吴茱萸碱通过激活人卵巢癌细胞中的 JNK 和 PERK 诱导细胞凋亡。
Evodiamine from Evodia rutaecarpa induces apoptosis via activation of JNK and PERK in human ovarian cancer cells.
机构信息
Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan; Department of Obstetrics and Gynecology, Mackay Memorial Hospital, Taipei, Taiwan.
Department of Nephrology, Chi-Mei Medical Center, Tainan, Taiwan; Department of Food Nutrition, Chung Hwa University of Medical Technology, Tainan, Taiwan.
出版信息
Phytomedicine. 2016 Jan 15;23(1):68-78. doi: 10.1016/j.phymed.2015.12.003. Epub 2015 Dec 22.
BACKGROUND
Evodiamine (EVO; 8,13,13b,14-tetrahydro-14-methylindolo[2'3'-3,4]pyrido[2,1-b]quinazolin-5-[7H]-one derived from the traditional herbal medicine Evodia rutaecarpa was reported to possess anticancer activity; however, the anticancer mechanism of EVO against the viability of human ovarian cancer cells is still unclear.
PURPOSE
A number of studies showed that chemotherapeutic benefits may result from targeting the endoplasmic reticular (ER) stress signaling pathway. The objective of the study is to investigate the mechanism by which ER stress protein PERK plays in EVO-induced apoptosis of human ovarian cancer cells.
METHODS
Cell death analysis was performed by MTT assay, DNA fragmentation assay, and Giemsa staining. DiOC6 staining was used for mitochondrial membrane potential measurement. Protein levels were analyzed by Western blotting. Pharmacological studies using MAPK inhibitors and PERK inhibitor GSK2606414 were involved.
RESULTS
The viability of human ovarian cancer cells A2780, A2780CP, ES-2, and SKOV-3 was inhibited by EVO at various concentrations in accordance with increases in the percentage of apoptotic cells, DNA ladders, and cleavage of caspase 3 and poly(ADP ribose) polymerase (PARP) proteins. Decreased viability of cells was reversed by adding caspase inhibitors VAD and DEVD in SKOV-3 and A2780CP cells, and incubation of cells with JNK inhibitor SP600125 (SP) and JNKI, but not other MAPK and AKT inhibitors including PD98059, SB203580, significantly prevented the apoptosis elicited by EVO in human ovarian cancer cells. Furthermore, increased expression of phospho-eIF2α (peIF2α) and phospho-PERK (pPERK) proteins was detected in EVO-treated human ovarian cancer cells, and that was inhibited by adding JNK inhibitors SP600125 and JNKI. Application of a PERK inhibitor GSK2606414 showed a significant protection of human ovarian cancer cells A2780 and A2780CP from EVO-induced apoptosis. EVO disruption of mitochondrial membrane potential (MMP) was also inhibited by adding JNK or PERK inhibitors. The structure-activity relationship study indicated that the alkyl group at position 14 in EVO is important for apoptosis induction via activation of JNK and PERK in human ovarian cancer cells.
CONCLUSION
Evidence supporting EVO induction of apoptosis via activation of JNK and PERK to disrupt MMP in human ovarian cancer cells is provided, and the alkyl at position 14 is a critical substitution for the apoptotic actions of EVO.
背景
从传统中药吴茱萸中提取的吴茱萸碱（EVO；8,13,13b,14-四氢-14-甲基吲哚[2'3'-3,4]吡啶并[2,1-b]喹唑啉-5-[7H]-酮）已被报道具有抗癌活性；然而，EVO 对人卵巢癌细胞活力的抗癌机制仍不清楚。
目的
许多研究表明，化疗益处可能源于靶向内质网（ER）应激信号通路。本研究的目的是探讨 ER 应激蛋白 PERK 在 EVO 诱导人卵巢癌细胞凋亡中的作用机制。
方法
通过 MTT 测定、DNA 片段化测定和吉姆萨染色法进行细胞死亡分析。使用 DiOC6 染色法测量线粒体膜电位。通过 Western blot 分析蛋白水平。涉及使用 MAPK 抑制剂和 PERK 抑制剂 GSK2606414 的药理学研究。
结果
EVO 以不同浓度抑制人卵巢癌细胞 A2780、A2780CP、ES-2 和 SKOV-3 的存活率，同时增加凋亡细胞的百分比、DNA 梯状条带以及 caspase 3 和多聚（ADP 核糖）聚合酶（PARP）蛋白的裂解。在 SKOV-3 和 A2780CP 细胞中加入 caspase 抑制剂 VAD 和 DEVD，以及在人卵巢癌细胞中加入 JNK 抑制剂 SP600125（SP）和 JNKI，可逆转细胞活力降低，而加入其他 MAPK 和 AKT 抑制剂，包括 PD98059、SB203580，均不能显著阻止 EVO 诱导的人卵巢癌细胞凋亡。此外，在 EVO 处理的人卵巢癌细胞中检测到磷酸化 eIF2α（peIF2α）和磷酸化 PERK（pPERK）蛋白表达增加，加入 JNK 抑制剂 SP600125 和 JNKI 可抑制该表达。应用 PERK 抑制剂 GSK2606414 可显著保护人卵巢癌细胞 A2780 和 A2780CP 免受 EVO 诱导的凋亡。EVO 也抑制了线粒体膜电位（MMP）的破坏，加入 JNK 或 PERK 抑制剂也可抑制该破坏。结构活性关系研究表明，EVO 中 14 位的烷基对于通过激活 JNK 和 PERK 在人卵巢癌细胞中诱导凋亡是重要的。
结论
提供了证据支持 EVO 通过激活 JNK 和 PERK 破坏人卵巢癌细胞的 MMP 诱导细胞凋亡，并且 14 位的烷基是 EVO 凋亡作用的关键取代基。
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