蒲公英萜内酯 2α-羟化物：抗多药耐药肿瘤细胞的活性及其作用模式。

2α-Hydroxyalantolactone from Pulicaria undulata: activity against multidrug-resistant tumor cells and modes of action.

机构信息

Department of Pharmaceutical Biology, Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz, Germany; Phytochemistry Department, National Research Centre, 33 El-Bohouth St., Dokki, Giza 12622, Egypt.

Department of Pharmaceutical Biology, Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz, Germany.

出版信息

Phytomedicine. 2021 Jan;81:153409. doi: 10.1016/j.phymed.2020.153409. Epub 2020 Nov 17.

DOI:10.1016/j.phymed.2020.153409

PMID:33341310

Abstract

BACKGROUND

Sesquiterpene lactones having α-methylene-γ-lactone moiety are promising natural metabolites showing various biological activity. One of the major metabolites isolated from Pulicaria undulata, 2α-hydroxyalantolactone (PU-1), has not been investigated in detail yet. Multidrug resistance (MDR) represents a major obstacle for cancer chemotherapy and the capability of novel natural products to overcoming MDR is of great interest.

PURPOSE

Exploring the molecular modes of action for potent natural product metabolites.

METHODS

The resazurin reduction assay was employed to evaluate the cytotoxicity of PU-1 on sensitive and their corresponding drug-resistant cell lines (overexpressing P-glycoprotein, BCRP, ABCB5, ΔEGFR, or TP53 knockout). Gene expression profiling was performed by transcriptome-wide mRNA microarray in the human CCRF-CEM leukemic cells after treatment with PU-1. The top significantly up- or down-regulated genes were identified by Chipster program and analyzed using Ingenuity Pathway Analysis (IPA) software. Finally, flow cytometry and Western blotting were performed for cell cycle analyses and apoptosis detection.

RESULTS

The sesquiterpene lactone, PU-1, showed potent cytotoxicity towards the drug-sensitive and -resistant cell lines. Transcriptome-wide mRNA expression profiling and pathway analysis pointed to genes involved in DNA damage response and G2/M cell cycle arrest. G2/M arrest was verified by flow cytometry and further confirmed by the upregulation of p21 and downregulation of p-CDC25C expression in Western blotting. Moreover, the suggested DNA damage checkpoint regulation was confirmed by immunofluorescence and Western blotting by upregulation of pS345 Chk1, p-H3 and γ-H2AX. Furthermore, PU-1 inhibited PI3K/AKT pathway, which is involved in signaling DNA damage and G2/M arrest. Cells ultimately induced apoptosis upon PU-1 treatment.

CONCLUSIONS

PU-1 is a potent natural product inhibiting otherwise drug-resistant human tumor cell growth through DNA damage, G2/M cell cycle arrest and apoptosis.

摘要

背景

具有α-亚甲基-γ-内酯部分的倍半萜内酯是具有各种生物活性的有前途的天然代谢产物。从 Pulicaria undulata 中分离出的一种主要代谢产物 2α-羟基白头翁内酯（PU-1）尚未得到详细研究。多药耐药（MDR）是癌症化疗的主要障碍，新型天然产物克服 MDR 的能力引起了极大的关注。

目的

探索有效天然产物代谢物的分子作用模式。

方法

采用 Resazurin 还原试验评估 PU-1 对敏感及其相应耐药细胞系（过表达 P-糖蛋白、BCRP、ABCB5、ΔEGFR 或 TP53 缺失）的细胞毒性。用 PU-1 处理人 CCRF-CEM 白血病细胞后，通过全转录组 mRNA 微阵列进行基因表达谱分析。使用 Chipster 程序鉴定并通过 IPA 软件分析上调或下调最显著的基因。最后，通过流式细胞术和 Western blot 进行细胞周期分析和凋亡检测。

结果

倍半萜内酯 PU-1 对敏感和耐药细胞系均表现出强大的细胞毒性。全转录组 mRNA 表达谱分析和通路分析表明，基因参与 DNA 损伤反应和 G2/M 细胞周期阻滞。通过流式细胞术验证了 G2/M 期阻滞，并通过 Western blot 进一步证实了 p21 的上调和 p-CDC25C 的下调。此外，通过免疫荧光和 Western blot 证实了 DNA 损伤检查点调节，上调了 pS345 Chk1、p-H3 和 γ-H2AX。此外，PU-1 抑制了参与信号转导 DNA 损伤和 G2/M 阻滞的 PI3K/AKT 通路。细胞最终在 PU-1 处理后诱导凋亡。