A Role for H2O2 and TRPM2 in the Induction of Cell Death: Studies in KGN Cells

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.

Sirtuin 1 and Sirtuin 3 in Granulosa Cell Tumors

“…TMAs were assembled from archived, no-longer-used residual paraffin material, as described earlier [ 28 ]. All the cases were pseudoanonymized.…”

“…The staining procedures were conducted as previously described [ 28 ]. The antibodies used for the study are listed in Supplementary Table S5 .…”

“…The commercial Annexin V -FITC Apoptosis Detection Kit (eBioscience TM , San Diego, CA, USA) was used to determine occurring apoptosis and was implemented according to the manufacturer’s instructions. FACS analysis was performed at the Core Facility Flow Cytometry at the Biomedical Center of the LMU Munich, as described previously [ 28 ].…”

“…In particular, KGN cells [ 25 ] are widely used as an in vitro cell culture model of common, adult GCTs [ 26 , 27 ]. As in our previous studies [ 15 , 28 ], we used KGN cells in the present study. Furthermore, samples from patients and a tissue microarray (TMA) were available to explore whether and how SIRTs may be involved in the growth of GCTs.…”

Free Radical Research in Cancer

“…Mechanisms, by which cellular processes are changed in cancer, spread on different molecules (such as enzymes, transcription factors, or ion channels). An example of an ion channel is the transient receptor potential melastatin 2 (TRPM2), a Ca 2+ channel that can be activated by H 2 O 2 [14]. A study presented by Hack et al [14] showed parallel expression of NOX4 and TRPM2 in human granulosa cell tumor samples, suggesting that induction of oxidative stress could be beneficial for the therapy, as activation of this channel by H 2 O 2 increased Ca 2+ levels and apoptotic cell death.…”