Mechanistic Investigation of Resveratrol-, Pterostilbene-, and δ-Tocotrienol-Mediated Anti-Nf-κb Activity and the Effect of δ-Tocotrienol on Colitis-Promoted Colon Tumorigenesis in Mice

Chao Yang, Purdue University

Abstract

Nuclear factor-κB (NF-κB) is a key transcription factor that regulates inflammatory responses and plays an important role in cancer development. Inhibition of NF-κB has been shown to attenuate inflammation and cancer progression and thus has become a strategy for prevention of chronic inflammation and cancer. This dissertation focused on the mechanistic investigation of the inhibitory effect on NF-κB activation by three natural compounds including resveratrol (Res), pterostilbene (Pte), and δ-tocotrienol (δTE), in macrophages and further evaluating the anti-cancer effect of δTE in a preclinical model. Res, a polyphenol rich in grapes and red wine, has been reported to inhibit NF-κB signaling in macrophages. However, the underlying mechanism is not completely understood. Pte, a natural dimethylated analog of Res found in blueberries, is shown to have higher bioavailability than Res. However, few studies have been conducted to evaluate the effect of Pte on NF-κB signaling. Therefore, in the first project we compared the effect of Res and Pte on modulating NF-κB signaling and investigated potential underlying mechanisms in Raw 264.7 macrophages. Both Res and Pte inhibited TNF-α induced NF-κB activation in a time- and dose- dependent manner. Res showed stronger inhibitory effect than Pte in this activity. Furthermore, we found that both Res and Pte potently inhibited TNF-α-induced phosphorylation of TAK1, a key upstream kinase essential for NF-κB activation. In search for the underlying mechanisms, we observed that Res treatment induced intracellular elevation of ceramides, sphingosine and dihydrosphingosine, which resulted in an elevation of cellular stress, as was reflected by the enhanced phosphorylation of JNK. Importantly, Res and Pte disrupted the formation of TRAF-2 and TAK1 complex that is essential for the activation of TAK1. Our study elucidates that Res and Pte inhibit NF-κB and its upstream signaling TAK1 via modulation of sphingolipid metabolism that leads to cellular stress and disruption of TRAF2-TAK1 interaction. δ-Tocotrienol (δTE) is a natural vitamin E form rich in palm oil and annatto seeds and has been shown to inhibit NF-κB in pancreatic cancer cells. However, whether δTE inhibits NF-κB activation in immune cells or the underlying mechanism have not been investigated. In the second project, we found that δTE inhibited TNF-α induced activation of NF-κB in a dose-dependent manner. Furthermore, δTE potently inhibited TNF-α-induced phosphorylation of TAK1, an upstream kinase that is required for the activation of NF-κB. Interestingly, δTE significantly increased the expression of A20 and CYLD, both endogenous negative regulators of NF-κB. Knockout of A20 partially diminished δTE's anti-NF-κB effect, supporting the notion that induction of A20 plays a key role in δTE's anti-NF-κB effect. Our previous work indicates that γ-tocotrienol, an analog of δTE, induced A20 via cellular stress and modulation of sphingolipids. Here we found that δTE treatment had a profound impact on de novo sphingolipids synthesis as was reflected by elevation of both dihydroceramides and ceramides. The modulation of sphingolipids was accompanied by increased phosphorylation of JNK, indicating induction of cellular stress. Importantly, δTE's induction of A20 and inhibitory effect on NF-κB activation were partially reversed by myriocin, a potent inhibitor of sphingosine biosynthesis, further confirming the critical role of sphingolipid metabolism in δTE's anti-NF-κB activity. It is well-established that inflammation promotes the development of colorectal cancer. My second project showed that δTE exerts potent anti-inflammatory effect by inhibiting NF-κB, a key transcription factor in inflammatory responses, in macrophages. δTE-13'-carboxychromanol (δTE-13') is a long chain metabolite of δTE that is found abundantly present in feces. Mechanistic studies showed that δTE-13' potently inhibited pro-inflammatory cycloxygenase-2 (COX-2) and induced the apoptosis and autophagy of colon cancer cells. To translate these mechanistic findings to a physiological environment, we further investigated the chemopreventive effect of δTE and δTE-13' on azoxymethane (AOM) induced and colitis-promoted colon tumorigenesis in male BALB/c mice. We found that δTE-rich mixed tocotrienols (δTE /γTE ~8/1, at 0.035% diet) inhibited colon tumorigenesis, as indicated by 36% reduction of multiplicity of large adenomatous polyps (> 2mm2, P < 0.05). Strikingly, δTE-13' (at 0.04% diet) significantly suppressed total polyps by 25% (P<0.05) and large polyps by 54% (P < 0.01). Our ongoing analyses indicate that δTE and δTE-13' appeared to modulate gut microbiota including elevation of Lactococcus. In addition, we identified that δTE-13' was the most abundant metabolite in the feces of mice fed with both δTE and δTE-13'. Our study demonstrates that δTE and its metabolite, δTE-13', effectively suppressed colitis-promoted colon tumorigenesis in mice. The modulation of gut microbiota probably contributes to their anti-cancer activity.

Degree

Ph.D.

Advisors

Jiang, Purdue University.

Subject Area

Molecular biology|Biochemistry|Nutrition

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