Induction of Endoplasmic Reticulum Stress Response by the Indole-3-Carbinol Cyclic Tetrameric Derivative CTet in Human Breast Cancer Cell Lines

Luca Galluzzi,Giorgio Brandi,Andrea Duranti,Rita Crinelli,Cinzia De Marco,Mauro Magnani,Nadia Zaffaroni,Mauro De Santi,Aamir Ahmad

doi:10.1371/journal.pone.0043249

Luca Galluzzi, Giorgio Brandi + Show 7 more

Open Access

https://doi.org/10.1371/journal.pone.0043249

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Abstract

BackgroundIndole-3-carbinol and its metabolic products are considered promising chemopreventive and anticancer agents. Previously we have shown that the indole-3-carbinol cyclic tetrameric derivative CTet induces autophagy and inhibits cell proliferation via inhibition of Akt activity and overexpression of p21/CDKN1A and GADD45A, in both estrogen receptor-positive (MCF-7) and triple negative (MDA-MB-231) breast cancer cell lines. In the present study, we further characterize the autophagic response and investigate the mechanism through which CTet regulates these events.Methodology/Principal FindingsAnalysis of gene expression microarray data and subsequent confirmation by quantitative real-time PCR, showed that CTet is able to induce up-regulation of key signaling molecules involved in endoplasmic reticulum (ER) stress response (e.g. DDIT3/CHOP, CHAC1, ATF3, HSPA5/BiP/GRP78, CEBPB, ASNS) and autophagy (e.g. MAP1LC3B), in both MCF-7 and MDA-MB-231 cell lines. Moreover, the monitoring of Xbp-1 splicing confirmed the activation of IRE1/Xbp-1 ER stress response branch after CTet treatment. The role of autophagic processes (known to be induced by ER stress) was investigated further through ATG5 gene silencing and pharmacological inhibition of AVOs formation. CTet was shown to induce an autophagy-related cell death. Moreover, CTet-treated cells stained with Hoechst/PI revealed the presence of necrotic processes without evidence of apoptosis.Conclusions/SignificanceThe ER stress response was identified as the main upstream molecular mechanism through which CTet acts in both hormone-responsive and triple-negative breast cancer cells. Because of its important role in cancer development, ER stress is a potential target in cancer therapy. The abiltiy of CTet to induce ER stress response and subsequently activate a death program in tumor cells confirms this molecule as a promising anticancer agent.

Highlights

Indole-3-carbinol (I3C) and its derivatives exhibit antitumor activity in various cancer cell lines
We have recently shown that CTet induces autophagy and inhibits cell proliferation in both estrogen receptor-positive (MCF7) and triple negative (MDA-MB-231) breast cancer cell lines
The endoplasmic reticulum (ER) stress is caused by perturbation of ER functions, and it is sensed by three ER-transmembrane transducers: ATF6, IRE1 and PERK [4,5]

Summary

Introduction

Indole-3-carbinol (I3C) and its derivatives exhibit antitumor activity in various cancer cell lines. The I3C derivatives characterized by antiproliferative properties, such as DIM (3,39diindoylmethane) and CTet (a cyclic tetramer), have been shown to induce a wide spectrum of signaling targets and cellular responses such as apoptosis and/or cell cycle arrest [1]. We have recently shown that CTet induces autophagy and inhibits cell proliferation in both estrogen receptor-positive (MCF7) and triple negative (MDA-MB-231) breast cancer cell lines. The inhibition of Akt activity and p53-independent p21/CDKN1A and GADD45A overexpression have been identified as the main molecular events responsible for CTet activity in both cell lines [2]. We have shown that the indole-3-carbinol cyclic tetrameric derivative CTet induces autophagy and inhibits cell proliferation via inhibition of Akt activity and overexpression of p21/CDKN1A and GADD45A, in both estrogen receptor-positive (MCF-7) and triple negative (MDA-MB-231) breast cancer cell lines. We further characterize the autophagic response and investigate the mechanism through which CTet regulates these events

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