Selected flavonoids potentiate the toxicity of cisplatin in human lung adenocarcinoma cells: a role for glutathione depletion

Abstract

Adjuvant therapies that enhance the anti-tumor effects of cis-diammineplatinum(II) dichloride (cisplatin, CDDP) are actively being pursued. Growing evidence supports the involvement of mitochondrial dysfunction in the anti-cancer effect of cisplatin. We examined the potential of using selective flavonoids that are effective in depleting tumor cells of glutathione (GSH) to potentiate cisplatin-mediated cytotoxicity in human lung adenocarcinoma (A549) cells. We found that cisplatin (40 microM, 48-h treatment) disrupts the steady-state levels of mitochondrial respiratory complex I, which correlates with elevated mitochondrial reactive oxygen species (ROS) production and cytochrome c release. The flavonoids, 2',5'-dihydroxychalcone (2',5'-DHC, 20 microM) and chrysin (20 microM) potentiated the cytotoxicity of cisplatin (20 microM), which could be blocked by supplementation of the media with exogenous GSH (500 microM). Both 2',5'-DHC and chrysin were more effective than the specific inhibitor of GSH synthesis, L-buthionine sulfoximine (BSO, 20 microM), in inducing GSH depletion and potentiating the cytotoxic effect of cisplatin. These data suggest that the flavonoid-induced potentiation of cisplatin's toxicity is due, in part, to synergetic pro-oxidant effects of cisplatin by inducing mitochondrial dysfunction, and the flavonoids by depleting cellular GSH, an important antioxidant defense.

Figure 1

Molecular structures of chalcone (no hydroxyl group), 2′-hydro-xychalcone (2′-HC), 4′-hydroxychalcone (4′-HC), 2′,5′-dihydroxychalcone (2′,5′-DHC) and chrysin.

Figure 2

Cisplatin (CDDP) disrupted the steady-state level mitochondrial respiratory complex I and increased mitochondrial superoxide (O₂^•−) formation in A549 cells. (A) Lower levels of the mitochondrial respiratory complex I were detected when compared to complex II as shown by immunoblotting in CDDP-treated A549 cells (40 μM, 48-h treatment) versus control (Co). Each figure is representative of three samples and the experiment repeated twice. (B) CDDP (20 μM, 48-h treatment) induced an increase of O₂^•− formation in the mitochondria as detected by flow cytometry using the mitochondrial O₂^•− sensitive dye MitoSOX. (C) Flow cytometry analysis of CDDP-mediated mitochondrial O₂^•− over time at 24 and 48 h. Bars with different letters are statistically different from one another (n=3, P<0.05).

Figure 3

2′,5′-DHC and chrysin induced GSH depletion and potentiated cisplatin-mediated cytotoxicity in A549 cells. (A) At 6-h treatment, the abilities of 2′,5′-DHC (DHC, 20 μM) and chrysin (Chr, 20 μM) to deplete GSH were more important compared to the inhibitor of γ-glutamylcysteine synthetase BSO (20 μM) (n=3). In the case of 2,5-DHC, however, a rebound of GSH levels was observed at 24 h. (B) 2,5-DHC and chrysin potentiated the toxicity of cisplatin (CDDP, 20 μM) in A549 cells. (C) Exogenous GSH (500 μM) restored cytosolic GSH levels (n=3). (D) Exogenous GSH (500 μM) partially protected the cells towards the toxicity of cisplatin and blocked the 2′,5′-DHC-mediated potentiation of cisplatin’s toxicity. Similar results were obtained with chrysin (data not shown). Bars with different letters are statistically different from one another (n=4, P<0.05).

Figure 4

2′,5′-DHC increased the cisplatin-mediated depolarization of the mitochondrial membrane and cytochrome c release. (A and B) As shown using flow cytometry and JC-1 as dye, cisplatin treatment (CDDP, 20 μM, 48 h) induced a depolarization of the mitochondrial membrane, effect that was amplified by adding 2′,5′-DHC (DHC, 20 μM). The FL2/FL1 ratio was measured using the mean fluorescence in each channel. Bars with different letters are statistically different from one another (n=3, P<0.05). (C) As shown by immunoblotting using GAPDH as internal standard, cisplatin treatment (CDDP, 20 μM, 48 h) induced the release of cytochrome c (Cyt. c), which was increased by adding 2′,5′-DHC (DHC, 20 μM). 2′,5′-DHC alone had no significant effect (not shown). Each figure was representative of three samples and the experiment was repeated at least twice.

Figure 5

Working hypothesis. The formation of cisplatin-mitochondrial DNA (CDDP-mtDNA) adducts disrupts the assembly of the apoproteins of the mitochondrial respiratory chain, which in turn induces the formation of mitochondrial superoxide (O₂^•−). The latter is converted by superoxide dismutase (SOD) into hydrogen peroxide (H₂O₂), which mediates the depolarization of the mitochondrial membrane and the release of cytochrome c, thus triggering the cascade of events leading to nuclear DNA fragmentation and apoptosis. Flavonoid-induced GSH depletion adds to the mitochondrial membrane depolarization by mechanisms that remain to be clarified.