Original Contribution
Vitamin E metabolite 13′-carboxychromanols inhibit pro-inflammatory enzymes, induce apoptosis and autophagy in human cancer cells by modulating sphingolipids and suppress colon tumor development in mice

https://doi.org/10.1016/j.freeradbiomed.2016.03.018Get rights and content

Highlights

  • 13′-Carboxychromanols are dual inhibitors of cyclooxygenase and 5-lipoxygenase.

  • 13′-Carboxychromanols induced cancer cell death via modulation of sphingolipids.

  • δTE-13′-carboxychromanol, a δ-tocotrienol derivative and found in Garcinia Kola, suppressed colon tumorigenesis in mice.

Abstract

Vitamin E forms are substantially metabolized to various carboxychromanols including 13′-carboxychromanols (13′-COOHs) that are found at high levels in feces. However, there is limited knowledge about functions of these metabolites. Here we studied δT-13′-COOH and δTE-13′-COOH, which are metabolites of δ-tocopherol and δ-tocotrienol, respectively. δTE-13′-COOH is also a natural constituent of a traditional medicine Garcinia Kola. Both 13′-COOHs are much stronger than tocopherols in inhibition of pro-inflammatory and cancer promoting cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX), and in induction of apoptosis and autophagy in colon cancer cells. The anticancer effects by 13′-COOHs appeared to be partially independent of inhibition of COX-2/5-LOX. Using liquid chromatography tandem mass spectrometry, we found that 13′-COOHs increased intracellular dihydrosphingosine and dihydroceramides after short-time incubation in HCT-116 cells, and enhanced ceramides while decreased sphingomyelins during prolonged treatment. Modulation of sphingolipids by 13′-COOHs was observed prior to or coinciding with biochemical manifestation of cell death. Pharmaceutically blocking the increase of these sphingolipids partially counteracted 13′-COOH-induced cell death. Further, 13′-COOH inhibited dihydroceramide desaturase without affecting the protein expression. In agreement with these mechanistic findings, δTE-13′-COOH significantly suppressed the growth and multiplicity of colon tumor in mice. Our study demonstrates that 13′-COOHs have anti-inflammatory and anticancer activities, may contribute to in vivo anticancer effect of vitamin E forms and are promising novel cancer prevention agents.

Introduction

Colorectal cancer is one of the leading causes of cancer death worldwide [1]. There is, however, no effective therapy for the late-stage cancer. Chemoprevention that prevents or delays the onset of late-stage cancer is an important strategy for reducing cancer-related mortality. To this end, specific forms of vitamin E may be potentially good candidates for chemoprevention. The vitamin E family consists of α-, β-, γ- and δ-tocopherol (αT, βT, γT and δT) and α-, β-, γ- and δ-tocotrienol (αTE, βTE, γTE and δTE). αT is the only vitamin E that has been tested in large intervention trials, but failed to show consistent beneficial effects in prevention of cancer in many clinical or preclinical studies [2]. On the other hand, we and others have documented that other vitamin E forms appear to be superior to αT in cancer prevention. For instance, γT, δT and γT/δT-rich mixed tocopherols but not αT inhibit development of colon cancer in preclinical animal model [3], [4], [5], [6], [7].

Despite these interesting findings, the anticancer effects of γT or δT may not solely be rooted in tocopherol themselves because these vitamin E forms are readily metabolized in the whole body environment. Except for αT, tocopherols and tocotrienols are substantially metabolized by cytochrome P450-catalyzed ω-hydroxylation and oxidation to generate 13′-carboxychromanols (13′-COOH), which are further catabolized to shorter chain carboxychromanols and sulfated carboxychromanols [3], [8], [9]. Carboxychromanols and their sulfated counterparts have been detected in the plasma and liver of rodents supplemented with γT and γTE [9], [10]. Recently, 13′-COOHs are found at high levels in feces from mice fed diet supplemented with γT, δT or mixed tocopherols [4], [11], [12], [13].

Emerging evidence suggests that long-chain carboxychroamols have bioactivities that are relevant to prevention and therapy of inflammation-associated diseases including cancer [3]. Specifically, we have demonstrated that δT-13′-COOH, a long-chain metabolite from δT, competitively inhibits cyclooxygenases (COX-1 and COX-2) and is much stronger than tocopherols or short-chain metabolites in these effects [14]. δT-13′-COOH, but not tocopherols, inhibits 5-lipoxygenase (5-LOX) [15]. It has been well recognized that COXs and 5-LOX are pro-inflammatory enzymes that play significant roles in inflammation and promote development of colon cancer [16], [17]. COX inhibitors have proven effective in decreasing colon cancer incidence in numerous clinical studies [16], [17]. Since high levels of long-chain carboxychromanols are found in feces, we reason that 13′-COOHs may play a role in prevention of colon cancer.

To test this hypothesis, we investigated anti-inflammatory and chemopreventive activities of long-chain carboxychromanols including δT-13′-COOH and δTE-13’-COOH (Fig. 1A). δT-13′-COOH used in this study is the synthetic counterpart of δ-tocopherol-derived 13′-COOH [14], [15]. δTE-13′-COOH is a metabolite from δ-tocotrienol and also found in the African Garcinia Kola, a traditional medicine used to treat osteoarthritis in West Africa [18], [19]. In our studies, we examined the effect of 13′-COOHs on COX-2 and 5-LOX, their impact on the growth and survival of various colon cancer cells and the mechanisms underlying observed anticancer actions. To translate mechanistic findings to a whole body environment, we examined the anticancer efficacy of δTE-13′-COOH against colon tumorigenesis induced by AOM/DSS (azoxymethane /dextran sodium sulfate) in mice.

Section snippets

Materials and reagents

δT-13′-COOH and δTE-13′-COOH (>95% pure) were synthesized as previously described [20]. δTE-13′-COOH (>92%) used in animal studies was isolated from the Garcinia Kola seeds based on a published procedure [18]. γT (≥96%) and δT (93-97%) were purchased from Sigma (St Louis, MO) and Suppleco (Bellefonte, PA). Arachidonic acid (AA), human recombinant COX-2 and 5-LOX were from Cayman Chemical (Ann Arbor, MI). C8-cyclopropenylceramide (C8-CPPC) was purchased from Matreya LLC (Pleasant Gap, PA). All

The effect of 13′-COOHs on the COX-2 and 5-LOX activity

We have previously shown that δT-13′COOH, which was isolated from conditioned media from cells incubated with δT, is an inhibitor of COX-1/COX-2 and 5-LOX [14], [15]. Here we found that chemically synthesized δT-13′-COOH showed similar inhibitory potency toward COX-2 and 5-LOX compared to that generated from δT metabolism in cells [14], [15] (Table 1). δTE-13′-COOH, an analog of δT-13′-COOH with three double bonds in the side chain (Fig. 1A), appeared to be slightly stronger than δT-13′-COOH in

Discussion

We demonstrate that vitamin E long-chain metabolites δT-13′-COOH and δTE-13′-COOH are dual inhibitors of COX-2 and 5-LOX and decrease viability of human colon cancer cells by induction of apoptosis and autophagy. For the anti-proliferative effect, δT-13′-COOH appears to be more potent than δTE-13′-COOH, and both 13′-COOHs are much stronger than tocopherols. Using a lipidomic approach with LC–MS/MS, we show for the first time that 13′-COOHs profoundly modulate sphingolipid metabolism.

Conflict of interest

None.

Acknowledgments

The authors would like to thank Amber S Jannasch for helps with LC-MS/MS analyses of sphingolipids. This study was in part supported by grants R21CA152588 and R01AT006882 (to QJ) from National Institutes of Health. This project was also partially supported by NIH grant P30CA023168 via Purdue Center for Cancer Research.

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