The Isoxazole Derivative of Usnic Acid Induces an ER Stress Response in Breast Cancer Cells That Leads to Paraptosis-like Cell Death.

Agnieszka Pyrczak-Felczykowska,Aleksandra Hać,Beata Guzow-Krzemińska,Marcin Jąkalski,Marcelina Malinowska,Anna Pawlik,Anna Herman-Antosiewicz,Tristan A Reekie,Kamil Ryś

doi:10.3390/ijms23031802

Abstract

Derivatives of usnic acid (UA), a secondary metabolite from lichens, were synthesized to improve its anticancer activity and selectivity. Recently we reported the synthesis and activity of an UA isoxazole derivative, named 2b, against cancer cells of different origins. Herein, the molecular mechanisms underlying its activity and efficacy in vivo were tested. The viability of breast cancer or normal cells has been tested using an MTT assay. Cell and organelle morphology was analyzed using light, electron and fluorescence microscopy. Gene expression was evaluated by RNAseq and protein levels were evaluated by Western blotting. In vivo anticancer activity was evaluated in a mice xenograft model. We found that 2b induced massive vacuolization which originated from the endoplasmic reticulum (ER). ER stress markers were upregulated both at the mRNA and protein levels. ER stress was caused by the release of Ca2+ ions from the ER by IP3R channels which was mediated, at least partly, by phospholipase C (PLC)-synthetized 1,4,5-inositol triphosphate (IP3). ER stress led to cell death with features of apoptosis and paraptosis. When applied to nude mice with xenografted breast cancer cells, 2b stopped tumour growth. In mice treated with 2b, vacuolization was observed in tumour cells, but not in other organs. This study shows that the antiproliferative activity of 2b relates to the induction of ER stress in cancer, not in healthy, cells and it leads to breast cancer cell death in vitro and in vivo.

Highlights

The rational design of anticancer therapies relies on the identification and the use of the weak points of cancer cells
Our previous work demonstrated that usnic acid (UA) derivative 2b induced massive cytoplasmic vacuolization (Figure 1A), which was partly reversed by dynasore, an inhibitor of dynamin [25]
The present work shows that 2b induced endoplasmic reticulum (ER) stress in MCF-7 breast cancer cells, which led to ER dilatation, massive cytoplasmic vacuolization and cell death, with features of paraptosis

Summary

Introduction

The rational design of anticancer therapies relies on the identification and the use of the weak points of cancer cells. The constitutive activation of mTORC1 found in many cancers leads to uncontrolled protein synthesis and ER overload [2]. It induces an adaptive response called the unfolded protein response (UPR), which, through activation of ER stress sensors, PERK, IRE1 and ATF6, leads to attenuation of global protein synthesis. Depending on the intensity and duration of ER stress, UPR may promote cell survival or death [5,6,7]

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Conclusion