Abstract
Reactive oxygen species (ROS) mediates cisplatin-induced cytotoxicity in tumor cells. However, when cisplatin-induced ROS do not reach cytotoxic levels, cancer cells may develop chemoresistance. This phenomenon can be attributed to the inherited high expression of antioxidant protein network. H-Ferritin is an important member of the antioxidant system due to its ability to store iron in a nontoxic form. Altered expression of H-Ferritin has been described in ovarian cancers; however, its functional role in cisplatin-based chemoresistance of this cancer type has never been explored. Here, we investigated whether the modulation of H-Ferritin might affect cisplatin-induced cytotoxicity in ovarian cancer cells. First, we characterized OVCAR3 and OVCAR8 cells for their relative ROS and H-Ferritin baseline amounts. OVCAR3 exhibited lower ROS levels compared to OVCAR8 and greater expression of H-Ferritin. In addition, OVCAR3 showed pronounced growth potential and survival accompanied by the strong activation of pERK/pAKT and overexpression of c-Myc and cyclin E1. When exposed to different concentrations of cisplatin, OVCAR3 were less sensitive than OVCAR8. At the lowest concentration of cisplatin (6 μM), OVCAR8 underwent a consistent apoptosis along with a downregulation of H-Ferritin and a consistent increase of ROS levels; on the other hand, OVCAR3 cells were totally unresponsive, H-Ferritin was almost unaffected, and ROS amounts met a slight increase. Thus, we assessed whether the modulation of H-Ferritin levels was able to affect the cisplatin-mediated cytotoxicity in both the cell lines. H-Ferritin knockdown strengthened cisplatin-mediated ROS increase and significantly restored sensitivity to 6 μM cisplatin in resistant OVCAR3 cells. Conversely, forced overexpression of H-Ferritin significantly suppressed the cisplatin-mediated elevation of intracellular ROS subsequently leading to a reduced responsiveness in OVCAR8 cells. Overall, our findings suggest that H-Ferritin might be a key protein in cisplatin-based chemoresistance and that its inhibition may represent a potential approach for enhancing cisplatin sensitivity of resistant ovarian cancer cells.
Highlights
The oxygen-containing reactive species (ROS) are unstable by-products of cellular metabolism that are essential for several biological processes including mitochondrial and plasma membrane functioning, cell signalling and immune response [1]
Through a series of assays including FHC knockdown and forced overexpression, we demonstrate for the first time that ferritin heavy subunit, through its ability to modulate Reactive oxygen species (ROS) amounts, is a key element in determining the response of ovarian cancer cells to cisplatin exposure
MitoSOX staining showed only a slightly different intensity between the two cell lines. These results suggest that OVCAR8 cells are characterized by higher levels of total ROS content compared to OVCAR3 cells
Summary
The oxygen-containing reactive species (ROS) are unstable by-products of cellular metabolism that are essential for several biological processes including mitochondrial and plasma membrane functioning, cell signalling and immune response [1]. The rate and magnitude of ROS production are tightly controlled by an antioxidant defense system. The imbalance in the circuitries of ROS production and removal leads to impairment of cell signalling, oxidative damage of cell components, and cytotoxicity [1,2,3,4]. A persistent ROS overproduction may induce cellular adaptation as it occurs in many diseases, especially in cancer. To keep ROS in the prooncogenic zone, cancer cells are provided by an extensive supply of antioxidant molecules that reduce the efficacy of prooxidant drugs and enable tumor cells to acquire chemoresistance [11, 12]
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