Elsevier

Free Radical Biology and Medicine

Volume 39, Issue 6, 15 September 2005, Pages 742-751
Free Radical Biology and Medicine

Original Contribution
Differential sensitization of cancer cells to doxorubicin by DHA: A role for lipoperoxidation

https://doi.org/10.1016/j.freeradbiomed.2005.04.023Get rights and content

Abstract

Polyunsaturated fatty acids have been reported to enhance the cytotoxic activity of several anticancer drugs. In the present study, we observed that doxorubicin chemosensitization of breast cancer cell lines by docosahexaenoic acid (DHA, a long-chain ω-3 polyunsaturated fatty acid) was cell-line selective, affecting MDA-MB-231 and MCF-7dox (a doxorubicin-resistant cell line) but not the parental MCF-7 cell line. DHA supplementation led to an increase in membrane phospholipid DHA level, but did not induce changes in intracellular [14C]doxorubicin accumulation. In MDA-MB-231, doxorubicin efficacy enhancement by DHA was linked to an increase in malondialdehyde level, a final product of lipid peroxidation. DHA elicited by itself a 3.7-fold malondialdehyde level increase, additive to that induced by doxorubicin. Addition of doxorubicin to DHA further increased the glutathione level, indicative of the generation of an oxidative stress. In contrast to MDA-MB-231, doxorubicin did not increase the malondialdehyde level in MCF-7, although DHA induced lipid peroxidation. Therefore in MCF-7, lipid peroxidation induced by DHA itself was not sufficient to trigger an oxidative stress and to subsequently increase sensitivity to doxorubicin. These data indicate that the differential effect of DHA among cells on drug toxicity results from a differential oxidative response to doxorubicin. Chemosensitization through fatty acids appears as a new promising adjuvant therapeutic paradigm, since ω-3 fatty acids are physiological molecules found in food and are nontoxic in vivo.

Introduction

Several epidemiological studies have indicated that populations consuming high amounts of ω-3 (n-3) fatty acids display a lower breast cancer risk [1], [2]. This effect seems to be related to specific n-3 polyunsaturated fatty acids and/or to the ratio of n-6 to n-3 fatty acids [3], [4], [5], [6]. Long-chain n-3 fatty acids found in fatty cold-water fish, particularly eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), have been shown to inhibit the growth of human breast cells both in culture and in xenografts [7], [8] and to increase the efficacy of anticancer drugs [9], [10].

Several mechanisms have been proposed to account for the activity of n-3 polyunsaturated fatty acids (n-3 PUFAs) against cancer cells, including alteration in gene expression [11], [12], modulation of cellular proliferation, apoptosis and differentiation [13], increase in drug transport across the cell membrane [14], [15], generation of free oxygen radicals, and lipid peroxidation [16], [17]. These biological mechanisms could explain that n-3 fatty acid-mediated inhibition of mammary carcinogenesis is not independent. For example, incorporation of n-3 polyunsaturated fatty acids into cell membrane phospholipids can alter membrane fluidity (and increase intracellular drug accumulation), modulate cell signaling [18], and enhance the production of free oxygen radicals [19] and lipid peroxidation [20].

Anthracyclines are among the most active anticancer agents, widely used in the treatment of solid tumors and leukemia. The cytotoxic action of doxorubicin has been mainly related to the inhibition of topoisomerase II and to the production of oxygen-reactive species [21], [22]. Among polyunsaturated fatty acids, DHA, with its 6 double bonds, is very prone to oxidation. The increased membrane unsaturation index consequently would provide more abundant targets for oxygen-reactive species generated by doxorubicin metabolism. Products of lipid peroxidation such as hydroperoxides and aldehydes have been implicated in the cytotoxic process and the increase in drug efficacy. Thus, conditions favoring an increased lipid peroxidation in response to doxorubicin would lead to a higher activity of the drug. Among PUFAs studied, DHA was the most potent to enhance the cytotoxic effect of doxorubicin [9]. In vivo and in vitro studies on the influence of DHA supplementation have reported higher peroxidation and oxidative stress, as demonstrated by an increase of thiobarbituric acid-reactive substances, conjugated dienes, or malondialdehydes and a decrease of antioxidant vitamins [23], [24], [25], [26]. Protection against oxidative damages is normally ensured by nonenzymatic (especially vitamins provided by diet) and enzymatic (catalases, superoxide dismutases, and glutathione peroxidases) defenses. Changes in the activity of these antioxidant enzymes have also been described during PUFA supplementation [27], [28].

Chemotherapy has improved during the last decades, largely due to the introduction of effective drug combinations and treatment schemes. However resistance is frequently observed in tumors undergoing primary therapy or, more frequently, is a result of treatment with various antitumor drugs. This multidrug resistance, the so-called phenotype MDR, remains a major obstacle for successful chemotherapeutic cure [29]. Among drug resistance mechanisms, the most common are: (i) an increase in drug efflux, associated with overexpression of the mdr-1 gene product, a Mr 170,000 plasma membrane glycoprotein (P-gp) that functions as an energy (ATP)-dependent efflux pump for cytotoxic drugs, and (ii) an abnormal redox status developed secondarily after drug exposure, in which glutathione and antioxidant enzyme activities are elevated and protect the cell against free radical aggression [30]. Attention has been focused recently on the study of MDR reversing agents. Although hundreds of compounds have been found in vitro to be able to modulate the MDR phenotype, their clinical application was limited owing to high toxicity in vivo such as cardiotoxicity, nephrotoxicity, or immunosuppression [29].

To determine to what extent an oxidative stress could account for the sensitization of cancer cells to doxorubicin by DHA, we have used three cell lines: a doxorubicin-resistant one (MCF-7dox) and two sensitive lines (MCF-7 and MDA-MB-231), and took advantage of their differing properties. We found that DHA was able to enhance doxorubicin cytotoxicity in MDA-MB-231 or in MCF-7dox cell lines, but not in the parental MCF-7 cell line. In order to understand the differential effects of DHA on these three cell lines, fatty acid composition of membrane phospholipids and intracellular accumulation of doxorubicin were examined. The cellular oxidative status was also evaluated through the measure of two parameters of oxidative stress, malondialdehyde level (a final product of lipid peroxidation) and glutathione levels, cofactors for major antioxidant enzymes which modulate the cell's response to redox changes and vitamin E level.

Section snippets

Drugs and chemicals

Unless otherwise stated, all chemicals were purchased from Sigma (Sigma-Aldrich Chimie, France). Doxorubicin (chlorhydrate de doxorubicine Teva®, 10 mg/5 ml) was purchased from Teva Pharma S.A. (France) and [14-14C]doxorubicin hydrochloride (2 GBq/mmol) was purchased from Amersham Pharmacia Biotech (France). Stock solutions of doxorubicin (1 mM) were stored at −80°C and dilutions of doxorubicin were freshly prepared in Hanks' medium (Hanks' balanced salts without sodium bicarbonate and phenol

Fatty acid composition of membrane phospholipids

Table 1 shows the composition (in mol%) of the membrane phospholipids fatty acids (pooled in series) of MDA-MB-231, MCF-7, and MCF-7dox. Under control conditions (ethanol: 0.02%), no statistical difference existed among the fatty acid compositions of these three cell lines (ANOVA, P > 0.05). DHA (30 μM) supplementation led to an increased level of total n-3 fatty acids for MDA-MB-231 and MCF-7dox cell lines (Student's t test, P < 0.05). This increase involved both 22:6n-3 and 20:5n-3 fatty

Discussion

Our study demonstrates that the sensitivity of breast cancer cells to doxorubicin can be increased when an oxidative stress is triggered, a condition brought about by a supplementation with DHA. We have taken advantage of the differential sensitivity of three breast cancer cell lines to doxorubicin and DHA in order to investigate mechanisms responsible for the chemosensitization induced by DHA. We found it to be cell-line selective, affecting MDA-MB-231 and resistant MCF-7dox cells but not the

Acknowledgments

We are grateful to Dr. K. Cowan (National Cancer Institute, Bethesda) for the gift of resistant cell line MCF-7dox used in this study. We thank Lysiane Boulay for excellent technical assistance. This work was supported in part by grants from Ligue Nationale contre le Cancer (Comités d'Indre et Loire, Loir et Cher, Indre), by Institut National de la Santé et de la Recherche Médicale (INSERM: Action Thématique Concertée "Nutrition"), by Conseil Régional (Région Centre), and by Cancéropôle

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