白花蛇舌草黄酮化合物 Rhoifolin 通过下调上皮间质转化过程中的 Podocalyxin-Ezrin 相互作用抑制乳腺癌细胞迁移的体外研究

In vitro anti-motile effects of Rhoifolin, a flavonoid extracted from Callicarpa nudiflora on breast cancer cells via downregulating Podocalyxin-Ezrin interaction during Epithelial Mesenchymal Transition.

机构信息

School of Pharmacy, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China.

Network and Educational Technology Center, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China.

出版信息

Phytomedicine. 2021 Dec;93:153486. doi: 10.1016/j.phymed.2021.153486. Epub 2021 Jan 28.

DOI:10.1016/j.phymed.2021.153486

PMID:34649211

Abstract

BACKGROUND

Callicarpa nudiflora (C. nudiflora), which is a medical herb in genus of Callicarpa, widely grows in the southern part of China. Several investigations had shown that this herb exerts anti-tumor effects. Ezrin is an important membrane-cytoskeleton-binding protein. By organizing membrane proteins and orchestrating their signal transduction, Ezrin contributes to modulation of cytoskeleton rearrangement in cell motility.

PURPOSE

To investigate the anti-motile properties of Rhoifolin (RFL), a flavonoid from C. nudiflora, and to determine whether its effects are related to the inhibition on Podocalyxin (PODXL)-Ezrin signal transduction.

METHODS

To determine suitable concentration of RFL and exposure time on breast cancer cells, the effects of RFL on viability of breast cancer cells were evaluated by MTT assay. Then, the anti-migratory properties of RFL were determined by AP 48 chamber system and ORIS cell migration assay. F-actin in MDA-MB-231 cells was visualized by Alexa Fluor™ 488 conjugated Phalloidin. Immunoprecipitation was involved to access the effects of RFL on the interaction between Ezrin and PODXL. In addition, several EMT markers, including E-cadherin, Vimentin, Snail and Slug, were measured by Western Blotting assay and cell immunofluorescent analysis. Finally, the effects of RFL on cell migration, expression of Ezrin and EMT markers were verified by small interfering RNA (siRNA) mediated gene silencing.

RESULTS

We showed here that treatments with 10 and 40 μM of RFL induced significant inhibitions on cell migration and alterations on the location and organization of actin cytoskeleton in breast cancer cells. Next, it was found that RFL suppressed Ezrin phosphorylation and consequent interaction with PODXL, significantly. Also, this compound showed an obvious inhibitory effect on TGF-β1-induced EMT in MDA-MB-231 cells. Furthermore, data from RNA interfering assay confirmed that the inhibitory effects of RFL on Ezrin was enhanced by the deletion of Ezrin.

CONCLUSION

RFL shows anti-motile properties on breast cancer cells, which is due to its potential to downregulate Podocalyxin-Ezrin interaction during Epithelial Mesenchymal Transition.

摘要

背景

作为马鞭草科紫珠属的药用植物，乌桕（C. nudiflora）在中国南方广泛分布。多项研究表明，该草药具有抗肿瘤作用。埃兹蛋白（Ezrin）是一种重要的膜-细胞骨架结合蛋白。通过组织膜蛋白并协调其信号转导，Ezrin 有助于调节细胞运动中的细胞骨架重排。

目的

研究来自乌桕的黄酮类化合物 Rhoifolin（RFL）的抗迁移特性，并确定其作用是否与抑制 Podocalyxin（PODXL）-Ezrin 信号转导有关。

方法

为了确定 RFL 对乳腺癌细胞的适宜浓度和暴露时间，通过 MTT 分析评估了 RFL 对乳腺癌细胞活力的影响。然后，通过 AP 48 室系统和 ORIS 细胞迁移测定法测定了 RFL 的抗迁移特性。用 Alexa Fluor™ 488 缀合的鬼笔环肽可视化 MDA-MB-231 细胞中的 F-肌动蛋白。免疫沉淀用于评估 RFL 对 Ezrin 和 PODXL 之间相互作用的影响。此外，通过 Western Blotting 分析和细胞免疫荧光分析测量了 EMT 标志物，包括 E-钙粘蛋白、波形蛋白、Snail 和 Slug。最后，通过小干扰 RNA（siRNA）介导的基因沉默验证了 RFL 对细胞迁移、Ezrin 和 EMT 标志物表达的影响。

结果

我们在这里表明，用 10 和 40 μM 的 RFL 处理可显著抑制乳腺癌细胞的迁移，并改变细胞骨架肌动蛋白的位置和组织。接下来，发现 RFL 显著抑制 Ezrin 磷酸化和随后与 PODXL 的相互作用。此外，该化合物对 MDA-MB-231 细胞中的 TGF-β1 诱导的 EMT 表现出明显的抑制作用。此外，RNA 干扰测定的结果证实，RFL 对 Ezrin 的抑制作用通过 Ezrin 的缺失而增强。