Effects of ICI 182,780, an ERα and ERβ antagonist, and G-1, a GPER agonist, on autophagy in breast cancer cells

Mari Luminosa Muler,Soraya Soubhi Smaili,Rodrigo Portes Ureshino,Gustavo José Da Silva Pereira,Fernanda Antunes,Claudia Bincoletto,Gabriel Cicolin Guarache,Rafaela Brito Oliveira

doi:10.31744/einstein_journal/2020ao4560

Abstract

ABSTRACTObjectiveTo investigate if ICI 182,780 (fulvestrant), a selective estrogen receptor alpha/beta (ERα/ERβ) antagonist, and G-1, a selective G-protein-coupled receptor (GPER) agonist, can potentially induce autophagy in breast cancer cell lines MCF-7 and SKBr3, and how G-1 affects cell viability.MethodsCell viability in MCF-7 and SKBr3 cells was assessed by the MTT assay. To investigate the autophagy flux, MCF-7 cells were transfected with GFP-LC3, a marker of autophagosomes, and analyzed by real-time fluorescence microscopy. MCF-7 and SKBr3 cells were incubated with acridine orange for staining of acidic vesicular organelles and analyzed by flow cytometry as an indicator of autophagy.ResultsRegarding cell viability in MCF-7 cells, ICI 182,780 and rapamycin, after 48 hours, led to decreased cell proliferation whereas G-1 did not change viability over the same period. The data showed that neither ICI 182,780 nor G-1 led to increased GFP-LC3 puncta in MCF-7 cells over the 4-hour observation period. The cytometry assay showed that ICI 182,780 led to a higher number of acidic vesicular organelles in MCF-7 cells. G-1, in turn, did not have this effect in any of the cell lines. In contrast, ICI 182,780 and G-1 did not decrease cell viability of SKBr3 cells or induce formation of acidic vesicular organelles, which corresponds to the final step of the autophagy process in this cell line.ConclusionThe effect of ICI 182,780 on increasing acidic vesicular organelles in estrogen receptor-positive breast cancer cells appears to be associated with its inhibitory effect on estrogen receptors, and GPER does notseem to be involved. Understanding these mechanisms may guide further investigations of these receptors’ involvement in cellular processes of breast cancer resistance.

Highlights

Breast cancer is one major cause of death among women, in Brazil and other countries.[1]
❚❚RESULTS Expression of estrogen receptors in MCF-7 and SKBr3 cell lines Initially we conducted a Western blot assay to identify any receptors differentially expressed in the two different breast cancer cell lines
The decrease in the number of cells could be due to inhibition of proliferation, or cell death.[15]. To proceed with the autophagy assessment, we first checked how long it took for cell viability to decrease after treatment with ICI, G-1 and RAP in MCF-7 cells

Summary

Introduction

Breast cancer is one major cause of death among women, in Brazil and other countries.[1]. Evidence points to ERα expression as a potential modulator of cancer cell response to different therapies, by means of autophagy.[6,7] Macroautophagy, referred to as “autophagy”, is a lysosomal pathway for recycling of macromolecules and cell organelles sequestered into autophagosomes, bounded by a double membrane. They merge with lysosomes to form autolysosomes, within which degradation takes place.[8]

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Conclusion