Targeting AKT-Dependent Regulation of Antioxidant Defense Sensitizes AKT-E17K Expressing Cancer Cells to Ionizing Radiation

Abstract

Aberrant activation of the phosphatidyl-inositol-3-kinase/protein kinase B (AKT) pathway has clinical relevance to radiation resistance, but the underlying mechanisms are incompletely understood. Protection against reactive oxygen species (ROS) plays an emerging role in the regulation of cell survival upon irradiation. AKT-dependent signaling participates in the regulation of cellular antioxidant defense. Here, we were interested to explore a yet unknown role of aberrant activation of AKT in regulating antioxidant defense in response to IR and associated radiation resistance. We combined genetic and pharmacologic approaches to study how aberrant activation of AKT impacts cell metabolism, antioxidant defense, and radiosensitivity. Therefore, we used TRAMPC1 (TrC1) prostate cancer cells overexpressing the clinically relevant AKT-variant AKT-E17K with increased AKT activity or wildtype AKT (AKT-WT) and analyzed the consequences of direct AKT inhibition (MK2206) and inhibition of AKT-dependent metabolic enzymes on the levels of cellular ROS, antioxidant capacity, metabolic state, short-term and long-term survival without and with irradiation. TrC1 cells expressing the clinically relevant AKT1-E17K variant were characterized by improved antioxidant defense compared to TrC1 AKT-WT cells and this was associated with increased radiation resistance. The underlying mechanisms involved AKT-dependent direct and indirect regulation of cellular levels of reduced glutathione (GSH). Pharmacologic inhibition of specific AKT-dependent metabolic enzymes supporting defense against oxidative stress, e.g., inhibition of glutathione synthase and glutathione reductase, improved eradication of clonogenic tumor cells, particularly of TrC1 cells overexpressing AKT-E17K. We conclude that improved capacity of TrC1 AKT-E17K cells to balance antioxidant defense with provision of energy and other metabolites upon irradiation compared to TrC1 AKT-WT cells contributes to their increased radiation resistance. Our findings on the importance of glutathione de novo synthesis and glutathione regeneration for radiation resistance of TrC1 AKT-E17K cells offer novel perspectives for improving radiosensitivity in cancer cells with aberrant AKT activity by combining IR with inhibitors targeting AKT-dependent regulation of GSH provision.

Keywords: AKT; antioxidant defense; glutathione reductase; glutathione synthase; glycolysis; hexokinase 2; radioresistance.

Figure 1

Expression of AKT-E17K is associated with increased levels of reduced cellular antioxidants and improved defense against ROS. TrC1 AKT-E17K and AKT-WT cells were treated with the AKT inhibitor MK2206 (4 µM) 1 h prior 5 Gy radiation and then analyzed 12 h after IR treatment or without IR treatment. GSH levels were analyzed using a luminescence-based assay upon indicated treatments at 12 h after IR (A–B). ROS levels were determined by DHE using flow cytometry 24 h after non-irradiated controls (C) and after irradiation with 5 Gy (D). Mean values and standard error of the mean (SEM) were scaled for data from at least 3 independent biological experiments with * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, ns p > 0.05 using two-way ANOVA followed by Tukey’s multiple comparison test.

Figure 2

Glycolytic activity is increased in TrC1 AKT-E17K cells and supports recovery from metabolic inhibition induced by IR. (A) Akt phosphorylation on S473 for unirradiated TrC1 AKT-WT and AKT-E17K cells measured in western blot and normalized on total AKT expression and β-actin. Representative pictures are shown in (A) with quantification in (B). Basal glycolytic activity of TrC1 AKT-WT and AKT-E17K cells at 0 Gy and 5 Gy with and without MK2206 (4 µM) treatment was measured in an extracellular flux assay (Seahorse technology) and extracellular acidification rate (ECAR) values are shown for 0 Gy and 12 h after 5 Gy (C–D), time dependent regulation of basal glycolysis in the first 24 h after 5 Gy (E), the acute response after radiation (F) and the recovery phase after IR (G). In an extracellular flux assay (Seahorse technology) glucose capacity (H) was determined using the fuel flex assay. Mean values and standard error of the mean (SEM) were scaled for data from n=3 independent biological experiments (A, B) or n = 8-16 wells from 2 independent biological experiments (C-H) with * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, ns p > 0.05 using standard unpaired t-test (A, B), two-way ANOVA followed by Tukey’s multiple comparison test (C–E, H) or mixed effect analysis multiple comparison with Sidak’s correction (F-G).

Figure 3

Inhibition of glycolytic activity by treatment with 2-deoxyglucose (2DG) affects antioxidant levels and enhances radiosensitivity. TrC1 AKT-WT and AKT-E17K cells were pre-treated for 1 h with the solvent control or the hexokinase inhibitor 2DG (10 mM) and subsequently exposed to irradiation with 0 Gy (controls) or 5 Gy as indicated. 12 h after the irradiation time point we determined (A, B) extracellular acidification rate (ECAR) by an extracellular flux assay; (C, D) NADPH/NADH ratio using FLIM measurement; and (F, G) GSH levels using a luminescence assay. Photomicrographs in (E) depict FLIM pictures for 0 Gy and 12 h after 5 Gy showing tm with representative pictures of one representative experiment out of three. ROS levels were measured in flow cytometry using DHE-staining for (H) 0 Gy and (I) 24 h after 5 Gy. Bar diagrams in (J, K) depict cell death levels detected by flow cytometry using PI-staining. (L) Long term survival of TrC1 AKT-WT and AKT-E17K cells pre-treated with 0 or 10 mM 2DG and subsequently irradiated with 0-8 Gy was determined by indirect colony formation assays; data depict the surviving fraction upon irradiation with 8 Gy. Mean values and standard error of the mean (SEM) were scaled for data from at least 3 independent biological experiments with * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, ns p > 0.05 using two-way ANOVA followed by Tukey’s multiple comparison test (A, B, F–L) or one way ANOVA followed by multiple test (C, D).

Figure 4

Pharmacologic inhibition of glutathione synthetase (GS) enhances sensitivity of TrC1 AKT-E17K cells to ionizing radiation (IR) by disturbing antioxidant defense. (A) Schematic representation of the GSH generation pathways via γ-glutamylcysteine synthetase (γ-GCS) and GS and its inhibition by buthionine sulfoximin (BSO) on the GS. Different parameters for radiation response were quantified for TrC1 AKT-E17K and AKT-WT cells treated with the GS inhibitor BSO (200 µM) 1 h before 5 Gy radiation or without IR. GSH levels were analyzed in a luminescence-based assay 12 h after 0 Gy (B) and 5 Gy (C) treatments. A flow cytometry-based analysis of ROS-positive cells 24 h after IR (E) compared to non-irradiated controls (D) was used for ROS quantification. Cell death levels were determined in non-irradiated 0 Gy controls (F) and 48 h after 5 Gy (G) treatment. The number of γH2A.X foci 30 min controls was analyzed via fluorescence microscopy 24 h after 3 Gy and BSO (200 µM) treatment in comparison with the controls (H-I). In an indirect colony formation assay, TrC1 AKT-WT and AKT-E17K cells were treated with BSO (20 µM) and irradiated with doses from 0-8 Gy and SF was plotted dose dependently in (J-K) and in a bar chart for 5 Gy treatment with additional glutathione ethyl ester (GEE, 4 mM) supplementation 1 h prior IR (L). Mean values and standard error of the mean (SEM) were scaled for data from at least 3 independent biological experiments with * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, ns p > 0.05 using two-way ANOVA followed by Tukey’s multiple comparison test (A-G, J-L) or one way ANOVA followed by multiple test (H).

Figure 5

Pharmacologic inhibition of glutathione reductase (GR) disturbs cellular antioxidant defense and sensitizes TrC1 E17K cells to ionizing radiation (IR). (A) Schematic representation of the mechanism of reduced glutathione (GSH) generation and the action of the inhibitor of GR AAPA. The effects on TrC1 AKT-E17K and AKT-WT cells of the inhibition of GR by AAPA (40 µM) treatment 1 h before 5 Gy or 0 Gy were analyzed. GSH levels were quantified in a luminescence-based assay 12 h after IR (C) or without IR (B). In flow cytometry ROS levels were determined by DHE-staining 24 h after respective treatments (D, E) whereas cell death levels were accessed by quantification of the % of PI-positive cells 48 h after treatment (F, G). The number of γH2A.X foci normalized on 30 min controls was measured 24 h after 3 Gy and AAPA (40 µM) treatment in comparison with the controls (H, I). TrC1 AKT-WT and AKT-E17K cells were treated with AAPA (4 µM) 1 h prior to irradiation with doses from 0-8 Gy and colony formation assay was performed determining survival fractions in doses-dependent curves (J, K) and in a bar chart for 5 Gy treatment with additional glutathione ethyl ester (GEE, 4 mM) supplementation 1 h prior IR (L). Mean values and standard error of the mean (SEM) were scaled for data from at least 3 independent biological experiments with * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, ns p > 0.05 using two-way ANOVA followed by Tukey’s multiple comparison test (A-G, J-L) or one way ANOVA followed by multiple test (H).