Sodium-Glucose Cotransporter 2 (SGLT2) Inhibition Induces DNA Damage and Radiosensitization in Glioblastoma Cell Lines

Abstract

Glioblastomas (GBM) are highly dependent on glucose for energy to promote cell proliferation and tumor growth. Preclinical studies have shown that sodium-glucose cotransporter 2 (SGLT2) inhibitors, drugs that are FDA approved for diabetes, reduce the viability and invasiveness of GBM cells. We hypothesize that SGLT2 inhibitors decrease GBM cell growth and viability by promoting DNA damage. The SGLT2 inhibitors canagliflozin, dapagliflozin and empagliflozin were used and cell studies were performed under low glucose (0.6 mM) media conditions. In the cell proliferation assays, cells were counted in triplicate after 24-, 48- and 72-hour treatment with SGLT2 inhibitors. Glucose transport assays were performed using a 6-fluorescent iodinated glucose analog (6FIGA) which is selectively transported by SGLTs and not by glucose transporters (GLUTs). Immunoblots were used to measure protein expression of SGLT2 and markers of DNA damage (phospho-ATM and gamma-H2AX). Cellular radiosensitization was evaluated by clonogenic survival assays. SGLT2 inhibition with canagliflozin, dapagliflozin and empagliflozin decreased cell proliferation in LN229, U118MG and LN18 GBM cells lines. In LN229 cells, 72-hour treatment with canagliflozin (30 µM) decreased cell proliferation by 60% compared to vehicle control cells (p<0.0001) whereas dapagliflozin (30 µM) and empagliflozin (30 µM) decreased cell proliferation compared to vehicle control cells by 17% (p<0.0001) and 19% (p<0.001), respectively. Glucose transport assays show that 6FIGA uptake is reduced by canagliflozin and dapagliflozin in LN229 and U87 GBM cells. Canagliflozin treatment (50 µM) for 24 hours in LN229 cells results in increased phosphorylated ATM protein and increased gamma-H2AX protein, markers of DNA damage, by immunoblot. SGLT2 protein expression was not affected by canagliflozin. Treatment with canagliflozin (50 µM) 48 hours prior to 2, 4 and 6 Gy of ionizing radiation reduced clonogenic survival of LN229, LN18 and U118MG GBM cells. SGLT2 inhibitors canagliflozin, dapagliflozin and empagliflozin decrease GBM cell proliferation, with the highest inhibitory effect seen with canagliflozin. Canagliflozin treatment alone increases DNA damage signaling in GBM cells. Canagliflozin radiosensitizes GBM cells. This work provides preliminary preclinical rationale for canagliflozin as a radiosensitizer in GBM.