Annurca apple polyphenol extract promotes mesenchymal-to-epithelial transition and inhibits migration in triple-negative breast cancer cells through ROS/JNK signaling

Daniela Cristina Vuoso,Giovanna Cacciapuoti,Stefano Guido,Marcella Cammarota,Stefania D’Angelo,Sergio Caserta,Marina Porcelli,Rosalia Ferraro

doi:10.1038/s41598-020-73092-2

Abstract

Aberrant activation of epithelial-to-mesenchymal transition has been shown to correlate with triple-negative breast cancer (TNBC) progression and metastasis. Thus, the induction of the reverse process might offer promising opportunities to restrain TNBC metastatic spreading and related mortality. Recently, the Annurca apple polyphenol extract (APE) has been highlighted as a multi-faceted agent that selectively kills TNBC cells by ROS generation and sustained JNK activation. Here, by qualitatively and quantitatively monitoring the real-time movements of live cells we provided the first evidence that APE inhibited the migration of MDA-MB-231 and MDA-MB-468 TNBC cells and downregulated metalloproteinase-2 and metalloproteinase-9. In MDA-MB-231 cells APE decreased SMAD-2/3 and p-SMAD-2/3 levels, increased E-cadherin/N-cadherin protein ratio, induced the switch from N-cadherin to E-cadherin expression and greatly reduced vimentin levels. Confocal and scanning electron microscopy imaging of APE-treated MDA-MB-231 cells evidenced a significant cytoskeletal vimentin and filamentous actin reorganization and revealed considerable changes in cell morphology highlighting an evident transition from the mesenchymal to epithelial phenotype with decreased migratory features. Notably, all these events were reverted by N-acetyl-l-cysteine and JNK inhibitor SP600125 furnishing evidence that APE exerted its effects through the activation of ROS/JNK signaling. The overall data highlighted APE as a potential preventing agent for TNBC metastasis.

Highlights

Aberrant activation of epithelial-to-mesenchymal transition has been shown to correlate with triplenegative breast cancer (TNBC) progression and metastasis
Our data exhibited a strong dose-dependent downregulation of N-cadherin and a noticeable up-regulation of E-cadherin resulting in a marked increase of E-cadherin/N-cadherin ratio that, at 300 μM EqC apple polyphenol extract (APE), increased about 25-fold compared to untreated cells (Fig. 2b). These results indicated that in MDA-MB-231 cells APE decreased the levels of the mesenchymal markers vimentin and N-cadherin, and modulated the TGF-β pathway via downregulation of SMAD-2/3 expression and phosphorylation causing the inhibition of epithelial-mesenchymal transition (EMT) in turn responsible for the suppression of cell migration
Aberrant activation of EMT and stemness features have been reported to be responsible for TNBC cell invasion, metastasis, and resistance to treatments[34]

Summary

Introduction

Aberrant activation of epithelial-to-mesenchymal transition has been shown to correlate with triplenegative breast cancer (TNBC) progression and metastasis. Confocal and scanning electron microscopy imaging of APE-treated MDA-MB-231 cells evidenced a significant cytoskeletal vimentin and filamentous actin reorganization and revealed considerable changes in cell morphology highlighting an evident transition from the mesenchymal to epithelial phenotype with decreased migratory features. All these events were reverted by N-acetyl-l-cysteine and JNK inhibitor SP600125 furnishing evidence that APE exerted its effects through the activation of ROS/JNK signaling. (b) Representative phase-contrast microscopy images showing the wound closure process at three different time points in MDA-MB-231 (left) and MDA-MB-468 (right) cells incubated or not with APE at the indicated concentrations. EMT is related to an acquired ability by tumor cells to evade senescence, apoptosis, and anoikis, which are needed for tumor dissemination and m etastasis[2,3,4]

Methods

Results

Conclusion