Abstract
Recent studies showed that KGN cells, derived from a human granulosa cell tumor (GCT), express NADPH oxidase 4 (NOX4), an important source of H2O2. Transient receptor potential melastatin 2 (TRPM2) channel is a Ca2+ permeable cation channel that can be activated by H2O2 and plays an important role in cellular functions. It is also able to promote susceptibility to cell death. We studied expression and functionality of TRPM2 in KGN cells and examined GCT tissue microarrays (TMAs) to explore in vivo relevance. We employed live cell, calcium and mitochondrial imaging, viability assays, fluorescence activated cell sorting (FACS) analysis, Western blotting and immunohistochemistry. We confirmed that KGN cells produce H2O2 and found that they express functional TRPM2. H2O2 increased intracellular Ca2+ levels and N-(p-Amylcinnamoyl)anthranilic acid (ACA), a TRPM2 inhibitor, blocked this action. H2O2 caused mitochondrial fragmentation and apoptotic cell death, which could be attenuated by a scavenger (Trolox). Immunohistochemistry showed parallel expression of NOX4 and TRPM2 in all 73 tumor samples examined. The results suggest that GCTs can be endowed with a system that may convey susceptibility to cell death. If so, induction of oxidative stress may be beneficial in GCT therapy. Our results also imply a therapeutic potential for TRPM2 as a drug target in GCTs.
Highlights
In a recent study, we described expression of NADPH oxidase 4 (NOX4) [1] in vivo in human granulosa cells (GCs) of ovarian follicles and in vitro in granulosa-lutein cells derived from in vitro fertilization patients
Our findings suggest that induction of oxidative stress in granulosa cell tumor (GCT) may result in cell death
Trolox-treatments were performed in parallel (n = 3), derived samples were run next to each other on evidence for basal H2O2 production and release by untreated KGN cells, resulting in increasing levels the gels and results of clCASP3 Western blots were analyzed using paired t-test
Summary
We described expression of NADPH oxidase 4 (NOX4) [1] in vivo in human granulosa cells (GCs) of ovarian follicles and in vitro in granulosa-lutein cells derived from in vitro fertilization patients. Activity of this enzyme is linked to the generation of H2 O2 [2], which is a diffusible reactive oxygen species (ROS) and has been postulated to be an important signaling molecule within the follicle (e.g., [3]). A specific NOX4 blocker [2] These results are in line with the changing view of ROS. The results implicate a therapeutic potential of TRPM2 as a possible drug target
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