Decreased insulin secretion, associated with pancreatic β-cell failure, plays a critical role in many human diseases including diabetes, obesity, and cancer. While numerous studies linked β-cell failure with enhanced levels of reactive oxygen species (ROS), the development of diabetes associated with hereditary conditions that result in iron overload, e.g., hemochromatosis, Friedreich’s ataxia, and Wolfram syndrome type 2 (WFS-T2; a mutation in CISD2, encoding the [2Fe-2S] protein NAF-1), underscores an additional link between iron metabolism and β-cell failure. Here, using NAF-1-repressed INS-1E pancreatic cells, we observed that NAF-1 repression inhibited insulin secretion, as well as impaired mitochondrial and ER structure and function. Importantly, we found that a combined treatment with the cell permeant iron chelator deferiprone and the glutathione precursor N-acetyl cysteine promoted the structural repair of mitochondria and ER, decreased mitochondrial labile iron and ROS levels, and restored glucose-stimulated insulin secretion. Additionally, treatment with the ferroptosis inhibitor ferrostatin-1 decreased cellular ROS formation and improved cellular growth of NAF-1 repressed pancreatic cells. Our findings reveal that suppressed expression of NAF-1 is associated with the development of ferroptosis-like features in pancreatic cells, and that reducing the levels of mitochondrial iron and ROS levels could be used as a therapeutic avenue for WFS-T2 patients.

We recently reported on the development of a unique cancer-targeting peptide called NAF-1 ^44-67 (derived from CISD2/NAF-1). NAF-1 ^44-67 selectively permeates the plasma membrane (PM) of cancer cells, but not healthy cells, causing the activation of apoptotic and ferroptotic cell death pathways specifically in cancer cells. NAF-1 ^44-67 also targets and shrinks human breast and ovarian cancer tumors in a xenograft mice model system without any apparent side effects. Although the specific permeation of NAF-1 ^44-67 through cancer cell PMs was studied, and its cancer killing effects validated in vitro and in vivo , little is known about how NAF-1 ^44-67 exerts its biological activity once it enters cancer cells. Here, we report that NAF-1 ^44-67 targets the CISD2/NAF-1 protein of cancer cells and disrupts its homodimeric structure. We further reveal that a peptide derived from the same domain of the human CISD1 (mitoNEET; mNT ^19-42 ) protein, a close family member to CISD2, has no killing activity towards cancer cells, and that dimers of NAF-1 ^44-67 (at two different orientations) have higher anticancer activity compared to monomeric NAF-1 ^44-67 . Our findings shed new light on the biological activity of NAF-1 ^44-67 and bring it closer to becoming a potential new anticancer drug.