Bitter gourd seed fatty acid rich in 9c,11t,13t-conjugated linolenic acid induces apoptosis and up-regulates the GADD45, p53 and PPARγ in human colon cancer Caco-2 cells

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Abstract

Bitter gourd (Momordica charantia) seed oil (BGO) is a unique oil which contains 9cis, 11trans, 13trans-conjugated linolenic acid (9c,11t,13t-CLN) at a high level of more than 60%. In this study, we investigated the anti-proliferative and apoptosis-inducing effects of free fatty acids prepared from BGO (BGO-FFA) using colon cancer Caco-2 cells. BGO-FFA and purified 9c,11t,13t-CLN remarkably reduced the cell viability of Caco-2. In Caco-2 cells treated with BGO-FFA, DNA fragmentation of apoptosis indicators was observed in a dose-dependent manner. The expression level of apoptosis suppressor Bcl-2 protein was also decreased by BGO-FFA treatment. The GADD45 and p53, which play an important role in apoptosis-inducing pathways, were remarkably up-regulated by BGO-FFA treatment in Caco-2 cells. Up-regulation of PPARγ mRNA and protein were also observed during apoptosis induced by BGO-FFA. These results suggest that BGO-FFA rich in 9c,11t,13t-CLN may induce apoptosis in Caco-2 cells through up-regulation of GADD45, p53 and PPARγ.

Introduction

Conjugated fatty acids with conjugated double bonds in the molecule has been the focus of many studies because of their beneficial effects for human health. It is well known that conjugated linoleic acid (CLA) can inhibit chemically induced carcinogenesis in the colon [1] and forestomach [2]. Furthermore, CLA inhibits the growth of human colon [3], lung [4], and breast cancer cell lines [5]. CLA is a naturally occurring substance in foods such as milk fat and meats from ruminant animals. The 9cis,11trans-CLA (9c,11t-CLA) isomer is the primary dietary form of CLA in food. However, the content of 9c,11t-CLA and other CLA isomers in food is usually less than 1%.

In contrast to CLA, conjugated linolenic acid (CLN) isomers are present at much higher levels in certain seed oils [6], [7], [8]. Takagi and Itabashi [7] reported that the seed oil of tung and bitter gourd contains 67.7% and 56.2% of 9c,11t,13t-CLN, respectively, while those of pomegranate, catalpa and pot marigold contains 83% 9c,11t,13c-CLN, 42.3% 9t,11t,13c-CLN, and 62.2% 8t,10t,12c-CLN, respectively. The study at our laboratory has recently demonstrated that dietary bitter gourd seed oil (BGO), which contains a high level of 9c,11t,13t-CLN, remarkably inhibits the development of AOM-induced colonic aberrant crypt foci (ACF) [9]. Furthermore, BGO and pomegranate seed oil (PGO) suppressed AOM-induced colon carcinogenesis in 32-week long-term experiments [10], [11]. It is noteworthy that BGO administration increased apoptotic cells in ACF, without affecting the surrounding crypts [9]. Tsuzuki et al. [12] have also reported that 9c,11t,13t-CLN exhibited growth inhibition and apoptosis induction in colon cancer DLD-1 cells. However, there are a few reports regarding gene regulation by CLN during apoptosis induction in colon cancer cells.

The p53 and the growth arrest and DNA damage-inducible gene 45 (GADD45) are known to have important roles in the pathways of growth inhibition and apoptosis induction in many types of cancer cells [13], [14]. Increased expression of GADD family genes is frequently observed in response to agents that trigger apoptosis [15], [16]. In addition, GADD45 are regulated by multiple signaling such as the p53-dependent pathway [13] and the MAP kinase cascade [17]. The GADD45 and p53 gene, therefore, may be target genes for apoptosis induction by BGO-FFA rich in 9c,11t,13t-CLN.

On the other hand, peroxisome proliferator-activated receptor (PPAR)γ has been focused on as one of the target molecules to preventing cancer [18], [19], [20]. Up-regulation of PPARγ by γ-tocopherol and troglitazone has been reported in colon cancer cells [21], [22]. To elucidate the up-regulation of PPARγ by BGO, we performed real-time quantitative RT-PCR analysis in the present study.

Our results reveal that BGO-FFA rich in 9c,11t,13t-CLN induced apoptosis through up-regulation of GADD45, p53 and PPARγ in colon cancer Caco-2 cells.

Section snippets

Materials

Bitter gourd (Momordica charantia) seeds were kindly donated by Tohoku Seed Co. (Tochigi, Japan). The seeds were ground to a powder with electric mill and extracted with n-hexane three times at room temperature. BGO-FFA was prepared from BGO as described in our previous report [23]. 9c,11t,13t-CLN was purified from BGO-FFA by high-performance liquid chromatography. 9c,11t-CLA was purchased from Cayman Chemical Co. (Denver, CO, USA).

Effect of conjugated fatty acids on the viability of Caco-2 cells

The growth inhibition of conjugated fatty acids was examined using human colon cancer cell line Caco-2. BGO-FFA reduced cell viability in a dose-dependent manner (Fig. 1). The viability of Caco-2 cells treated with 25 and 50 μM of BGO-FFA were approximately 48% and 19% after 24 h incubation, respectively. 9c,11t,13t-CLN, which was purified from BGO-FFA, also markedly reduced the viability to 19% after 24 h incubation at 12.5 μM (Fig. 1). On the contrary, 9c,11t-CLA did not reduce viability of

Discussion

In the current study, we demonstrated that BGO-FFA rich in 9c,11t,13t-CLN reduced cell viability and induced apoptosis through decrease of Bcl-2 protein level in human colon cancer cell line Caco-2. Furthermore, BGO-FFA up-regulated GADD45, p53 and PPARγ in Caco-2 cells during apoptosis induction.

We recently reported that dietary administration of BGO effectively suppressed the AOM-induced ACF and adenocarcinoma in F344 rats [9], [10]. Since body and liver weights of rats given BGO diet were

Acknowledgements

This study was supported by the 21st Century COE Program “Marine Bio-Manipulation Frontier for Food Production” of the Ministry of education, Culture, Sports, Science and Technology of Japan.

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