Ovarian cancer is a leading cause of cancer death in women in the United States. 
While the majority of ovarian cancers are serous, some rarer subtypes (i.e. 
clear cell) are often associated with endometriosis, a benign gynecological 
disease. Iron is rich in the cyst fluid of endometriosis-associated ovarian 
cancers and 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 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.