Medical College, Hunan Normal University, Changsha 410013, China.
Cancer Chemother Pharmacol. 2012 Jan;69(1):195-206. doi: 10.1007/s00280-011-1686-9. Epub 2011 Jun 10.
5, 7-dimethoxyflavone (DMF) has been reported to induce apoptosis in various cancer cells. The aim of this study was to examine whether DMF sensitizes human hepatocellular carcinoma (HCC) cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis and its mechanism.
Human hepatocellular carcinoma cell lines Hep3B, Huh-7, and Hep G2 and human embryo liver L-02 cells were cultured in vitro. The cytotoxic activities were determined using MTT assay. The apoptotic cell death was examined using Flow cytometry using PI staining and DNA agarose gel electrophoresis. The activities of caspase-3, caspase-8, and caspase-9 were measured using ELISA. Intracellular ROS was measured by FCM using the fluorescent probe DCHF-DA, and the expression of DR4, DR5, CHOP, GPR78, and ATF4 proteins was analyzed using Western blot.
Our results demonstrated subtoxic concentrations of DMF sensitize HCC cells to TRAIL-induced apoptosis and induce the death receptor 5 (DR5) expression level, accompanying the generation of reactive oxygen species (ROS) and the upregulation of CHOP, GPR78, and ATF4 protein expression. Pretreatment with N-acetylcysteine (NAC) inhibited DMF-induced upregulation of DR5, CHOP, GPR78, and ATF4 protein expression and blocked the cotreatment-induced apoptosis. Furthermore, DMF-mediated sensitization of HCC cells to TRAIL was reduced by administration of a blocking antibody or small interfering RNAs for DR5, salubrinal, an inhibitor of ER stress, and the small interfering RNAs for CHOP. However, DMF could not induce the upregulation of DR5 expression, generation of ROS, and sensitization of TRAIL-induced apoptotic cell death in human embryo liver L-02 cells or normal human peripheral blood mononuclear cells (PBMCs).
The present study demonstrates that DMF selectively enhances TRAIL-induced apoptosis by ROS-stimulated ER-stress triggering CHOP-mediated DR5 upregulation in HCC.
5,7-二甲氧基黄酮（DMF）已被报道能诱导多种癌细胞凋亡。本研究旨在探讨 DMF 是否能增强人肝癌细胞（HCC）对肿瘤坏死因子相关凋亡诱导配体（TRAIL）诱导的细胞凋亡的敏感性及其机制。
体外培养人肝癌细胞系 Hep3B、Huh-7 和 Hep G2 以及人胚肝 L-02 细胞。采用 MTT 法测定细胞毒性。采用碘化丙啶（PI）染色和 DNA 琼脂糖凝胶电泳流式细胞术检测凋亡细胞死亡。采用 ELISA 法测定 caspase-3、caspase-8 和 caspase-9 的活性。采用荧光探针 DCHF-DA 通过 FCM 法测定细胞内 ROS 的含量，并用 Western blot 法分析 DR4、DR5、CHOP、GPR78 和 ATF4 蛋白的表达。
我们的结果表明，亚毒性浓度的 DMF 可增强 HCC 细胞对 TRAIL 诱导的凋亡的敏感性，并诱导死亡受体 5（DR5）表达水平，同时产生活性氧（ROS）并上调 CHOP、GPR78 和 ATF4 蛋白表达。用 N-乙酰半胱氨酸（NAC）预处理可抑制 DMF 诱导的 DR5、CHOP、GPR78 和 ATF4 蛋白表达上调，并阻断联合处理诱导的细胞凋亡。此外，用 DR5 阻断抗体或小干扰 RNA（siRNA）、内质网应激抑制剂 salubrinal 和 CHOP 的 siRNA 处理可降低 DMF 介导的 HCC 细胞对 TRAIL 的敏感性。然而，DMF 不能诱导人胚肝 L-02 细胞或正常人外周血单个核细胞（PBMCs）中 DR5 表达的上调、ROS 的产生以及 TRAIL 诱导的凋亡细胞死亡的敏感性。
本研究表明，DMF 通过 ROS 刺激内质网应激触发 CHOP 介导的 DR5 上调选择性增强 TRAIL 诱导的凋亡。
