Basic nutritional investigationInhibition of proliferation and induction of apoptosis by γ-tocotrienol in human colon carcinoma HT-29 cells
Introduction
Tocotrienols and tocopherols are two subclasses of vitamin E compounds that are abundant in food ingredients such as palm oil, rice bran oil, barley, corn, oats, rye, and wheat [1], [2]. Dietary intake of palm oil, the richest known source of natural tocotrienols and tocopherols [3], [4], suppresses chemically induced mammary tumorigenesis in female rats [5], [6]. Furthermore, the tocotrienol-rich fraction (TRF) from palm oil (36% γ-tocotrienol, 18% α-tocotrienol, 12% δ-tocotrienol, and 22% α-tocopherol) [7] also has anticarcinogenic effects on human colon carcinoma [8] and prostate cancer [9] cells in vitro. The composition of TRF is approximately 75% tocotrienols and 25% α-tocopherol, but previous studies have demonstrated that its antiproliferative effects are mediated by tocotrienols, not α-tocopherol [10], [11], [12]. It is not completely understood why tocotrienols are more potent than tocopherols, but greater cellular accumulation is at least part of the reason [13]. Natural tocotrienols exist in four different forms or isomers, i.e., α-, β-, γ-, and δ-tocotrienol, which contain different numbers of methyl groups on the chromanol ring. Although all the isomers are effective antioxidants because a hydrogen atom from the hydroxyl group on the chromanol ring can readily be donated to reduce free radicals, each has its own biological activity. In particular, γ-tocotrienol is one of the most abundant forms of tocotrienol in foods [7]. Furthermore, various studies have indicated that γ-tocotrienol has significant anticancer activity [14], [15], [16], [17]. In vivo, dietary γ-tocotrienol suppressed murine melanoma growth and increased host survival time [7]. In vitro, γ-tocotrienol proved cytotoxic to various human tumor cell lines [18], [19], [20] but had no toxic effect on the proliferation of normal cells [21], [22]. For instance, it inhibits proliferation and induces apoptosis in MDA-MB-231 [17] and Hep3B [19] cells. Recent results from our laboratory have demonstrated that γ-tocotrienol induces apoptosis and metastasis in the human gastric adenocarcinoma cell line SGC-7901 by downregulation of the extracellular signal-regulated kinase signaling pathway [14], [20]. However, details of the mechanism by which γ-tocotrienol inhibits proliferation and induces apoptosis in tumor cell lines remain unclear.
Colon carcinoma is a serious health problem and one of the leading causes of cancer mortality worldwide, especially in developed countries [23]. Chemoprevention is a major strategy in cancer prevention, because therapies have not proved effective to date in controlling the high incidence or the low survival rate of human colon carcinoma [24]. Dietary factors that inhibit cell proliferation are an exciting prospect for cancer prevention and treatment. A 4-y study provided convincing data that a high vitamin E intake was associated with a reduced risk of colon carcinoma, particularly for women younger than 65 y [25]. It has been reported recently that the TRF has antiproliferative effects and induces apoptosis in human colon carcinoma RKO cells [8]. However, the effects of individual TRF components on human colon carcinoma cell proliferation and the possible mechanisms involved remain unclear. γ-Tocotrienol has potent biological and pharmacologic activities. The objectives of our present study were to evaluate the effects of γ-tocotrienol on proliferation and apoptosis in HT-29 cells and to investigate the underlying molecular mechanism. Our results suggest that a possible molecular mechanism involves the suppression of nuclear factor-κB (NF-κB) p65 protein expression and its nuclear translocation, resulting in a direct effect on cell-cycle progression and activation of the proapoptotic pathway.
Section snippets
Materials
The human colon carcinoma HT-29 cell line was obtained from the Cancer Institute of the Chinese Academy of Medical Science. The Cycle Test PLUS DNA reagent kit was bought from Becton-Dickinson (Franklin Lanes, NJ, USA). γ-Tocotrienol was from Davos (Singapore). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethylsulfoxide were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Giemsa stain was purchased from Amresco (USA). Rabbit polyclonal antibodies for
Effect of γ-tocotrienol on growth of HT-29 cells
The effects of γ-tocotrienol on the viability of HT-29 cells are shown in Figure 1. After treatment with 15, 30, 45, and 60 μmol/L of γ-tocotrienol for 48 h, cell viability decreased by 26.8%, 52.3%, 64.2%, and 68.4% (Fig. 1A), respectively. The viability of cells cultured in 30 μmol/L of γ-tocotrienol for 24, 48, 72, and 96 h decreased with time (Fig. 1B) compared with the control group. These results showed that γ-tocotrienol inhibits the growth of HT-29 cells in a dose- and time-dependent
Discussion
Chemoprevention by natural compounds from plant or in diets appears to be a practical approach to preventing and treating carcinoma, and it is estimated that diets rich in phytochemicals can reduce the cancer risk by 20% [35], [36]. γ-Tocotrienol is one of the most important components of vitamin E, which is enriched in high-lipid plants. Previous studies have shown that γ-tocotrienol in very low doses exerts antiproliferative and apoptotic activities on various types of cancer cells including
Conclusion
Our data suggest that γ-tocotrienol may potently inhibit cell growth and induce apoptosis of HT-29 cells by up- or downregulation of the expression of Bcl-2 family proteins. Furthermore, these results indicate that NF-κB p65 protein may participate in the regulation of cell survival and induction of apoptosis by γ-tocotrienol.
Acknowledgments
The authors thank Prof. Ying Ma from the School of Food Science and Engineering, Harbin Institute of Technology, for her helpful suggestions and assistance. They also thank Neal Okarter from Food Science, Cornell University, for reading and revising this manuscript.
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This project was supported by the National Natural Science Foundation of China (grant 30471444).