Non-Antioxidant Properties of α-Tocopherol Reduce the Anticancer Activity of Several Protein Kinase Inhibitors In Vitro

The antioxidant properties of α-tocopherol have been proposed to play a beneficial chemopreventive role against cancer. However, emerging data also indicate that it may exert contrasting effects on the efficacy of chemotherapeutic treatments when given as dietary supplement, being in that case harmful for patients. This dual role of α-tocopherol and, in particular, its effects on the efficacy of anticancer drugs remains poorly documented. For this purpose, we studied here, using high throughput flow cytometry, the direct impact of α-tocopherol on apoptosis and cell cycle arrest induced by different cytotoxic agents on various models of cancer cell lines in vitro. Our results indicate that physiologically relevant concentrations of α-tocopherol strongly compromise the cytotoxic and cytostatic action of various protein kinase inhibitors (KI), while other classes of chemotherapeutic agents or apoptosis inducers are unaffected by this vitamin. Interestingly, these anti-chemotherapeutic effects of α-tocopherol appear to be unrelated to its antioxidant properties since a variety of other antioxidants were completely neutral toward KI-induced cell cycle arrest and cell death. In conclusion, our data suggest that dietary α-tocopherol could limit KI effects on tumour cells, and, by extent, that this could result in a reduction of the clinical efficacy of anti-cancer treatments based on KI molecules

Schematic representation of the potential non-antioxidant properties of α-tocopherol on different pharmacological classes of anticancer drugs.

<p>Two hypotheses may be arisen in regards to the non-antioxidant protective role of α-tocopherol against the action of preclinical and clinical protein kinase inhibitors. The vitamin may either act outside the cells by preventing the different drugs to enter within the cell (extracellular effects) or inside by blocking the signalling pathway leading to death (intracellular effect). In the first case, we could imagine that α-tocopherol directly binds to the cytotoxic compounds or change the permeability of the cytoplasmic membrane to render it impermeable to this kind of inhibitors. In the second case, α-tocopherol would either directly act on (i) the kinase inhibitor, (ii) their targets or (iii) a common signalling partner engaged upon kinase inhibitors treatment and supposedly leading to death ( = “x” protein).</p

α-tocopherol blocks the anti-proliferative and pro-apoptotic properties of other kinase inhibitors independently of its antioxidant capacities.

<p><b>A</b> Benzoyl-STS dose-response curves in presence or absence of 100 µM of α-tocopherol or 100 µM of trolox in a clone of HeLa cells stably expressing a C3/7-DAP. EC₅₀ values are shown below. <b>B</b> HeLa cells were treated with various concentrations of benzoyl-STS in presence or not of 100 µM of α-tocopherol or 100 µM of trolox, and stained for cell cycle profile. The percentage of cells in G2M is reported here. <b>C, D</b> HeLa cells were treated with increasing amount of flavopiridol (<b>C</b>) or roscovitine (<b>D</b>) with or without 100 µM of α-tocopherol for 24 hours. Cell cycle profile was determined by flow cytometry using propidium iodide staining and the ratio of the percentage of cells in G2M on the percentage of cells in G0G1 was calculated and reported. <b>E</b> S37614 dose-response curves in presence or absence of 100 µM of α-tocopherol, 100 µM of trolox or 3 mM of NAC were determined using a clone of HeLa cells stably expressing a C3/7-DAP. <b>F</b> DiI-stained HeLa cells were treated with increasing amounts of S37614 supplemented with vehicle or 100 µM of tocopherol, 100 µM of trolox or 3 mM of NAC. Cell proliferation dose response curves were established from the median intensity fluorescence of DiI obtained for each point and normalized to the untreated control cells. EC₅₀ values are shown in the table below the curves.</p

α-tocopherol inhibits staurosporine but not camptothecin, TNF-α or etoposide-induced apoptosis.

<p>In all cases, α-tocopherol was added simultaneously to drug treatment. <b>A–D</b> Dose response curves obtained for camptothecin (A), TNF-α supplemented with 10 µM of cycloheximide (CHX) (B), etoposide (C) and STS (D) in response to α-tocopherol treatment (100 µM) in a clone of HeLa cells stably expressing a caspase3/7-differential anchorage probe (C3/7-DAP). <b>E</b> Dose response of STS in a clone of DU145 (prostate carcinoma) expressing a C3/7-DAP was determined in presence or absence of α-tocopherol treatment (100 µM). In each case, EC₅₀ were determined and are presented below the dose-response curves.</p

α-tocopherol prevents staurosporine-mediated cell cycle arrest in an antioxidant independent manner. A, B

<p>DiI-stained HeLa cells were treated with increasing amounts of STS supplemented with vehicle or 30, 100 and 300 µM of αtocopherol (A) or trolox (B) for 48 hours. Cell proliferation dose response curves were established from the median intensity fluorescence of DiI obtained for each point and normalized to the untreated control cells. EC₅₀ values are shown in the table below the curves. <b>C</b> HeLa cells, co-treated with STS (1, 10, 30, 100 nM) and α-tocopherol (0, 100 or 300 µM) were stained with propidium iodide and cell cycle profile was determined by flow cytometry. The ratio of the percentage of cells in G2M on the percentage of cells in G0G1 was calculated and is shown here as a measure of the cell cycle profile variation. <b>D</b> HeLa cells were co-treated with 0.1 µM of STS and vehicle, α-tocopherol (100 µM) or NAC (3 mM) for 24 h or 48 h. Cells were fixed and colored with Hoechst. Images were acquired using the LSM510 confocal microscope. <b>E</b> HeLa cells were left untreated (black bars) or treated with 100 nM of STS for 24 h (grey bars). Antioxidants (100 µM of tempol, 10 µM of SB203580, 100 µM of propofol, 100 µM of α-tocopherol, 100 µM of trolox or 50 µM of euk8) were added simultaneously to STS. The percentage of cells in G0G1 phase was measured after propidium iodide staining and reported in the graph. <b>F</b> Dose response functions was obtained for STS in response to α-tocopherol treatment (100 µM) in presence or not of cycloheximide (CHX 10 µM) in a clone of HeLa cells stably expressing a C3/7-DAP. EC₅₀ values were determined and presented in the table below the dose-response curves. <b>G</b> Growth curves of HeLa cells stained with DiI and treated with vehicle or 30, 100 and 300 µM of α-tocopherol for 24, 48 and 72 hours. Proliferation index was determined as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036811#s4" target="_blank">materials and methods</a> section.</p

α-tocopherol protects from staurosporine-induced cell death independently of its antioxidant properties.

<p>When co-treatments were needed, antioxidants were added simultaneously to STS. <b>A, C, E</b> HeLa cells stably expressing a C3/7-DAP were treated with increasing amounts of STS in presence or not of different concentrations of trolox (A), NAC (C) or propofol (E). Dose response curves were established accordingly and EC₅₀ values are presented below the curves. <b>B, D</b> HeLa cells were treated for 24 h with increasing amounts of STS in presence or not of trolox (B) and NAC (D) before measuring the percentage of cytolyzed cells using EMA staining. Dose response curves and EC₅₀ values are shown below. <b>F</b> HeLa cells were treated with increasing amounts of STS in presence or not of propofol for 24 h. The percentage of subG1 cells was quantified after propidium iodide staining and dose response curves done accordingly. EC₅₀ values are shown below. <b>G</b> DEVDase activity- and cytolysis- dose response curves in response to STS with or without the addition of zVAD, a pan caspase inhibitor. The number of cells was determined 24 h post-drugs treatment. <b>H</b> Quantification of ROS production in HeLa cells treated with vehicle or 30 nM of STS in presence or not of different antioxidants such as propofol (30 µM), α-tocopherol (100 µM), and tempol (100 µM) measured 24 hours after treatment using DCFDA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036811#s4" target="_blank">Materials and Methods</a>. <b>I</b> Hela cells were treated with DMSO or 100 nM of STS in presence or not of 100 µM of α-tocopherol, 10 mM of NAC or 100 µM of zVAD for 24 hours. Apoptotic cell death was then evaluated by immunoblotting using an anti-cleaved PARP antibody. Actin was used as a loading control.</p