Yamada Yasushi, Miyamoto Tsutomu, Kashima Hiroyasu, Kobara Hisanori, Asaka Ryoichi, Ando Hirofumi, Higuchi Shotaro, Ida Koichi, Shiozawa Tanri
a Department of Obstetrics and Gynecology , Shinshu University School of Medicine , Matsumoto , Japan.
Free Radic Res. 2016;50(4):414-25. doi: 10.3109/10715762.2015.1134795. Epub 2016 Feb 12.
Ovarian clear cell carcinoma (CCC) arises from ovarian endometriosis. Intra-cystic fluid contains abundant amounts of free iron, which causes persistent oxidative stress, a factor that has been suggested to induce malignant transformation. However, the mechanisms linking oxidative stress and carcinogenesis in CCC currently remain unclear. Lipocalin 2 (LCN2), a multifunctional secretory protein, functions as an iron transporter as well as an antioxidant. Therefore, we herein examined the roles of LCN2 in the regulation of intracellular iron concentrations, oxidative stress, DNA damage, and antioxidative functions using LCN2-overexpressing (ES2), and LCN2-silenced (RMG-1) CCC cell lines. The results of calcein staining indicated that the up-regulated expression of LCN2 correlated with increases in intracellular iron concentrations. However, a DCFH-DA assay and 8OHdG staining revealed that LCN2 reduced intracellular levels of reactive oxygen species and DNA damage. Furthermore, the expression of LCN2 suppressed hydrogen peroxide-induced apoptosis and prolonged cell survival, suggesting an antioxidative role for LCN2. The expression of mRNAs and proteins for various oxidative stress-catalyzing enzymes, such as heme oxygenase (HO), superoxide dismutase (SOD), and glutathione peroxidase, was not affected by LCN2, whereas the intracellular concentration of the potent antioxidant, glutathione (GSH), was increased by LCN2. Furthermore, the expression of xCT, a cystine transporter protein, and CD44 variant 8-10 (CD44v), a stem cell marker, was up-regulated by LCN2. Although LCN2 increased intracellular iron concentrations, LCN2-induced GSH may catalyze and override oxidative stress via CD44v and xCT, and subsequently enhance the survival of CCC cells in oxidative stress-rich endometriosis.
卵巢透明细胞癌（CCC）起源于卵巢子宫内膜异位症。囊内液含有大量游离铁，可导致持续的氧化应激，这一因素被认为可诱导恶性转化。然而，目前尚不清楚氧化应激与CCC致癌作用之间的联系机制。脂质运载蛋白2（LCN2）是一种多功能分泌蛋白，具有铁转运蛋白和抗氧化剂的功能。因此，我们在此使用LCN2过表达（ES2）和LCN2沉默（RMG-1）的CCC细胞系，研究了LCN2在调节细胞内铁浓度、氧化应激、DNA损伤和抗氧化功能中的作用。钙黄绿素染色结果表明，LCN2表达上调与细胞内铁浓度增加相关。然而，DCFH-DA检测和8-羟基脱氧鸟苷染色显示，LCN2降低了细胞内活性氧水平和DNA损伤。此外，LCN2的表达抑制了过氧化氢诱导的细胞凋亡并延长了细胞存活时间，表明LCN2具有抗氧化作用。LCN2对各种氧化应激催化酶，如血红素加氧酶（HO）、超氧化物歧化酶（SOD）和谷胱甘肽过氧化物酶的mRNA和蛋白表达没有影响，而强效抗氧化剂谷胱甘肽（GSH）的细胞内浓度则因LCN2而增加。此外，胱氨酸转运蛋白xCT和干细胞标志物CD44变体8-10（CD44v）的表达因LCN2而上调。虽然LCN2增加了细胞内铁浓度，但LCN2诱导的GSH可能通过CD44v和xCT催化并克服氧化应激，随后提高CCC细胞在富含氧化应激的子宫内膜异位症中的存活率。
