MnSOD in response to the exposure to bile salts in an esophageal epithelial cell line. Het-1A cells were seeded at 5 x 10(5) and 10(7) and incubated with taurocholate, cholate, glycocholate, deoxycholate, and the mixture of these bile salts. Mitochondria and cytoplasma were separated, and the expression and localization of MnSOD was determined by Western blot and immunocytochemical assay. Proliferation rates were strongly inhibited in the groups with taurocholate and bile salts mixture at 4 h, with 0.367 +/- 0.042 and 0.396 +/- 0.046, respectively, compared to 0.684 +/- 0.054 in untreated groups (P < 0.05). An increased apoptotic rate compared to untreated group (3.65 +/- 0.59) were significantly increased in taurocholate group and in bile salts mixture group were 33.62 +/- 10.25 and 31.52 +/- 8.97 at 4 h, respectively (P < 0.05). The protein level of MnSOD in mitochondria was increased at 4 h, but with a decreased enzymatic activity after bile salts treatment. Cytoplasmic MnSOD was detected in the cells with bile salts treatment. Immunocytochemical staining demonstrated that esophageal epithelial cell underwent morphological alteration and MnSOD relocalization after bile salts treatment. This is the first study to demonstrate cellular cytosolic MnSOD expression and that this relocalization to the cytosol is a cause for decreased MnSOD enzymatic activity. This suggests that bile salts may contribute to the dysfunction of mitochondria, by enzymatically inhibiting of MnSOD localization and thus activation in the mitochondria.
锰超氧化物歧化酶功能的丧失与 Barrett 食管和食管腺癌的发病率增加有关。在之前的研究中，我们已经证明，MnSOD 的丧失会导致内源性和外源性胆汁引起严重的食管损伤。然而，MnSOD 在食管上皮细胞中的改变方式在很大程度上尚不清楚。在这项研究中，我们研究了在食管上皮细胞系中暴露于胆盐时 MnSOD 的表达和定位。Het-1A 细胞以 5 x 10(5)和 10(7)的密度接种，并与牛磺胆酸钠、胆酸钠、甘胆酸钠、去氧胆酸钠和这些胆盐的混合物孵育。分离线粒体和细胞质，通过 Western blot 和免疫细胞化学测定法确定 MnSOD 的表达和定位。与未处理组相比，牛磺胆酸钠组和混合胆盐组在 4 小时时增殖率受到强烈抑制，分别为 0.367 +/- 0.042 和 0.396 +/- 0.046，而未处理组为 0.684 +/- 0.054（P < 0.05）。与未处理组相比，牛磺胆酸钠组和混合胆盐组的凋亡率在 4 小时时显著增加，分别为 33.62 +/- 10.25 和 31.52 +/- 8.97（P < 0.05）。与胆盐处理前相比，线粒体中 MnSOD 的蛋白水平在 4 小时时增加，但胆盐处理后酶活性降低。在有胆盐处理的细胞中检测到细胞质 MnSOD。免疫细胞化学染色显示，胆盐处理后食管上皮细胞发生形态改变，MnSOD 重新定位。这是第一项研究表明细胞细胞质 MnSOD 表达，并表明这种重新定位到细胞质是 MnSOD 酶活性降低的原因。这表明，胆盐可能通过酶抑制 MnSOD 的定位和因此在线粒体中的激活，导致线粒体功能障碍。
