Energy and endoplasmic reticulum stress induction by gold(III) dithiocarbamate and 2-deoxyglucose synergistically trigger cell death in breast cancer

Abstract

The elusiveness of triple-negative breast cancer from targeted therapy has redirected focus toward exploiting the metabolic shortcomings of these highly metastatic subtypes of breast cancer. Cueing from the metabolic heterogeneity of TNBC and the exposition of the dual dependence of some TNBCs on OXPHOS and glycolysis for ATP, we herein report the efficacy of cotreatment of TNBCs with an OXPHOS inhibitor, 2a and 2DG, a potent glycolysis inhibitor. 2a-2DG cotreatment inhibited TNBC cell proliferation with IC₅₀ of ∼5 to 36 times lower than that of 2a alone and over 5000 times lower than IC₅₀ of 2DG alone. 2a-2DG cotreatment suppressed mitochondrial ATP production and significantly induced AMPK activation. Mechanistic studies revealed the distinct yet synergistic contributions of 2a and 2DG to the antiproliferative effect of the cotreatment. While 2a induced apoptotic cell death, 2DG sensitized TNBCs to the antiproliferative effects of 2a via endoplasmic reticulum stress induction. Strikingly, the combination of 2a-2DG ablated SUM159 tumors in an orthotopic xenograft mouse model. This study highlights the synergistic effect of a gold-based complex with 2DG and the potential benefit of multimetabolic pathways targeting as an effective therapeutic strategy against TNBCs.

Keywords: TNBC; breast cancer; endoplasmic reticulum; energy; gold; mitochondria.

Figure 1

Effect of 2a and/or 2DG on viability and proliferation in TNBC. Chemical structures of (A) 2a and (B) 2DG., (C) a dose-response curve showing % cell viability in response to varying concentrations of 2DG in MDA-MB-468 and SUM 159 after 72 h treatment, (D) 2a only and 2a-2DG combinations in MDA-MB-468 after 72 h treatment, and (E) 2a only and 2a-2DG combinations in SUM 159 after 72 h treatment. Mean ± SD. (n = 6) Cell viability evaluated using MTT assay. Effects of 2a and/or 2DG on colony formation following 2a and/or 2DG treatment on (F) and (G) (MDA-MB-468, (H) and (I) SUM 159. Mean ± SD. n = 3. Data presented in G and I were analyzed by ordinary one-way ANOVA followed by Tukey’s multiple comparison test (∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). 2DG, 2-deoxyglucose; MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide); TNBC, triple-negative breast cancer.

Figure 2

Effect of 2a and/or 2DG on spheroid formation. Effects of 2a and/or 2DG on spheroid formation 5 days following 2a and/or 2DG treatment on (A) MDA-MB-468 mammospheres, (B) SUM159 mammospheres (C) MCF10A mammospheres and (D) HEK293 spheroids. The scale bar represents 200 μm. 2DG, 2-deoxyglucose.

Figure 3

Mechanism of cell death in 2a and/or 2DG treated TNBC. Annexin V-PI flow cytometric analysis to assess the effect of 2a (10 μM) and/or 2DG (10 mM) after 18 h treatment on (A) and (C) MDA-MB-468, (B and D) SUM 159. n = 3. E, Western blot analyses of apoptosis-related proteins in MDA-MB-468 cells treated with 2a (1 μM) and/or 2DG (10 mM) for 6 h. (F) Effect of 2a (1 μM) and/or 2DG (5 mM) on cell cycle of MDA-MB-468 cells after 12, 24, and 48 h treatment. Data are plotted as mean ± SD. n = 3. Data presented in B and D were analyzed by ordinary one-way ANOVA followed by Tukey’s multiple comparison test (∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). TNBC, triple-negative breast cancer; 2DG, 2-deoxyglucose.

Figure 4

Effects of combined treatment of TNBC cells with 2a and 2DG. The Fa-CI plots demonstrate the synergism of 2a and 2DG combinations in (A) MDA-MB-468 and (E) SUM159, calculated with the Compusyn software. Fa-DRI plots show the reducibility of the 2a and 2DG doses in the combination treatment against MDA-MB-468 (B–D) and SUM159 (F–H). Data points represent mean values. 2DG, 2-deoxyglucose; DRI, dose reduction index; TNBC, triple-negative breast cancer.

Figure 5

Combination of 2a and 2DG inhibits oxygen consumption and mitochondria-ATP production. (A) OCR data following treatment of MDA-MB-468 cells with 2a and/or 2DG for 24 h. Extrapolated parameters from mito stress test upon treatment of MDA-MB-468 cells with 2a and/or 2DG showing (B) basal respiration, (C) maximal respiration, (D) ATP production, and (E) rate of glycolysis. Western blot showing key energy stress marker, AMPK, and proteins associated with AMPK pathway following treatment with 2a and/or 2DG for 6 h in (F) MDA-MB-468 and (G) SUM159. H, mechanism of AMPK and ACC phosphorylation. Data are plotted as the mean ± SD. (n = 8). Data presented in B–G were analyzed by ordinary one-way ANOVA followed by Tukey’s multiple comparison test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. n.s. = not significant). 2DG, 2-deoxyglucose; ACC, acetyl CoA carboxylase; AMPK, AMP-activated protein kinase; OCR, oxygen consumption rate.

Figure 6

Cotreatment of 2a and 2DG intensifies induction of ER stress in TNBC. Western blot showing the expression of phosphorylated protein kinase R-like ER Kinase (p-PERK), protein kinase R-like ER kinase (PERK), binding immunoglobulin protein (BiP), activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), activating transcription factor 6 (ATF6), phosphorylated inositol-requiring enzyme 1 alpha (p-IRE1α), and x-box binding protein 1, spliced form (XBP1s) in (A) and (C) MDA-MB-468 cells and (B) and (D) SUM159 after 6 h treatment with 2a and/or 2DG (E) mechanism of ER stress activation through the three arms of the pathway. Data presented in A–D were analyzed by ordinary one-way ANOVA followed by Tukey’s multiple comparison test (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). 2DG, 2-deoxyglucose; TNBC, triple-negative breast cancer.

Figure 7

Combination of 2a and 2DG inhibits TNBC progression in vivo.A, change in SUM159 tumor volume following 2a and/or 2DG administration for 27 days. B, body weight of mice following 2a and/or 2DG administration for 27 days. C, representative images of the tumor across all the mice groups. Data are plotted as mean ± SEM. n = 3. Data were analyzed by two-sided Student’s t test (∗p < 0.05, ∗∗p < 0.01). 2DG, 2-deoxyglucose; TNBC, triple-negative breast cancer.