Fluorizoline is a new pro‐apoptotic agent that binds to prohibitins (PHB1 and PHB2) in the mitochondria and induces the integrated stress response (ISR). With phosphorylation of eukaryotic translation initiation factor 2α as the central event of this signaling pathway, fluorizoline induces ATF4 and ATF3. We demonstrated the key role of NOXA in fluorizoline‐induced apoptosis and identified ATF3 and ATF4 transcription factors as key mediators of NOXA upregulation and fluorizoline‐induced apoptosis.

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.

No abstract