Jacaric acid, a linolenic acid isomer with a conjugated triene system, has a strong antitumor effect in vitro and in vivo

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Abstract

In this study, we compared the cytotoxic effects of natural conjugated linolenic acids (CLnAs) on human adenocarcinoma cells (DLD-1) in vitro, with the goal of finding CLnA isomers with strong cytotoxic effects. The antitumor effect of the CLnA with the strongest cytotoxic effect was then examined in mice. The results showed that all CLnA isomers have strong cytotoxic effects on DLD-1 cells, with jacaric acid (JA) having the strongest effect. Examination of the mechanism of cell death showed that CLnAs induce apoptosis in DLD-1 cells via lipid peroxidation. The intracellular levels of incorporated CLnAs were measured to examine the reason for differences in cytotoxic effects. These results showed that JA was taken into cells efficiently. Collectively, these results suggest that the cytotoxic effect of CLnAs is dependent on intracellular incorporation and induction of apoptosis via lipid peroxidation. JA also had a strong preventive antitumor effect in vivo in nude mice into which DLD-1 cells were transplanted. These results suggest that JA can be used as a dietary constituent for prevention of cancer.

Highlights

► Conjugated linolenic acid is a linolenic acid isomer with a conjugated triene system. ► Conjugated linolenic acid had a strong cytotoxic effect in tumor cells. ► Conjugated linolenic acid induced apoptosis via lipid peroxidation. ► Cytotoxic effects of fatty acids were dependent on intracellular incorporation. ► Jacaric acid had a strong preventive antitumor effect in vivo.

Introduction

Cancer is a major cause of mortality worldwide. Many anticancer therapies are available, but these are not always successful. Cancer prevention is critically important and may be achievable through dietary components with a cancer preventive effect, including conjugated linoleic acid (CLA) [1]. CLA is a fatty acid that has a conjugated double bond system and is a geometric and positional isomer of linoleic acid. CLA has various beneficial effects, including anticarcinogenic, antiobesity, antiallergic properties [2], [3], [4], [5]. CLA is found naturally, particularly in ruminant fats such as beef tallow and milk fat. However, the CLA level in the fatty acids of these foodstuffs is only about 0.5%, which prevents the use of natural fats as health-promoting food containing CLA.

Natural seed oils of certain plants include conjugated trienoic fatty acids that are isomers of α-linolenic acid (LnA, 9c12c15c-18:3) (Fig. 1) [6], [7], [8]. We have previously shown that α-eleostearic acid (α-ESA, 9c11t13t-18:3) has a stronger antitumor effect than CLA in human tumor cells [9], through a mechanism of selective induction of apoptosis in tumor cells via lipid peroxidation. Therefore, α-ESA may be useful as a food supplement for cancer prevention. Moreover, since seed oils of tung and karela (bitter gourd) include 60–80% α-ESA, it may be easier to use α-ESA in food products compared to CLA [6]. Various conjugated linolenic acids (CLnAs) also occur naturally, in addition to α-ESA (Fig. 1) [6], [7], [8]. Pomegranate seed oil contains punicic acid (PA, 9c11t13c-18:3), catalpa seed oil contains catalpic acid (CPA, 9t11t13c-18:3), tung oil and karela seed oil contain α-ESA and β-eleostearic acid (β-ESA, 9t11t13t-18:3), jacaranda seed oil contains jacaric acid (JA, 8c10t12c-18:3), and calendula seed oil contains α-calendic acid (α-CDA, 8t10t12c-18:3) and β-calendic acid (β-CDA, 8t10t12t-18:3). Each of these oils includes CLnAs at high levels and several reports have suggested cytotoxic effects of the CLnAs in tumor cells [10], [11]. However, the differences in activities among the CLnAs have not been studied.

In this study, we compared the cytotoxic effects of CLnAs from natural plant sources in human adenocarcinoma cells in vitro, with the goal of finding CLnA isomers with strong cytotoxic effects. Differences in the cytotoxic effects of CLnAs were examined and the antitumor effect of JA, which had the strongest in vitro cytotoxic effect, was also examined in vivo. Strong cytotoxic effects on DLD-1 human adenocarcinoma cells were obtained with all CLnA isomers (PA, CPA, α-ESA, β-ESA, JA, α-CDA, and β-CDA). JA and CPA exerted stronger effects than that of α-ESA, and JA had the strongest effect. Annexin V staining indicated that cell death was induced via apoptosis. Lipid peroxidation was significantly increased in DLD-1 cells exposed to CLnAs and cell death was inhibited by addition of antioxidants. These results showed that CLnAs induce apoptosis via lipid peroxidation. We also found a strong positive correlation between cytotoxic effects and cellular uptake of CLnAs. JA had a strong preventive antitumor effect in vivo in nude mice with transplanted DLD-1 cells. These results suggest that JA may be a useful dietary constituent that may serve as a cancer prevention agent.

Section snippets

Materials

RPMI-1640 medium containing 0.3 mg/mL l-glutamine and 2.0 mg/mL sodium bicarbonate, fetal bovine serum (FBS), and LnA were obtained from Sigma (St Louis, MO). Penicillin and streptomycin were products of Gibco BRL (Rockville, MD). Punicic acid (99% purity), catalpic acid (99% purity), α-eleostearic acid (99% purity), β-eleostearic acid (99% purity), jacaric acid (99% purity), α-calendic acid (99% purity), and β-calendic acid (99% purity) were purchased from Larodan Fine Chemicals AB (Malmö,

Cytotoxic effects of conjugated linolenic acids (CLnAs) in DLD-1 human adenocarcinoma cells

To find CLnAs with stronger cytotoxic effects than α-ESA, the survival of DLD-1 cells exposed to various CLnAs was measured (Fig. 2A). The survival rate of DLD-1 cells exposed to 0 μmol/L fatty acids for 24 h was defined as 100%. DLD-1 cells showed significantly decreased survival when exposed to CPA, α-ESA, JA and β-CDA at 5 μmol/L and to all CLnAs at 10 μmol/L. The half maximal inhibitory concentrations (IC50, μmol/L) were PA: 7.9, CPA: 3.6, α-ESA: 6.7, β-ESA: 8.1, JA: 3.4, α-CDA: 7.4, β-CDA:

Discussion

In this study, we showed that CLnAs induce apoptosis in human adenocarcinoma cells via lipid peroxidation and that the cytotoxic effect of CLnAs was dependent on cellular uptake. We have previously shown that α-ESA has a stronger antitumor effect than CLA [9], [25], indicating that α-ESA may be a superior health supplement. However, there are many other natural CLnA isomers, in addition to α-ESA (Fig. 1), and some of these isomers may have even stronger cytotoxic effects than α-ESA. In this

Conclusions

In this study, the results showed that all CLnA isomers have strong cytotoxic effects on DLD-1 cells, with JA having the strongest effect and that CLnAs induce apoptosis in DLD-1 cells via lipid peroxidation. JA and CPA were taken into cells more effectively than other CLnAs. Collectively, these results suggest that the cytotoxic effect of CLnAs is dependent on intracellular incorporation and induction of apoptosis via lipid peroxidation. JA also had a strong preventive antitumor effect in vivo

Acknowledgements

This study was supported by KAKENHI of JSPS, Japan (20157639). The authors declare that there are no conflicts of interest.

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