Elsevier

Journal of Functional Foods

Volume 35, August 2017, Pages 428-435
Journal of Functional Foods

δ-Tocotrienol suppresses tumorigenesis by inducing apoptosis and blocking the COX-2/PGE2 pathway that stimulates tumor–stromal interactions in colon cancer

https://doi.org/10.1016/j.jff.2017.06.002Get rights and content

Highlights

  • δ-Tocotrienol-rich diet suppressed colorectal cancer in a mouse model.

  • δ-Tocotrienol showed the most significant anti-cancer effect in colon cancer cells.

  • δ-Tocotrienol induced apoptosis by stimulating caspase-3 and caspase-9.

  • δ-Tocotrienol affected cancer stromal cells by reducing COX-2 protein expression.

Abstract

Anticancer effects of δ-tocotrienol have been reported for several types of cancer, but have not been fully elucidated in colorectal cancer. We investigated the anti-proliferative effect of tocotrienols in vitro, in colon epithelial cells and stromal cells, and in vivo, in an induced colorectal cancer mouse model. Of the four isoforms tested, δ-tocotrienol exerted the most potent anti-proliferative effect on colon adenocarcinoma cells. δ-Tocotrienol reduced the nitrite and prostaglandin E2 (PGE2) concentrations in mouse embryonic fibroblasts (MEFs) pretreated with δ-tocotrienol and stimulated with lipopolysaccharide (LPS) and interferon γ. Furthermore, supernatants of LPS-stimulated MEFs promoted adenocarcinoma cell proliferation, while δ-tocotrienol treatment suppressed this effect. Additionally, a δ-tocotrienol-enriched diet significantly suppressed tumor formation in azoxymethane and dextran sulfate sodium-treated mice. Taken together, these data suggest that a δ-tocotrienol-enriched diet prevents colorectal cancer. At the molecular level, tocotrienols exert a direct anti-proliferative effect on colon adenocarcinoma cells, and an indirect, stromal cell-mediated, anti-proliferative effect.

Introduction

Tocotrienols are more bioactive than tocopherols and positively affect cardiovascular disease, angiogenesis, bone health, gastric injury, inflammation, and life extension (Tan, Watson, & Preedy, 2012). Several studies have reported that dietary vitamin E intake reduces the risk of colorectal cancer (Park et al., 2010, Williams et al., 2010); however, in those instances, vitamin E was defined as α-tocopherol. The effects exerted specifically by tocotrienol have been rarely reported.

While in vitro studies have shown that γ-tocotrienol or δ-tocotrienol displays a more pronounced anti-cancer activity than α-tocotrienol, reports on β-tocotrienol bioactivity are limited (Eitsuka et al., 2006, Wada et al., 2005). Reports on anti-cancer effects of tocotrienols in mammary cancer (Ahn et al., 2007, Shah et al., 2003) or prostate cancer (Luk et al., 2011) have been published. On the other hand, few studies have focused on anti-cancer effect of tocotrienol in colorectal cancer (Wada & Naito, 2012, Chap. 7).

Tocotrienols function as anti-proliferative molecules through the induction of apoptosis (Agarwal et al., 2004, Shibata et al., 2010), cell cycle arrest (Shibata et al., 2010), and telomerase inhibition (Eitsuka et al., 2006), which are all considered direct anti-cancer effects. Recently, indirect anti-cancer effects have been investigated. The involvement of fibroblasts in tumor–stromal interactions has been associated with carcinogenesis during all cancer progression stages (Kalluri & Zeisberg, 2006). Paracrine secretion of cyclooxygenase-2 (COX-2) by stromal cells plays a critical role in tumorigenesis (Ko et al., 2002). In colorectal adenomas, elevated COX-2 levels were observed in myofibroblasts, while at early cancer stages, characterized by normal mucosa and hyperplastic polyps, COX-2 was detected only in macrophages and endothelial cells (Adegboyega et al., 2004). These results indicate that myofibroblast-derived COX-2 may play a critical role in cancer progression.

It has been reported that vitamin E and its metabolites suppress COX activity in macrophages and epithelial cells (Jiang et al., 2000, Jiang et al., 2008). However, the effect of δ-tocotrienol on COX-2 or on tumor–stromal interactions has yet to be fully elucidated (Jiang, 2014).

Although the potential of tocotrienols for cancer prevention has been increasingly investigated since the 2000s, animal studies are still limited (Wada, 2012). However, in one animal study, a γ-tocopherol-rich tocopherol diet mixture inhibited colon inflammation and carcinogenesis (Ju et al., 2009). Shibata et al. (2009) have shown that α-tocopherol inhibits tocotrienol activity in vitro, and α-tocopherol content in the diet may impact the bioavailability of tocotrienols in vivo.

In this paper, we evaluated the anti-colorectal cancer activity of δ-tocotrienol, and elucidated the mechanisms of its direct and indirect anti-proliferative effect in vitro. We also investigated the preventative anti-colorectal cancer effect of tocotrienols in the presence of low and high α-tocopherol concentrations in an induced colorectal cancer mouse model.

Section snippets

Colon cancer cell culture

Mouse rectal polyploid carcinoma CMT93 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Nissui Pharmaceutical Co., Tokyo, Japan) with 10% fetal bovine serum (FBS). HT29 cells were obtained from Cell Bank, RIKEN BioResource Center (Ibaraki, Japan), and were maintained in monolayer culture in McCoy’s 5A Medium Modified (Sigma-Aldrich, Tokyo, Japan) with 10% FBS. The Colon 26 murine colon adenocarcinoma cell line (Corbett,

Anti-proliferative activity of tocotrienols in colon cancer cells

We analyzed the anti-proliferative effects of δ-tocotrienols and other vitamin E isoforms in colon cancer cells. In CMT93 cells, treatment with 20 μM δ-tocotrienol induced the most significant cell proliferation-suppressive effect, although there was no statistically significant difference with β-tocotrienol (Fig. 1A). As the effect exerted by δ-tocotrienol was the most pronounced in comparison to the other vitamin E isoforms tested, we analyzed the anti-proliferative effect of δ-tocotrienol at

Discussion

We here evidenced the anti-cancer activity of δ-tocotrienol in colorectal cancer in vitro and in vivo. From the eight vitamin E isoforms tested in colorectal cancer cells, δ-tocotrienol displayed the most pronounced anti-proliferative effect. One mechanism of this phenomenon involved the induction of apoptosis through caspase-3 and caspase-9 activation. δ-Tocotrienol also affected the tumor stromal environment, suppressing the tumor cell proliferation-promoting COX-2/PGE2 pathway. Using the

Competing interests

The authors declare that they have no conflict of interest.

Funding

This work was supported by Japan Society for the Promotion of Science Grant-in-Aid for Young Scientists (B) to SW (Grant No. 20790428), and Japan Science and Technology Agency Grant for the Program for Supporting Research Activities of Female Researchers to SW.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

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