Lin You-shi, Zhu Ming-yue, Zhou Sheng, Xie Xie-ju, Li Meng-sen
Department of Laboratory Medicine, Affiliated Hospital of Hainan Medical University, Haikou 570102, China.
Zhonghua Gan Zang Bing Za Zhi. 2010 Oct;18(10):745-50. doi: 10.3760/cma.j.issn.1007-3418.2010.10.006.
To explore the mechanism of Alpha-fetoprotein (AFP) effects on hepatocellular carcinoma cells (HCC) resistances apoptosis induced by tumor necrosis factor-related apoptosis inducing-ligand (TRAIL).
The expressed alteration of TRAIL receptor-2 (DR5) after the human hepatoma cells line Bel 7402 (AFP-producing) and HLE cells (non-AFP producing) were treated with all trans retinoic acid (ATRA) were determined by Western blot; Interaction of AFP with RAR-beta was analyzed by co-immunoprecipitation (Co-IP); Laser confocal microscopy was used to observe co-localization of AFP and RAR-beta; Short small RNA interfering (RNAi) was applied to knock down the expression of AFP in Bel 7402 cells; The full AFP gene cDNA was inserted into pcDNA3.1 vector and constructed the expressed vector of AFP (named pcDNA3.1-afp); The growth of hepatoma cells was analyzed by MTT.
Bel 7402 and HLE cells expressed DR5, lowed dosage of ATRA (40mumol/L) had no influence on the expression of DR5 in Bel 7402 cells, but ATRA (160mumol/L) could inhibit the expression of AFP and promote the expression of DR5 significantly; Co-IP indicated that AFP had a property for interacting with RAR-beta; The results also demonstrated AFP co-localization with RAR-beta in cytoplasm of Bel 7202 cells; The expression of DR5 was enhanced while the expression of AFP was knocked down by RNAi. pcDNA3.1-afp vector was transfected into HLE cells, the growth of HLE cells were stimulated and TRAIL cytotoxicity of HLE cells were reduced. But when the expression of AFP was knocked down the sensitivity of Bel 7402 cells to TRAIL was enhanced.
These data provided that AFP had a capability to interact with RAR-beta and suppressed the expression of DR5. AFP could play pivotal role on hepatoma cells resistance-induced apoptosis by TRAIL.
探讨甲胎蛋白（AFP）对肿瘤坏死因子相关凋亡诱导配体（TRAIL）诱导的肝癌细胞（HCC）抗凋亡作用的机制。
采用蛋白质免疫印迹法检测全反式维甲酸（ATRA）处理人肝癌细胞系Bel 7402（产AFP）和HLE细胞（不产AFP）后TRAIL受体-2（DR5）表达的变化；通过免疫共沉淀（Co-IP）分析AFP与视黄酸受体β（RAR-β）的相互作用；利用激光共聚焦显微镜观察AFP与RAR-β的共定位；应用小干扰RNA（RNAi）敲低Bel 7402细胞中AFP的表达；将全长AFP基因cDNA插入pcDNA3.1载体，构建AFP表达载体（命名为pcDNA3.1-afp）；采用MTT法分析肝癌细胞的生长情况。
Bel 7402和HLE细胞均表达DR5，低剂量ATRA（40μmol/L）对Bel 7402细胞中DR5的表达无影响，但ATRA（160μmol/L）可显著抑制AFP表达并促进DR5表达；Co-IP表明AFP与RAR-β具有相互作用；结果还显示在Bel 7202细胞的细胞质中AFP与RAR-β共定位；RNAi敲低AFP表达后DR5表达增强。将pcDNA3.1-afp载体转染至HLE细胞，可促进HLE细胞生长并降低HLE细胞对TRAIL的细胞毒性。但敲低AFP表达后Bel 7402细胞对TRAIL的敏感性增强。
这些数据表明AFP能够与RAR-β相互作用并抑制DR5的表达。AFP在TRAIL诱导的肝癌细胞抗凋亡过程中可能起关键作用。
