Dietary phenolic acids act as effective antioxidants in membrane models and in cultured cells, exhibiting proapoptotic effects in leukaemia cells

Abstract

Caffeic, syringic, and protocatechuic acids are phenolic acids derived directly from food intake or come from the gut metabolism of polyphenols. In this study, the antioxidant activity of these compounds was at first evaluated in membrane models, where caffeic acid behaved as a very effective chain-breaking antioxidant, whereas syringic and protocatechuic acids were only retardants of lipid peroxidation. However, all three compounds acted as good scavengers of reactive species in cultured cells subjected to exogenous oxidative stress produced by low level of H(2)O(2). Many tumour cells are characterised by increased ROS levels compared with their noncancerous counterparts. Therefore, we investigated whether phenolic acids, at low concentrations, comparable to those present in human plasma, were able to decrease basal reactive species. Results show that phenolic acids reduced ROS in a leukaemia cell line (HEL), whereas no effect was observed in normal cells, such as HUVEC. The compounds exhibited no toxicity to normal cells while they decreased proliferation in leukaemia cells, inducing apoptosis. In the debate on optimal ROS-manipulating strategies in cancer therapy, our work in leukaemia cells supports the antioxidant ROS-depleting approach.

Figure 1

Chemical structures of studied phenolic acids.

Figure 2

Oxygen uptake traces during AAPH (17 mM) induced peroxidation of PC (15 mM) unilamellar vesicles at 37°C and pH 7.2 in the absence of inhibitor (control) and in the presence of one of the various phenolic antioxidants, each at the same concentration (5 μM). The arrow shows antioxidant injection. Inset: relationship between CAF concentrations (1.3 to 10 μM) and inhibition periods measured.

Figure 3

Antioxidant effect of phenolic compounds in HEL cells and HUVEC exposed to oxidative stress generated by 50 μM H₂O₂. Cells were preincubated with different compounds (5 or 10 μM for HEL cells, (a); 10 μM for HUVEC, (b)) for 20 hours, treated with H₂O₂ for 30 min, then ROS levels were measured by means of H₂DCFDA assay as described in Section 2. Results are expressed as means ± SD of three independent experiments, each performed in octuplicate. **P < 0.005, significantly different from control cells; ***P < 0.0005, significantly different from control cells.

Figure 4

Comparison between basal ROS levels in HEL cells and in HUVEC. ROS levels were measured by means of H₂DCFDA assay as described in Section 2. Results are expressed as means ± SD of three independent experiments, each performed in octuplicate. Significant difference ***P < 0.0001.

Figure 5

Effect of phenolic compounds on basal ROS levels in HEL cells and HUVEC. Cells were treated with different compounds (5 or 10 μM for HEL cells, (a); 20 μM for HUVEC, (b)) for 20 hours, then ROS levels were measured by means of H₂DCFDA assay as described in Section 2. Results are expressed as means ± SD of four independent experiments, each performed in octuplicate. **P < 0.005, significantly different from control cells; ***P < 0.0005, significantly different from control cells.

Figure 6

Effect of phenolic compounds on cell viability/proliferation. Cells were treated with different compounds (5 to 100 μM for HEL cells, (a), (b) and (d); 20 μM for HUVEC, (c)) for 20 hours. (a): Viability was estimated by Trypan Blue exclusion test. (b) and (c): Viability/proliferation was evaluated by MTT assay as described in Section 2. Results are expressed as means ± SD of three independent experiments, each performed in quadruplicate. *P < 0.05, significantly different from control cells;  **P < 0.01, significantly different from control cells;  ***P < 0.001, significantly different from control cells. (d): HEL cells viability, after 20-h treatment with 10, 50, or 100 μM compounds, was determined by the ATPlite 1step luminescence kit as described in Section 2. Results are expressed as means ± SD of three independent experiments, each performed in triplicate. All values are significantly different from control (***P < 0.001).

Figure 7

Caspase activity in HEL cells after phenolic compound treatment. HEL cells were incubated with different compounds (5, 10, 50, or 100 μM) for 20 hours, then cell lysates were incubated with three different substrates at 37°C for 15 min, that is Ac-DEVD-AMC as specific fluorogenic substrate for caspase 3 (a), Ac-IETD-AMC for caspase 8 (b), and Ac-LEHD-AFC for caspase 9 (c), as described in Section 2. Results are expressed as means ± SD of four independent experiments, each performed in triplicate.  *P < 0.05, significantly different from control cells;  **P < 0.01, significantly different from control cells;  ***P < 0.001, significantly different from control cells.

Figure 8

Effect of phenolic compounds on apoptosis. HEL cells were incubated with different compounds (5, 10, 50, or 100 μM) for 20 hours, then cell lysates were subjected to SDS-PAGE and Western blotting with the indicated antibodies as described in Section 2. Tubulin detection was used as a control. Representative immunoblots are shown. (a): anti-caspase 3; (b): anti-Bax and anti-Bcl-2; (e): anti-p-Akt. (c): Densitometric analysis of three independent Western blot assays for cleaved caspase 3 (17 kDa fragment). (d): Bax/Bcl-2 ratio from densitometric analysis of three independent experiments.  *P < 0.05, significantly different from control cells; **P < 0.01, significantly different from control cells; ***P < 0.001, significantly different from control cells.