Concentration Dependence of the Antioxidant and Prooxidant Activity of Trolox in HeLa Cells: Involvement in the Induction of Apoptotic Volume Decrease

Abstract

Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a hydrophilic analog of vitamin E, is known for its strong antioxidant activity, being a high radical scavenger of peroxyl and alkoxyl radicals. Under particular conditions, Trolox may also exhibit prooxidant properties. The present work aimed at studying the dual antioxidant/prooxidant behavior of Trolox over a wide range of concentrations (from 2.5 to 160 µM) in HeLa cells. In particular, the study addressed the dose-dependent effects of Trolox on the oxidative cell status and vitality of HeLa cells, focusing on the potential role of the vitamin E analog in the induction of one of the first steps of the apoptotic process, Apoptotic Volume Decrease (AVD). In HeLa cells, Trolox showed significant antioxidant activity, expressed as the ability to reduce the endogenous ROS production detected by the ROS-sensitive probe 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA), at low concentrations (range: 2.5-15 µM), but exerted a dose-dependent prooxidant effect at higher concentrations after 24 h exposure. The prooxidant effect was paralleled by the reduction in cell viability due to the induction of the apoptotic process. The dual behavior, antioxidant at lower concentrations and prooxidant at higher concentrations, was evident also earlier after 2 h incubation, and it was paralleled by the isotonic shrinkage of the cells, ascribed to AVD. The use of SITS, known Cl^- channel blocker, was able to completely inhibit the Trolox-induced isotonic cell shrinkage, demonstrating the involvement of the vitamin E analog in the alteration of cell volume homeostasis and, in turn, in the AVD induction. In conclusion, the study shed light on the concentration dependence of the Trolox antioxidant/prooxidant activity in HeLa cells and revealed its role in the induction of one of the first events of apoptosis, AVD, at high concentrations.

Keywords: AVD; HeLa; Trolox; antioxidant; apoptosis; prooxidant.

Figure 1

Percentage variation of fluorescence intensity in HeLa cells exposed for 24 h to increasing concentrations of Trolox (from 2.5 to 160 µM) and then charged with 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA). The ordinates indicate the percentage variation of the probe fluorescence intensity, which was calculated as follows: (fluorescence of control cells − fluorescence of treated cells)/(fluorescence of control cells) × 100. Data are expressed as mean ± SEM of 3 independent experiments.

Figure 2

(A) The vitality of HeLa cells exposed for 24 h to increasing concentrations of Trolox (from 2.5 to 160 µM) assessed by MTT test. Data are expressed as percentage vs. control. (B) The vitality of HeLa cells exposed for 24 h to increasing concentrations of Trolox (from 2.5 to 160 µM) assessed by the fluorescence intensity (% vs. control) of propidium iodide. Data are expressed as mean ± SEM of 3 independent experiments. The statistical significance of data was analyzed by one-way ANOVA and Dunnett test. * = p < 0.05; ** = p < 0.01; *** = p < 0.001.

Figure 3

Effect of Trolox after 14 h exposure on annexin V and propidium iodide fluorescence (expressed as percentage variations of control) in HeLa cells. Details as Figure 1. Data are expressed as mean ± SEM of 3 independent experiments. Statistical significance of differences was assessed by one-way ANOVA and Dunnett test. ** = p < 0.01; *** = p < 0.001.

Figure 4

(A) Percentage variation of fluorescence intensity in HeLa cells exposed for 2 h to increasing concentrations of Trolox (from 2.5 to 160 µM) and then charged with CM-H₂DCFDA. Details as Figure 1. (B) Effect of increasing concentrations of Trolox on cell volume after 2 h incubation. Data are expressed as cell volume percentage variation calculated as follows: (cell size of control cells − cell size of treated cells/cell size of control cells) × 100. Data are expressed as mean ± SEM of 3 independent experiments.

Figure 5

Effect of SITS (4-acetamido-4’-isothiocyanostilbene) (0.5 mM) on the Trolox-induced cell volume decrease. Details as Figure 4B. Data are reported as mean ± S.E.M of 3 independent experiments. Statistical significance was assessed by one-way ANOVA and Dunnett test. ** = p < 0.01.