Cyclophosphamide Induces Glioblastoma Tumor Cell Death and Oxidative Stress Through the Increase of TRPM2 Channel Stimulation: The Role of Carvacrol.

Abstract

Cyclophosphamide (CP) damages glioblastoma cells by producing an excessive amount of intracellular (iROS) and mitochondrial (mROS) reactive oxygen species. Both iROS and mROS are produced when TRPM2 is activated, but they are decreased when carvacrol (CAR) and N-(p-amylcinnamoyl) anthranilic acid (ACA) inhibit it. Therefore, iROS, and mROS via upregulating Ca²⁺ influx and apoptosis in glioblastoma (DBTRG-05MG) cells, CP-mediated TRPM2 stimulation may cause oxidant and apoptotic activities. We investigated how TRPM2 activation not only promotes DBTRG-05MG death but also modifies oxidative damage and apoptosis to counteract the effects of ACA and CAR. The groups of control (CN), CAR (200 μM for 24 h), CP (2 mM for 24 h), and CP + CAR were induced in the DBTRG-05MG. While cytosolic free Ca²⁺ levels decreased in the cells as a result of the CAR and ACA treatments, they were further elevated in the CP group by the stimulation of TRPM2 (H₂O₂). The cells in the CP group had higher levels of dead cell percentage, apoptosis, mitochondrial membrane dysfunction, mROS, iROS, and caspases -3, -8, and -9 than the CN and CAR cells, although their levels were lower in the CP + CAR than in the CP only. CAR incubation increased the CP-induced glutathione concentration and cell viability percentage declines. In summary, the anticancer effect of CP was enhanced by TRPM2 stimulation, while CP-induced oxidative stress and DBTRG-05MG death were reduced by TRPM2 suppression when CAR was treated. TRPM2 activation may be a possible tumor killer channel due to oxidative glioma damage caused by CP.