PPARα signaling mediates the synergistic cytotoxicity of clioquinol and docosahexaenoic acid in human cancer cells

doi:10.1016/j.bcp.2009.02.002

Biochemical Pharmacology

Volume 77, Issue 9, 1 May 2009, Pages 1480-1486

https://doi.org/10.1016/j.bcp.2009.02.002 Get rights and content

Abstract

This study investigated the involvement of PPARγ and PPARα signaling in the synergistic anticancer activity of clioquinol (5-chloro-7-iodo-8-hydroxyquinoline) and docosahexaenoic acid (DHA) in human cancer cells. The synergistic cytotoxicity of DHA and clioquinol was demonstrated in nine human cancer cell lines representing different tissues of origin. A2780, a well-established ovarian cancer model system, was chosen for further characterization because of its sensitivity to DHA and clioquinol. Both PPARα and PPARγ were expressed in A2780 cells when analyzed with western blotting and reporter gene technique. Treatment of the cells with clofibrate (a PPARα agonist) and clioquinol for three days mimicked the synergy of DHA and clioquinol, whereas this synergy could not be seen with the use of troglitazone (a PPARγ agonist) and clioquinol, suggesting that PPARα signaling is involved in the synergistic action. When used alone, the IC₅₀ of clofibrate was 513 μM in A2780 cells. However, the addition of 5 μM clioquinol to clofibrate-treated cells led to a dramatic reduction of its IC₅₀ value (148 μM). The combination effects index (CI) analysis confirmed the synergy of clioquinol and clofibrate on inhibiting cancer cell viability. Using inhibitors to block PPARα signaling diminished the synergistic cytotoxicity of clioquinol and DHA. These results provide pharmacological evidence that the synergistic anticancer action of clioquinol and DHA is mediated by PPARα signaling in human cancer cells.

Graphical abstract

Introduction

The biological differences between normal and cancer cells are too subtle to easily achieve selective cancer cell targeting [1]. Recently, drug combinations have been utilized in an attempt to more effectively kill cancer cells. Combinations of drugs with different modes of action may enhance individual drugs’ anticancer action yet protect the host from side effects. Such synergistic interactions of anticancer drugs have been reported in studies of cultured tumor cell lines, animal models, and cancer patients [2], [3], [4], [5], [6], [7], [8].

The mechanisms by which a combination of drugs may selectively target tumor cells have not been well understood, although different modes of action are proposed to be responsible for the observed synergy [1]. Several cellular signaling molecules or pathways are proposed to be involved in the drug synergy on cancer cells, including Akt, NF-κB, apoptosis-related proteins, and the COX-2 pathway [9]. It is apparent that each drug combination may have distinct mechanisms of action because they may target different molecules or signaling pathways [1]. Since anticancer drugs are often toxic to normal cells, drug combinations, in some cases, may also be synergistically toxic to the host, parallel to their enhanced anticancer effects [1]. Therefore, more insight into the cellular mechanisms responsible for the synergistic action is critical to the development of an effective drug combination against cancer.

DHA is a long-chain n-3 polyunsaturated fatty acid, and it has been recognized to have anticancer activity in cultured tumor cells and various animal models [10], [11], [12], [13], [14], [15]. Its synergy with chemotherapeutics has also been reported [16], [17], [18], [19]. Since DHA is an essential fatty acid and protects neuronal cells from apoptosis [20], [21], the combination of DHA and chemotherapeutics may achieve enhanced toxic effects selectively towards cancer cells. Indeed, DHA has been shown to enhance the cytotoxicity of paclitaxel in experimental models and in human clinical trials without seeing enhanced side effects in patients [3], [4]. These observations justify the use of DHA as combination therapy with chemotherapeutics. We have recently demonstrated that DHA acts synergistically with clioquinol, a metal binding compound, to kill tumor cells [22]. Clioquinol has been recently tested for the treatment of Alzheimer's disease in clinical trials [23], [24] and is a newly discovered anticancer agent [25], [26], [27], [28]. Understanding the mechanisms of the synergistic action is essential for further development of DHA and clioquinol as a novel drug combination for chemotherapy. Because DHA is a well-established peroxisome proliferator activated receptor (PPAR) ligand [29], the present study examined the involvement of PPAR signaling in the synergistic anticancer action. We report here that PPARα, but not PPARγ, mediates the synergistic anticancer action of DHA and clioquinol in human cancer cells.

Section snippets

Materials

3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was purchased from Promega (Madison, WI). Antibodies for PPARα and PPARγ were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody was obtained from ProMab Biotechnologies, Inc. (Albany, CA). The PPRE-lu reporter construct was obtained from Dr. Bruce Spiegelman at the Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA [30]

Effects of DHA and clioquinol on cell viability in human cancer cells

We first determined the effects of clioquinol and DHA on the viability of nine different human cancer cell lines. Treatment of the cells with clioquinol and DHA for three days inhibited cell viability in a concentration-dependent manner. The IC₅₀s of clioquinol and DHA used alone or in combination were calculated (Table 1). The combination of these two compounds significantly lowered the IC₅₀ value in each of the cell lines examined but the magnitude of the change differed among cell lines,

Discussion

The present study demonstrates, through pharmacological characterization, that PPARα signaling mediates the synergistic anticancer action of clioquinol and DHA in human cancer cells. These observations provide novel information on the synergistic anticancer action of clioquinol and DHA and suggest that PPARα ligands could potentially be used to enhance anticancer activity of chemotherapeutics.

Both DHA and clioquinol have been recognized to have anticancer activity [25], [26], [27], [28], [47],

Acknowledgement

Financial support was obtained from the Oklahoma Center for the Advancement of Science and Technology, the American Cancer Society (Institutional Seed Grant), the China Scholarship Council, and the College of Medicine, University of Oklahoma Health Sciences Center.

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: To study the association of n-3 polyunsaturated fatty acid (n-3 PUFA) intake from diet with depressive symptoms in midlife women.
: Data for this cross-sectional study were retrieved from baseline assessment of the Study of Women's Health Across the Nation (SWAN). Logistic regression and restricted cubic spline models were performed to examine the association n-3 PUFA intake with depressive symptoms.
: A total of 3054 women aged 42–52 years were included in the present study. In overall midlife women, n-3 PUFA intake was not significantly associated with depressive symptoms (CES-D score ≥16) after adjustment for potential confounders. In early perimenopausal women, n-3 PUFA intake was inversely associated with depressive symptoms after adjustment for potential confounders. The fully adjusted OR with 95% CI of depressive symptoms were 0.062 (0.009–0.457) in the highest quartile (n-3 PUFAs intake was categorized by quartiles) compared with the lowest quartile for n-3 PUFA intake. Restricted cubic spline analyses showed that n-3 PUFA intake were dose–response inversely associated with depressive symptoms in early perimenopausal women. However, in premenopausal women, no significant association between depressive symptoms and n-3 PUFA intake was observed.
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However, erastin mediates this process through a different triggering mechanism-the inhibition of system xc- cystine-glutamate transport [10]. The anticancer actions of PUFA, like DHA, have often been attributed to PPAR activation [24,25]. In this study we also sought to determine if these nuclear receptors also mediate LDL-DHA's tumor cytotoxicity.
Low-density lipoprotein nanoparticles reconstituted with the natural omega-3 fatty acid, docosahexaenoic acid (LDL-DHA), have been reported to selectively kill hepatoma cells and reduce the growth of orthotopic liver tumors in the rat. To date, little is known about the cell death pathways by which LDL-DHA nanoparticles kill tumor cells. Here we show that the LDL-DHA nanoparticles are cytotoxic to both rat hepatoma and human hepatocellular carcinoma (HCC) cell lines. Following LDL-DHA treatment both rat and human HCC cells experience pronounced lipid peroxidation, depletion of glutathione and inactivation of the lipid antioxidant glutathione peroxidase-4 (GPX4) prior to cell death. Inhibitor studies revealed that the treated HCC cells die independent of apoptotic, necroptotic or autophagic pathways, but require the presence of cellular iron. These hallmark features are consistent and were later confirmed to reflect ferroptosis, a novel form of nonapoptotic iron-dependent cell death. In keeping with the mechanisms of ferroptosis cell death, GPX4 was also found to be a central regulator of LDL-DHA induced tumor cell killing. We also investigated the effects of LDL-DHA treatments in mice bearing human HCC tumor xenografts. Intratumoral injections of LDL-DHA severely inhibited the growth of HCC xenografts long term. Consistent with our in vitro findings, the LDL-DHA treated HCC tumors experienced ferroptotic cell death characterized by increased levels of tissue lipid hydroperoxides and suppression of GPX4 expression. Conclusion: LDL-DHA induces cell death in HCC cells through the ferroptosis pathway, this represents a novel molecular mechanism of anticancer activity for LDL-DHA nanoparticles.
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The fundamental difference between immortalized and primary cardiac cells dictates the importance of obtaining a better understanding of DHA-mediated biological events, which is important for the development of DHA-based therapeutic strategies to treat various pathogenic conditions. DHA has been shown to initiate a cytotoxic response in several cancer cell lines through peroxisome proliferator-activated receptors (PPARs) (Tuller et al., 2009). PPARs are ligand-activated transcription factors and members of the nuclear hormone receptor superfamily (Issemann and Green, 1990; Kliewer et al., 1999).
Docosahexaenoic acid (22:6n3, DHA) is an n-3 polyunsaturated fatty acid (PUFA) known to affect numerous biological functions. While DHA possesses many properties that impact cell survival such as suppressing cell growth and inducing apoptosis, the exact molecular and cellular mechanism(s) remain unknown. Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear receptors that regulate many cell pathways including cell death. As DHA acts as a ligand to PPARs the aim of this study was to examine the involvement of PPARδ in DHA-mediated cytotoxicity toward H9c2 cells. Treatment with DHA (100 μM) resulted in a significant decline in cell viability, cellular metabolic activity and total antioxidant capacity coinciding with increased total proteasome activities and activity of released lactate dehydrogenase (LDH). No changes in reactive oxygen species (ROS) production or accumulation of lipid peroxidation products were observed but DHA promoted apoptotic cell death as detected by flow cytometry, increased caspase-3 activity and decreased phosphorylation of Akt. Importantly, DHA enhanced PPARδ DNA binding activity in H9c2 cells strongly signifying that the cytotoxic effect of DHA might be mediated via PPARδ signaling. Co-treatment with the selective PPARδ antagonist GSK 3787 (1 μM) abolished the cytotoxic effects of DHA in H9c2 cells. Cytotoxic effects of DHA were attenuated by co-treatment with myriocin, a selective inhibitor of serine palmitoyl transferase (SPT), preventing de novo ceramide biosynthesis. LC/MS analysis revealed that treatment with DHA resulted in the accumulation of ceramide, which was blocked by GSK 3787. Interestingly, inhibition of cytochrome P450 (CYP) oxidase with MS-PPOH (50 μM) abolished DHA-mediated cytotoxicity suggesting downstream metabolites as the active mediators. We further demonstrate that CYP oxidase metabolites of DHA, methyl epoxy docosapentaenoate (EDP methyl esters, 1 μM) (mix 1:1:1:1:1:1; 4,5-, 7,8-, 10,11-, 13,14-, 16,17- and 19,20-EDP methyl esters) and 19,20-EDP cause cytotoxicity via activation of PPARδ signaling leading to increased levels of intracellular ceramide. These results illustrate novel pathways for DHA-induced cytotoxicity that suggest an important role for CYP-derived metabolites, EDPs.
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Second, DHA is an established PPAR ligand which can activate the PPAR signaling pathway, thereby regulating expression of many target genes [64]. We have previously shown that DHA activates PPARα signaling in A2780 cells and PPARα mediates the suppression of superoxide dismutase 1 expression [39] and the cytotoxicity of DHA [40]. Third, there are well-established AREs and PPREs in the human HO-1 gene promoter, and both DNA elements were previously described to regulate HO-1 gene transcription [14,35,37].
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Recent studies demonstrated that these two drugs have anticancer properties in various model systems (9–11), but the mechanisms behind their actions remain to be elucidated. We have reported previously that activation of PPARα mediates the anticancer action of docosahexaenoic acid, likely through down-regulation of hypoxia signaling (12, 13). We therefore hypothesized that the anticancer properties of PPARα agonists may similarly be due to suppression of HIF-1α signaling in human cancer cells.
Activation of peroxisome proliferator-activated receptor α (PPARα) has been demonstrated to inhibit tumor growth and angiogenesis, yet the mechanisms behind these actions remain to be characterized. In this study, we examined the effects of PPARα activation on the hypoxia-inducible factor-1α (HIF-1α) signaling pathway in human breast (MCF-7) and ovarian (A2780) cancer cells under hypoxia. Incubation of cancer cells under 1% oxygen for 16 h significantly induced HIF-1α expression and activity as assayed by Western blotting and reporter gene analysis. Treatment of the cells with PPARα agonists, but not a PPARγ agonist, prior to hypoxia diminished hypoxia-induced HIF-1α expression and activity, and addition of a PPARα antagonist attenuated the suppression of HIF-1α signaling. Activation of PPARα attenuated hypoxia-induced HA-tagged HIF-1α protein expression without affecting the HA-tagged HIF-1α mutant protein level, indicating that PPARα activation promotes HIF-1α degradation in these cells. This was further confirmed using proteasome inhibitors, which reversed PPARα-mediated suppression of HIF-1α expression under hypoxia. Using the co-immunoprecipitation technique, we found that activation of PPARα enhances the binding of HIF-1α to von Hippel-Lindau tumor suppressor (pVHL), a protein known to mediate HIF-1α degradation through the ubiquitin-proteasome pathway. Following PPARα-mediated suppression of HIF-1α signaling, VEGF secretion from the cancer cells was significantly reduced, and tube formation by endothelial cells was dramatically impaired. Taken together, these findings demonstrate for the first time that activation of PPARα suppresses hypoxia-induced HIF-1α signaling in cancer cells, providing novel insight into the anticancer properties of PPARα agonists.
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PPARα signaling mediates the synergistic cytotoxicity of clioquinol and docosahexaenoic acid in human cancer cells

Abstract

Graphical abstract

Introduction

Section snippets

Materials

Effects of DHA and clioquinol on cell viability in human cancer cells

Discussion

Acknowledgement

Trends Pharmacol Sci

Biochem Biophys Res Commun

Prostaglandins Leukot Essent Fatty Acids

Blood

Free Radic Biol Med

Leukemia

Cancer Lett

J Lipid Res

Toxicology

Biochem Pharmacol

J Nutr

Cancer Cell

Neoplasia

Laboratory and clinical evidence of synergistic cytotoxicity of sequential treatment with gemcitabine followed by docetaxel in the treatment of sarcoma

J Clin Oncol

Tumor targeting by covalent conjugation of a natural fatty acid to paclitaxel

Clin Cancer Res

Phase I study of docosahexaenoic acid-paclitaxel: a taxane-fatty acid conjugate with a unique pharmacology and toxicity profile

Clin Cancer Res

Molecular evidence for increased antitumor activity of gemcitabine by genistein in vitro and in vivo using an orthotopic model of pancreatic cancer

Cancer Res

Inactivation of nuclear factor kappaB by soy isoflavone genistein contributes to increased apoptosis induced by chemotherapeutic agents in human cancer cells

Cancer Res

Therapeutic synergy between irinotecan and 5-fluorouracil against human tumor xenografts

Clin Cancer Res

Using chemopreventive agents to enhance the efficacy of cancer therapy

Cancer Res

Effect of diets high in omega-3 and omega-6 fatty acids on initiation and postinitiation stages of colon carcinogenesis

Cancer Res

Effects of dietary perilla oil, soybean oil and safflower oil on 7,12-dimethylbenz[a]anthracene (DMBA) and 1,2-dimethyl-hydrazine (DMH)-induced mammary gland and colon carcinogenesis in female SD rats

Carcinogenesis

Effect of omega-3 fatty acids on growth of a rat mammary tumor

J Natl Cancer Inst

Dietary fish oil inhibits human breast carcinoma growth: a function of increased lipid peroxidation

Lipids

Effects of dietary omega-3 fatty acids on human breast cancer growth and metastases in nude mice

J Natl Cancer Inst