Antiproliferative and apoptotic effects of selective phenolic acids on T47D human breast cancer cells: potential mechanisms of action

Abstract

Introduction: The oncoprotective role of food-derived polyphenol antioxidants has been described but the implicated mechanisms are not yet clear. In addition to polyphenols, phenolic acids, found at high concentrations in a number of plants, possess antioxidant action. The main phenolic acids found in foods are derivatives of 4-hydroxybenzoic acid and 4-hydroxycinnamic acid.

Methods: This work concentrates on the antiproliferative action of caffeic acid, syringic acid, sinapic acid, protocatechuic acid, ferulic acid and 3,4-dihydroxy-phenylacetic acid (PAA) on T47D human breast cancer cells, testing their antioxidant activity and a number of possible mechanisms involved (interaction with membrane and intracellular receptors, nitric oxide production).

Results: The tested compounds showed a time-dependent and dose-dependent inhibitory effect on cell growth with the following potency: caffeic acid > ferulic acid = protocatechuic acid = PAA > sinapic acid = syringic acid. Caffeic acid and PAA were chosen for further analysis. The antioxidative activity of these phenolic acids in T47D cells does not coincide with their inhibitory effect on tumoral proliferation. No interaction was found with steroid and adrenergic receptors. PAA induced an inhibition of nitric oxide synthase, while caffeic acid competes for binding and results in an inhibition of aryl hydrocarbon receptor-induced CYP1A1 enzyme. Both agents induce apoptosis via the Fas/FasL system.

Conclusions: Phenolic acids exert a direct antiproliferative action, evident at low concentrations, comparable with those found in biological fluids after ingestion of foods rich in phenolic acids. Furthermore, the direct interaction with the aryl hydrocarbon receptor, the nitric oxide synthase inhibition and their pro-apoptotic effect provide some insights into their biological mode of action.

Figure 1

The phenolic acids used in the present study.

Figure 2

Time effect and dose effect of phenolic acids on the proliferation of T47D breast cancer cells. (a) Cells (2 × 10⁴) were seeded in 24-well culture plates, and incubated for the indicated number of days with 10^-8 M of different phenolic acids. In another series of experiments, cells were cultured in the absence of phenolic acids. The figure presents the ratio of the cell number in the presence/absence of the different phenolic acids. A typical experiment is presented, which was performed three more times with similar results. Inset: Data presented in the figure were analyzed by the application of the exponential decay equation , in which y is the observed ratio of the cell number in the presence/absence of the phenolic acid, y₀ is the maximal observed decrease of cell proliferation (plateau), A₁ is 1 - y₀, x₀ is the initial time of observation (= 0), and x is the time (in days) of the y observation (abscissa of the y observation). The equation after transformation is rearranged as ln [(y - y₀) / (1 - y₀)] = -xt, the left side of which is the logit of the earlier quantity. From this equation one can obtain the calculation of t, which is an estimate of the half-life (t_1/2) of the effect of the applied agent. In the case of the monotonous curves (sinapic acid, syringic acid, protocatechuic acid, and 3,4-dihydroxy-phenylacetic acid), this t_1/2 was varying between 0.9 and 1.3 days. In the case of ferulic acid, in which the observed curve follows that of sinapic acid and ferulic acid for short incubation times and jumps to that of protocatechuic acid and 3,4-dihydroxy-phenylacetic acid for longer incubation times, the calculated t_1/2 was 5.7 days. Finally, for caffeic acid, in which a sigmoidal curve was observed, data were not well fitted to the curve and the calculated t_1/2 was 4.1 days. (b) Dose effect of phenolic acids on T47D cell proliferation. Cells (2 × 10⁴) were incubated for 5 days in the absence or the presence of the indicated concentrations of phenolic acids, ranging from 10^-12 to 10^-6 M. The figure presents the ratio of the number of cells in the presence/absence of the indicated phenolic acids. A typical experiment is presented, repeated three more times with similar results.

Figure 3

Effect of caffeic acid and 3,4-dihydroxy-phenylacetic acid (PAA) on the cell cycle and apoptosis after long (two cell cycles) incubation times. Cells were incubated with the indicated phenolic acids for 5 days. The medium, containing 10^-7 M of the agents, was changed every other day. At the end of the incubation period, cells were removed from plates by scraping, stained with annexin V and/or propidium iodide (PI), as described in Materials and methods, and analyzed by flow cytometry. Early apoptotic changes were identified as the cell population having a normal (diploid) DNA content and annexin V staining. Late apoptotic cells were those presenting a hypodiploid DNA content identified by PI staining. After permeabilization of cells, staining with PI and flow cytometry revealed cell cycle phases. Necrotic cells were identified by forward and side scatter. (a) Typical flow cytometric analysis of the cell cycle. Cells were stained with PI. (b) Cumulative cell cycle phases of nonapoptotic cells (mean ± standard error of the mean of three measurements). (c) Cumulative effects of phenolic acids on apoptosis (mean ± standard error of the mean of three experiments). Cells were stained with annexin V and PI. (d) Effect of phenolic acids on apoptosis-related proteins. Semiquantitative western blot analysis on cell homogenates from T47D cell cultures treated with 10^-7 M various phenolic acids for 5 days. At the end of the incubation period, cells were removed from plates by scraping and apoptosis-related proteins were measured with western blot analysis, as described in Materials and methods. Quantification results are depicted, expressed as the percentage of nontreated control values. Data are mean ± standard error of the mean of three independent experiments.

Figure 4

Effect of phenolic acids on the inhibition of H₂O₂ cytotoxicity. (a) Cells (15 × 10⁴) were incubated for 24 hours with the different phenolic acids (10^-7 M). The medium was then discarded and replaced with a fresh one, in which fetal bovine serum was omitted, containing the indicated concentrations of H₂O₂. Incubation was followed for 3 hours, and then the viability of cells was determined by the MTT method. Results are presented of an experiment (in triplicate) performed four times with similar results. (b) The difference of effective concentration 50% values of H₂O₂ obtained in the presence of phenolic acids, as compared with control experiments. Bars represent the standard error of the mean of four different experiments performed in triplicate. IC₅₀, inhibitory concentration 50%; PAA, 3,4-dihydroxy-phenylacetic acid.

Figure 5

(a) Effect of 24 hours of incubation with phenolic acids (10^-7M) on the activity of nitric oxide synthase (NOS) in the cytoplasm of T47D cells. (b) Time course of the effect of 3,4-dihydroxy-phenylacetic acid (PAA) (10^-7 M) on the transcript of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS). RT-PCR data for either enzyme were normalized for actin, and divided by the corresponding control values (control = 1 in all cases).

Figure 6

Interaction of phenolic acids with the aryl hydrocarbon receptor (AhR). (a) Interaction of 10^-7 M phenolic acids on the AhR against TCDD. (b) Competition of caffeic acid on aryl hydrocarbon binding. Homologous displacement with TCDD given for comparison. (c) Inhibition of basal and TCDD-stimulated CYP1A1 (O-dealkylation of ethoxyresorufin [EROD]) activity by 10^-7 M caffeic acid. (d) Dose effect of caffeic acid on the (EROD) activity of CYP1A1, after 24 hours of incubation. The time course of (e) the basal and (f) the TCDD-stimulated CYP1A1 transcript, after application of 10^-7 M caffeic acid. RT-PCR data were normalized for actin, and divided by the corresponding control values (control = 1 in all cases). PAA, 3,4-dihydroxy-phenylacetic acid.