Figure 8.

Reconstitution of TRPM2 and Nrf2 modulates ROS in TRPM2 depletion. (a) SH-SY5Y cells depleted of TRPM2 and transfected with empty vector (KO-1-v), wild type TRPM2 (KO-1-FL), or the E960D pore mutant (KO-1-E960D) were loaded with MitoSOX Red and studied at baseline and 24 hours after treatment with 0.3 μM doxorubicin. Scrambled control cells were transfected with empty vector (Scr-1-v). Intensity of MitoSOX fluorescence was quantitated with confocal microscopy. Representative fields of untreated or doxorubicin treated cells are shown. Means ± SEM fluorescence intensity of a minimum of 100 cells in at least 10 fields in each group was quantified. Results from a representative experiment of two are shown on the right. (b) SH-SY5Y cells depleted of TRPM2 were transfected with empty vector (KO-1-v) or Nrf2 (KO-1-Nrf2). Cells were loaded with MitoSOX Red and studied as described in a and results from a representative experiment of three is shown. (a,b) *indicates p < 0.05, ** < 0.01, *** < 0.001, one-way ANOVA. (c) Schema of impact of TRPM2 inhibition on antioxidant response and ROS levels in neuroblastoma. TRPM2 inhibition or depletion lowers calcium entry. Nrf2 is reduced by down modulation of IQGAP1 and downstream enzymes involved in glutamine/GSH production, antioxidant defense, and folate and the pentose phosphate pathways are decreased. This leads to decreased antioxidant cofactors and impaired antioxidant response, increasing susceptibility to chemotherapeutic agents and decreasing cell survival and tumor growth.