Supplementary MaterialsSupplementary Figure 1a. induces persistent oxidative stress. The role of iron, an essential nutrient involved in multiple cellular functions, in normal ovarian cell survival and ovarian cancer remains unclear. Iron, presented as ferric ammonium citrate (FAC), dramatically inhibits cell survival in ovarian cancer cell types associated with Ras mutations, while it AR-231453 is without effect in immortalized normal ovarian surface epithelial (T80) and endometriotic epithelial cells (lacking Ras mutations). Interestingly, FAC induced changes in cytoplasmic vacuolation concurrently with increases in LC3-II levels (an autophagy marker); these changes occurred in an ATG5/ATG7-dependent, beclin-1/hVps34-independent, and Ras-independent manner. Knockdown of autophagy mediators in HEY ovarian cancer cells reversed FAC-induced LC3-II levels, but there was little effect on reversing the cell death response. Intriguingly, transmission electron microscopy of FAC-treated T80 cells demonstrated abundant lysosomes (confirmed using Lysotracker) rich in iron particles, which occurred in a Ras-independent manner. Although the mitogen-activated protein kinase (MAPK) inhibitor, U0126, reversed FAC-induced LC3-II/autophagic punctae and lysosomes in a Ras-independent manner, it was remarkable that U0126 reversed cell death in malignant ovarian cells associated with Ras mutations. Moreover, FAC increased heme oxygenase-1 expression in H-Ras-overexpressing T80 cells, which was associated with increased cell death when overexpressed in T80 cells. Disruption of intracellular iron levels, via chelation of intracellular iron (deferoxamine), was also detrimental to malignant ovarian cell survival; thus, homeostatic intracellular iron levels are essential for cell survival. Collectively, our results implicate iron in modulating cell death in a Ras- and MAPK-dependent manner in ovarian cancer cells. strong class=”kwd-title” Keywords: AR-231453 iron (ferric ammonium citrate), lysosomes, Ras, ovarian cancer, MAP kinase Ovarian carcinoma is the fifth most common cancer for women in the United States and is usually diagnosed at an advanced stage when the cancer has already spread.1 Several ovarian cancer subtypes exist that elicit differential responses to chemotherapy. Clear cell ovarian carcinoma (CCC, a rare subtype) LRRC48 antibody is more resistant to chemotherapy compared with serous epithelial ovarian cancers, the major epithelial ovarian carcinoma (EOC).2 Endometriotic cysts, considered a precursor to endometriosis-associated ovarian cancers, contain a high level of heme,3, 4 which can be broken down via the action of heme oxygenase-1 (HO-1) to release iron, biliverdin, and carbon monoxide; these products increase oxidative stress that alters cell survival and contribute to cancer development.3, 4 Treating normal ovarian surface epithelial cells with redox-active iron promotes acquisition of a CCC signature.5 Iron can also induce cell death in cell types associated with Ras mutations.6 Thus, iron may elicit dual functional roles in cancer development. Reactive oxygen species (ROS) can also be generated via hypoxia, correlated with elevated LC3A (a marker of autophagy) expression in CCC associated with hypoxic regions and poor patient outcome.7 Autophagy is a self-eating process where damaged and oxidized cellular material are sequestered in autophagosomes and then degraded within lysosomes.8 Autophagy elicits tumor suppressive effects in normal cells, while under conditions of oxidative stress, autophagy sustains survival of cancer cells. It is presently unknown whether oxidative stress induced by iron alters autophagy to modulate cell survival in normal and malignant ovarian cells. Herein, we present data implicating iron in inhibiting cell survival in ovarian cancer cell types associated with Ras mutations. Iron elevates LC3-II levels in multiple cell types in an ATG5/ATG7-dependent and beclin-1/hVps34-independent fashion. However, knockdown of AR-231453 autophagy mediators resulted in only a modest reversal of cell death. Iron also induced an increase in lysosome numbers in a Ras-independent manner. Inhibition of the mitogen-activated protein kinase (MAPK) pathway in ovarian cancer cells dramatically reversed iron-induced LC3-II levels and lysosome numbers. Strikingly, this inhibitor reversed the cell death response in cell lines associated with Ras mutations. Iron also induced cell death via upregulation of HO-1 in AR-231453 a nuclear factor (erythoid-derived 2)-like 2 (NRF2)-independent but Ras-dependent manner. Modulation of intracellular levels of iron (via chelation with deferoxamine (DFO)) also disrupts cell survival, implicating a need to critically monitor and maintain appropriate levels of.