Inhibition of proliferation and induction of apoptosis by γ-tocotrienol in human colon carcinoma HT-29 cells

Abstract

Objective

γ-Tocotrienol is a major component of the tocotrienol-rich fraction of palm oil, but there is limited evidence that it has antitumor activity. In particular, the effects of γ-tocotrienol on human colon carcinoma cells have not been reported. To investigate the chemopreventive effects of γ-tocotrienol on colon cancer, we examined its capacity to inhibit proliferation and induce apoptosis in HT-29 cells and explored the mechanism underlying these effects.

Methods

We cultured HT-29 cells in the presence of γ-tocotrienol. The effect of γ-tocotrienol on cell proliferation was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, mitotic index, and colony formation. The cell-cycle distribution was investigated by flow cytometry. We measured apoptosis by nuclear staining, transmission electron microscopy, and DNA fragmentation. Apoptosis-related proteins and the nuclear factor-κB p65 protein were determined by western blotting and immunofluorescence.

Results

γ-Tocotrienol inhibited cell growth and arrested HT-29 cells in G₀/G₁ phase. The 50% inhibitory concentration was 31.7 μmol/L (48 h). γ-Tocotrienol–induced apoptosis in HT-29 cells was accompanied by downregulation of Bcl-2, upregulation of Bax, and activation of caspase-3. Furthermore, we found that γ-tocotrienol reduced the expression level of total nuclear factor-κB p65 protein and inhibited its nuclear translocation.

Conclusion

The results indicated that γ-tocotrienol inhibits cell proliferation and induces apoptosis in HT-29 cells in a time- and dose-dependent manner, and that this process is accompanied by cell-cycle arrest at G₀/G₁, an increased Bax/Bcl-2 ratio, and activation of caspase-3. Our data also indicated that nuclear factor-κB p65 protein may be involved in these effects.

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.