Activation of the Integrated Stress Response and ER Stress Protect from Fluorizoline-Induced Apoptosis in HEK293T and U2OS Cell Lines.

José Saura-Esteller,Daniel Iglesias-Serret,Ana M Cosialls,Ismael Sánchez-Vera,Pablo M Garcia-Roves,Sonia Núñez-Vázquez,Pau Gama-Pérez,Michael R Duchen,Gabriel Pons,Rodolfo Lavilla,Lorena Mendive-Tapia,Gauri Bhosale,Joan Gil

doi:10.3390/ijms22116117

Abstract

The prohibitin (PHB)-binding compound fluorizoline as well as PHB-downregulation activate the integrated stress response (ISR) in HEK293T and U2OS human cell lines. This activation is denoted by phosphorylation of eIF2α and increases in ATF4, ATF3, and CHOP protein levels. The blockage of the activation of the ISR by overexpression of GRP78, as well as an increase in IRE1 activity, indicate the presence of ER stress after fluorizoline treatment. The inhibition of the ER stress response in HEK293T and U2OS led to increased sensitivity to fluorizoline-induced apoptosis, indicating a pro-survival role of this pathway after fluorizoline treatment in these cell lines. Fluorizoline induced an increase in calcium concentration in the cytosol and the mitochondria. Finally, two different calcium chelators reduced fluorizoline-induced apoptosis in U2OS cells. Thus, we have found that fluorizoline causes increased ER stress and activation of the integrated stress response, which in HEK293T and U2OS cells are protective against fluorizoline-induced apoptosis.

Highlights

Fluorizoline is a synthetic molecule that presents a fluorinated thiazoline scaffold and induces apoptosis in cancer cell lines and primary cells, even in those that present p53 mutations [1,2,3]
We investigated whether fluorizoline and PHB downregulation caused the activation of the integrated stress response (ISR) in HEK293T, U2OS, and Jurkat cell lines
We analyzed the effect of fluorizoline treatment in the expression of other proteins related to the ISR and observed that fluorizoline treatment resulted in increased protein expression of C/EBP homologous protein (CHOP) and ATF3 in HEK293T

Summary

Introduction

Fluorizoline is a synthetic molecule that presents a fluorinated thiazoline scaffold and induces apoptosis in cancer cell lines and primary cells, even in those that present p53 mutations [1,2,3]. (PHBs) [1], which regulate multiple processes within the cell, including cell signaling, gene transcription, or cell cycle progression [4,5,6,7,8]. Prohibitins are conserved proteins that locate to the mitochondria, the plasma membrane, and the nucleus. The most studied functions of PHBs take place in the mitochondria, where these proteins form large heteromultimeric ring-like complexes [9,10]. Mitochondrial PHB complexes participate in the quality control of mitochondria and regulate mitochondrial fusion and fission. PHBs are important in the regulation of cristae morphology, reactive oxygen species formation, or mitophagy [4,5,11]. Different molecules have been described to interact with PHBs [12,13,14]

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Conclusion