Differential effects of 2-and 3-series E-prostaglandins on in vitro expansion of Lgr5+ colonic stem cells
Differential effects of 2-and 3-series E-prostaglandins on in vitro expansion of Lgr5+ colonic stem cells
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DOI：
10.1093/carcin/bgt412
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发表时间：
2014-03-01
期刊：
影响因子：
4.7
通讯作者：
Chapkin, Robert S.
中科院分区：
文献类型：
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作者：
Fan, Yang-Yi;Davidson, Laurie A.;Chapkin, Robert S.
花生四烯酸（20：4（5，8，11，14），AA）衍生的前列腺素E-2（PGE（2））促进结肠癌的发展。相比之下，化学保护性n-3多不饱和脂肪酸取代AA，从而降低了结肠细胞中PGE（2）的生物合成，特别是二十碳五烯酸（20：5（5，8，11，14，17），EPA）被代谢为新的3系列E-前列腺素（PGE（3）），一种推定的抗肿瘤环加氧酶代谢物。由于成体干细胞的转化是引发肠癌的极其重要的途径，我们利用富含亮氨酸重复序列的G蛋白偶联受体5（Lgr 5）增强的绿色荧光蛋白-内部核糖体进入位点（IRES）-creER（T2）敲入小鼠模型分离和培养结肠类器官，以记录对外源性PGE（2）和PGE（3）的离体反应。从转基因小鼠中分离结肠隐窝，并在基于Matrigel的三维平台中培养。用外源性PGE（2）、PGE（3）或二甲亚砜（载体对照）处理类器官5天，每天记录活类器官的数量。随后，对样品进行免疫组织化学、流式细胞术和实时PCR分析。与对照组和PGE（3）组相比，PGE（2）组能促进类器官的生长，并诱导细胞增殖（P < 0.05）。相反，与PGE（3）和对照组相比，PGE（2）处理的培养物中Lgr 5-绿色荧光蛋白阳性干细胞数量独特地增加了2倍以上。这与干细胞相关的Sox 9，Axin 2和Cd 44信使RNA的上调相一致。我们的研究结果表明，相对于AA衍生的PGE（2），一种已知的结肠肿瘤发生的启动子，EPA衍生的PGE（3）在小鼠结肠类器官中支持结肠干细胞扩增的能力减弱。
Arachidonic acid (20:4(5,8,11,14), AA)-derived prostaglandin E-2 (PGE(2)) promotes colon cancer development. In contrast, chemoprotective n-3 polyunsaturated fatty acids supplant AA, thereby decreasing PGE(2) biosynthesis in colonocytes, with eicosapentaenoic acid (20:5(5,8,11,14,17), EPA) in particular being metabolized to a novel 3-series E-prostaglandin (PGE(3)), a putative anti-tumorigenic-cyclooxygenase metabolite. Because transformation of adult stem cells is an extremely important route toward initiating intestinal cancer, we utilized the leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5)-enhanced green fluorescent protein-internal ribosome entry site (IRES)-creER(T2) knock-in mouse model to isolate and culture colonic organoids, in order to document ex vivo responses to exogenous PGE(2) and PGE(3). Colonic crypts were isolated from transgenic mice and cultured in a Matrigel-based three-dimensional platform. Organoids were treated with exogenous PGE(2), PGE(3) or dimethyl sulfoxide (vehicle control) for 5 days and the number of viable organoids was recorded daily. Subsequently, samples were processed for immunohistochemistry, flow cytometry and real-time PCR analyses. PGE(2) promoted optimal organoid growth and induced significantly higher levels of cell proliferation (P < 0.05) compared with PGE(3) and control. In contrast, the Lgr5-green fluorescent protein-positive stem cell number was uniquely elevated by > 2-fold in PGE(2)-treated cultures compared with PGE(3) and control. This coincided with the upregulation of stem-cell-related Sox9, Axin2 and Cd44 messenger RNAs. Our results demonstrate that relative to AA-derived PGE(2), a known promoter of colon tumorigenesis, EPA-derived PGE(3) has diminished ability to support colonic stem cell expansion in mouse colonic organoids.