2',5'-Dihydroxychalcone-induced glutathione is mediated by oxidative stress and kinase signaling pathways

Abstract

Hydroxychalcones are naturally occurring compounds that continue to attract considerable interest because of their anti-inflammatory and antiangiogenic properties. They have been reported to inhibit the synthesis of the inducible nitric oxide synthase and to induce the expression of heme oxygenase-1. This study examines the mechanisms by which 2',5'-dihydroxychalcone (2',5'-DHC) induces an increase in cellular glutathione (GSH) levels using a cell line stably expressing a luciferase reporter gene driven by antioxidant-response elements (MCF-7/AREc32). The 2',5'-DHC-induced increase in cellular GSH levels was partially inhibited by the catalytic antioxidant MnTDE-1,3-IP(5+), suggesting that reactive oxygen species (ROS) mediate the antioxidant adaptive response. 2',5'-DHC treatment induced phosphorylation of the c-Jun N-terminal kinase (JNK) pathway, which was also inhibited by MnTDE-1,3-IP(5+). These findings suggest a ROS-dependent activation of the AP-1 transcriptional response. However, whereas 2',5'-DHC triggered the NF-E2-related factor 2 (Nrf2) transcriptional response, cotreatment with MnTDE-1,3-IP(5+) did not decrease 2',5'-DHC-induced Nrf2/ARE activity, showing that this pathway is not dependent on ROS. Moreover, pharmacological inhibitors of mitogen-activated protein kinase (MAPK) pathways showed a role for JNK and p38MAPK in mediating the 2',5'-DHC-induced Nrf2 response. These findings suggest that the 2',5'-DHC-induced increase in GSH levels results from a combination of ROS-dependent and ROS-independent pathways.

Fig. 1

Structures of chalcone, 2′-hydroxychalcone (2′-HC), 2′,5′-dihydroxychalcone (2′,5′-DHC) and the flavonoid chrysin.

Fig. 2

2′,5′-DHC induces oxidative stress. (A) 2′,5′-DHC (40 μM)-induced cytotoxicity was increased by the GSH synthesis inhibitor BSO (20 μM) and diminished by the catalytic antioxidant MnTDE-1,3-IP⁵⁺ (MnP, 10–20 μM) (48 h treatment). N-acetyl cysteine (NAC, 1 mM) did not protect the cells against 2′,5′-DHC-induced cytotoxicity. Bars with different letters were statistically different from one another (n = 4, P < 0.05). (B) As shown by microscopy using MitoSOX as dye, 2′,5′-DHC (40 μM) induced an increase in ROS-induced fluorescence that was diminished by MnTDE-1,3-IP⁵⁺ (MnP, 10 μM) (24 h treatment). (C) As shown by flow cytometry using MitoSOX as dye, a non-toxic concentration of 2′,5′-DHC (10 μM) induced a temporary increase in fluorescence at 1 h treatment (n = 3).

Fig. 3

2′,5′-DHC-induced increase in iGSH levels is inhibited by MnTDE-1,3-IP⁵⁺. (A) 2′,5′-DHC (10 μM) induced a drop (6 h) followed by an increase (24 h) of iGSH levels, whereas chrysin (Chr, 10 μM) induced only a drop of iGSH levels (n = 3). (B) MnTDE-1,3-IP⁵⁺ (MnP, 10 μM) significantly inhibited the 2′,5′-DHC-induced increase in iGSH levels, while NAC (1 mM) did not (n = 3). (C) When compared to 2′,5′-DHC (DHC, 10 μM), the non-hydroxylated chalcone (Cha, 10 μM) and 2′-HC (10 μM) had intermediate effects in increasing iGSH levels. Bars with different letters were statistically different from one another (n = 3, P < 0.05).

Fig. 4

2′,5′-DHC induces an increase in GCL activity. (A) Cytosolic fractions of untreated (Con) and 2′,5′-DHC-treated AREc32 cells (15 μM, 16 h) were analyzed in their ability to catalyze the formation of γ-glutamyl-cysteine (γ-GC). 2′,5′-DHC induced a 20% increase in GCL activity. The reaction time was 30 min. Bars with different letters were statistically different from one another (n = 3, P < 0.05). (B) As shown by immunoblotting, 2′,5′-DHC (10 μM) induced the expression of HO-1 (34 kDa) (24 h treatment), an effect that was inhibited by MnTDE-1,3-IP⁵⁺ (MnP, 10 μM). 2′,5′-DHC had no significant effect on GCLC (73 kDa) levels. The experiment was repeated once. (C) As shown by immunoblotting, AREc32 cells treated (24 h) with 2′,5′-DHC (10 μM) alone or in combination with MnTDE-1,3-IP⁵⁺ (MnP, 10 μM) had no apparent effect on GCLC, GCLM nor GCL holoenzyme (holo-GCL) levels.

Fig. 5

2′,5′-DHC-induced Nfr2/ARE response activity is ROS-independent. (A) The hydroxychalcones 2′-HC and 2′,5′-DHC (10 and 20 μM) induced the Nrf2/ARE response in AREc32 cells, as assessed by measuring luciferase activity (18 h treatment). (B) The Nrf2/ARE response was not inhibited by the antioxidants NAC (5 mM) nor MnTDE-1,3-IP⁵⁺ (MnP, 20 μM). Bars with different letters were statistically different from one another (n = 3, P < 0.05).

Fig. 6

2′,5′-DHC-induced Nfr2/ARE response activity is inhibited by inhibitors of protein kinase pathways. (A-D) Luciferase activity was measured in 2′,5′-DHC-treated AREc32 cells co-treated with pharmacological inhibitors of various protein kinase pathways, namely SP600125 (JNK), SB203580 (p38MAPK), wortmannin (PI3K) and U0126 (ERK) (18 h treatment). (A) SP600125 (SP, 20 μM) and (B) SB203580 (SB, 20 μM) inhibited 15 μM 2′,5′-DHC-induced luciferase activity by 43% and 61%, respectively, while (C) wortmannin (Wort, 0.1 μM) had no significant effect. (D) The inhibitor of the ERK pathway U0126 (10 μM) induced the activation of the Nrf2/ARE pathway and this effect was additive to 2′,5′-DHC co-treatment. (E) SB203580 (SB, 20 μM) and SP600125 (SP, 20 μM) and wortmannin (Wort, 0.1 μM) were tested in their ability to inhibit 2′,5′-DHC-induced GSH synthesis (24 h treatment). Both SB203580 and SP600125 had an important inhibitory effect, while wortmannin had a slight effect. Bars with different letters were statistically different from one another (n = 3, P < 0.05).

Fig. 7

2′,5′-DHC-induced phosphorylation of c-Jun is ROS-dependent. (A) 2′,5′-DHC (DHC, 15 μM) induced the phosphorylation of c-Jun (48 kDa) while c-Jun levels remained the same (39 kDa) in AREc32 cells treated for 2 h, an effect that was inhibited by MnTDE-1,3-IP⁵⁺ (MnP, 10 μM), as shown by immunoblotting. The experiment was repeated once. (B) 2′,5′-DHC (DHC, 30 μM) induced an increase in luciferase activity in transformed 16HBE cells, an effect that was inhibited by MnTDE-1,3-IP⁵⁺ (MnP, 20 μM) (24 h treatment). Bars with different letters were statistically different from one another (n = 3, P < 0.05).